Signaling Pathways in Squamous Cancer

Signaling Pathways in Squamous Cancer Adam B. Glick Carter Van Waes ● Editors Signaling Pathways in Squamous Can...

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Signaling Pathways in Squamous Cancer

Adam B. Glick Carter Van Waes ●

Editors

Signaling Pathways in Squamous Cancer

Editors Adam B. Glick, Ph.D. Associate Professor Center for Molecular Toxicology and Carcinogenesis Department of Veterinary and Biomedical Sciences The Pennsylvania State University and Department of Dermatology Penn State Milton S. Hershey Medical Center [email protected]

Carter Van Waes, M.D., Ph.D. Clinical Director and Chief, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, Senior Investigator, Radiation Oncology Branch, National Cancer Institute National Institutes of Health [email protected]

ISBN 978-1-4419-7202-6 e-ISBN 978-1-4419-7203-3 DOI 10.1007/978-1-4419-7203-3 Springer New York Dordrecht Heidelberg London  Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Squamous epithelia form the lining surface of tissues in contact with the environment, including the skin, upper aerodigestive, respiratory and genital tracts, and several other specialized tissues. Cancers that form in squamous epithelia are among the most common human solid tumors due to increased exposure to environmental carcinogens such as ultraviolet light, tobacco smoke, and other genotoxic compounds, as well as infectious agents such as human papilloma virus. Late stage cancers of the upper aerodigestive tract, esophagus and cervix have high morbidity and there has been little improvement in survival. Thus there is compelling need to identify critical signaling pathways that regulate the development of squamous cancer and translate these findings into therapeutic targets to improve patient survival. In general, squamous epithelia are multilayered or stratified epithelia in which proliferation is confined to the basal layer in contact with the basement membrane, and squamous differentiation occurs as cells move away from the basal layer. As in any epithelium, proliferation and differentiation are tightly regulated by signaling pathways that respond to the external tissue and cellular microenvironment, and become dysregulated during progression to malignancy. This text addresses some of the most important signaling pathways that regulate normal growth and differentiation in squamous epithelia; how they are altered during progression to carcinoma; and their potential as therapeutic targets. The reviews include studies from human squamous cancers and cancer cell lines, as well as mouse two-stage skin carcinogenesis and genetically engineered mice, which provide meaningful animal models for the development of squamous cancers in multiple tissues. Because these different squamous tissues likely share similar regulatory networks, studies in one tissue or animal model are likely to have general significance for cancer development and therapy in other squamous epithelia. While each chapter focuses on a specific pathway and its role in squamous cancer, it is clear that these represent a network of interacting pathways that control many different aspects of normal keratinocyte homeostasis. Alterations in any one pathway during cancer progression are likely to impact several others. Interaction of epithelial cells with the extracellular matrix in the basement membrane through integrin receptors is critical for tissue integrity and control of epithelial cell proliferation. Chapters by Dr. Kramer, University of Pennsylvania and Dr. DiPersio, Albany Medical College and colleagues review recent studies on the alterations in v

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expression and adhesive interactions between integrins and their ECM ligands that drive local tissue invasion and progression squamous cell carcinoma. Equally important, extracellular signals that regulate cell growth come from both positive and negative growth factor signaling pathways. The epidermal growth factor receptor family and its ligands are critical regulators of both normal keratinocyte proliferation and differentiation, and aberrant expression and activation of this pathway is a consistent feature of squamous cancers. Chapters by Dr. Hansen, Creighton University and Dr. Grandis, University of Pittsburgh and their colleagues discuss recent data on the role of the epidermal growth factor receptor in mouse models of squamous cancer, human head and neck squamous cell carcinoma (HNSCC) and targeting this signaling pathway for therapy of HNSCC. The role of another important growth factor pathway, HGF/cMet, in the development of squamous cell cancer, and therapeutic targeting of this pathway is also discussed in a chapter by Dr. Zhong Chen, NIDCD, NIH. Transforming growth factor beta (TGFb1) is a critical negative regulator of keratinocyte proliferation but with important autocrine and paracrine roles in cancer pathogenesis that may both inhibit and enhance the malignant phenotype. The chapter by Drs. Reiss and Xie, UMDNJ-Robert Wood Johnson Medical School, examines the role of the TGFb1 signaling pathway and mutations in this pathway in HNSCC. Many of these growth factor pathways activate intracellular signaling molecules that are the center of important regulatory nodes controlling proliferation differentiation and inflammatory signaling in keratinocytes. Thus, this book contains several chapters which review studies in humans and mouse models that indicate an important role of Ras (Drs. Cataisson and Yuspa, NCI), Protein Kinase C (Dr. Denning, Loyola University), AKT and mTOR (Dr. Nathan et al., LSU; Drs. Lin and Rocco, Harvard Medical School; Dr. Gukind et al., NIDCR) and Cox-2 (Drs. Rundaug and Fischer, UT M.D. Anderson Cancer Center) in the development of squamous cancer, and the potential of these molecules as therapeutic targets. These signaling pathways converge on two transcription factor families AP-1 and NF-kB that play critical roles in gene expression that regulates keratinocyte proliferation, differentiation and inflammatory signaling. Drs. Bowden and Alberts, University of Arizona, and Hess and Angel, German Cancer Research Center review studies on ultraviolet activation of AP-1 signaling and potential therapeutic targets in this pathway and the role of AP-1 in mouse skin carcinogenesis, while Dr. Karin and colleagues, University of California, San Diego discuss recent studies on the role of NF-kB and IkB kinases in squamous cancer and the interaction with other signaling pathways. Other nuclear transcription factor families play critical roles in normal keratinocyte homeostasis and are frequently altered during progression to squamous cell carcinoma. Chapters on PPARs (Drs. Peters and Gonzalez, Pennsylvania State University and NCI), p. 63 (Drs. Roop and Koster, University of Colorado, Denver), retinoic acid receptors (Drs. Kadara and Lotan, UT M.D. Anderson Cancer Center) and vitamin D receptors review the important role of these transcription factor families in the regulation of epidermal proliferation and differentiation their role in squamous cancer. Finally the chapter by Drs. Zhou, Hu, and Wong, University of California at Los Angeles, describes

Preface

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recent advances in high throughput molecular profiling as a means to identify new genomic alterations and therapeutic targets for oral cancer. While this is not an exhaustive survey of all signaling pathways that regulate squamous cancer development we hope that pulling together this diverse research into one monograph will provide potential for cross-fertilization between researchers studying different aspects of squamous cancer, stimulate new research directions and highlight potential new targets for therapeutic intervention. The editors would like to thank their colleagues who contributed chapters to this book and to everyone in the research community that have made significant contributions to our understanding of this disease. Adam B. Glick Carter Van Waes

Acknowledgments

CVW supported by NIDCD Intramural Research Projects ZIA-DC-000016, 000073 and 000074.

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Contents

1 Cell Adhesion Molecules in Carcinoma Invasion and Metastasis......... Barry L. Ziober, Joseph O. Humtsoe, and Randall H. Kramer

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2 Roles of Integrins in the Development and Progression of Squamous Cell Carcinomas.................................................................. John Lamar and C. Michael DiPersio

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3 Alterations of Transforming Growth Factor-b Signaling in Squamous Cell Carcinomas.................................................................. Wen Xie and Michael Reiss

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4 Aberrant Activation of HGF/c-MET Signaling and Targeted Therapy in Squamous Cancer........................................... Zhong Chen

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5 The Epidermal Growth Factor Receptor in Normal and Neoplastic Epithelia............................................................................ 113 Susan K. Repertinger, Justin G. Madson, Kyle J. Bichsel, and Laura A. Hansen 6 Cyclooxygenase-2 Signaling in Squamous Cell Carcinomas................. 131 Joyce E. Rundhaug and Susan M. Fischer 7 Interacting Signaling Pathways in Mouse Skin Tumor Initiation and Progression............................................................ 149 Christophe Cataisson and Stuart H. Yuspa 8 Protein Kinase C and the Development of Squamous Cell Carcinoma........................................................................................... 165 Mitchell F. Denning

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Contents

9 The Transcription Factor AP-1 in Squamous Cell Carcinogenesis: Lessons from Mouse Models of Skin Carcinogenesis............................................................................. 185 Jochen Hess and Peter Angel 10 NF-kB, IkB Kinase and Interacting Signal Networks in Squamous Cell Carcinomas................................................................ 201 Antonio Costanzo, Giulia Spallone, and Michael Karin 11 Regulation of Squamous Cell Carcinoma Carcinogenesis by Peroxisome Proliferator-Activated Receptors.................................. 223 Jeffrey M. Peters and Frank J. Gonzalez 12 p63 in Squamous Differentiation and Cancer....................................... 241 Dennis R. Roop and Maranke I. Koster 13 Effects of Natural and Synthetic Retinoids on the Differentiation and Growth of Squamous Cancers................... 261 Humam Kadara and Reuben Lotan 14 Regulation of Keratinocyte Differentiation by Vitamin D and Its Relationship to Squamous Cell Carcinoma.............................. 283 Arnaud Teichert and Daniel D. Bikle 15 Epidermal Growth Factor Receptor-Targeted Therapies.................... 305 Sun M. Ahn, Seungwon Kim, and Jennifer R. Grandis 16 Targeting UVB Mediated Signal Transduction Pathways for the Chemoprevention of Squamous Cell Carcinoma...................... 335 G. Tim Bowden and David S. Alberts 17 Molecular-Targeted Chemotherapy for Head and Neck Squamous Cell Carcinoma..................................................... 365 Harrison W. Lin and James W. Rocco 18 Effects and Therapeutic Potential of Targeting Dysregulated Signaling Axes in Squamous Cell Carcinoma: Another Kinase of Transcription and Mammalian Target of Rapamycin............................................................................... 383 Cheryl Clark, Oleksandr Ekshyyan, and Cherie-Ann O. Nathan 19 Head and Neck Cancer and the PI3K/Akt/mTOR Signaling Network: Novel Molecular Targeted Therapies................... 407 Panomwat Amornphimoltham, Vyomesh Patel, Alfredo Molinolo, and J. Silvio Gutkind

Contents

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20 High Throughput Molecular Profiling Approaches for the Identifications of Genomic Alterations and Therapeutic Targets in Oral Cancer............................................... 431 Xiaofeng Zhou, Shen Hu, and David T. Wong Index.................................................................................................................. 453

Contributors

Sun M. Ahn Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA David S. Alberts Department of Medicine, College of Medicine, University of Arizona, Tucson, AZ, USA and Arizona Cancer Center, University of Arizona, Tucson, AZ, USA Panomwat Amornphimoltham Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, Bethesda, MD, USA Peter Angel DKFZ-ZMBH Alliance, Division of Signal Transduction and Growth Control (A100), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany [email protected] Kyle J. Bichsel Biomedical Science, Creighton University School of Medicine, Omaha, NE, 68178, USA Daniel D. Bikle Endocrine Unit, University of California, San Francisco, CA, USA G. Tim Bowden Department of Cell Biology and Anatomy, Arizona Cancer Centre, University of Arizona, College of Medicine, University of Arizona, Tucson, AZ, USA and Arizona Cancer Center, University of Arizona, Tucson, AZ, USA [email protected] xv

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Contributors

Christophe Cataisson Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH, Bethesda, MD, 20892, USA Zhong Chen Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 10 Center Drive, Building 10, 5D55, Bethesda, MD 20892, USA [email protected] Cheryl Clark Department of Otolaryngology-Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, USA and Feist-Weiller Cancer Center, Shreveport, LA, USA Antonio Costanzo Department of Dermatology, University of Rome “Tor Vergata”, 00133, Rome, Italy Mitchell F. Denning Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60153, USA [email protected] C. Michael DiPersio Center for Cell Biology & Cancer Research, Albany Medical College, 47 New Scotland Avenue, MC 165, Albany, NY 12208, USA [email protected] Oleksandr Ekshyyan Department of Otolaryngology-Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, USA and Feist-Weiller Cancer Center, Shreveport, LA, USA Susan M. Fischer The University of Texas M.D. Anderson Cancer Center, Science Park – Research Division, Smithville, TX 78957, USA [email protected] Frank J. Gonzalez Laboratory of Metabolism, National Cancer Institute, Bethesda, MD, 20892, USA

Contributors

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Jennifer R. Grandis Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA [email protected] J. Silvio Gutkind Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, 30 Convent Drive, Building 30, Room 212, Bethesda, MD 20892-4340, USA [email protected] Laura A. Hansen Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA [email protected] Jochen Hess DKFZ-ZMBH Alliance, Division of Signal Transduction and Growth Control (A100), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany Shen Hu School of Dentistry, Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, CA, USA Joseph O. Humtsoe Department of Cell and Tissue Biology, University of California San Francisco, 521 Parnassus Avenue, Room C-640, San Francisco, CA, 94143-0640, USA Humam Kadara Department of Thoracic/Head and Neck Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA Michael Karin Laboratory of Gene Regulation and Signal Transduction, School of Medicine, University of California, San Diego, 9500 Gilman Drive MC 0723, La Jolla, CA 92093-0723, USA [email protected] Seungwon Kim Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

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Contributors

Maranke I. Koster Department of Dermatology, Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado Denver, Aurora, CO 80045, USA [email protected] Randall H. Kramer Department of Cell and Tissue Biology, University of California San Francisco, 521 Parnassus Avenue, Room C-640, San Francisco, CA 94143-0640, USA [email protected] John Lamar Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, E17-23002139, USA [email protected] Harrison W. Lin Department of Otolaryngology (H.W.L., J.W.R.), Massachusetts Eye and Ear Infirmary, Boston, MA, USA and Department of Otology and Laryngology (H.W.L., J.W.R), Harvard Medical School, Boston, MA, USA and Department of Surgery (J.W.R.), Massachusetts General Hospital, Boston, MA, USA Reuben Lotan Department of Thoracic/Head and Neck Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA [email protected] Justin G. Madson Departments of Biomedical Science, Creighton University School of Medicine, Omaha, NE, 68178, USA and Department of Dermatology, The University of Oklahoma College of Medicine, Oklahoma City, OK, 73126, USA Alfredo Molinolo Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, Bethesda, MD, USA

Contributors

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Cherie-Ann O. Nathan Department of Otolaryngology-Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, USA and Feist-Weiller Cancer Center, Shreveport, LA, USA [email protected] Vyomesh Patel Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, Bethesda, MD, USA Jeffrey M. Peters Department of Veterinary and Biomedical Sciences, The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802, USA [email protected] Michael Reiss Division of Medical Oncology, Department of Internal Medicine, The Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA [email protected] Susan K. Repertinger Department of Pathology, Creighton University School of Medicine, Omaha, NE, USA James W. Rocco Department of Surgery, Massachusetts General Hospital, Jackson 904G, 55 Fruit Street, Boston, MA 02114, USA [email protected] Dennis R. Roop Department of Dermatology, Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado Denver, Aurora, CO, 80045, USA Joyce E. Rundhaug The University of Texas M.D. Anderson Cancer Center, Science Park – Research Division, Smithville, TX, 78957, USA Giulia Spallone Department of Dermatology, University of Rome “Tor Vergata”, 00133, Rome, Italy

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Contributors

Arnaud Teichert Endocrine Unit, University of California, San Francisco, CA, USA [email protected] David T. Wong School of Dentistry, Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, CA, USA Wen Xie Division of Medical Oncology, Department of Internal Medicine, The Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ, 08901, USA Stuart H. Yuspa Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH, Bethesda, MD 20892, USA [email protected] Xiaofeng Zhou Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois at Chicago, Chicago, IL, USA [email protected] Barry L. Ziober Oncology, Centocor Ortho Biotech Inc, Radnor, PA, 19087, USA and Department of Otorhino-laryngology: Head and Neck Surgery, University of Pennsylvania, Philadelphia, PA, 19104, USA

Chapter 1

Cell Adhesion Molecules in Carcinoma Invasion and Metastasis Barry L. Ziober, Joseph O. Humtsoe, and Randall H. Kramer

Abstract The primary reason for treatment failure in patients with head and neck squamous cell carcinoma (HNSCC) is local tumor cell invasion. HNSCC invasion is a necessary component of metastasis where tumor cells infiltrate into adjacent tissues, degrading basement membranes and extracellular matrix (ECM), and disrupting tissue architecture. These adhesive interactions of integrins with their ECM ligands are important not only in physically modulating HNSCC migration and invasion, but also in regulating the pathways required for survival and continued tumor expansion. During tumor progression, tumor cells must overcome a hostile microenvironment that can include hypoxia, growth factor deprivation, and loss of adhesion to the ECM. A second class of receptors expressed in HNSCC, the cadherins, form intercellular adhesions and are also relevant to the invasive process. These cell–cell adhesions are responsible for forming stratifying cell layers, but also influence the differentiated state of the tumor cells, and tend to restrain invasion. In these epithelial tumors, cadherin engagement can promote cell survival by a process termed “synoikis” that involves the receptor tyrosine kinase, EGFR. The complex signaling pathways transduced by integrin and cadherin receptors are poorly understood but are known to coordinately regulate such diverse cellular processes as apoptosis, proliferation, and the invasive phenotype.

1.1 Introduction Despite significant advancements in surgery, radiation, and chemotherapy, only about half of individuals diagnosed with head and neck squamous cell carcinoma (HNSCC) will survive for 5 years. HNSCC spreads by local and distant metastasis, and it is this aspect of the disease that is the most challenging for developing

R.H. Kramer (*) Department of Cell and Tissue Biology, University of California San Francisco, 521 Parnassus Avenue, Room C-640, San Francisco, CA 94143-0640, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_1, © Springer Science+Business Media, LLC 2011

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s uccessful clinical approaches to therapy. The rich lymphatic drainage from the oral cavity and the highly invasive behavior of HNSCC facilitate the early spread to regional lymph nodes. Timely detection of malignant lesions is the most critical step at the present time to reduce the morbidity and mortality of HNSCC, but it is important that we gain a better understanding about the basic mechanisms of invasion that lead to recurrence and metastasis. The process of squamous cell carcinoma invasion and dissemination requires active cell migration through the extracellular matrix with the simultaneous remodeling of intercellular adhesions. These cellular processes are poorly understood and more effort is needed to identify signaling pathways regulating the invasive tumor phenotype, including how specific adhesion receptors are involved. Integrins are one class of adhesion receptors that mediate interactions with the surrounding extracellular matrix (ECM) and are clearly important in the invasive process. The intercellular adhesion receptors, predominately the cadherins, are another class of adhesion receptors that may restrain invasion and promote a more differentiated phenotype. In various carcinomas, cadherins can modulate cell locomotion by contact inhibition that can also lead to suppression of cell growth. Importantly, there is evidence for cross-talk and synergy between the two types of receptors whereby they act together to control various cellular functions.

1.2 Overview of Cell–ECM Adhesion Epithelial cell–matrix adhesion complexes mechanistically and functionally link cells to their underlying basement membrane. The underlying basement membrane is rich in ligands such as collagens, laminins, and fibronectin. It acts as an adhesive scaffolding to help maintain anchorage and organize cell layers and tissue architecture. The basement membrane also provides an important barrier between the epithelium and lamina propria. The integrins, a family of cell transmembrane receptors, are responsible for mediating the cell’s adhesive interactions with the ECM ligands (Patarroyo et al. 2002). Integrins provide a linkage between the cell cytoskeleton and the extracellular environment. Each integrin is a heterodimer composed of a noncovalently associated a and b subunit. In the case of head and neck squamous cell carcinoma (SCC), the major integrin receptors include the a2b1, a3b1, a5b1, a6b1/a6b4, and the av complexes (reviewed Ziober and Kramer 2003; Ziober et al. 2001). It is now well recognized that integrin interactions with the ECM can transduce signaling cascades that are not only important for adhesive and migratory functions, but are also important for cell growth, apoptosis, epithelial-mesenchymal transition, angiogenesis, protease production, differentiation, and gene expressions – all properties involved in malignant conversion and invasion (Aumailley et al. 2003; Goldfinger et al. 1998, 1999; Kosmehl et al. 1999; Niki et al. 2002; Shang et al. 2001). The interaction between integrins and laminins can regulate most, it not all, of these properties.

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1.3 Laminin-332 and SCC 1.3.1 Laminin-332 Expression in SCC Laminins are large, heterotrimeric extracellular glycoproteins composed of an a, b, and g subunit. To date, more than 12 different laminin heterotrimers have been identified (Colognato and Yurchenco 2000). The primary laminin expressed in stratified epithelium and derived carcinomas is laminin-5 (new nomenclature, laminin 332 Aumailley et al. 2005). Laminin-332 is composed of an a3, b3, and g2 subunit (Nguyen et al. 2000; Rousselle et al. 1991) and plays a significant role in SCC tumor biology. In SCC, laminin-332 is overexpressed primarily at the invasive front (Pyke et al. 1995). It was determined by immunohistochemistry and in situ hybridization, that in normal mucosa and lichen planus, laminin-332 was present as a thin, continuous line located in the basement membrane region. In epithelial dysplasia, this staining was discontinuous and more diffuse (Kainulainen et al. 1997). However, there was a strikingly intense cytoplasmic staining of the carcinoma cells along the invasive border and in the individual infiltrating carcinoma cells in invasive carcinomas and lymph node metastasis (Kainulainen et al. 1997). As tumor invasion is typically associated with hypoxic environments, recent evidence has determined that the hypoxia induced transcription factor HIF-1 contributes to cell motility and invasion by up-regulating laminins-5 expression and deposition (Fitsialos et al. 2008). These observations indicate that in SCC tumor progression, synthesis and secretion of laminin-332 are altered; the most intense expression occurs at the hypoxic invasive front and in the lymph node metastasis. Furthermore, it has been demonstrated that along with tumor cells, mesenchymal cells contribute to the synthesis and deposition of laminin-332 at the invasive front (Franz et al. 2007). Finally, it has been demonstrated that within distinct domains of laminin-332 reside sites for normal tumor adhesion as well as sites required for SCC tumorigenesis (Waterman et al. 2007). In addition to being expressed and contributing to SCC development and progression, laminin-332 has also been identified as a clinically unique tumor marker. Ono et al. found a significant correlation between laminin-332- immunopositive SCC cells and increased infiltrative growth and poorer differentiation (Ono et al. 1999). In addition, these same researchers, by analyzing patient survival data, found that increased laminin-332 expression was significantly associated with poorer patient outcome. Furthermore, several reports have indicated that the laminin-332 g2 chain is expressed in SCC of the skin, colon, esophagus, larynx, oral cavity, and recurrent lesions of these cancers (Ginos et al. 2004; Patarroyo et al. 2002; Patel et al. 2002). Additionally, as described above, immunohistochemical staining indicates that the g2 chain of laminin-332 is dispersed in the invasive front and is a good indicator of a less favorable outcome (Kosmehl et al. 1999; Niki et al. 2002; Pyke et al. 1995). As a monomer, the g2 chain has been found frequently expressed in several cancers without the expression of the α3 or b3 chains (Koshikawa et al. 1999; Seftor et al. 2001). As such, the g2 chain of laminin-332

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has been detected circulating in patients’ serum with several cancer types and in particular invasive carcinomas (Katayama et al. 2005). Furthermore, oral mucosal lesions that express the laminin-332 g2 chain show an increased risk for tumor progression (Nordemar et al. 2003). Recently, a monoclonal antibody to the g2 chain has been developed that can specifically detect the monomer g2 chain and not the laminin-332 heterotrimer (Koshikawa et al. 2008). This antibody should be a powerful diagnostic tool for detecting potential oral cancer lesions, g2-monmer expressing tumor cells and can also be a possible agent in cancer therapy. Overall, increased laminin-332 expression may provide a significant marker for SCC and tumor invasion, and be indicative of a poorer outcome for patients with SCC.

1.3.2 Role of Laminin-332 Processing in SCC The laminin-332 (Ln-5) heterotrimer is secreted as a 460-kDa precursor that undergoes specific proteolytic processing after secretion (Fig. 1.1). Ln-5 extracted from tissue is composed of a3 (165 or 145 kDa), b3 (145 kDa), and g2 (105 kDa) chains. In culture, keratinocytes synthesize a precursor form of a3 (190 kDa) and g2 (155 kDa) chains, which are processed to the tissue forms extracellularly (Ebihara et al. 2000; Koshikawa et al. 2000). Ln-5 is considered fully processed when the heterotrimer contains a 145-kDa a3 chain, a 145-kDa b3 chain, and a 105-kDa g2 chain. Recently

Cadherin Integrin

BM

Fig. 1.1 Adhesion receptors in head and neck squamous cell carcinoma cells. Integrins and c adherins mediate cell–matrix and cell–cell adhesions, respectively. Integrins are heterodimeric adhesion receptors and provide a linkage between the cell cytoskeleton and the extracellular environment. Cadherins form junctional adhesions between adjacent epithelial cells and involve the linker proteins, catenins, and the cytoskeleton. During tumor invasion, migrating cell penetrate the underlying laminin-rich basement membrane matrix (BM) and interact with the lamina propria interstitial extracellular matrix.

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it has been shown that the laminin-332 b3 chain is also proteolytically processed. The processing of all 3 chains of laminin-332 suggests another level of complexity in regards to the functions and activities of laminin-332. The function of Ln-5 processing is somewhat controversial. It appears that different proteolytically cleaved products of laminin-332 can yield diverse cellular responses when secreted and deposited. In normal tissues, the G4–G5 region of the laminin-332 a3 chain is proteolytically removed. However, in SCC, this a3 region is retained and plays a role in tumorigenesis by activating cell signaling pathways, metalloproteinase production, invasion, and tumorigenicity in vivo (Tran et al. 2008). In agreement, Bachy et al. have shown that retention of the a3 precursor chain in laminin-332 provides a binding site for integrin a3b1 and syndecan-1 which when bound together triggers intracellular events required for keratinocyte migration (Bachy et al. 2008). The a3 chain of laminin-332 can also be cleaved by several enzymes including plasmin and astacins (bone morphogenic protein-1 and m-tolloid) (Amano et al. 2000; Veitch et al. 2003). Cleavage by these enzymes occurs between the globular modules 3 and 4 (LG3 and LG4) of the a3 chain favoring hemidesmosome assembly and stability. In support, the recombinant LG3 module of the a3 chain has been shown to support cell adhesion and migration in an integrin a3b1-dependent manner (Shang et al. 2001). Unlike the a3 chain, cleavage of the g2 chain by matrix metalloproteinase-2 (MMP-2), membrane type-1 matrix metalloproteinase (MT1-MMP) or astacins can promote binding of a3b1 to the a3 chain of laminin-332 and induction of cell migration (Giannelli et al. 1997; Koshikawa et al. 2000). Expression of both MMP-2 and MT1-MMP are up-regulated (Dumas et al. 1999; Ondruschka et al. 2002; Yoshizaki et al. 1997), while reports have indicated both a loss and a gain of the astacins in head and neck SCC (Veitch et al. 2003; Ziober et al. 2006). Recent work has demonstrated that only when g2 is completely processed is Ln-5 incorporated into the ECM, resulting in cells that are more resistant to enzymatic detachment (Gagnoux-Palacios et al. 2001). These results suggest that complete processing of Ln-5 is required for ECM development and the formation of stable adhesions. Conversely, unprocessed g2 subunits may support cell motility until processed. In support of this concept, we have recently shown in the oral cancer cell lines JHU022 and UM-SCC1 that the g2 chain is fully processed in JHU-022 while it is not processed in UM-SCC1 (Fig. 1.2) (Yuen et al. 2005; Ziober in preparation). Furthermore, UM-SCC1 cells contain higher levels of GTP bound Rac1 and are more mobile and invasive through MatrigelTM than the JHU-022 cells (Yuen et al. 2005; Ziober in preparation; Fig. 1.2). Interestingly, UM-SCC1 invasion can be inhibited by infection with an adenoviral vector expressing a dominant negative Rac1. Together, these results suggest that failure to process Ln-5, in particular the g2 chain, may lead to activation of Rac1, cell motility, and tumor invasion. Finally and in contrast, it has been suggested that cell motility requires the processing of the g2 chain to the 105-kDa form (Veitch et al. 2003). It is believed that the g2 chain can negatively regulate the phosphorylation of laminin-332 binding integrin a6b4 and hemidesmosome formation (cell adhesion). This activity of the g2 chain involves syndecan-1 binding and its signaling which eventually leads to enhanced

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γ2

β3

Laminin 332

integrin

1 2

155 kDa 5 3 4

G domain

c

UM-SCC1

b

α3

JHU-022

a

Laminin-5 γ2 chain

105 kDa

d

Invasion through Matrigel

Rac1-GTP Total cellular Rac1

Fig. 1.2 Unprocessed Ln-5 correlates with increased Rac1 activity and head and neck squamous cell carcinoma (HNSCC) invasion. (a) Structure of Laminin-332 (Laminin-5). Laminin 5 is a heterotrimer consisting of the a3, b3, and g2 chains forming the typical laminin cross-shaped structure. Integrin receptors bind to the base region of the molecule. Both the a3 and g2 chains can be processed by proteolytic cleavage as indicated. (b) Conditioned medium from the poorly invasive JHU-022 and the more invasive UM-SCC1 HNSCC cell lines were immunoblotted with an anti-human laminin-332 g2 chain antibody. (c) Cell lysates from JHU-022 and UM-SCC1 HNSCC cell lines were assayed in the GTPase pulldown assay for Rac1 activity. GTP bound Rac1 (top) and total cellular Rac1 (bottom) were immunoblotted with an anti-human Rac1 antibody. (d) SCC1 cells were infected with an adenoviral vector expressing green fluorescent protein (GFP) or dominant negative Rac1 (N17; DN-Rac1). Expression of DN-Rac1 in the UM-SCC1 cells inhibited cellular invasion in the Matrigel invasion assay as compared to both the control (uninfected) and GFP-infected SCC1 cells.

cell adhesion and inhibition of cell motility (Ogawa et al. 2007). Thus, it is possible that the binding of syndecan-1 to the g2 chain may be an important factor influencing cell migration or adhesion. More work is required to fully understand g2 processing and cell motility. Finally, the b3 chain of laminin-332 has recently been found to be cleaved as well. The processing of the b3 chain can occur by matrilysin (MMP-7) and hepsin, (Nakashima et al. 2005; Remy et al. 2006; Tripathi et al. 2008) and appears to be important for matrix assembly as well as cell adhesion and motility. More work is required to identify the regulatory factors responsible for laminin-332 cleavage and to understand how these cleavage products regulate the static and dynamic mechanisms of cell movement and tumor invasion.

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1.4 Laminin-332 Receptors and SCC Invasion and Motility 1.4.1 Signaling Pathways Activated by a3b1 Integrin SCC cells from the oral cavity express a number of integrin receptors for laminin, including a3b1, a6b1, and a6b4. The integrin a3b1 is now considered to be the major laminin-332 receptor (Baker et al. 1996; Mizushima et al. 1997) and has been reported to be responsible for mediating cell spreading and motility on laminin-332 substrates (Carter et al. 1991; DiPersio et al. 1997; Zhang and Kramer 1996). a3b1 is up-regulated in several SCC tumor cell lines and biopsies (Jones et al. 1996). In particular, a3b1 is increased in the suprabasilar area during development of SCC (Van Waes 1995). In contrast, reduced expression of a3b1 has been reported to correlate with poor histological differentiation in SCC (Shang et al. 2001). Based on these studies, it is likely that a3b1 plays a significant role in SCC tumor development and tumor invasion; however, more thorough studies are required. Previously, it has been shown that laminin-332 promoted rapid cell scattering in HNSCC cells, whereas fibronectin and collagen-I did not (Kawano et al. 2001). The role of GTPases is probably important in such integrin-mediated responses to specific ECM ligands and may be important during tumor invasion. On laminin-332 substrate, a3b1 integrin preferentially inactivated RhoA and induced activation of Cdc42 and PAK1, and thereby promoted migration of oral SCC cells (Zhou and Kramer 2005). In contrast, on type I collagen, a3b1 integrin strongly activated RhoA, leading to enhanced focal contact formation, and thereby hindered cell migration. These results suggest that Rho signaling in SCC may be important in defining a cell’s invasive phenotype. Activation of RhoA/ROCK may be important in regulating invasion of HNSCC cells. For example, laminin-332 and a3b1 integrin have the ability to inactivate the RhoA pathway (Zhou and Kramer 2005) and enhance cell motility and invasion. Clearly, further studies are needed to establish whether inactivation of RhoA/ ROCK promotes invasion and metastasis in head and neck cancer. However, the RhoA/ROCK pathway plays an important role in metastasis in other malignancies including those of the bladder and breast cancer, and melanoma (Kamai et al. 2003; Nakajima et al. 2003; Nishimura et al. 2003). The influence of RhoA/ROCK in this process can be explained by work done by Sahai and Marshall (2003) where they identified two types of tumor cell motility in 3-dimensional matrices that involved different Rho signaling functionality and mode of invasion. Rho signaling through ROCK promoted a rounded bleb-associated mode of motility that does not require pericellular proteolysis. In contrast, elongated cell motility did not require Rho, ROCK, or ezrin function but was rather dependent on Rac for cell movement. Thus, it is apparent that the RhoA/ROCK pathway is a strong regulator of cell motility and invasion. a3b1 appears to be the major receptor for activating this pathway as well as other pathways required for SCC tumor development and progression. Adhesion of a3b1 to laminin-332 can stimulate a signaling pathway involving mitogen-activated protein kinase (MAPK), which appears to be important in

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r egulating cell growth and survival, thus presumably contributing to the development of SCC (Gonzales et al. 1999). In addition, a recent study has indicated that a3b1, when bound to laminin-332, can induce cross talk between the beta-catenin and Smad signaling pathways. When activated by laminin-332, these two pathways along with TGF-b1 can activate epithelial-mesenchymal transition during injury, tumor development, and tumor invasion (Giannelli et al. 2005; Kim et al. 2009). Similarly, a3b1 has also been shown to contribute to tumor invasion by up-regulating MMP-9 during p53 null and activated H-Ras epithelial cell transformation (Lamar et al. 2008). Likewise, a3b1 when attached to laminin-332 regulates Src kinase signaling through FAK. This pathway eventually activates Rac1 and promotes lamellipodium extension which is indicative of migratory/invasive cells (Choma et al. 2004). Together, it appears that laminin-332 and a3b1 binding stimulates the phenotypic changes and pathways required for cell motility and invasion. However, work using mice that lack a3b1 specifically in the basal layer of the epidermis has indicated that a3b1 can also delay keratinocyte migration during wound healing (Margadant et al. 2009). Thus, it will take further investigations to decipher how a3b1 contributes to the migratory/invasive properties of SCC. For example, the migratory/invasive phenotype displayed by SCC tumor cells may be a result of signaling pathways activated by engagement of both a3b1 and the a6 integrin subunits to laminin-332.

1.4.2 Signaling Pathways Activated by a6b4 Integrin The a6 integrin is another important laminin-binding receptor in SCC cells (Zhang et al. 1996; Van Waes et al. 1991). a6 can pair with b1 or b4 in SCC cells, potentially giving rise to two laminin receptors. Because a6 preferentially combines with b4, this usually is the dominant complex found in SCC. In contrast to a3b1, a6b4 was originally believed to be involved in the static structures known as hemidesmosomes and to contribute little to cell migration. The extracellular laminin-332 anchoring molecules are directly linked with the keratin filament network within the cell by hemidesmosomes (Nguyen et al. 2000). During wound healing, the dermal-epidermal adhesive structure provided by a6b4 is undesirable. Thus, for epithelial cells to migrate, they must first disassemble their hemidesmosomes (Goldfinger et al. 1999). In fact, SCC tumor invasion may represent a normal wound repair process, involving laminin-332 and its two primary receptors a6b4 and a3b1 that are no longer properly controlled. a6 has been reported to show a higher expression in non-metastatic cells than in metastatic SCC cell lines (Jones et al. 1996). However, this subunit is upregulated in tumor biopsies from invasive and metastatic cases (Jones et al. 1996). Analysis of a6b4 levels in poorly differentiated SCCs revealed pronounced expression of this receptor along with laminin-332 at the invasive front (Mercurio et al. 2001). Furthermore, in tumors of patients with SCC, a6b4 levels have also been reported to be increased, localizing along the invasive border (Ilic and Damsky 2002).

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Earlier studies indicated that in normal squamous epithelium, a6b4 expression is limited to the contact sites of the basement membrane. In aggressive SCC, recurrent tumors, and metastatic tumor cell lines, this normal basal polarity of a6b4 is lost and there is a more intense suprabasal staining of a6b4 (Juliano 2002; Wolf and Carey 1992). This suggests that cytoplasmic or membrane components, possibly CD151 and bullous pemphigoid antigens 1 and 2, which function in a6b4 polarity and hemidesmosome formation, may be defective or lacking during SCC tumor progression (St Croix et al. 1996; St Croix and Kerbel 1997). Recent work using mouse tumor-initiating cells has begun to answer some of the questions regarding a6b4’s role in tumor development and progression. In this study, it was shown that a6b4 by not binding to laminin-332 but rather by recruiting the cytoplasmic linker protein, plectin, to the plasma membrane can suppress tumor growth (Raymond et al. 2007). In contrast, when mouse tumor-initiating cells were further transformed with Ras, a6b4 stimulated tumor growth. Thus, depending on the transformation context, a6b4 can either mediate adhesion-independent tumor suppression or act as a tumor promoter. An interesting question arises as to the regulation of a6b4 in normal epithelial architecture versus tumor invasion: if a6b4 is primarily involved in inhibiting cell migration through the formation of hemidesmosomes, why does an increased expression of this receptor not inhibit SCC tumor invasion? Studies directed at understanding the signaling pathways elicited by a6b4, including the signaling pathways that disrupt the hemidesmosome structure, the factors responsible for proper cell polarity, and the proteolytic regulation of the motility-inducing laminin-332 isoforms, are required to answer this question. For example, in keratinocytes phosphorylation of the cytoplasmic tail of a6b4 by protein kinase C-delta redistributes this integrin from the hemidesmosome to the cytosol (Alt et al. 2001). It should also be noted that the cytoplasmic interactions of a6b4 may also play important roles in whether this receptor functions in cell adhesion or motility. In this regard, BPAG1, which localizes to the inner surface of hemidesmosomal structures containing a6b4, positively influences cell migration when removed by homologous recombination (Day et al. 1999). In agreement with this work, it has recently been shown that BPAGe1 is required for efficient regulation of keratinocyte polarity and migration by activating Rac1 (Hamill et al. 2009). This body of work suggests that a6b4 is probably a more important regulator of SCC cell motility and tumor invasion than originally thought. a6b4, by signaling to Rac1 and the actin-severing protein, cofilin, regulates the assembly of laminin-332 tracks required for keratinocyte migration (Kligys et al. 2007). Furthermore, a6b4 via activation of Rac1, 14-3-3 proteins, and slingshot phosphatase family members (SSH) can regulate cell polarity and migration (Kligys et al. 2007). Interestingly, that cofilin is a major regulator and is involved in invadopodia, suggests that a6b4, laminin-332, and the Rac1/SSH pathway may be instrumental in regulating SCC tumor invasion. Finally, more recent studies have confirmed this work by showing that 14-3-3zeta/tau heterodimers regulate the activity of SSH and cytoskeleton remodeling during cell migration in keratinocytes (Kligys et al. 2009).

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Decreased expression of a6b4, in direct contrast to the reports above, has also been described in SCC. Normally, a6b4 expression shows a basally polarized distribution. Lack of a restricted basal polarity of a6b4, by absence of b4 expression, has been suggested to be an early marker of oral malignancy (Garzino-Demo et al. 1998). Examination of normal and epithelial SCC tissue sections for a6 expression demonstrated that the staining intensity of this subunit was significantly reduced in SCC compared to normal epithelium (Maragou et al. 1999). Similarly, a6b4 was found to be down-regulated in oral SCC, and this reduction in a6b4 correlated with poor histological differentiation (Shang et al. 2001). Thus, loss of a6b4 and the absence of hemidesmosome formation may result in a more motile and invasive SCC tumor. This notion is supported by the fact that blocking antibodies to b4 can result in stimulation of cell migration and an increase in MMP-2 activity (Daemi et al. 2000). Finally, transfection of the b4 subunit into a neoplastic keratinocyte cell line failed to restore differentiation capacity or proliferation properties, suggesting that a6b4 is not required for these properties in SCC (Jones et al. 1996).

1.5 Intercellular Adhesion and Signaling In normal epithelial cells, survival, growth, and proliferation are dependent on the coordinated involvement of ECM and growth factors (Cabodi et al. 2004; Miranti and Brugge 2002). The loss of attachment to ECM can result to cellular stress that eventually may trigger programmed cell death or anoikis (Frisch and Screaton 2001; Gilmore 2005). However, during tumor progression, SCC cells have the ability to adapt to adverse microenvironmental conditions that include hypoxia as well as nutrient, growth factor, and anchorage deprivation. For tumor cells to continue to survive and grow, they may use adaptive mechanisms that favor survival through inter- and intracellular signaling pathways. The cellular processes controlling these signaling pathways remain complex and poorly understood. Understanding how SCC cells become insensitive to anoikis is important because such survival mechanism may not only permit continued tumor expansion, but may also favor tissue invasion and metastasis. One mechanism by which carcinoma cells may overcome these diverse biological stresses is through their ability to form intercellular adhesions (Alt-Holland et al. 2005; Bates et al. 2000).

1.5.1 E-Cadherin Mediated Signaling In the absence of proper cell attachment to ECM, acquisition of cell–cell adhesion may induce critical signaling pathways to promote survival and growth regulating responses (Bates et al. 2000; Santini et al. 2000). Typical SCCs are characterized as nests of 3-dimensional aggregates or tumor islands with an extensive network of intercellular adhesions. Many of the cells in the tumor nests lack a direct interaction

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with the surrounding extracellular matrix and these cells could depend on the survival signaling generated by cell–cell contacts. In some SCCs, cell aggregation induces adhesion-mediated signaling that is dependent on the engagement of cellular adhesion receptors, such as E-cadherin. For example, previous work in a 3-dimensional multicellular model showed that E-cadherin-dependent intercellular adhesion mediated the anchorage independent survival and growth of SCC cells (Kantak and Kramer 1998). Another study further demonstrated that cell–cell adhesion through E-cadherin could trigger a ligand-independent transactivation of EGFR to promote survival primarily through activation of the ERK signaling pathway (Shen and Kramer 2004). These studies have led to the use of the Greek term, synoikis, to describe the process of intercellular adhesion-dependent cell survival (Kramer et al. 2005; Shen and Kramer 2004). Other studies in Ewing tumor (Kang et al. 2007; Lawlor et al. 2002) and breast epithelial cells (Fournier et al. 2008) have shown that E-cadherin can mediate survival and growth in anchorage independent cultures. The E-cadherin mediated Ewing cell spheroids induce PI3-kinase through the ErbB4 tyrosine kinase receptor (Kang et al. 2007). In different experimental settings where the cells in study were subjected to ECM-adherent condition, E-cadherin engagement has been shown to activate various signaling components that also affect cellular function. For example, E-cadherin was found to mediate the induction of MAPK (Pece and Gutkind 2000; Reddy et al. 2005), Rac (Betson et al. 2002; Kovacs et al. 2002), STAT3 (Onishi et al. 2008), and PI3-kinase (Pang et al. 2005) activity following cell–cell contact formation in monolayer adherent conditions. This intercellular adhesion-mediated signal transduction appears to involve several tyrosine and non-tyrosine protein kinases. The activation of MAPK and Rac appears to occur through EGFR signaling (Betson et al. 2002; Pece and Gutkind 2000; Reddy et al. 2005), while PI3-kinase is controlled by c-Src kinases (McLachlan et al. 2007; Pang et al. 2005). The JAK and Src signaling pathway also seems to play a major role in regulating E-cadherin induced STAT3 activation (Onishi et al. 2008). Using recombinant E-cadherin-Fc to ligate E-cadherin, Liu et al. (2006) have shown that direct E-cadherin engagement alone is sufficient to promote proliferation through Rac activation. Besides E-cadherin’s role in serving as a mechanical support between neighboring cells, it is strongly evident that E-cadherin can also directly influence and modulate distinct signaling pathways potentially dictating cellular functions in a context dependent manner. A recent study showed that intercellular adhesion is sufficient to induce STAT3 activation in HNSCC (Onishi et al. 2008). This observation is in support of other previous studies in monolayer adherent culture conditions where cell–cell contact induced enhanced STAT3 activation (Vultur et al. 2004). STAT3, a member of the STAT family proteins, is generally activated by multiple receptor and nonreceptor tyrosine kinases in response to various cytokines, hormones, and growth factors (Levy and Darnell 2002). Constitutive activation of STAT3 has been demonstrated in several human cancers, including HNSCC (Grandis et al. 2000). It has been suggested that STAT3 signaling induced by E-cadherin-mediated cellular adhesions may also play a role in conferring resistance to anoikis in SCC cells (Onishi et al. 2008). Understanding how intercellular adhesions in SCC cells activate STAT3 is

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important since targeting STAT3 is being viewed as an effective therapeutic strategy in SCC of the head and neck (Boehm et al. 2008; Leeman et al. 2006). Recent studies add more to the growing evidence that suggests that intercellular adhesion mediated signaling may recruit different signaling components depending on the cell type. For example, aggregations of colonic epithelial cells mediate cell survival through Src and PI3-kinase signaling that involves beta-catenin (Hofmann et al. 2007). Aggregates of malignant pleural mesothelioma cells have also been shown to exhibit the ability to resist anoikis (Daubriac et al. 2009). This study showed that the inactivation of the SAPK/JNK signaling pathway due to loss of anchorage may be involved. Because of its role in survival and growth, further studies to elucidate the molecular mechanism of intercellular adhesion-mediated signaling is needed.

1.5.2 E-Cadherin Mediated EGFR Activation The molecular signaling networks and mechanisms promoting synoikis appear to be complex and remain to be understood. One of the prime signaling axes that appears to regulate this process in SCCs is through the E-cadherin-mediated ligandindependent activation of EGFR (Shen and Kramer 2004). Despite the existing evidence of a molecular crosstalk between E-cadherin and EGFR signaling, the mechanisms by which E-cadherin transactivates EGFR still remain unclear. Earlier work from Kemler’s group (Hoschuetzky et al. 1994) showed that the cytoplasmic E-cadherin/EGFR association is mediated through interaction with b-catenin. Subsequent reports from others provide evidence that the extracellular region of E-cadherin is necessary for its interaction with EGFR (Fedor-Chaiken et al. 2003; Qian et al. 2004). Several studies carried out in adherent or anchorage independent condition have demonstrated ligand-independent EGFR activation and the ability of EGFR to associate with E-cadherin (Fedor-Chaiken et al. 2003; Pece and Gutkind 2000; Shen and Kramer 2004). These studies point to the competence of EGFR to associate with E-cadherin as an essential event in the process of E-cadherin induced EGFR activation (Gavard and Gutkind 2008). In addition to this finding, our laboratory has analyzed the EGFR/E-cadherin interactions following chemical cross-linking and resolution in two-dimensional gel (Fig. 1.3). First, it is evident that for efficient physical interactions between the two receptors, a stable cell–cell contact is essential. Secondly, it reveals that EGFR can exist in multiple structural complexes. The ability of EGFR to form these ligand-independent higher order structures appears to be dependent on the extent of cell–cell contact formation. These structures may represent the different EGFR oligomers that are formed during adhesion-mediated receptor activation. Generally, EGFR undergoes homo- and heterodimerization in response to ligand binding, an initial event in the EGFR signaling pathway (Jorissen et al. 2003; Schlessinger 2002). It is possible that upon initiation of stable cell–cell adhesions through E-cadherin, EGFR may dimerize or heterodimerize with other EGFR family members,

High density (ML) Ca++ Switch Assay

Low density (ML)

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EGFR

E-Cad

EGFR

EGTA

E-Cad EGFR

Ca++

E-Cad

MCA

EGFR E-Cad

Fig. 1.3 Two-dimensional gel analysis of EGFR structural complexes induced during cell–cell adhesions. The cell membrane impermeable protein crosslinker, DTSSP, was added to HSC-3 cells cultured at very low- and high-density or as multicellular aggregates (MCA) (Onishi et al. 2008). The high-density monolayer cell culture was subjected to a Ca++-switch assay prior to DTSSP treatment. Cell lysates were then prepared for immunoprecipitation with an EGFR antibody. The EGFR immunocomplex was then resolved in the first dimension (4% non-reduced tube gel), followed by incubation in b-mercaptoethanol containing buffer, and then resolved in the second dimension (7% reducing slab gel). Western blotting was then performed for EGFR and E-cadherin. Arrowheads indicate monomers, while the arrows indicate the slower mobile structures representing homo- and heteroligomerization.

or become physically associated with other cell surface molecules, including E-cadherin (Fig. 1.3). The formation of such hetero-oligomers could then lead to higher EGFR aggregation, and thus EGFR transactivation in a ligand-independent manner (Gavard and Gutkind 2008). Nevertheless, the presence of multiple EGFR forms reflects the complexity of the mechanism of EGFR signaling that is induced by E-cadherin mediated intercellular adhesions. In addition, the nature of E-cadherin dependent EGFR signaling needs to be further addressed. For example, such mode of EGFR signaling appears to be distinct from that of ligand-induced signaling events (Humtsoe and Kramer, submitted). While ligand treatment can stimulate efficient phosphorylation of EGFR tyrosine residues, intercellular adhesion alone produces inefficient tyrosine phosphorylation at residue EGFR-Y1086. Furthermore, in the multicellular spheroid model, intercellular adhesion-mediated EGFR

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a ctivation alone fails to transduce efficient PI3K/AKT signaling which eventually regulates cell proliferation (Humtsoe and Kramer, submitted). Perhaps, in tumor islands of cadherin-mediated compact cell aggregates, the nature and specificity of EGFR signaling may be determined by the extensiveness of cell–cell junctions and the microenvironment. Unraveling the signaling mechanism that drives the process of synoikis can be greatly challenging. Signals produced by intercellular adhesion-mediated activation maybe important for prolonged survival and growth of tumor cell aggregates that are commonly present in SCC lesions in vivo. The dissemination of metastatic cells occurs when malignant cells detach from their primary tumor site and infiltrate to surrounding tissues. The malignant cells develop mechanisms to suppress anoikis while transiting through the ECM-deficient lymphatic and vascular systems. Thus, in malignant cell aggregates, cell survival through synoikis may be advantageous in rendering a greater likelihood of implanting and forming metastatic lesions. Selective targeting of cell–cell adhesion-induced signaling pathways during tumor metastasis may represent an effective therapeutic approach. Interestingly, maintenance of cadherin-mediated cell–cell interactions not only promotes cell survival, but enhances resistance to chemotherapeutic agents (Kang et al. 2007; Nakamura et al. 2003). Future studies are needed to elucidate the complicated crosstalk of cell surface molecules in cell–cell and cell–ECM adhesion and in understanding how these interactions induce a complex array of important downstream signaling pathways.

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Kligys K, Claiborne JN, DeBiase PJ, Hopkinson SB, Wu Y, Mizuno K, Jones JC (2007) The slingshot family of phosphatases mediates Rac1 regulation of cofilin phosphorylation, laminin-332 organization, and motility behavior of keratinocytes. J Biol Chem 282:32520–32528 Kligys K, Yao J, Yu D, Jones JC (2009) 14-3-3zeta/tau heterodimers regulate Slingshot activity in migrating keratinocytes. Biochem Biophys Res Commun 383:450–454 Koshikawa N, Moriyama K, Takamura H, Mizushima H, Nagashima Y, Yanoma S, Miyazaki K (1999) Overexpression of laminin gamma2 chain monomer in invading gastric carcinoma cells. Cancer Res 59:5596–5601 Koshikawa N, Giannelli G, Cirulli V, Miyazaki K, Quaranta V (2000) Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5. J Cell Biol 148:615–624 Koshikawa N, Minegishi T, Nabeshima K, Seiki M (2008) Development of a new tracking tool for the human monomeric laminin-gamma 2 chain in vitro and in vivo. Cancer Res 68:530–536 Kosmehl H, Berndt A, Strassburger S, Borsi L, Rousselle P, Mandel U, Hyckel P, Zardi L, Katenkamp D (1999) Distribution of laminin and fibronectin isoforms in oral mucosa and oral squamous cell carcinoma. Br J Cancer 81:1071–1079 Kovacs EM, Ali RG, McCormack AJ, Yap AS (2002) E-cadherin homophilic ligation directly signals through Rac and phosphatidylinositol 3-kinase to regulate adhesive contacts. J Biol Chem 277:6708–6718 Kramer RH, Shen X, Zhou H (2005) Tumor cell invasion and survival in head and neck cancer. Cancer Metastasis Rev 24:35–45 Lamar JM, Iyer V, DiPersio CM (2008) Integrin alpha3beta1 potentiates TGFbeta-mediated induction of MMP-9 in immortalized keratinocytes. J Invest Dermatol 128:575–586 Lawlor ER, Scheel C, Irving J, Sorensen PH (2002) Anchorage-independent multi-cellular spheroids as an in vitro model of growth signaling in Ewing tumors. Oncogene 21:307–318 Leeman RJ, Lui VW, Grandis JR (2006) STAT3 as a therapeutic target in head and neck cancer. Expert Opin Biol Ther 6:231–241 Levy DE, Darnell JE Jr (2002) Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3:651–662 Liu WF, Nelson CM, Pirone DM, Chen CS (2006) E-cadherin engagement stimulates proliferation via Rac1. J Cell Biol 173:431–441 Maragou P, Bazopoulou-Kyrkanidou E, Panotopoulou E, Kakarantza-Angelopoulou E, Sklavounou-Andrikopoulou A, Kotaridis S (1999) Alteration of integrin expression in oral squamous cell carcinomas. Oral Dis 5:20–26 Margadant C, Raymond K, Kreft M, Sachs N, Janssen H, Sonnenberg A (2009) Integrin alpha3beta1 inhibits directional migration and wound re-epithelialization in the skin. J Cell Sci 122:278–288 McLachlan RW, Kraemer A, Helwani FM, Kovacs EM, Yap AS (2007) E-cadherin adhesion activates c-Src signaling at cell-cell contacts. Mol Biol Cell 18:3214–3223 Mercurio AM, Rabinovitz I, Shaw LM (2001) The alpha6beta4 integrin and epithelial cell migration. Curr Opin Cell Biol 13:541–545 Miranti CK, Brugge JS (2002) Sensing the environment: a historical perspective on integrin signal transduction. Nat Cell Biol 4:E83–E90 Mizushima H, Takamura H, Miyagi Y, Kikkawa Y, Yamanaka N, Yasumitsu H, Misugi K, Miyazaki K (1997) Identification of integrin-dependent and -independent cell adhesion domains in COOHterminal globular region of laminin-5 alpha 3 chain. Cell Growth Differ 8:979–987 Nakajima M, Hayashi K et al (2003) Effect of Wf-536, a novel ROCK inhibitor, against metastasis of B16 melanoma. Cancer Chemother Pharmacol 52:319–324 Nakamura T, Kato Y, Fuji H, Horiuchi T, Chiba Y, Tanaka K (2003) E-cadherin-dependent intercellular adhesion enhances chemoresistance. Int J Mol Med 12:693–700 Nakashima Y, Kariya Y, Yasuda C, Miyazaki K (2005) Regulation of cell adhesion and type VII collagen binding by the beta3 chain short arm of laminin-5: effect of its proteolytic cleavage. J Biochem 138:539–552 Nguyen BP, Ryan MC, Gil SG, Carter WG (2000) Deposition of laminin 5 in epidermal wounds regulates integrin signaling and adhesion. Curr Opin Cell Biol 12:554–562 Niki T, Kohno T et al (2002) Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas. Am J Pathol 160:1129–1141

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Shang M, Koshikawa N, Schenk S, Quaranta V (2001) The LG3 module of laminin-5 harbors a binding site for integrin alpha3beta1 that promotes cell adhesion, spreading, and migration. J Biol Chem 276:33045–33053 Shen X, Kramer RH (2004) Adhesion-mediated squamous cell carcinoma survival through ligandindependent activation of epidermal growth factor receptor. Am J Pathol 165:1315–1329 St Croix B, Kerbel RS (1997) Cell adhesion and drug resistance in cancer. Curr Opin Oncol 9:549–556 St Croix B, Florenes VA, Rak JW, Flanagan M, Bhattacharya N, Slingerland JM, Kerbel RS (1996) Impact of the cyclin-dependent kinase inhibitor p27Kip1 on resistance of tumor cells to anticancer agents. Nat Med 2:1204–1210 Tran M, Rousselle P, Nokelainen P, Tallapragada S, Nguyen NT, Fincher EF, Marinkovich MP (2008) Targeting a tumor-specific laminin domain critical for human carcinogenesis. Cancer Res 68:2885–2894 Tripathi M, Nandana S, Yamashita H, Ganesan R, Kirchhofer D, Quaranta V (2008) Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression. J Biol Chem 283:30576–30584 Van Waes C (1995) Cell adhesion and regulatory molecules involved in tumor formation, hemostasis, and wound healing. Head Neck 17:140–147 Van Waes C, Kozarsky KF, Warren AB, Kidd L, Paugh D, Liebert M, Carey TE (1991) The A9 antigen associated with aggressive human squamous carcinoma is structurally and functionally similar to the newly defined integrin alpha 6 beta 4. Cancer Res 51:2395–2402 Van Waes C, Surh DM et al (1995) Increase in suprabasilar integrin adhesion molecule expression in human epidermal neoplasms accompanies increased proliferation occurring with immortalization and tumor progression. Cancer Res 55:5434–5444 Veitch DP, Nokelainen P et al (2003) Mammalian tolloid metalloproteinase, and not matrix metalloprotease 2 or membrane type 1 metalloprotease, processes laminin-5 in keratinocytes and skin. J Biol Chem 278:15661–15668 Vultur A, Cao J, Arulanandam R, Turkson J, Jove R, Greer P, Craig A, Elliott B, Raptis L (2004) Cell-to-cell adhesion modulates Stat3 activity in normal and breast carcinoma cells. Oncogene 23:2600–2616 Waterman EA, Sakai N, Nguyen NT, Horst BA, Veitch DP, Dey CN, Ortiz-Urda S, Khavari PA, Marinkovich MP (2007) A laminin-collagen complex drives human epidermal carcinogenesis through phosphoinositol-3-kinase activation. Cancer Res 67:4264–4270 Wolf GT, Carey TE (1992) Tumor antigen phenotype, biologic staging, and prognosis in head and neck squamous carcinoma. J Natl Cancer Inst Monogr 13:67–74 Yoshizaki T, Sato H, Maruyama Y, Murono S, Furukawa M, Park CS, Seiki M (1997) Increased expression of membrane type 1-matrix metalloproteinase in head and neck carcinoma. Cancer 79:139–144 Yuen HW, Ziober AF, Gopal P, Nasrallah I, Falls EM, Meneguzzi G, Ang HQ, Ziober BL (2005) Suppression of laminin-5 expression leads to increased motility, tumorigenicity, and invasion. Exp Cell Res 309:198–210 Zhang K, Kramer RH (1996) Laminin 5 deposition promotes keratinocyte motility. Exp Cell Res 227:309–322 Zhang K, Kim JP, Woodley DT, Waleh NS, Chen YQ, Kramer RH (1996) Restricted expression and function of laminin 1-binding integrins in normal and malignant oral mucosal keratinocytes. Cell Adhes Commun 4:159–174 Zhou H, Kramer RH (2005) Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1. J Biol Chem 280:10624–10635 Ziober BL, Kramer RH (2003) Adhesion receptors in oral cancer invasion. In: Ensley JF, Gutkind JS (eds) Head and neck cancer. Elsevier Science, New York, pp 65–79, Chap 6 Ziober BL, Silverman SS Jr, Kramer RH (2001) Adhesive mechanisms regulating invasion and metastasis in oral cancer. Crit Rev Oral Biol Med 12:499–510 Ziober AF, Falls EM, Ziober BL (2006) The extracellular matrix in oral squamous cell carcinoma: friend or foe? Head Neck 28:740–749

Chapter 2

Roles of Integrins in the Development and Progression of Squamous Cell Carcinomas John Lamar and C. Michael DiPersio

Abstract The identification of therapeutic targets for inhibiting malignant p rogression and metastasis remains a critically important step in combating cancer mortality in the clinic. Integrins, the major cell surface receptors for cell adhesion to the extracellular matrix, are involved in all stages of carcinogenesis and are promising targets for anti-cancer therapies. Indeed, roles for integrins in cancer have been the focus of intense investigation since this family of cell adhesion receptors was first discovered in the early 1980s, and many studies during the past three decades have described critical functions for integrins expressed on carcinoma cells in controlling proliferation, survival, migration, and angiogenesis. In addition to mediating cell adhesion, integrins serve as conduits of signal transduction across the plasma membrane, thereby mediating information flow between the interior of the tumor cell and the extracellular microenvironment that promotes angiogenesis and drives malignant growth and metastasis. Although a number of integrin antagonists are currently in pre-clinical and clinical development, the repertoire of integrins that is expressed by tumor cells varies considerably among different types of cancer. Therefore, the most effective combination of integrins to target will vary among cancer types and must be determined in each case. In this chapter, we will provide an overview of current knowledge regarding tumor-promoting functions of integrins that are expressed in squamous cell carcinoma (SCC), and we will consider the prospect of exploiting these integrins as therapeutic targets for inhibiting SCC in the clinic.

C.M. DiPersio (*) Center for Cell Biology & Cancer Research, Albany Medical College, 47 New Scotland Avenue, MC 165, Albany, NY 12208, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_2, © Springer Science+Business Media, LLC 2011

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2.1 Introduction Normal structure, function, and repair of stratified epithelial tissues are regulated by adhesive interactions of individual cells with both neighboring cells and the extracellular matrix (ECM), and abnormal changes in cell adhesion contribute to the development of squamous cell carcinoma (SCC) (Janes and Watt 2006). Integrins are the major receptors for cell adhesion to the ECM (Hynes 2002), while epithelial cell-cell interactions are controlled largely through cadherins, although there is considerable crosstalk between these two receptor families, as reviewed elsewhere (Chen and Gumbiner 2006; DiPersio 2008). While integrins are well known for their roles in cell adhesion, they can also modulate signal transduction pathways that control a variety of cell functions important in normal and pathological tissue remodeling, including proliferation, survival, motility, cytoskeletal dynamics, and gene expression (Hynes 2002; Giancotti and Ruoslahti 1999; Ridley et al. 2003). Indeed, integrins are important at every stage of carcinogenesis (Janes and Watt 2006; Kramer et al. 2005; Ziober et al. 2001), and they are potential therapeutic targets for inhibiting cancer progression (Rust et al., 2002; Stupp and Ruegg 2007). This chapter provides an overview of current knowledge regarding the roles that integrins play in the development and malignant progression of SCC. Initial sections offer a brief review of known integrin functions in normal stratified epithelia, which provides the foundation for subsequent sections that are focused on integrin functions in SCC. The latter sections are structured to emphasize key concepts regarding various mechanisms that are used by different integrins to regulate SCC cell functions, and they draw on specific examples to illustrate these concepts. As space limitations preclude a complete discussion of the numerous published studies that have contributed to this field, the reader is directed to several excellent reviews for further coverage of relevant topics (Janes and Watt 2006; Kramer et al. 2005; Ziober et al. 2001).

2.2 Integrins as Potential Targets for Anticancer Therapies All members of the integrin family are heterodimeric, transmembrane glycoproteins consisting of an a and a b subunit (Hynes 2002). Eighteen a subunits and eight b subunits can dimerize in various combinations to form at least 24 different integrins with distinct, though often overlapping, ligand-binding specificities (Hynes 2002). As a group, integrins bind to a wide variety of extracellular ligands, many of which are associated with the ECM. Simultaneously, integrin cytoplasmic domains can interact with cytoskeletal proteins to mediate a transmembrane linkage of the ECM to the cytoskeleton, which is critical for controlling cell shape, polarization, and motility (Chen and Gumbiner 2006; Delon and Brown 2007; Litjens et al. 2006; Liu et al. 2000; Ridley et al. 2003). In addition, although integrins lack intrinsic enzymatic activity, they are conduits of bidirectional signal transduction across the plasma membrane (Hynes 2002; Schwartz and Ginsberg 2002). Indeed,

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through interactions of their cytoplasmic domains with a wide variety of signaling effectors inside the cell (Legate and Fassler 2009; Liu et al. 2000), integrins regulate intracellular pathways in response to extracellular cues (i.e., “outside-in” signal transduction). In addition, some cytoplasmic interactions can regulate the activation state of an integrin, thereby modulating its binding affinity for extracellular ligands (i.e., “inside-out” signal transduction) (Askari et al. 2009; Hynes 2002). Signaling functions of some integrins can be modulated by lateral interactions with other cell surface proteins, such as tetraspanins, urokinase receptor (uPAR), or caveolin, at sites of cell adhesion or from within specialized membrane microdomains (reviewed in Berditchevski 2001; Chapman et al. 1999; Del Pozo and Schwartz 2007; Hemler 2005; Porter and Hogg 1998). As discussed later in Sect. 2.4.3, some integrins signal through cooperative interactions with cell surface receptors for growth factors or cytokines (reviewed in Comoglio et al. 2003; French-Constant and Colognato 2004; Giancotti and Ruoslahti 1999; Guo and Giancotti 2004). Integrins regulate many cell functions associated with epithelial-to-mesenchymal transition (EMT), and they have important roles in the development and malignant progression of SCC and other carcinomas (Brakebusch et al. 2002; Janes and Watt 2006; White et al. 2004). In their capacity as bidirectional signaling receptors, integrins regulate both tumor cell-mediated changes to the microenvironment that promote cancer progression, and tumor cell responses to such changes, implicating them as potential targets for antagonistic agents in anti-cancer therapies (Mulgrew et al. 2006; Rust et al. 2002; Stupp and Ruegg 2007; Wu et al. 1998). However, most integrin inhibitors in clinical development are thought to alter angiogenesis by targeting integrins on endothelial cells of the tumor vasculature (Alghisi and Ruegg 2006; Stupp and Ruegg 2007), and there remains a critical need to identify and validate specific integrin targets on tumor cells. As will be discussed, several integrins have been shown to regulate skin tumorigenesis, malignant progression, and metastasis, identifying them as possible therapeutic targets for SCC (Felding-Habermann 2003; Janes and Watt 2006; Ziober et al. 2001).

2.3 Roles of Integrins in Stratified Epithelia Expression patterns of individual integrins in normal epidermis and other epithelia are well documented, and several integrins are known to have critical roles in regulating epithelial growth, differentiation, and wound repair (Watt 2002). Importantly, some integrin functions that are involved in maintenance of the stem cell compartment, or that promote epithelial regeneration during wound healing, also contribute to SCC (Janes and Watt 2006; Ziober et al. 2001). Indeed, it has long been recognized that wound healing and carcinogenesis share intriguing similarities regarding epithelial cell behaviors and microenvironmental factors that drive each process (Dvorak 1986). We will briefly review the expression and functions of specific integrins in normal stratified epithelia, and how they change during wound healing, to provide a foundation for our discussions in later sections on integrin functions in SCC.

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2.3.1 Integrin Functions in Normal Stratified Epithelia Stratified epithelia are continually renewed by stem cells that give rise to committed progenitor cells, or transit-amplifying cells, which in turn give rise to differentiated keratinocytes (Fuchs 2008; Owens and Watt 2003; Watt 2002). Proliferating keratinocytes, including stem cells and transit-amplifying cells, are normally restricted to the basal cell layer where they are adhered through integrins to the underlying basement membrane (BM), a specialized ECM that separates epithelial cell layers from adjacent connective tissue. In the epidermis, integrin expression is normally restricted to cells of the basal layer and outer root sheath of the hair follicle (Hertle et al. 1991; Watt 2002). Differentiating keratinocytes down-regulate integrin expression as they detach from the BM and are displaced into the suprabasal layers (Watt 2002). Several integrins are expressed constitutively in normal, unwounded epidermis and oral squamous epithelium, including a3b1 and a6b4 (both laminin-332 receptors), a2b1 (a collagen receptor), a9b1 (a fibronectin and tenascin receptor) and avb5 (a vitronectin receptor) (Thomas et al. 2006; Watt 2002). Integrin a6b4 is an essential component of hemidesmosomes, which are adhesion structures on the basal surfaces of keratinocytes that anchor the epidermis to the dermis (Litjens et al. 2006). Consistently, deletion of a6b4 (through null mutation of either the Itga6 or Itgb4 gene, encoding the a6 or b4 subunit, respectively) leads to extensive epidermal blistering (Dowling et al. 1996; Georges-Labouesse et al. 1996; van der Neut et al. 1996). Deletion of a3b1 (through null mutation of the Itga3 gene encoding the a3 subunit) causes minor perinatal blistering at the epidermal-dermal junction, but this is caused by rupture of the BM, which is disorganized in a3-null mice (DiPersio et al. 1997). Interestingly, mice that lack a3b1 and a6b4, either alone or in combination, show essentially normal epidermal stratification (DiPersio et al. 1997; Dowling et al. 1996; Georges-Labouesse et al. 1996; van der Neut et al. 1996; DiPersio et al., 2000a). Similarly, individual deletion of integrin a2b1, a9b1, or avb5 does not substantially alter epidermal differentiation (Grenache et al. 2007; Huang et al. 2000; Singh et al. 2009; Zweers et al. 2007). In contrast, ablation of all b1 integrins from epidermis (through null mutation of the Itgb1 gene encoding the b1 subunit) leads to proliferation defects, loss of hair follicles and sebaceous glands and, depending on the genetic model, a modest increase in terminally differentiating keratinocytes (Brakebusch et al. 2000; Grose et al. 2002; Raghavan et al. 2000). Thus, there appears to be overlap in the roles of different integrins in maintaining epidermal homeostasis. The level of integrin expression in keratinocytes is thought to control the balance between stem cell renewal and terminal differentiation, which is important for maintaining tissue homeostasis (Fuchs 2008; Jones et al. 1995; Watt 2002; Zhu et al. 1999). Indeed, b1 integrins and a6b4 are expressed at relatively high levels in epidermal stem cells (Janes and Watt 2006; Jones and Watt 1993; Jones et al. 1995; Terunuma et al. 2007), and some integrin signaling pathways have been linked to maintenance of the epidermal stem cell compartment, such as those

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involving mitogen-activated protein kinases (MAPKs), or the Rho family guanosine triphosphatase (GTPase), Rac1 (Benitah et al. 2005; Haase et al. 2001; Zhu et al. 1999). Presumably, it is these resident stem cells that accumulate mutations in oncogenes and tumor suppressor genes that lead to tumorigenesis, since differentiated keratinocytes are eventually shed from the outer layer of stratified epithelia (Owens and Watt 2003; Watt 2002). Therefore, changes in integrins that disrupt the balance between stem cell renewal and differentiation are likely to greatly influence SCC development and progression. It is important to point out, however, that altered integrin expression on differentiating cells of the suprabasal layers, as occurs in SCC and other hyperproliferative states such as wound healing and psoriasis, can also influence the stem cell compartment and, therefore, affect tumor growth and progression (reviewed in Janes and Watt 2006). Indeed, skin carcinogenesis studies performed in transgenic mice showed that forced integrin expression in suprabasal keratinocytes can influence both the clonal expansion of tumor progenitor cells and malignant progression of resulting tumors to SCC (Nguyen et al. 2000; Owens and Watt 2001; Owens et al. 2003, 2005). Interestingly, suprabasal expression of different integrins had distinct effects. For example, suprabasal a2b1 had no effect on SCC progression, while suprabasal a3b1 suppressed malignant conversion of papillomas (Owens and Watt 2001). In contrast, suprabasal a6b4 or a5b1 each increased tumor incidence and progression to SCC, although through distinct mechanisms (Owens and Watt 2003; Owens et al. 2005). Thus, new integrin signals that are turned on in suprabasal cells can influence nearby tumor progenitor/stem cells.

2.3.2 Integrin Functions in Wound Healing As mentioned above, there are compelling similarities between wound healing and SCC progression (Dvorak 1986), and integrins regulate a number of epithelial functions important in both processes, including migration, proliferation, and the production of proangiogenic factors. Consistently, expression patterns of integrins in SCC often mirror those that occur in wound healing (Thomas et al. 2006; Watt 2002). During cutaneous wound healing, several integrins that are expressed in unwounded epidermis at high or moderate levels (i.e., a3b1, a9b1, a6b4) or low levels (i.e., a5b1) show sustained or increased expression, while avb5 is downregulated and replaced by avb6. As a group, these integrins can bind multiple ligands that are present in the provisional ECM of the wound, including fibronectin (a5b1, a9b1, avb6), vitronectin (avb6), and tenascin (a9b1, avb6), as well as laminin-332 (a3b1, a6b4) that is newly deposited by migrating keratinocytes (Nguyen et al. 2000; Thomas et al. 2006; Watt 2002). Furthermore, numerous studies in cultured cells have shown that these integrins regulate keratinocyte adhesion and migration on their respective ECM ligands [for example, (Grose et al. 2002; Carter et al. 1990a, b; Choma et al. 2004; Frank and Carter 2004; Pilcher et al. 1997;

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Sehgal et al. 2006)]. Therefore, it is perhaps not surprising that wound healing studies in integrin knockout mice have indicated considerable overlap in the abilities of different integrins to mediate epidermal migration. Indeed, while mice with epidermis-specific deletion of all b1 integrins showed impaired wound reepithelialization (Grose et al. 2002), mice that lack certain b1 integrins individually (a2b1, a3b1, or a9b1) did not show such a defect (Grenache et al. 2007; Zweers et al. 2007); Singh et al. 2009; (Margadant et al. 2009). Similarly, absence of integrin avb6 did not cause impaired wound healing in young adult mice (although it caused delayed wound healing in old mice) (AlDahlawi et al. 2006). On the other hand, epidermis-specific deletion of individual integrins has revealed important roles in regulating other aspects of wound healing. For example, deletion of a3b1 from epidermis was associated with reduced wound angiogenesis, indicating a3b1-dependent secretion of pro-angiogenic factors (Mitchell et al. 2009). In addition, deletion of a9b1 from epidermis caused proliferation defects in wound keratinocytes (Singh et al. 2009). Thus, the repertoire of distinct integrins expressed in wounded epidermis is important for coordinating diverse keratinocyte functions (migration, proliferation, ECM remodeling, secretion of pro-angiogenic factors) that collectively ensure efficient wound repair and epidermal regeneration. Importantly, these same integrin-mediated cell functions are also likely to contribute to SCC progression.

2.4 Roles of Integrins in SCC Roles for integrins in promoting both early and late stages of SCC have been investigated extensively (reviewed in Janes and Watt 2006; Kramer et al. 2005; Marinkovich 2007; Thomas et al. 2006; Ziober et al. 2001). Integrins regulate a number of tumor cell functions that facilitate initial tumor growth, including proliferation, survival, and secretion of pro-angiogenic factors. In addition, integrinmediated cell survival, migration, invasion, and ECM proteolysis are important for later stages of malignant tumor progression and metastasis (Brakebusch et al. 2002; Felding-Habermann 2003). Integrins can influence tumor cell behavior directly through their cell adhesion and signaling functions, or indirectly through effects on ECM remodeling. Indeed, there are many reports of integrins regulating expression or activities of extracellular proteases, such as matrix metalloproteinases (MMPs) or urokinase plasminogen activator (uPA), that can promote tumor angiogenesis and carcinoma progression [for example, (Brooks et al. 1996; Ellerbroek et al. 1999; Morini et al. 2000; Thomas et al. 2001a; Ghosh et al. 2000, 2006; Gu et al. 2002; Han et al. 2002; Iyer et al. 2005; Symowicz et al. 2007)]. In this section, we will review what is currently known about integrin expression and function in SCC. Although changes in relevant ECM ligands that occur in SCC will be mentioned where appropriate, the reader is directed to several excellent reviews for further details on this subject (Marinkovich 2007; Ziober et al. 2001). Because of space limitations, our discussion is concentrated on tumor cell-autonomous functions

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of integrins on SCC cells and their potential value as therapeutic targets. However, integrins expressed on stromal cells, such as endothelial cells, macrophages, and fibroblasts, also regulate the abilities of these cells to alter the tumor microenvironment and influence carcinoma progression. Therefore, the importance of integrins on these nontumor cells as therapeutic targets should not be overlooked (Hofmeister et al. 2008).

2.4.1 Integrin Expression in SCC There is evidence that the expression levels of certain integrins in SCC may serve as useful biomarkers for clinical outcome (Kurokawa et al. 2008). As already mentioned, altered integrin expression in SCC bears similarities to that which occurs during wound healing and includes sustained expression, increased expression, or loss of expression (Bagutti et al. 1998; Jones et al. 1993). For example, integrins a5b1 and avb6 are expressed at low or negligible levels in normal epidermis but are increased in SCC (Gomez and Cano 1995; Shinohara et al. 1999), while avb5 is downregulated (Janes and Watt 2004, 2006). On the other hand, expression of a3b1 and a6b4 often persists in SCC (Janes and Watt 2006), although reduced expression has also been reported in some cases (Bagutti et al. 1998; Maragou et al. 1999). Colocalization of a9b1 and its ligand, tenascin, has also been reported in SCC tumors, although inflamed areas often showed focal loss of both at the BM zone (Hakkinen et al. 1999). While some studies reported increased expression of a2b1 in metastatic SCC cell lines and tumor biopsies (Shinohara et al. 1999), others reported that loss of a2b1 and its collagen ligands is correlated with SCC progression (reviewed in (Ziober et al. 2001)). Importantly, there can also be considerable variation in expression of an individual integrin either within a tumor or between different SCC tumors, possibly reflecting differential expression in distinct cellular compartments of the tumor and/or at distinct stages of tumor progression (Janes and Watt 2006; Watt 2002). However, while the expression pattern of an individual integrin might reflect its involvement in SCC, by itself it reveals no information about the functional role of the integrin at a particular stage of carcinogenesis. In fact, there is increasing evidence that some integrins that are already expressed on normal epithelial cells acquire new functions during SCC progression (see Sect. 4.5). As discussed in the following sections, numerous preclinical studies have identified key roles for specific integrins in SCC, and they also suggest that the malignant phenotype is influenced by the cumulative roles of several integrins, rather than by any particular integrin alone.

2.4.2 Integrin Signaling in SCC Integrins expressed on tumor cells can relay signals bidirectionally across the plasma membrane that control basic cell functions important for cancer progression,

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invasion, and metastasis, as reviewed in detail elsewhere (Brakebusch et al. 2002; Felding-Habermann 2003; Giancotti and Ruoslahti 1999; Gilcrease 2007; Guo and Giancotti 2004). As mentioned above and discussed in several reviews (Berditchevski 2001; Chapman et al. 1999; Del Pozo and Schwartz 2007; Hemler 2005; Porter and Hogg 1998; Salanueva et al. 2007), integrin signaling functions can be modulated through lateral associations with other cell surface proteins, including growth factor receptors (see Sect. 2.4.3). This discussion is focused on integrin-mediated outsidein signaling; however, changes in integrin activation state that are regulated by inside-out signals also control cell functions that promote carcinoma progression (Legate and Fassler 2009; Schwartz and Ginsberg 2002). Focal adhesion kinase (FAK) has emerged in recent years as a particularly important effector of integrin-mediated signal transduction in tumor cells, and its regulation serves as a useful paradigm of outside-in integrin signaling that promotes malignant cell behavior (Brunton and Frame 2008; McLean et al. 2005; Mitra and Schlaepfer 2006; Zhao and Guan 2009). FAK is a nonreceptor tyrosine kinase that associates with several integrins at focal adhesions or other cell-matrix contacts, and its activation by cell adhesion is the initial enzymatic step in several integrin-dependent signaling pathways. Integrin-mediated FAK activation can contribute to many different tumor cell functions, including proliferation, survival, motility, and invasiveness, and it has been linked to the stimulation of various pathways involving the MAPKs, extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK), certain Rho family GTPases (CDC42, Rho, RAC1), and the serine/threonine kinase AKT (Felding-Habermann 2003). FAK can also be activated by growth factor receptors, identifying it as a potential integrator of growth factor and integrin signaling (Brunton and Frame 2008; Sieg et al. 2000). Importantly, FAK expression is enhanced in invasive SCCs (Kornberg 1998), where it appears to regulate both early and late stages of cancer progression (reviewed in (Ziober et al. 2001)). Indeed, deletion of FAK from the epidermis suppresses carcinogen-induced skin tumorigenesis, as well as malignant progression of benign papillomas to carcinomas (McLean et al. 2004). An early step in many integrin-FAK signaling pathways is the direct binding of a SRC-family kinase (SFK) to activated FAK at sites of cell adhesion (Schaller et al. 1999), as described in detail in several excellent reviews (Brunton and Frame 2008; Cary and Guan 1999; McLean et al. 2005; Mitra and Schlaepfer 2006). Briefly, integrin binding to ECM ligands leads to FAK clustering and auto-phosphorylation of Y397, creating a high-affinity binding site for the Src-homology 2 (SH2) domain of SRC (or another SFK) and leading to formation of a FAK/SRC complex. Subsequent phosphorylation of other FAK tyrosines by SRC creates binding sites for a number of signaling intermediates, such as GRB2, p130CAS, and phosphatidylinositol 3¢-kinase (PI3-K). These intermediates link the FAK/SRC complex to different downstream effectors, including the RAS-to-ERK pathway, AKT, and JNK (Giancotti and Ruoslahti 1999; Grille et al. 2003; Mitra and Schlaepfer 2006), thereby activating several pathways that promote EMT by enhancing proliferation, survival, migration, invasion, and expression of ECMdegrading proteases and pro-angiogenic factors. As mentioned above, FAK/SRC

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can also signal through Rho family GTPases (CDC42, Rho, and RAC1) to modulate cytoskeletal dynamics and cell migration (Felding-Habermann 2003). Furthermore, RAC1 plays important roles in keratinocyte proliferation and migration in vivo (Tscharntke et al. 2007), and it is critical for maintaining epidermal stem cell compartments (Benitah et al. 2005; Castilho et al. 2007; Chrostek et al. 2006), suggesting that enhanced FAK/SRC-to-RAC1 signaling may contribute to clonal expansion of tumor progenitor/stem cells. Consistently, mice that lack Tiam1, a guanine nucleotide-exchange factor (GEF) that activates RAC1, are resistant to RAS-induced skin tumors (Malliri et al. 2002). Given the importance of the FAK/SRC complex as a major signaling nexus that links integrin-mediated adhesion to pathways that promote several cancer cell functions, it is not surprising that small molecule inhibitors of both FAK and SRC have been the focus of recent clinical studies to inhibit tumor progression (reviewed in (Brunton and Frame 2008; McLean et al. 2005; Mitra and Schlaepfer 2006)). However, integrins in epithelial cells can also signal through effectors other than FAK, such as integrin-linked kinase (ILK) and phospholipase C (PLC), as reviewed in detail elsewhere (Gilcrease 2007). Therefore, FAK-independent pathways should not be overlooked as potential therapeutic targets for SCC.

2.4.3 Functions of Individual Integrins in SCC In the following subsections we will discuss tumor cell-autonomous functions of individual integrins that are expressed on SCC cells. This discussion is focused on integrins avb6, a3b1, and a6b4, since their roles have been studied most extensively. However, several other integrins with less-defined roles should also be mentioned briefly. For example, increased expression of integrin a9b1 and two of its ECM ligands, fibronectin and tenascin, has been reported in some SCCs (Hakkinen et al. 1999; Ziober et al. 2001). However, functional roles for a9b1 in SCC are poorly defined, in part because this integrin is down-regulated in cultured keratinocytes and has been largely overlooked in studies of integrin-mediated keratinocyte function. Although high expression of a2b1 (a receptor for certain laminins and collagens) and a5b1 (a receptor for fibronectin) has been reported in some SCC cell lines and tumor biopsies (Shinohara et al. 1999), expression patterns are quite variable and roles for these integrins in SCC require further study (Hakkinen et al. 1999; Ziober et al. 2001). 2.4.3.1 Integrin avb6 Regulatory roles for integrin avb6 in SCC have been studied quite extensively, as reviewed in (Thomas et al. 2006). Although not expressed constitutively by normal epithelium, avb6 is upregulated during wound healing and in many carcinomas (Breuss et al. 1995; Hamidi et al. 2000; Jones et al. 1997), and it has been correlated

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with malignant progression of SCC (Hazelbag et al. 2007). Furthermore, numerous studies have demonstrated roles for this integrin in promoting SCC cell motility and invasion (for example, (Thomas et al. 2001a, b; Ramos et al. 2002)). avb6 binds to a tripeptide motif, arginine-glycine-aspartic acid (RGD), that occurs within several of its ECM ligands including fibronectin, tenascin, and vitronectin (Hynes 2002; Thomas et al. 2006). In addition to mediating cell migration on these ligands, avb6 may promote invasion by regulating expression of extracellular proteases that degrade or remodel ECM. For example, avb6 promotes invasion of oral keratinocytes through up-regulation of MMP-9 and, to a lesser extent, MMP-2 (Thomas et al. 2001a). Other invasion-promoting proteases that can be regulated by avb6 in carcinoma cells include MMP-3 (Ramos et al. 2002) and uPA (Ahmed et al. 2002). Signaling intermediates that have been implicated in avb6-mediated SCC cell invasion include cyclooxygenase-2 (COX-2) (Nystrom et al. 2006), RAC1 (Yap et al. 2009), and the SRC-family member FYN (Li et al. 2003). In addition to promoting an invasive phenotype, the de novo expression of avb6 has been shown to prevent oral SCC cells from undergoing differentiation, as well as protect them from anoikis (i.e., apoptosis caused by reduced or inappropriate cell adhesion) when they are deprived of normal attachments to BM (Janes and Watt 2004). The latter function involves avb6- mediated activation of an AKT survival pathway (Janes and Watt 2004). One of the most important functions of avb6 in SCC cells may be its ability to activate the ECM-associated pool of latent TGFb, thereby initiating TGFb signaling pathways that influence tumor progression (Sheppard 2005; Thomas et al. 2006). As discussed further in Sect. 2.4.4.1, avb6 binds to an RGD motif within the latent TGFb complex, thereby inducing a conformational change that activates TGFb (Munger et al. 1999). Although this mechanism has been best characterized in colon cancer cells, it also occurs in keratinocytes and is likely to be important for regulating TGFb-mediated signaling in SCC progression (Munger et al. 1999). Finally, it is important to note that some studies have indicated that avb6 has tumor suppressing roles, rather than tumor promoting roles, in SCC. For example, mice that are doubly-deficient for avb6 and thrombospondin showed increased incidence of skin papillomas and SCCs, suggesting that avb6 suppresses tumor formation in this model (Ludlow et al. 2005). Similarly, genetic deletion of av integrins in epithelial cells of the eyelid skin and conjunctiva lead to increased SCC (McCarty et al. 2008). In another study, increased expression of avb6 suppressed the invasive phenotype of transformed oral keratinocytes (Mogi et al. 2005). The paradoxical findings regarding roles for avb6 in SCC may, to some extent, be reflective of the well known biphasic roles of TGFb, which suppresses early stages of skin tumorigenesis but promotes progression to malignancy at later stages (Wakefield and Roberts 2002; Wang 2001), such that effects of avb6-mediated TGFb activation on tumor cells are dependent on the stage of cancer development at which this activation occurs (Thomas et al. 2006). 2.4.3.2 Laminin-332-Binding Integrins, a6b4 and a3b1 In recent years, a prominent role in SCC growth and invasion has emerged for laminin-332 (previously known as laminin-5, kalinin, nicein, or epiligrin) [for a

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review, see (Marinkovich 2007)]. Laminin-332 expression is enhanced at the invasive fronts of SCCs (Pyke et al. 1995) and is correlated with poor prognosis in SCC patients (Ono et al. 1999). Laminin-332 also disrupts cell-cell adhesions and induces scattering in SCC and other carcinoma cells (Kawano et al. 2001; Miyazaki et al. 1993), suggesting that it can act as a pro-invasive autocrine factor (Marinkovich 2007). Laminin-332 can be cleaved by MMPs or other proteases (Ziober et al. 2001), and specific proteolytic events have been linked to carcinoma cell migration and invasion [for example, see (Gianelli et al. 1997; Goldfinger et al. 1998; Schenk et al. 2003)]. The effects of laminin-332 on behaviors of both normal keratinocytes and carcinoma cells are mediated largely through its main integrin receptors, a3b1 and a6b4 (Carter et al. 1991; Nguyen et al. 2000; Giannelli et al. 2002a; Dajee et al. 2003), although other receptors such as syndecan-1 also contribute (Okamoto et al. 2003). For simplicity, functions of a3b1 or a6b4 are discussed individually below, although as receptors for a common ECM ligand these two integrins are likely to function coordinately to regulate some aspects of SCC growth and invasion (Marinkovich 2007). There is also substantial evidence that some signaling functions of a3b1 or a6b4 occur independently of binding to laminin-332, and instead involve lateral interactions with other cell surface proteins (see below). Integrin a6b4 Expression of integrin a6b4 is often high in SCCs and has been correlated with malignant conversion and poor prognosis (Jones et al. 1993; Rabinovitz and Mercurio 1996; Tennenbaum et al. 1993; Van Waes et al. 1991, 1995). Therefore, it is not surprising that roles for a6b4 in carcinogenesis have been studied extensively by several groups. These studies have revealed that a6b4 is critical for SCC formation (Dajee et al. 2003), and that it can promote survival and invasion of SCC and other carcinoma cells (Lipscomb and Mercurio 2005; Marinkovich 2007). a6b4 can also control organization of laminin-332 in the ECM, which is an important regulator of keratinocyte migration (Sehgal et al. 2006). However, a6b4 functions in carcinoma cells appear to be quite complex, and multiple mechanisms have been proposed for its effects on tumor cell behavior, as described below. As already mentioned, a6b4 in normal keratinocytes mediates stable adhesion through its association with the intermediate filaments in hemidesmosomes (Carter et al. 1990a; Litjens et al. 2006). In contrast, a6b4 in invasive carcinoma cells is mobilized out of hemidesmosomes and associates instead with the actin cytoskeleton in membrane protrusions (Mercurio and Rabinovitz 2001; Mercurio et al. 2001), where it facilitates migration and invasion rather than stable adhesion. Signaling pathways through which a6b4 regulates cell behavior are complex and have been shown to involve several effectors, including PI3-K, RAC and Rho GTPases, and Shc/RAS-to-MAPK pathways (Lipscomb and Mercurio 2005; Mainiero et al. 1997; O’Connor et al. 2000; Russell et al. 2003). Adding further to this complexity, a6b4 often signals in collaboration with receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) (Mariotti et al. 2001), the MET receptor for hepatocyte growth factor (HGF) (Trusolino et al. 2001), and the

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Ron receptor for macrophage stimulating protein (MSP) (Santoro et al. 2003) (see Sect. 2.4.3). Integrin a6b4 has also been implicated in the regulation of early stages of SCC progression, where it was shown to cooperate with RAS and IkBa to induce transformation of primary keratinocytes (Dajee et al. 2003). In this study, blocking antibodies against either a6b4 or laminin-332 blocked the formation of SCCs, and keratinocytes that lacked b4 were unable to form SCC after transformation with Ras. There is also evidence that a6b4 can regulate carcinoma cell survival (Chung et al. 2002; Zahir et al. 2003). While a6b4-mediated signaling pathways mentioned above are likely to contribute to this regulation, a6b4-mediated induction of VEGF protein translation in carcinoma cells can also regulate autocrine cell survival (Chung et al. 2002). The fact that a6b4 impacts SCC development and progression at multiple steps, and through multiple mechanisms, identifies this integrin as an attractive target for anticancer therapies, especially since at least some of these mechanisms are acquired by carcinoma cells (see Sect. 2.4.5.2). Integrin a3b1 Integrin a3b1 has also been implicated in invasion and metastasis of many cancer cell types (Morini et al. 2000; Tawil et al. 1996; Tsuji et al. 2002; Wang et al. 2004). In vivo and in vitro studies have identified roles for a3b1 in regulating several functions in epithelial cells that contribute to both wound healing and carcinogenesis, including ECM deposition and organization (deHart et al. 2003; Hamelers et al. 2005), cell polarization and migration (Choma et al. 2004; Frank and Carter 2004), adhesion-dependent survival (Manohar et al. 2004), proliferation (Gonzales et al. 1999), and secretion of ECM proteases and pro-angiogenic factors (Marinkovich 2007; Mitchell et al. 2009; Sugiura and Berditchevski 1999; DiPersio et al. 2000b). There is also solid evidence that some of these a3b1-mediated functions involve regulation of TGFb signaling (see Sect. 2.4.2). Integrin a3b1 in immortalized/transformed keratinocytes regulates the expression of MMP-9 (Iyer et al. 2005; Lamar et al. 2008a), a known promoter of both tumor angiogenesis and SCC invasion (Bergers et al. 2000; McCawley and Matrisian 2001). Using oncogenically-transformed keratinocytes generated from mice that either express or lack a3b1, we showed that a3b1 is required for MMP-9 gene expression, and also regulates tumor growth in vivo and cell invasion in vitro (Iyer et al. 2005; Lamar et al., 2008a). Although promigratory effects of a3b1laminin binding probably contribute to invasion, RNAi and overexpression studies indicated that MMP-9 was largely responsible for a3b1-dependent invasion (Lamar et al. 2008a). a3b1 may also be involved in later stages of metastasis, as it was able to promote arrest of circulating carcinoma cells in the lungs (Wang et al. 2004). Many functions of a3b1 are clearly dependent on its interactions with its laminin ligands in the ECM (Kreidberg 2000). However, some a3b1 functions have been reported to be mediated, or modulated, by direct or indirect interactions with other cell surface proteins, including tetraspanins (Berditchevski et al. 1996; Sugiura and

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Berditchevski 1999), uPAR (Wei et al. 2001), and components of adherens junctions (Chattopadhyay et al. 2003; Kim et al. 2009). Thus, roles of a3b1 in tumor cells appear complex and may involve both ECM-dependent and ECM-independent signaling. In particular, a3b1 binds directly and robustly to the tetraspanin CD151 (Yauch et al. 2000), and this interaction has been shown to regulate both a3b1mediated signaling (Yauch et al. 1998) and motility of epidermal carcinoma cells (Winterwood et al. 2006). Interestingly, recent studies have identified important roles for CD151-integrin interactions in several aspects of carcinoma progression and metastasis in vivo (Sadej et al. 2009; Yang et al. 2008; Zijlstra et al. 2008). Although the full extent to which CD151 modulates a3b1-dependent functions in tumor cells is still unclear, it seems likely that this interaction will prove to be important for SCC progression and metastasis.

2.4.4 Cooperative Functions of Integrins and Growth Factors in SCC Growth factors influence cancer growth and progression through both autocrine and paracrine effects on tumor cells and stromal cells. Multiple studies in both normal and cancer cells have revealed significant signaling crosstalk and complex formation between integrins and growth factor receptors (French-Constant and Colognato 2004; Gilcrease 2007; Guo and Giancotti 2004). In the following sections, we will discuss several examples that serve to illustrate different mechanisms whereby integrins can collaborate with growth factors to promote tumor growth and progression, including forming complexes with growth factor receptors, regulating the expression of growth factors or their receptors, and activating ECMbound growth factors. As described below, some of these mechanisms are best illustrated by roles that have been demonstrated for certain integrins in regulating cellular responses to TGFb. 2.4.4.1 Integrin-Dependent Activation of Latent Growth Factors In what is perhaps the best characterized example of direct activation of a latent growth factor by an integrin, avb6 plays a critical role in the activation of the latent TGFb complex (Sheppard 2005). Each of the three mammalian TGFb isoforms (TGFb1, 2, and 3) is secreted as an inactive complex consisting of the latencyassociated protein (LAP) and the latent TGFb binding protein (LTBP). This latent complex is covalently linked through LTBP to certain ECM proteins (e.g., fibronectin) (Sheppard 2005; Taipale et al. 1994), and it must be activated either through proteolytic release of TGFb from the LAP [for example, mediated by MMP-9 or plasmin (Lyons et al. 1990; Sato et al. 1990; Yu and Stamenkovic 2000)], or through a conformational change in the complex [for example, induced by thrombospondin-1 (Crawford et al. 1998; Schultz-Cherry et al. 1995)]. As already mentioned, avb6 can activate latent

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TGFb1 or TGFb3 (but not TGFb2) by binding to an RGD motif within the LAP and inducing a conformational change in the complex (Annes et al. 2004; Munger et al. 1999; Sheppard 2005). It is well known that TGFb acts as a tumor suppressor at early stages of tumorigenesis, but switches to a promoter of EMT at later stages of progression (for several excellent reviews, see Derynck et al. 2001; He et al. 2001; Wakefield and Roberts 2002; Wang 2001; Zavadil et al. 2001). Given that avb6 is upregulated in SCC, its ability to activate latent TGFb could play a role in these biphasic effects of TGFb on cancer progression. In addition to direct growth factor activation, there is evidence that some integrins can activate latent or ECM-sequestered growth factors through less direct mechanisms. For example, certain integrins, such as avb6 and a3b1, can induce the expression of MMP-9, uPA, or other extracellular proteases (Thomas et al. 2001a; Ghosh et al. 2000, 2006; Iyer et al. 2005), which can then degrade ECM and release reservoirs of ECM-associated growth factors (i.e., VEGF) that can promote tumor proliferation or angiogenesis (Bergers et al. 2000; McCawley and Matrisian 2001). 2.4.4.2 Integrin-Dependent Enhancement of Growth Factor Signaling Once activated, TGFb interacts with its type I and type II serine/threonine kinase receptors to initiate signaling pathways that modulate transcriptional or post- transcriptional gene regulation. Cellular responses to TGFb can be mediated by the Smad family of transcription factors, or by Smad-independent pathways of TGFb signaling, such as MAPK pathways (Derynck and Zhang 2003). Many studies have identified interactions between integrins and TGFb signaling pathways that regulate motility or invasiveness of keratinocytes or carcinoma cells (Gailit et al. 1994; Zambruno et al. 1995; Giannelli et al. 2002b; Decline et al. 2003; Galliher and Schiemann 2007; Jeong and Kim 2004; Reynolds et al. 2008). We recently discovered that integrin a3b1 potentiates the ability of TGFb to induce MMP-9 gene expression in immortalized keratinocytes through a mechanism that is independent of changes in the levels of TGFb or its receptors (Lamar et al. 2008b). However, a3b1 did not enhance all TGFb signaling pathways, since TGFb-mediated Smad phosphorylation remained intact in a3b1-deficient (Itga3-/-) keratinocytes, suggesting that this integrin is a selective modulator of a subset of TGFb signaling pathways (Lamar et al., 2008b). These findings raise the intriguing possibility that a3b1 is involved in the above-mentioned switch in TGFb function from tumor suppressor to tumor promoter during SCC progression. Although the mechanism whereby a3b1 enhances TGFb signaling is not yet clear, it is unlikely that this integrin activates latent TGFb as described above for avb6 (Sect. 2.4.1), since a3b1 does not bind RGD ligands efficiently, and such a mechanism was not indicated for b1 integrins (Munger et al. 1999). Rather, the ability of a3b1 to potentiate MMP-9 induction in response to exogenous pre-activated TGFb, and the dependence of this regulation on SRC (Lamar et al. 2008b), suggests similarities to a previously described mechanism used by integrin avb3 to modulate

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a subset of TGFb signaling pathways in breast cancer cells (Galliher and Schiemann 2007). In the latter study, avb3 was shown to activate the TGFb type II receptor in a SRC-dependent manner, which was required for TGFb-mediated activation of p38 MAPK, but not for TGFb-mediated stimulation of Smad2/3 (Galliher and Schiemann 2007). Further studies are required to determine if MMP-9 induction requires the formation of a a3b1/TGFb receptor signaling complex, or results from distinct pathways that are initiated independently by TGFb or a3b1 and converge on a common intermediate. In any case, it appears that different integrins may modulate TGFb activation and signaling through different mechanisms, thereby cooperating to determine the overall response of the tumor cell to TGFb. Although TGFb exerts its effects on tumor cells in large part through signaling pathways that ultimately regulate the transcription of target genes (Derynck et al. 2001; Derynck and Zhang 2003), it is well known that TGFb can also regulate the expression of EMT-associated genes through post-transcriptional mRNA stability (Dibrov et al. 2006). Integrin a3b1 promotes Mmp9 mRNA stability in immortalized mouse keratinocytes (Iyer et al. 2005), raising the intriguing possibility that TGFb and a3b1 cooperate to stabilize mRNA transcripts of EMT genes. The MAPK p38 is a potential effector for this regulation, since it has been implicated in both EMTpromoting effects of TGFb (Bakin et al. 2002; Zavadil and Bottinger 2005) and TGFb-mediated mRNA stability (Dibrov et al. 2006), and it can be activated through cooperative interactions between b1 integrins and TGFb (Bhowmick et al. 2001). The MAPK ERK is another potential intermediate in this regulation. Indeed, TGFb activates ERK pathways in cell culture models of EMT (Zavadil and Bottinger 2005), and ERK is both activated by a3b1, and required for induction of Mmp9 mRNA in immortalized keratinocytes (Iyer et al. 2005; Manohar et al. 2004). In addition, TGFb signaling through the type I receptor, ALK5, leads to MEK/ERK-dependent induction of MMP9 mRNA in human breast cancer cells (Safina et al. 2007). 2.4.4.3 Formation of Integrin-Growth Factor Receptor Signaling Complexes Association of integrin a6b4 with the MET receptor provides a compelling example of an integrin-growth factor receptor interaction that regulates signal transduction in carcinoma cells, most likely in a manner that is independent of a6b4 binding to laminin-332 (Trusolino et al. 2001). Indeed, in a complex formed with activated MET, a6b4 acts as an essential adapter protein that facilitates HGF-mediated cell invasion through a signaling mechanism that involves Shc and PI3-K (Trusolino et al. 2001). a6b4 can also form a complex with the activated Ron receptor, dependent on 14-3-3 binding, which displaces a6b4 from hemidesmosomes and activates new signaling pathways that promote keratinocyte migration (Santoro et al. 2003). Other studies in carcinoma cells have revealed crosstalk between a6b4 and EGFR that leads to Rho activation (Gilcrease et al. 2009), as well as complex formation between a6b4 and ERBB2 (a binding partner of EGFR) that enhances activation of the transcription factors STAT3 and c-Jun (Guo et al. 2006). In a less direct

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mechanism of enhanced growth factor signaling, a6b4 can also regulate translation of ERBB2 (Yoon et al. 2006) and VEGF (Chung et al. 2002).

2.4.5 Integrin Switches That Promote SCC As described in the following section, SCC cells can acquire new adhesion properties and signaling pathways either through changes in the expression of particular integrins, or through alterations in the signaling functions of integrins that were already expressed in normal epithelial cells. Elucidating the mechanisms that control these integrin switches may identify novel targets for therapeutic agents that inhibit cancer cell-specific integrin functions with minimal off-target effects on normal cells. Figures 2.1, 2.2, and 2.3 illustrate three different ways in which SCC cells can acquire new integrin functions: (1) expression of new integrins (Fig. 2.1), (2) changes in functions of pre-existing integrins (Fig. 2.2), and (3) integrin mutations (Fig. 2.3). The following sections will focus on examples of each mechanism from studies performed in keratinocytes or SCC cells. There are several points to keep in mind when considering these examples. First, as discussed below, there is evidence that some integrin switches may be linked to specific stages of carcinogenesis, and perhaps associated with specific oncogene or tumor suppressor mutations. Second, new integrin expression that is acquired during the clonal expansion of tumor progenitor cells could contribute to both heterogeneous expression patterns within a tumor, and variations in expression between different SCC samples (Janes and Watt 2006; Jones et al. 1993). Third, it is still not clear whether a “new” integrin function observed in SCC cells arises as a result of de novo activation of the function, or reflects the clonal expansion of a stem cell population within which the function pre-existed.

Fig. 2.1 Integrin switches that promote SCC: expression of new integrins.

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Fig. 2.2 Integrin switches that promote SCC: altered function of pre-existing integrins.

Fig. 2.3 Integrin switches that promote SCC: integrin mutations.

2.4.5.1 av Integrin Switch Upregulation of avb6 in SCC occurs at the expense of avb5 expression, probably due to higher affinity of the av subunit for the b6 subunit (Janes and Watt 2004). Consequently, there is a switch from avb5 to avb6 as keratinocytes undergo malignant transformation, similar to that which has been described in wound healing (Clark et al. 1996). This switch in av integrin expression from avb5 to avb6 provides a

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clear example of new integrin expression that facilitates SCC progression(Fig. 2.1). Recent studies indicate that the switch from avb5 to avb6 protects SCC cells from undergoing differentiation or anoikis when they are deprived of normal attachments to BM, due to the very different effects that these two integrins have on both cell survival pathways and cell death pathways (Janes and Watt 2004). For example, protection of SCC cells from anoikis results in part from the ability of newly expressed avb6 to activate AKT survival pathways in cells that have lost adhesion to the BM and would otherwise undergo anoikis (Janes and Watt 2004). Loss of avb5 may also enhance cell survival, since this integrin had pro-apoptotic effects in its unligated form (Janes and Watt 2004). The switch from avb5 to avb6 may also provide temporal control of TGFb activation by avb6 (see Sect. 2.4.1), since avb5 binds to the latent TGFb complex with significantly lower avidity and probably does not support efficient TGFb activation (Sheppard 2005). Although the mechanism that triggers the switch in av integrins is not yet clear, possible effectors of avb6 induction include TNFa (Scott et al. 2004) and, interestingly, TGFb itself (Zambruno et al. 1995).

2.4.5.2 Functional Switch in a6b4 In addition to de novo integrin expression described above, there is increasing evidence that some pre-existing integrins undergo functional changes during carcinoma progression, leading to the activation of new signaling pathways that promote tumorigenesis and invasion. Integrin a6b4 provides a paradigm for this sort of regulation, as during malignant conversion it switches from a predominantly adhesive receptor to a proinvasive signaling protein that can form complexes with growth factor receptors (Lipscomb and Mercurio 2005; Mercurio et al. 2001) (Fig. 2.2). As already mentioned, a6b4 is associated with the intermediate filaments in hemidesmosomes of normal, undamaged epidermis (Litjens et al. 2006), but in invasive carcinoma cells this integrin relocates to actin-associated filopodia and lamelipodia through a mechanism that involves PKCa-mediated phosphorylation of the b4 cytoplasmic domain (Mercurio et al. 2001; Rabinovitz et al. 1999). There is also evidence that EGFR signaling through FYN kinase can disrupt a6b4 function in hemidesmosomes (Mariotti et al. 2001). This relocalization of a6b4 not only relieves tumor cells of stable adhesion that would presumably suppress invasive growth, but it also frees a6b4 to form new signaling complexes with growth factor receptors, such as those described above in Sect. 2.4.4.3, that promote tumor growth and malignant progression. Interestingly, the SCC-associated switch in a6b4 function can be influenced by mutations in key oncogenes and tumor suppressor genes. Indeed, a recent study showed that a6b4 acts as either a tumor suppressor or tumor promoter depending on specific mutations that are acquired by keratinocytes (Raymond et al. 2007). Specifically, a6b4 inhibited tumor growth in tumorigenic keratinocytes that harbor loss-of-function mutations in the genes that encode p53 (Trp53) and Smad4 (Smad4), but it promoted tumor growth when these same cells were transformed by oncogenic RAS, indicating that RAS-mediated transformation induces a switch in a6b4 function (Raymond et al.

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2007). Consistently, a6b4 was also shown to be essential for SCC formation caused by oncogenic RAS and the inhibitor of kBa (IkBa) (Dajee et al. 2003). 2.4.5.3 Functional Switch in a3b1 Integrin a3b1 provides another example of an integrin that can acquire novel signaling functions as keratinocytes accumulate cancer-promoting mutations (Fig. 2.2). We showed that a3b1-dependent Mmp9 gene expression (discussed above in Sect. 2.3.2) was acquired in mouse keratinocytes as a result of immortalization caused by loss of p53, and was retained in RASV12-transformed versions of these cells where it promoted cell invasion (Lamar et al. 2008a). Importantly, a3b1dependent MMP-9 expression was also observed in immortalized human keratinocytes and human SCC cell lines that harbor p53 mutations (Lamar et al. 2008a). Cancer-cell specific pathways whereby a3b1 induces expression of the MMP9 gene or other EMT-promoting genes, would be attractive therapeutic targets. However, it is not yet known whether the acquisition of a3b1-dependent MMP-9 expression by immortalized cultures of keratinocytes represents a switch in a3b1 function that occurred de novo within tumor progenitor cells that have lost p53, or reflects the a3b1-dependent outgrowth of stem/tumor-progenitor cells that already possess this pathway. That a3b1 may promote the expansion of a tumor progenitor/ stem cell compartment is an intriguing possibility, especially since a3b1 has been reported to be expressed at higher levels in epidermal stem cells (Jones et al. 1995). Consistent with such a role, a3b1 in keratinocytes is a known regulator of RAC1 signaling pathways (Choma et al. 2004), and RAC1 is essential for maintenance of the stem cell compartment in the epidermis (Benitah et al. 2005). 2.4.5.4 Gain-of-Function Mutations in Integrins Another potential mechanism whereby altered integrin function may contribute to SCC progression is through rare gain-of-function mutations in integrin genes that predispose keratinocytes to the transforming effects of oncogenes. To date, the best example of such a mutation is T188Ib1, which was first identified as a heterozygous mutation in the b1 gene (ITGB1) of a human cell line derived from a poorly differentiated SCC of the tongue (Evans et al. 2003). This mutation, which occurs in a region of the b1 I-like domain that determines ligand-binding specificity, leads to constitutive activation of all ab1 integrin heterodimers and causes enhanced cell spreading, sustained ERK signaling, and reduced differentiation (Evans et al. 2003; Ferreira et al. 2009). Nevertheless, transgenic expression of T188Ib1 did not alter normal architecture or homeostasis of the epidermis (Ferreira et al. 2009), consistent with the notion that this mutation is a genetic polymorphism that has no deleterious effects on epidermal development or function (Evans et al. 2003). However, following chemical carcinogenesis to induce skin tumors, mice expressing T188Ib1 in the epidermis showed an increase in the frequency and rate of papilloma conversion to

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SCCs, and also formed more poorly differentiated SCCs, compared with mice expressing only wild type b1 (Ferreira et al. 2009). These intriguing findings suggest that the T188Ib1 mutation both predisposes benign tumors to malignant conversion and promotes formation of less differentiated tumors, providing an example of an integrin mutation that may influence both susceptibility to SCC and disease progression. Although other polymorphisms in the genes that encode b integrin subunits have been reported to occur in SCCs or other human cancers (Evans et al. 2003, 2004), their effects on carcinogenesis are not yet known. It also remains to be determined whether activating mutations in other domains of the b1 subunit, or in other b or a integrin subunits, can similarly predispose epidermis to SCC.

2.5 Exploiting Integrins in the Clinic As described in the preceding sections, preclinical studies using cell culture and in vivo models of SCC have identified critical roles for integrins in the regulation of tumor growth, invasion, and metastasis. These studies provide a solid foundation for the development of therapeutic strategies to inhibit SCC using agents that target integrins, particularly since the location of integrins on the cell surface make them readily accessible to therapeutic compounds. Indeed, several types of integrin antagonists are currently in preclinical and clinical development, including humanized monoclonal antibodies (i.e., volociximab against a5b1, and Vitaxin against avb3), RGDcontaining peptides (i.e., cilengitide), and non-peptide antagonists (Mulgrew et al. 2006; Rust et al. 2002; Stupp and Ruegg 2007; Thomas et al. 2006; Van Waes et al. 2000; Wu et al. 1998). The majority of these compounds are intended for use as angiogenesis inhibitors with a demonstrated ability to target integrins expressed on endothelial cells in the tumor vasculature (reviewed in (Stupp and Ruegg 2007; Tucker 2006)). However, preclinical studies in mice suggest that these and other compounds also effectively target integrins on tumor cells to reduce tumor cell growth, survival and metastasis (Chen et al. 2008; Gramoun et al. 2007; Harms et al. 2004; Landen et al. 2008; Park et al. 2006; Stoeltzing et al. 2003). Importantly, investigators have also exploited the increased or newly acquired expression of integrins on tumor cells, or tumor vessels, to deliver chemotherapies specifically to tumors (Abraham et al. 2007; Arap et al. 1998; Hallahan et al. 2003). Integrin-targeted micelles or liposomes have also been utilized to specifically deliver genes and antisense oligonucleotides to tumors (Bachmann et al. 1998; Cemazar et al. 2002; Oba et al. 2007). Despite the success of some integrin antagonists in preclinical studies, these compounds have had only minimal success in early clinical trials (reviewed in (Stupp and Ruegg 2007; Tucker 2006)). In addition, recent evidence suggests that at least some integrin antagonists may enhance, rather than inhibit, tumor growth and angiogenesis under certain circumstances. For example, the avb3/avb5-specific RGD-mimetic, cilengitide, was shown to have antitumor activity in preclinical and clinical studies of certain forms of glioblastoma (Reardon et al. 2008), but it was recently reported to stimulate tumor growth and angiogenesis when administered at low doses in a murine model of tumor growth (Reynolds et al. 2009). These

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discordant findings could be due to dose-dependent effects of cilengitide, or they may reflect differences between cancer types in the roles that av integrins play on tumor cells and endothelial cells (Weller et al. 2009). In any case, they highlight the importance of understanding the roles of the intended integrin targets within distinct cellular compartments of the tumor type being tested, in order to predict the overall effect of a particular integrin antagonist. Future preclinical studies using inducible and cell-specific transgenic/knockout models, in which candidate integrins and signaling proteins can be manipulated with temporal and spatial precision, should reveal roles of individual integrins within different cellular compartments of the tumor, and help develop effective strategies to inhibit SCC by targeting integrins. Another potential challenge that must be overcome before integrins can be fully exploited as therapeutic targets is that most integrins expressed on SCC cells also perform essential functions in normal cells; therefore systemic delivery of integrin inhibitors may cause adverse side effects. This problem might be avoided by targeting integrins that are expressed at high levels on SCC cells but at low or negligible levels on normal cells, such as avb6 (Fig. 2.1). In addition, identifying mechanisms that trigger new functions of pre-existing integrins, such as a3b1 and a6b4 (Fig. 2.2), or determining how gain-of-function mutations in integrins predispose keratinocytes to SCC (Fig. 2.3), may reveal intermediate molecules or pathways that are activated specifically in cancer cells and can be targeted with minimal effects on normal cell function. Another challenge is that the malignant phenotype is probably influenced by the cumulative functions of the various integrins expressed on SCC cells, such that combinatorial targeting of more than one integrin may be necessary to effectively inhibit disease progression. Integrin antagonists may prove to be most useful in combination with other types of therapeutics. For example, preclinical studies have already demonstrated that cilengitide can synergize with either chemotherapy or radiotherapy (Albert et al. 2006; Burke et al. 2002; Tentori et al. 2008), and other compounds targeting integrins a5b1 and aVb3 were also demonstrated to enhance chemosensitivity (Menendez et al. 2005; Stoeltzing et al. 2003) and augment the effects of radiotherapy (Abdollahi et al. 2005). Whether administered alone or in combination with other therapies, the efficacy and specificity of integrin antagonists or inhibitors of integrin signaling pathways could be improved by exploiting newly developed strategies for targeted delivery of compounds to tumors and tumor stroma. It may also be advantageous to target integrin function in tumor cells by combining targeted delivery systems with antisense oligonucleotides or RNAi to inhibit specific integrin expression. In support of this approach, antisense oligonucleotides that target avb3 were found to be effective against hepatocellular and mammary carcinoma in preclinical studies (Li et al. 2007; Townsend et al. 2000). In summary, integrins represent promising targets for therapeutic strategies to inhibit SCC development and progression. Although most studies to test integrin antagonists have focused on cancers other than SCC, one preclinical study demonstrated that a non-peptide antagonist of integrin av, SM256, significantly inhibited the in vivo growth of a murine SCC (Van Waes et al. 2000), suggesting that integrin av antagonists are likely to be effective inhibitors of SCC in the clinic, as well. Future clinical and preclinical studies in SCC model systems, using antagonists or RNAi

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strategies developed against other integrins discussed in this chapter, should determine if these integrins will also serve as effective therapeutic targets to treat SCC. Acknowledgments The authors are grateful to the members of the DiPersio laboratory, and c olleagues at Albany Medical College, for valuable discussions and insights. Studies conducted by the authors were supported by grants from the NIH/NCI to C.M. DiPersio (R01CA84238, R01CA129637). In addition, J. Lamar was supported by a predoctoral training grant from the National Heart, Lung, and Blood Institute (NIH-T32-HL-07194) and a predoctoral fellowship from the National Cancer Center (06118). We offer our apologies to the many researchers whose valuable contributions to the field could not be cited due to space constraints.

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Chapter 3

Alterations of Transforming Growth Factor-b Signaling in Squamous Cell Carcinomas

Wen Xie and Michael Reiss

Abstract Genetic mouse models have clearly demonstrated that either activation or attenuation of the transforming growth factor-(TGF-)b and the TGF-b signaling pathway can have a major impact on either the genesis and/or the progression of squamous cell carcinomas (SCC) in the epidermis as well as in the head-and-neck region. In general, inactivation of the TGF-b signaling pathway in stratified squamous epithelium promotes the de novo emergence of benign papillomas that have the potential to progress to invasive SCC. On the other hand, activation of TGF-b signaling in established SCC clearly favors their progression to highly invasive and metastatic SCC. Furthermore, a large number of reports of structural and functional alterations in TGF-b pathway components in human SCC cell lines as well as tumor specimens strongly support the idea that this pathway in general, and TGF-b receptors in particular, play an important role in human SCC as well. Attenuation of either Type I TGF-b receptor (TbR)-I or -II signaling promotes SCC development in mice, and mutation and/or loss of expression of TbR-I or -II receptors are commonly seen in human SCC. Thus, approximately 10–15% of head and neck squamous cell carcinoma (HNSCC) display evidence of functional inactivation of TbR receptor signaling, as defined by the absence of pSmad2 and -3 or the presence of an inactivating TGFBR gene mutation. Patients with this tumor type appear to have a particularly favorable clinical outcome. On the other hand, in approximately 40–60% of human SCC TbR expression is reduced but not eliminated. In this context, exposure of the tumor cells to bioactive TGF-b will activate a proinvasive and -metastatic gene expression program, thereby conferring a worse clinical outcome. Therefore, we would like to propose that a structural and functional analysis of the TbR receptors potentially represents a powerful prognostic tool for the management of patients with SCC.

M. Reiss (*) Division of Medical Oncology, Department of Internal Medicine, The Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_3, © Springer Science+Business Media, LLC 2011

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Abbreviations DMBA ECM EMT HNSCC IHC MMP SCC SSCP TGFBR1, Tgfbr-1 TGFBR2, Tgfbr-2 TGF-b TPA TbR-I TbR-II

7,12-Dimethylbenz(a)anthracene Extracellular matrix Epithelial-to-mesenchymal transition Human head-and-neck squamous cell carcinoma(s) Immunohistochemistry Matrix metalloproteinase Squamous cell carcinoma(s) Single-strand conformation polymorphism Type I TGF-b receptor gene Type II TGF-b receptor gene Transforming growth factor-b 12-tetradecanoyl-phorbol-13-acetate Type I TGF-b receptor Type II TGF-b receptor

3.1 Transforming Growth Factor-b Signaling in Nonneoplastic Keratinocytes The transforming growth factor-(TGF)-b family of polypeptides comprises a group of highly conserved proteins with a molecular weight of about 25 kDa (Roberts and Sporn 1993). Following activation of latent TGF-b, the ligand binds to the type II TGF-b receptor (TbR-II). The type I receptor (TbR-I) is then recruited into the ligand/TbR-II complex and phosphorylated and activated by the TbR-II kinase. The activated TbR-I receptor then phosphorylates the receptor-associated Smads, Smad2, and Smad3. These, in turn, form complexes with the common Smad, Smad4, and accumulate in the nucleus. Along with coactivators and cell-specific DNA-binding factors, these nuclear activated Smad complexes regulate gene expression and cellular responses. It is important to realize that, besides this classical pathway, the TbR-II receptor is capable of partnering with other members of the type I receptor family, including Alk-1, Alk-2 and, possibly, Alk-3 (Bharathy et al. 2008; Daly et al. 2008; Konig et al. 2005; Lebrin et al. 2005; Liu et al. 2009). In these cases, TGF-b signals can also activate the BMP Smads 1, 5, and 8. This alternate pathway appears to activate a distinct genetic program (Bharathy et al. 2008; Daly et al. 2008; Konig et al. 2005; Lebrin et al. 2005; Liu et al. 2009). Furthermore, in the context of cancer, this second pathway can become constitutively activated and drive epithelial-to-mesenchymal transitions (EMT), cell motility and invasiveness (Bharathy et al. 2008). In keratinocytes of the skin and other stratified epithelia, TGF-b appears to exert two major functions, tissue homeostasis and the response to tissue injury.

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3.1.1 Homeostatic Functions of TGF-b In the epidermis, TGF-b plays a key role in maintaining the balance between cellular self renewal and differentiation and loss. This function is probably mediated by a low level of active TGF-b signaling. Several lines of evidence support this idea. First, using a transgenic mouse model, Cui et al. (1995) showed that constitutive expression of TGF-b1 in suprabasal epidermal keratinocytes protects against 12-tetradecanoyl-phorbol-13-acetate (TPA)-induced hyperplasia preceded by a strong induction of TbR-II expression. Thus, TGF-b1 and its type II receptor are components of the endogenous homeostatic regulatory machinery in the mouse epidermis. In addition, low levels of endogenous phosphorylated Smad2 (pSmad2) can usually be detected in human keratinocytes in culture (Kareddula et al. 2008). Furthermore, when these cells are treated with selective TbR-I kinase inhibitors, pSmad2 becomes dephosphorylated and cell growth is stimulated (Kareddula et al. 2008). In aggregate, these observations indicate that these cells are subject to a low level of endogenous TGF-b signaling and some degree of growth inhibition even in the absence of exogenous TGF-b. Consistent with these in vitro findings, pSmad2 is normally detectable in murine as well as human epidermis (Xie et al. 2003). This suggests that even though most of the TGF-b secreted into the extracellular matrix (ECM) remains latent, a small amount is activated at the cell surface, presumably to control normal cell proliferation and differentiation. Finally, it is likely through this homeostatic function that TGF-b suppresses tumor development. This is clearly illustrated by mice that are homozygous for a hypomorphic allele of the latent TGF-b binding protein, LTBP-4. These animals fail to express pSmad2 precisely in those epithelial tissues that normally express this particular LTBP isoform, such as colon and lung (Sterner-Kock et al. 2002). Furthermore, the mice are prone to developing colon cancer, supporting the idea of a tissue-specific failure of TGFb’s homeostatic function. Finally, we have found that in vivo most human head and neck squamous cell carcinomas (HNSCC) continue to express pSmad2 (Xie et al. submitted; Xie et al. 2003). As these tumors are actively growing, they have presumably escaped from TGF-b-mediated growth arrest. Besides its role in cell cycle control, TGF-b also maintains tissue homeostasis by regulating apoptosis and perhaps others forms of cell death. For example, TGFb-3 plays a key role in mediating the massive apoptosis of mammary glandular epithelium during postlactational involution, an effect that appears to be mediated by Smad3 (D’Cruz et al. 2002; Faure et al. 2000; Yang et al. 2002). A third important homeostatic function of TGF-b is to protect keratinocytes against DNA damage (Glick et al. 1996, 1999). For example, keratinocytes from Tgfb1 null animals displayed a higher frequency of N-phosphonoacetyl-l-aspartate (PALA)-induced gene amplification than those from wild type animals (Glick et al. 1996, 1999). Moreover, when these cells were transduced with a Ha-Ras oncogene, the frequency of aneuploidy and chromosomal abnormalities was higher than in keratinocytes from TGF-b1 wild-type littermates. Furthermore, exogenous TGF-b1 suppressed gene amplification, aneuploidy, and chromosome breaks in TGF-b1 null

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keratinocytes at lower concentrations than those required for cell cycle arrest, suggesting that the TGF-b signaling pathway directly mediates genomic stability (Glick et al. 1999). It has been demonstrated by more recent studies that genotoxic stress and DNA damage induced by ionizing radiation or cytotoxic chemotherapy are associated with dramatic activation of TGF-b (Biswas et al. 2007; Kirshner et al. 2006). In addition, TGF-b is required for activation of the ATM kinase, which, in turn, coordinates the cellular program of damage control to ionizing radiationinduced DNA damage (Ewan et al. 2002; Kirshner et al. 2006). Thus, in addition to its roles in homeostatic control of cell cycle and -survival, TGF-b1 plays an important role in regulating responses to genotoxic stress, the failure of which may well contribute to cancer development and progression (Andarawewa et al. 2007).

3.1.2 Role of TGF-b in Epidermal Wound Repair In addition to controlling homeostasis, the second major function of TGF-b is to orchestrate and mediate the local response to tissue injury. Wounding results in brisk local activation of TGF-b, which induces epithelial cells to detach from each other, assume a fibroblastoid and motile phenotype (EMT), and to secrete ECM proteins that become incorporated into scar tissue (Roberts et al. 2001). Phenotypically, EMT is characterized by realignment of the actin cytoskeleton from its submembranous location to a cytoplasmic stress-fiber network connected to focal adhesions, down-regulation of epithelial adhesion molecules such as E-cadherin and zonula occludens 1 (ZO-1), and up-regulation of mesenchymal markers such as fibronectin, Fsp1, a-smooth muscle actin and vimentin (Xu et al. 2009). Normally, this process is self-limited in space and time, so that epithelial cells eventually revert back to their cohesive epitheloid phenotype (Barcellos-Hoff 1998). TGF-b is a potent inducer of EMT in HaCaT human immortalized keratinocytes in vitro (Xu et al. 2009; Zavadil et al. 2001). Moreover, treatment of keratinocytes with TbR kinase inhibitors blocked TGF-b-induced EMT in keratinocytes, and enhanced the epitheloid phenotype (Ge et al. 2004; Peng et al. 2005). Consistent with this, mouse skin keratinocytes stably transfected with a dominant-negative Tgfbr-2 gene were unable to undergo the EMT switch in vivo, indicating that EMT was mediated directly by TGF-b signaling in vivo as well (Portella et al. 1998). Furthermore, in TGF-b1/dominant-negative Tgfbr-2 compound transgenic mice, loss of the TbR-II receptor was associated with decreased EMT (Han et al. 2005). In vitro and in vivo studies using dominant-negative Smad mutants, Smad RNAi, tissue-specific Smad knock-out or Tgfbr-1 mutants that cannot bind and activate R-Smads have clearly established the role of Smad signaling in TGF-b-induced EMT (Xu et al. 2009). For example, skin keratinocytes derived from Smad3 null mice have a reduced migration response to TGF-b in vitro (Ashcroft et al. 1999). In contrast, in a mouse model with a targeted deletion of Smad2 in skin keratinocytes, the absence of Smad2 promoted EMT and accelerated skin tumor formation, implying that Smad2 may function to maintain an epithelial phenotype (Hoot et al. 2008).

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The results suggest the possibility that the Smad3 to Smad2 ratio may be the main determinant of EMT. In addition to receptor-associated Smads, Smad4 is also indispensable for EMT. Knockdown expression of SMAD4 by RNAi or expression of a dominant negative mutant of SMAD4 resulted in preserving E-cadherin expression and in suppressing the profibrotic type I collagen in human epidermal keratinocytes in vitro (Valcourt et al. 2005). The Ras-Erk MAP kinase pathway, activated by either EGF or an oncogenic Ras gene, appears to cooperate with TGF-b in inducing EMT in keratinocytes. In cells expressing wild type Ras, TGF-b activates ERK and p38 MAPK, and levels of activation are increased further by treatment with EGF. Activation of MEK/Erk/ MAP kinase signaling enhances TGF-b induced transcription responses, leading to downregulation of E-cadherin, upregulation of N-cadherin and increased matrix metalloproteinase (MMP) expression (Janda et al. 2002; Lehmann et al. 2000). Conversely, inhibition of the MAPK pathway blocked the induction of EMT by TGF-b (Davies et al. 2005). Bae et al. (2009) recently examined the interactions between ras and TGF-b signaling in primary wild type and Smad3 null mouse epidermal keratinocytes. Oncogenic ras and hyperactivation of the ERK1/2 pathway did not affect Smad2 phosphorylation, nuclear translocation or regulation of Smad3 dependent homeostatic gene responses. In contrast, the induction of many extracellular matrix TGF-b/Smad3 target genes was attenuated by v-rasHa. Thus, ERK1/2 activation has distinct effects on the TGF-b transcriptome, especially favoring EMT and matrix remodeling. Besides the MAPK pathway, Notch signaling pathway has also been implicated in the regulation of EMT by TGF-b. In response to TGF-b, the immediate early induction of Hey1, a transcriptional target of Notch, followed by induction of the Notch-ligand, Jagged1 (Jag1), contribute to EMT by disassembly of E-cadherin adherens junctions, resulting in cell–cell separation, and increased cell motility of epidermal keratinocytes (Zavadil et al. 2004). Silencing of Jag1 or Hey1 expression using siRNA or chemical inactivation of Notch signaling block TGF-b-induced EMT (Zavadil et al. 2004). Activation of Hey1 and delayed expression of Jag1 by TGF-b is Smad3-dependent, as it does not occur in Smad3-deficient cells (Zavadil et al. 2004). In addition, Jag1 and Hey were activated in chemically induced squamous cell carcinomas (SCC) in Tgfb-1 transgenic mice, but not in Tgfb-1/ dominant-negative Tgfbr-2 bigenic mice in which the epidermal cells fail to undergo EMT in vivo (Han et al. 2005). In chronic inflammatory conditions, persistent activation of TGF-b causes epithelial cells to be permanently converted to myofibroblasts, eventually resulting in the loss of epithelial structures and progressive tissue fibrosis (Border and Noble 1994; Branton and Kopp 1999; Roberts et al. 2006). Similarly, persistent activation of TGF-b signaling in the context of cancer appears to drive invasion and metastasis via constitutive induction of EMT (Massague 2008). For example, in mouse models of skin carcinogenesis, the highly invasive and metastatic mesenchymal phenotype that is characteristic of late stage SCC appears to be associated with a persistent autocrine or intrinsic activation of TGF-b signaling (Oft et al. 2002; Portella et al. 1998). Thus, in concert with a Ha-ras oncogene,

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activated Smad2 drives the progression of well-differentiated skin SCC to highly invasive undifferentiated SCC by breaking down cell cohesion, increasing cell motility and inducing invasion (Oft et al. 2002). Furthermore, these spindle cells carcinoma with elevated levels of activated Smad2 acquire the capability to metastasize (Oft et al. 2002). Thus, in this case, constitutive activation of TGF-b’s tissue repair function represents an oncogenic event. Loss of E-cadherin expression is a key feature of EMT, particularly in tumor cells (Xu et al. 2009). Consistent with the genetic mouse models, E-cadherin expression is inversely correlated both with tumor grade and with the presence of lymph node metastases in human HNSCCs, suggesting that the loss of the cell adhesion molecule E-cadherin plays an important role in the progression of human HNSCC in vivo (Schipper et al. 1991). Using a dominant-negative form of E-cadherin to repress endogenous E-cadherin expression, Andl et al. (2006) demonstrated this to result in decreased cell adhesion, enhanced migration and invasion of human primary esophageal keratinocytes. Interestingly, overexpression of wildtype E-cadherin was associated with elevated TbR-II mRNA and protein levels. Moreover, the extracellular domains of E-cadherin and TbR-II appear to physically interact, suggesting a coordinated role for E-cadherin and TbR-II loss in epithelial carcinogenesis (Andl et al. 2006).

3.1.3 TGF-b Signaling and Angiogenesis In addition to its effects on normal and malignant keratinocytes, TGF-b exerts a wide range of effects on the normal host microenvironment that indirectly play an important role in the development and progression of SCC and other epithelial carcinomas. For example, both in vitro studies and mouse models have demonstrated that TGF-b1 is a potent inducer of angiogenesis associated with wound repair and tumor development (reviewed in Pardali and ten Dijke (2009)). Increased angiogenesis was observed in preneoplastic head and neck lesions in Tgfb-1 transgenic mice (Lu et al. 2004). Similarly, in xenografts of human HNSCC tumor cells, TGF-b1 appears to attract tumor associated macrophages into the tumor microenvironment, and to induce these cells to secrete angiogenic factors, such as VEGF and interleukin (IL)-8. Moreover, this process can be blocked by treatment with anti-TGF-b1 antibody, suggesting the possibility that such an antibody could be used as an antiangiogenic agent (Liss et al. 2001).

3.1.4 TGF-b Signaling and Immune Suppression Secretion and activation of TGF-b1 by tumor cells also stimulate tumor development and progression indirectly via immune evasion. TGF-b inhibits the proliferation and functional differentiation of T lymphocytes, lymphokine-activated killer cells,

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natural killer cells (NK cells), neutrophils, macrophages as well as B cells (Li and Flavell 2008; Wan and Flavell 2008; Wrzesinski et al. 2007). Consistent with TGFb1’s immune-suppressive but potent chemotactic effects on macrophages and neutrophils, HNSCC arising in Tgfbr-2 null mice are associated with increased expression of endogenous TGF-b and increased macrophage and neutrophil infiltration (Lu et al. 2006). Furthermore, TGF-b expression by tumor cells promotes tumorigenicity by locally repressing immune functions. Thus, Dasgupta et al. (2005, 2006) examined the effects of vaccination with a recombinant vaccinia virus expressing IL-2 (rvv-IL-2) on NK cell-mediated anti-tumor immunity in an orthotopic murine model of HNSCC (SCC VII/SF). SCC VII/SF tumors expressed high levels of TGFb-1, which were down modulated by vaccination with rvv-IL-2. Incubation of NK cells with tumor homogenate or cultured supernatant of SCC VII/ SF cells reduced the expression of NKG2D and CD16. This inhibition appeared to be mediated by TGFb-1, as it could be blocked by treatment with a TGF-bneutralizing antibody (Dasgupta et al. 2005, 2006).

3.1.5 TGF-b Signal Strength Determines Response Type in Human Keratinocytes As summarized above, TGF-b maintains keratinocyte homeostasis on the one hand, and orchestrates EMT and the response to tissue injury on the other. However, surprisingly little is known about how these two fundamentally different responses to TGF-b are regulated. We recently examined the TGF-b-regulated gene expression programs and cellular responses in human keratinocytes as a function of TbR-I kinase activity and TGF-b level (Kareddula et al. 2008). The TGF-b-mediated homeostatic gene response program and cellular growth arrest were extremely sensitive to a reduction in receptor kinase activity, while much stronger inhibition of TGF-b receptor activity was required to block the tissue injury response gene expression program and EMT. Both endogenous TGF-b and high exogenous levels of TGF-b controlled homeostasis, while higher levels of TGF-b were required to induce EMT. The results suggest the working model that two major changes in TGF-b signaling might occur during SCC development: First, a global reduction in receptor signaling and results in loss of homeostatic control and of TGF-b’s tumor suppressive activity. At a later stage of tumor progression, overproduction of bioactive TGF-b might result in activation of a proinvasive, -angiogenic, and -metastatic TGF-b-regulated gene expression program. On the other hand, complete inactivation of TGF-b signaling by, for example, inactivating mutations or deletion of one of the TGFBR genes, would result not only in loss of TGF-b-dependent homeostatic control, but also eliminate TGF-b’s proinvasive and -metastatic actions. Therefore, one might predict that tumors with somatic mutation or deletion of a TGFBR gene might be less aggressive than those in which TGF-b signaling is partly retained. Finally, this model predicts that TGF-b pathway antagonists that target the intracellular signaling machinery (for example, chemical receptor kinase

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inhibitors) may alter the cellular gene expression profile and phenotype in ways that are quite distinct from agents that trap excess ligand (for example, neutralizing TGF-b antibodies). While the former may mimic the effects of lowering receptor expression or receptor mutation, the latter may be more selective in blocking the metastasis-associated gene expression profile. These are important issues to consider in the design of clinical trials of these agents.

3.2 Alterations of TGF-b Signaling and Squamous Cell Cancer In humans, SCCs arise primarily in the skin, the aerodigestive tract (which includes the head and neck region, esophagus, and bronchi), and the uterine cervix. Based on the experimental evidence summarized in the earlier sections, one might predict that loss of TGF-b signaling, both by defects of TGF-b receptors or of Smads, would result in uncontrolled proliferation of squamous epithelial cells and promote the development of SCC. In addition, constitutive activation of TGF-b signaling in advanced stage SCC cell may play a role in SCC progression. In the following sections, we will review the changes in genomic sequence, expression and function of TGF-b, it’s receptors, and the Smads that have been found in human SCC and how these might contribute to the development of human SCC.

3.2.1 TGF-b Ligands 3.2.1.1 TGF-b in Mouse Models of Squamous Carcinogenesis Several studies have shown that TGF-b1 exerts potent effects on the malignant transformation of keratinocytes (Glick et al. 1993, 1994). For example, TGF-b1 null (TGF-b1−/−) primary mouse keratinocytes undergo spontaneous transformation at significantly higher frequency than wild type cells. Furthermore, v-Ha-Ras transformed Tgfb-1−/− keratinocytes transplanted onto the skin of athymic mice gave rise to papillomas with dysplasia that rapidly progressed to multifocal SCC, irrespective of the dermal fibroblast genotype (Tgfb-1 wild-type or null), while grafts from v-Ha-Ras transformed keratinocytes with wild-type Tgfb-1 did not progress beyond well-differentiated papillomas (Glick et al. 1993, 1994). Similarly, when TGF-b1 was conditionally overexpressed in skin keratinocytes and the mice were exposed to the 7,12-dimethylbenz(a)anthracene (DMBA)-TPA two-stage chemical carcinogenesis protocol, TGF-b1 suppressed skin papillomas formation (Cui et al. 1996). Conversely, ectopic expression of constitutively active Tgfb-1 in keratinocytes was associated with increased resistance to TPA-induced benign skin tumor formation (Cui et al. 1995, 1996). These studies clearly demonstrated

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that TGF-b1 acts as a potent tumor suppressor of skin carcinogenesis (Cui et al. 1995, 1996). In a genetic mouse model of HNSCC, in which TGF-b1 expression levels was selectively induceable in squamous epithelia in a dose-dependent manner by the synthetic progesterone inhibitor RU486, increased levels of TGF-b1 were associated with severe inflammation and angiogenesis (Lu et al. 2004). Moreover, Tgfb-1 transgenic epithelia exhibited a phenotype of hyperproliferation in the buccal mucosa, tongue and esophagus. This result appears to contradict previous in vivo studies that had suggested that induction of TGF-b1 inhibits proliferation of keratinocytes (Cui et al. 1996; Wang et al. 1999). One possible explanation is that the increased proliferation of keratinocytes was caused indirectly by inflammation and angiogenesis, which may override the antiproliferative effect of TGF-b. Therefore, some of the growth promoting effects associated with the overexpression of TGF-b at early stages of SCC might be attributed to a tumor-promoting microenvironment induced by TGF-b. In contrast to its ability to suppress initial papilloma and SCC development, TGF-b appears to promote progression of SCC once they have formed. Thus, when TGF-b1 is conditionally overexpressed in skin keratinocytes and the mice are exposed to the two-stage chemical carcinogenesis protocol, TGF-b1 increases the invasiveness and metastatic potential of the SCC once they arise (Cui et al. 1996). Under the influence of Tgfb-1 transgene expression, benign skin tumors underwent EMT, forming invasive spindle carcinoma cells in vivo, which expressed high levels of TGF-b3. Metastatic SN161 cells, derived from a chemically induced mouse skin carcinoma, underwent a reversible conversion to a fibroblastoid phenotype in vitro following treatment with TGF-b1. Furthermore, these SCC cells spontaneously converted to a fibroblastoid phenotype after subcutaneous inoculation in nude mice. Conversely, SN161 clones that were stably transfected with a dominant-negative TGFBR2 gene failed to develop into spindle cell carcinomas in vivo, demonstrating that the EMT was mediated directly by the TGF-b signaling pathway, and was sufficient to enhance tumorigenicity and invasive characteristics of the tumor in vivo (Portella et al. 1998). To address whether the two disparate effects of TGF-b, i.e., inhibition of papilloma formation and enhancement of tumor metastasis, are both dependent on TGF-b receptor signaling, Han et al. (2005) used a mouse skin cancer model that allows stage-specific overexpression of Tgfb-1 in the context of keratinocyte- specific overexpression of a dominant-negative Tgfbr-2 gene. These investigators found that, in a wild type Tgfbr-2 background, induction of TGF-b1 early during carcinogenesis suppressed tumor formation, while, at later stages, TGF-b1 not only failed to inhibit tumor growth but also induced EMT and metastasis. Conversely, in a dominant-negative Tgfbr-2 background, induction of TGF-b1 early in carcinogenesis failed to suppress benign tumor growth. However, even in the presence of TGF-b1, metastases failed to develop, indicating that TbR-II also mediates TGFb’s ability to promote metastasis in a cell autonomous manner (Han et al. 2005). Thus, the effects of TGF-b1 overexpression on tumor development and progression depend, in large part, on the tumor cells’ ability to respond to the ligand.

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3.2.1.2 TGF-b Expression in Human SCCs Cancer-associated increases in TGF-b expression either at the level of protein or mRNA expression have been reported for a number of different tumor types (reviewed in Gold, 1999). Several studies have addressed the question whether development and progression of human SCC are associated with changes in TGF-b expression (Table 3.1). In general, TGF-b expression tends to be increased in SCC compared to adjacent normal tissues (Eisma et al. 1996; Fukai et al. 2003; Hagedorn et al. 2001; Lu et al. 2004). On the other hand, several studies have suggested that SCC-associated TGF-b expression might decrease as a function of tumor grade (El-Sherif et al. 2000; Mincione et al. 2008; Natsugoe et al. 2002; Torng et al. 2003). Thus, these studies of TGF-b expression in SCC have yielded conflicting results and no clear pattern has emerged. This is likely a result of, at least in part, differences in the types of tissue specimens examined, in the specific comparisons reported, the different methods used to assess TGF-b expression levels, and specific TGF-b isoforms examined. However, the differences across studies could also be reconciled if one assumes that SCC can be classified into two major subgroups, one in which TGF-b signaling is completely inactivated, and one in which the signaling pathway is attenuated or altered. The signaling pathway appears to be fundamentally altered in tumor cells in such a way that the tumor cells interpret incoming signals as proinvasive, while they are no longer growth inhibited. Thus, a microenvironment rich in bioactive TGF-b would provide a selective pressure that favors growth and invasion of tumor cells with this particular phenotype.

3.2.2 TGF-b Type I Receptor (TbR-I) 3.2.2.1 TbR-I in Mouse Models of Squamous Carcinogenesis Bian et al. (2009) recently developed a genetic mouse model that allows the conditional deletion of the Tgfbr-1 gene in epithelia of the head-and-neck region. This was accomplished by crossing Tgfbr-1 floxed mice with K14-CreER(tam) mice in which a Cre recombinase is fused to a human estrogen receptor, which can be activated by treatment with tamoxifen in vivo. This fusion protein is driven by a keratin 14 (K14) promoter, which specifically targets gene expression to the basal layer of stratified epithelia. Applying tamoxifen to the oral cavity to induce Cre expression resulted in conditionally deleting Tgfbr-1 in the mouse head and neck epithelia. Four weeks following initiation with DMBA and tamoxifen treatment, basal epithelial cells of Tgfbr-1 knock-out mice displayed enhanced proliferation and loss of apoptosis, which were associated with a decrease in pSmad2 and -3 levels as well as activation of the phosphoinositide 3-kinase/Akt pathway. Moreover, almost half the Tgfbr-1 conditional knockout mice began developing SCC in the head and neck area at 16 weeks, while no tumors were observed in control littermates. These results confirmed the critical role of TGF-b signaling in general and of the TbR-I receptor in particular in suppressing head and neck carcinogenesis.

52

15

13

38

258

48

C

C

HN

HN

HN

C

N/A

148 (57)

N/A

0 (0)

N/A

N/A

Decrease

Decrease

No detectable change Increase

Decrease

Decrease

IHC

IHC

IHC

IHC

IHC, qRT-PCR

IHC

Table 3.1 Alterations in TGF-b signaling in human SCC cell lines and tumors Number with TGF-b/Smad signaling defect (%) Alteration type Detection method SCC type n TGF-b (in vivo) Expression HN 47 0 (0) No detectable IHC change HN 17 N/A Increase ELISA

Trend for increased TGF-b1, 2, 3 expression compared to adjacent stromal cells Only TGF-b1 examined. Decrease defined as £ 10% of cells positive TGF-b1 expression decreased in CIN 1, 2, 3 compared to normal (p < 0.05)

(continued)

Natsugoe et al. (2002) Torng et al. (2003)

Paterson et al. (2001) Hagedorn et al. (2001)

El-Sherif et al. (2000)

Eisma et al. (1996) Eisma et al. (1996) Xu et al. (1999)

TGF-b1, -2 and -3 examined TGF-b1 and -2 expression increased in HNSCC compared with normal TGF-b1 and -2 expression decreased in CIN 1, 2, 3 compared with normal TGF-b1, 2 and -3 expression progressively decreased in CIN 1, 2, 3 compared with normal (p < 0.001). TGF-b1, 2 and 3 mRNA decreased only in HPV16-positive CIN compared with normal (p = 0. 0034, 0.0033, and 0.029) TGF-b1, -2 and -3 examined

References

Comments

3 Alterations of Transforming Growth Factor-b Signaling in SCCs 63

Expression

TbR-I (in vitro) Genomic alteration

7

2

HN

8

HN

HN, E, C

14

22

HN

HN

140

HN

8

80

E

HN, E, C

n 32

SCC type HN

Table 3.1 (continued)

2 (100)

0 (0)

0 (0)

0 (0)

0 (0)

15 (68)

88 (63)

29 (36)

Number with TGF-b/Smad signaling defect (%) 25 (80)

Decrease or absence

No deleterious mutations detected No deleterious mutations detected No deleterious mutations detected Absence

Decrease

Decrease

Increase

Alteration type Increase

Immunoblot

Affinity labeling

Reiss and Stash (1990) Mincione et al. (2008)

Qiu et al. (2007)

RT-PCR and DNA sequencing

Mincione et al. (2008)

Logullo et al. (2003)

Fukai et al. (2003)

References Lu et al. (2004)

RT-PCR and DNA sequencing

TbR-I cell surface binding sites detectable in all SCC lines TbR-I expression decreased in CAL27 and absent in FaDu cells

TGF-b1 expression increased in HNSCC compared to normal control tissues TGF-b1 expression increased compared to normal adjacent tissue as a function of primary tumor size (p = 0.03) TGF-b1 expression decreased compared to normal adjacent tissue as a function of tumor grade (p < 0.001) TGF-b1 expression decreased as a function of tumor grade (p = 0.0028)

Comments

Reiss unpublished data Paterson et al. (2001)

RT-PCR and DNA sequencing

IHC

IHC

IHC

Detection method ELISA

64 W. Xie and M. Reiss

Expression

TbR-I (in vivo) Genomic alteration

47

38

13

38

48

80

22

HN

HN

HN

C

E

HN

30

HN

HN

21

HN

15 (68)

43 (54)

N/A

0 (0)

0 (0)

0 (0)

40 (85)

0 (0)

4 (19)

Decrease

No detectable change No detectable change No detectable change No detectable change Decrease

Intragenic deletion and missense mutations No deleterious mutations detected Absence

IHC

IHC

IHC

IHC

IHC

IHC

PCR-SSCP and DNA sequencing IHC

PCR-SSCP and DNA sequencing

Knobloch et al. (2001)

Chen et al. (2001b)

(continued)

Eisma et al. (1996) Muro-Cacho et al. (1999) Paterson et al. (2001) Trend for decreased TbR-I expression as Hagedorn et al. a function of tumor grade (2001) TbR-I expression unchanged in CIN 1, 2, Torng et al. 3 compared with normal tissue (2003) TbR-I expression decreased as a function Fukai et al. (2003) of depth of invasion (p = 0.0015), lymph node metastasis (p = 0.03), TNM stage (p = 0.01) and survival (p = 0.03) TbR-I expression decreased as a function Mincione et al. of tumor grade (p = 0.0013) (2008)

4 bp deletion (GACA, nt186189)Missense mutations (A230T, K223E, S387Y)

3 Alterations of Transforming Growth Factor-b Signaling in SCCs 65

Expression

7

2

2

HN

HN

8

HN

E

14

HN

7

20

E

HN, E, C

5

8

C

n

SCC type

TbR-II (in vitro) Genomic HN, E, C alteration

Table 3.1 (continued)

1 (50)

1 (50)

3 (43)

7 (100)

1 (13)

1 (7)

0 (0)

2 (40)

2 (25)

Number with TGF-b/Smad signaling defect (%)

Decrease

Decrease

Absence

Missense mutation Absence

No deleterious mutations detected Frameshift mutation

Intragenic deletion

Missense mutations

Alteration type

Immunoblot

Immunoblot

Immunoblot

RT-PCR and DNA sequencing Affinity labeling

PCR-SSCP and DNA sequencing PCR-SSCP and DNA sequencing PCR-SSCP and DNA sequencing RT-PCR and DNA sequencing

Detection method

Garrigue-Antar et al. (1995)

References

Paterson et al. (2001)

Qiu et al. (2007) TbR-II cell surface binding sites Reiss and Stash undetectable (1990) TbR-II expression markedly decreased in Fukuchi et al. TE-8, TT and TTn cells (2002a) TbR-II expression markedly decreased in Mincione et al. FaDu cells (2008) Wang et al. TbR-II expression decreased in Tb (2009) metastatic variant compared to parental Tca8113 cells

Single nucleotide insertion within the poly A tract (bases 709–718, codons 125–128) resulting in C-terminal truncation. R537P (A253)

Osawa et al. (2000)

Intragenic deletions of TbR-II in ME-180 Chu et al. and C-33A (1999)

E526Q (SqCC/Y1), R537P (A253)

Comments

66 W. Xie and M. Reiss

TbR-II (in vivo) Genomic alteration

21

28

16

15

26

10

21

12

E

HN

C

C

E

C

HN

HN

5 (41)

0 (0)

0 (0)

1 (4)

0 (0)

1 (6)

5 (18)

0 (0)

No deleterious mutations detected No deleterious mutations detected Missense and frameshift mutations

No deleterious mutations detected Missense mutation

No deleterious mutations detected Missense mutations, intragenic deletion Nonsense mutation

PCR-SSCP and DNA sequencing PCR-SSCP and DNA sequencing

PCR-SSCP and DNA sequencing PCR-SSCP and DNA sequencing PCR-SSCP and DNA sequencing DNA sequencing

PCR-SSCP and DNA sequencing PCR-SSCP and DNA sequencing

Chen et al. (2001b)

Herzog et al. (2001)

Tanaka et al. 2000)

(continued)

Missense mutations (D35V, T96I, N94D, Nerlich et al. (2003) C136R, A179D, S199stop, R254H, K260T, E282D, D379V, R433G, E440K, S441P, S449P, C520G, T530A, L547P)

Missense mutation (E526Q)

Chen et al. (1999)

Nonsense mutation (E142Stop)

Chu et al. (1999)

Wang et al. (1997a)

Missense mutations (V250A, M373I, S401F, Y448C, K488E)

Garrigue-Antar et al. (1996)

3 Alterations of Transforming Growth Factor-b Signaling in SCCs 67

Expression

n 47

21

23

38

13

38

10

48

80

114

32 22

SCC type HN

E

HN

HN

HN

HN

C

C

E

E

HN HN

Table 3.1 (continued)

N/A 15 (68)

86 (75)

23 (29)

N/A

6 (60)

0 (0)

N/A

N/A

20 (87)

6 (29)

Number with TGF-b/Smad signaling defect (%) 43 (91)

Decrease Decrease

Absent

No detectable change Decrease

No detectable change Decrease

Decrease

Decrease

Decrease

Absence

Alteration type Absence

qRT-PCR IHC

INH

IHC

IHC

RT-PCR

IHC

IHC

IHC

RT-PCR

RT-PCR

Detection method IHC

References Eisma et al. (1996) Garrigue-Antar et al. (1996) Compared to normal tissue Wang et al. (1997a) TbR-II expression decreased as a Muro-Cacho function of tumor grade et al. (1999) Normal versus HNSCC: p < 0.04HNSCC Paterson et al. versus metastasis: p < 0.01 (2001) Trend for decreased TbR-II expression as Hagedorn et al. a function of tumor grade (2001) Herzog et al. TbR-II mRNA expression decreased by ³50% (2001) TbR-II expression unchanged in CIN 1, Torng et al. 2, 3 compared to normal tissue (2003) Fukai et al. TbR-II expression decreased as a (2003) function of depth of invasion (p = 0.0012), lymph node metastasis (p = 0.006), TNM stage (p = 0.04), and survival (p = 0.02) Andl et al. Absence TbR-II expression coincided with loss of E-cadherin (2006) Normal versus HNSCC: p < 0.01 Lu et al. (2006) TbR-II expression decreased as a Mincione et al. function of tumor grade (p = 0.0028) (2008)

Comments

68 W. Xie and M. Reiss

Smad2 (in vivo) Genomic alteration

Phosphorylation

Smad2 (in vitro) Genomic alteration

20

45

E

C

5

E

8

HN

7

6

C

HN

8

108

HN

HN

1 (2)

0 (0)

2 (40)

2 (29)

1 (13)

1 (17)

0 (0)

71 (66)

No deleterious mutations detected Frameshift mutation

Missense mutation Absence (pSmad2) Absence (pSmad2)

Frameshift mutation

No deleterious mutations detected

Decrease

PCR-SSCP and DNA sequencing (RT)PCR-SSCP and DNA sequencing

Immunoblot

(RT)PCR-SSCP and DNA sequencing RT-PCR and DNA sequencing Immunoblot

RT-PCR and mutation detection by in vitro synthesis of protein assay

IHC

Single nucleotide insertion at codon 122

pSmad2 expression markedly decreased in TE-13 and TE-15 cells

S276L (SCC-15)

Single nucleotide deletion at codon 428 (C-33A)

Normal versus HNSCC: p < 0.05

(continued)

Maliekal et al. (2003)

Osawa et al. (2000)

Qiu et al. (2007) Yan et al. (2000) Fukuchi et al. (2002a)

Maliekal et al. (2003)

Riggins et al. (1997)

Wang et al. (2009)

3 Alterations of Transforming Growth Factor-b Signaling in SCCs 69

Phosphorylation

Expression

80

117

786

C

HN

787

HN

E

117

C

170

80

E

HN

10

C

13

167

HN

HN

n 13

SCC type HN

Table 3.1 (continued)

145 (19)

0 (0)

29 (36)

24 (14)

5 (38)

4 (1)

0 (0)

7 (9)

6 (60)

2 (1)

Number with TGF-b/Smad signaling defect (%) 5 (38)

Absence (pSmad2) Absence (pSmad2) Absence (pSmad2) Absence (pSmad2) Absence (pSmad2)

Absence

Absence

Absence

Absence

Absence

Alteration type Absence

IHC

IHC

IHC

IHC

IHC

IHC

IHC

IHC

RT-PCR

IHC

Detection method IHC

Absence of pSmad2 predicts for favorable outcome

Absence of pSmad2 predicts for poor outcome

Absence of pSmad2 in the context of Smad2 expression

Undetectable Smad2 mRNA in 1 of 4 premalignant samples, and in 6 of 10 SCC samples

Comments

Fukuchi et al. (2006) Kloth et al. (2008) Xie et al. (submitted) Muro-Cacho et al. (2001) Xie et al. (2003) Fukuchi et al. (2006) Kloth et al. (2008) Xie et al. (submitted)

References Muro-Cacho et al. (2001)) Xie et al. (2003) Maliekal et al. (2003)

70 W. Xie and M. Reiss

Smad4 (in vivo) Genomic alteration

Expression

Smad4 (in vitro) Genomic alteration

8

2

HN

HN

20

13

C

HN

7

8

HN

E

6

C

7

7

HN, E, C

HN, E, C

16

HN

1 (5)

1 (50)

2 (25)

4 (13)

2 (29)

3 (43)

2 (25)

0 (0)

1 (14)

2(13)

Missense mutation

Decrease

Absence

Absence

Absence

Nonsense mutations Homozygous deletion No deleterious mutations detected Nonsense mutation and Homozygous deletion Absence

RT-PCR and DNA sequencing

Immunoblot

Immunoblot

Immunoblot

Immunoblot

Qiu et al. (2007)

Kim et al. (1996) Yan et al. (2000) Maliekal et al. (2003)

Missense mutation (I525V)

(continued)

Kim et al. (1996)

Smad4 expression absent in FaDu, CE-48 Yan et al. and C4-I cells (2000) Smad4 expression markedly decreased in Fukuchi et al. TE-1 and TE-2 cells (2002b) Baldus et al. Smad4 deficiency due to an intronic (2005) rearrangement or deletions of 3¢ exons Qiu et al. (2007) Wang et al. Smad4 expression decreased in Tb (2009) metastatic variant compared to parental Tca8113 cells

Q245stop (CAL27), Homozygous deletion (FaDu)

(RT)PCR-SSCP and DNA sequencing RT-PCR and DNA sequencing

Immunoblot

Homozygous deletion (FaDu)

V526stop (UMSCC22A+B)

RT-PCR and DNA sequencing Southern blot

3 Alterations of Transforming Growth Factor-b Signaling in SCCs 71

Expression

13

258

80

172

10

41

117

108

HN

E

HN

C

C

C

HN

20

E

HN

n 5

SCC type HN

Table 3.1 (continued)

66 (61)

15 (13)

10 (25)

3 (30)

38 (22)

41 (51)

175 (68)

5 (38)

0 (0)

Number with TGF-b/Smad signaling defect (%) 0 (0)

Decrease

Absence

Absence

Absence

Absence

Absence

Decrease

Alteration type No deleterious mutations detected No deleterious mutations detected Absence

IHC

IHC

IHC

RT-PCR

IHC

IHC

IHC

PCR-SSCP and DNA sequencing IHC

Detection method Full sequencing

Smad4 loss associated with depth of invasion (p = 0.036), tumor size (p = 0.02), and HPV subtype (p = 0.02) Normal versus HNSCC p < 0.001, decreased as a function of tumor grade and stage

Smad4 loss associated with depth of invasion (p = 0.0008) and TNM stage (p = 0.008)

Concurrent loss of (p)Smad2 expression in 4 of 5 cases

Comments

Wang et al. (2009)

Xie et al. (2003) Maliekal et al. (2003) Baldus et al. (2005) Kloth et al. (2008)

Muro-Cacho et al. (2001) Natsugoe et al. (2002) Fukuchi et al. (2002b)

Osawa et al. (2000)

References Schutte et al. (1996)

72 W. Xie and M. Reiss

E

E

E

115

5

115

786

N/A

1 (20)

N/A

95 (12)

Decrease

Absence

Decrease

Absence

IHC

Immunoblot

IHC

IHC

Smad7 undetectable in TE-2 cells

Smad6 loss associated with depth of invasion (p = 0.0003)

Smad7 loss associated with depth of invasion (p = 0.0173) HN, HNSCC; E, Esophageal SCC; C, SCC of the uterine cervix; N/A, Not applicable; IHC, Immunohistochemistry

Smad7 (in vivo)

Smad7 (in vitro) Expression

Smad6 (in vivo)

HN

Osawa et al. (2004)

Fukuchi et al. (2002a)

Osawa et al. (2004)

Xie et al. (submitted)

3 Alterations of Transforming Growth Factor-b Signaling in SCCs 73

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W. Xie and M. Reiss

3.2.2.2 Tbr-I in Human SCC To date, to our knowledge, no genomic alterations of the TGFBR1 gene in human SCC cell lines have been reported (Table 3.1). However, a small number of somatic mutations of the TGFBR1 gene in human HNSCC have been described (Table 3.1). In our own study of fine-needle aspirates of 21 HNSCC metastases, we identified mutations of TGFBR1 in four cases (19%). These included one somatic intragenic 4-bp deletion that predicts for a truncation of the receptor protein (Chen et al. 2001a). In addition, we identified missense mutations located between the juxtamembrane- and serine-threonine kinase domains in three other cases. One of these mutations resulted in an alanine-tothreonine substitution at codon 230 (A230T) which disrupts receptor’s signaling activity by causing rapid protein degradation within the endoplasmatic reticulum. Moreover, a mutation resulting in a serine-to-tyrosine substitution at codon 387 (S387Y) was observed in a metastasis but not in the corresponding primary tumor. This same S387Y mutation had previously been detected in breast cancer metastases and is associated with a diminished ability to mediate TGF-b dependent signaling as compared with wild type TbR-I (Chen et al. 1998). In addition, it has recently been shown that the lysine at codon 389 of TbR-I is required for sumoylation, a posttranslational modification that enhances receptor signaling (Kang et al. 2008). The S387Y mutation affects a residue close to the sumoylation site and prevents sumoylation of TbR-I, thus attenuating TGF-b/Smad signaling and associated growth inhibition (Chen et al. 1998, 2001b; Kang et al. 2008). In contrast to our own study, Knobloch et al. (2001) failed to detect any deleterious mutations in the TGFBR1 gene in a series of 30 HNSCC. In summary, cancer-associated mutations of TGFBR1 that result in attenuation or inactivation of the TGF-b signaling pathway do occur in human SCC, albeit at a low (<10%) frequency. In contrast to the rare TGFBR1 gene mutations, detectable decreases in TbR-I expression appear to occur much more commonly in human SCC (Table 3.1). Several studies have reported a significant decrease or loss of TbR-I protein expression in the majority of SCC (Eisma et al. 1996; Fukai et al. 2003; Mincione et al. 2008), although other studies have failed to confirm these findings (Hagedorn et al. 2001; Muro-Cacho et al. 1999; Paterson et al. 2001; Torng et al. 2003). Furthermore, two studies have suggested that TbR-I protein expression decreases as a function of tumor grade, suggesting an association with tumor progression (Hagedorn et al. 2001; Mincione et al. 2008). Possible mechanisms of receptor down-regulation include epigenetic changes, such as DNA hypermethylation in gene promoter regions, as well as posttranscriptional or -translational modifications, although none of these mechanisms have been documented to occur in human SCC (Kang et al. 1999). In summary, while the incidence of TGFBR1 gene mutations in human SCC is probably <10%, published studies suggest that approximately 40% of these tumors are associated with a decrease in TbR-I expression. The biological and clinical implications of these findings remain to be determined.

3 Alterations of Transforming Growth Factor-b Signaling in SCCs

75

3.2.3 TGF-b Type II Receptor (TbR-II) There is strong evidence to suggest that genetic, epigenetic, or transcriptional silencing of the TbR-II receptor completely abrogates TGF-b signaling, leading to resistance to the growth-inhibitory effects of the TGF-b cytokine (Kareddula et al. 2008). Conversely, transgenic overexpression of Tgfbr-2 in mice is associated with enhanced tumor suppressor activity (Cui et al. 1996). Thus, Tgfbr-2 qualifies as a bona fide tumor suppressor gene. Consistent with this concept, many types of human cancers, including SCC, have been found to harbor either missense or frameshift mutations within the TGFBR2 receptor gene or a reduction in TbR-II receptor expression.

3.2.3.1 TbR-II in Mouse Models of SCC Several genetic mouse models have illuminated the role of TbR-II in SCC development and progression. For example, transgenic mice that expressed a dominantnegative Tgfbr-2 transgene exhibited a thickened and wrinkled skin (Wang et al. 1997b). Histologically, the epidermis was markedly hyperplastic and hyperkeratotic. In vivo BrdU labeling showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei found in both basal and suprabasal cells. Consistent with these findings, the growth rate of primary keratinocytes from these dominantnegative Tgfbr-2 mice was increased rate in comparison with non-transgenic controls and these cells were resistant to TGF-b-induced growth inhibition (Wang et al. 1997b). In an orthotopic mouse model of HNSCC, in which dominant-negative TGFBR2 expressing H400 human SCC cells were inoculated into the floor of the mouth of athymic mice, comparable numbers of primary tumors arose at the site of inoculation as in controls (Huntley et al. 2004; Prime et al. 2004). However, dominantnegative TGFBR2 tumors were less differentiated, as demonstrated by the absence of keratin 10. Furthermore, metastatic dissemination to the lungs and lymphatics was more evident in grafts of cells expressing dominant-negative TGFBR2 than of controls. In aggregate, these results demonstrate that attenuation of TGF-b receptor signaling in an established SCC cell line leads to an increase in metastatic potential without affecting tumor growth (Huntley et al. 2004; Paterson et al. 2001; Prime et al. 2004). Lu et al. (2006) recently developed an organ-specific inducible Tgfbr-2 knockout mouse model, which consists of two mouse lines, K5.CrePR1 mice and Tgfbr-2f/f mice. In the K5.CrePR1 line, a Cre recombinase is fused to a truncated human progesterone receptor that can be activated by RU486. This fusion protein is driven by a keratin 5 (K5) promoter, which targets gene expression specifically to the epidermis and the oropharyngeal epithelium (Lu et al. 2004). After crossing the K5. CrePR1 line with the Tgfbr-2If/f line, Tgfbr-2 deletion in head-and-neck epithelia was achieved by administering RU486. TbR-II and pSmad2 were undetectable in the

76

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buccal tissue or tongue of Tgfbr-2−/− mice in comparison with those of wild type control mice. Although ablation of Tgfbr-2 induced only modest epithelial hyperplasia at 1 year, crossing Tgfbr-2−/− mice with K-ras12D transgenic mice greatly accelerated the development of SCC as compared with a parallel group, in which DMBA was used to chemically induce H-ras mutations. Unlike their Tgfbr-2 wild type littermates, early lesions in K-ras12D/+/ Tgfbr-2−/− or DMBA-initiated Tgfbr-2−/− epithelia progressed from hyperplasia to dysplasia to invasive SCC (Lu et al. 2006). Of note, loss of TbR-II expression in head-and-neck epithelia also resulted in increased endogenous TGF-b1 expression (Lu et al. 2006), which enhanced the effect of TGF-b1 on tumor stroma, such as induction of basement membrane degradation by MMPs, inflammation by inflammatory cytokines/chemokines and angiogenesis. Using a similar experimental approach, Guasch et al. (2007) conditionally induced loss of Tgfbr-2 in mice by using a keratin 14 (K14) promoter, which is active in stratified squamous epithelia of back skin, oral cavity, and the anogenital region, as well as in some glandular and ductal epithelia, to express the Cre recombinase. The K14-Cre/Tgfbr-2fl/fl conditional knockout mice, which lacked TGF-b receptor signaling in epithelial cells as shown by absent TbR-II and pSmad2 expression, spontaneously developed invasive SCC in the anogenital epithelium, a region with intense mitogenic activity. In contrast, the Tgfbr-2 null epidermis from back skin remained phenotypically normal except for a mild increase in epithelial cell proliferation. These findings suggest that endogenous TGF-b normally provides a tonic growth inhibitory signal in surface epithelia, and that secondary events are required to induce progression to SCC in the anogenital region (Guasch et al. 2007). Grafts of epidermal cells transduced with a retroviral vector expressing an oncogenic Ha-Ras mutation (Ha-RasV12) onto mice develop into benign papilloma (Roop et al. 1986). However, in striking contrast, grafts of Ha-RasV12/Tgfbr-2−/− keratinocytes rapidly developed into large, aggressive tumors. Thus, the absence of TbR-II appeared to provide the epithelial cells with a selective advantage that leads to progression beyond the papilloma stage. In aggregate, these genetic mouse models have demonstrated that TbR-II has a key role for in mediating TGF-b-induced growth inhibition of the epidermis in vivo and in maintaining epidermal homeostasis, and that loss of TGF-b signaling destabilizes tissue homeostasis at multiple levels, thereby promoting tumor development and progression. Overall, these two Tgfbr-2 knock-out HNSCC mouse models represent close phenocopies of the recently reported conditional Tgfbr-1 knock-out mouse model (Bian et al. 2009) (see Sect. 3.2.2).

3.2.3.2 TbR-II in Human SCC Human SCC cell lines appear to be uniformly refractory to TGF-b-mediated growth arrest (Reiss and Sartorelli 1987; Reiss and Stash 1990). In two of seven SCC cell lines examined, TGF-b failed to activate pSmad2/3 (Yan et al. 2000). In each of these two HNSCC cell lines, missense mutations in the TGFBR2 gene were identified (Garrigue-Antar et al. 1995). Both were G:C to C:G transversions, which

3 Alterations of Transforming Growth Factor-b Signaling in SCCs

77

resulted in glutamic acid-to-glutamine (E526Q) and arginine-to-proline (R537P) substitutions, respectively. Moreover, both mutations were located within the serine-threonine kinase domain. The E526Q mutation was associated with inactivation of TbR-II kinase activity, thus blocking ligand-dependent activation of TbR-I and, consequently, Smad2/3 phosphorylation (Bharathy et al. 2008; Qiu et al. 2007). In contrast, the R537P mutant was associated with a constitutively active TbR-II kinase, which results in aberrant activation of Smad1/5 signaling (Bharathy et al. 2008; De et al. 1998; Garrigue-Antar et al. 1995). This, in turn, endowed the carcinoma cells with a highly motile and invasive fibroblastoid phenotype. The activated phenotype was TbR-I-independent and could be reversed by a dual TbR-I and -II kinase inhibitor (Bharathy et al. 2008). Thus, identification of such activated TbR-II receptor mutations in tumors may have direct implications for appropriately targeting these cancers with selective therapeutic agents. In addition to the missense mutations, several instances of single nucleotide insertions or deletions within the TGFBR2 coding sequence have been identified in SCC cell lines (Chu et al. 1999; Paterson et al. 2001) (Table 3.1). The frame shift mutations result in expression of truncated TbR-II receptors. By acting in a dominant negative fashion, the truncated receptors attenuate TGF-b signaling (Huntley et al. 2004; Paterson et al. 2001). Genomic alterations of the TGFBR2 gene have also been reported to occur in HNSCC in vivo (Table 3.1). Wang et al. (1997a) identified somatic mutations in the coding region of TbR-II in 6 of 28 primary HNSCCs. These mutations included five missense mutations (A:T to G:C transitions in codons 250, 401, 448, and 488, and a G:C to G:A transversion in codon 373), as well as a 38-bp deletion between nucleotides 1825–1862, which occurs within kinase subdomain XI. The latter appears to be critical for TbR-II catalytic activity because deletion of this region resulted in a reduction of in vitro kinase activity of the receptor (Wieser et al. 1993). Missense mutations in codons 448 (TAC to TGC) and 488 (AAG to GAG) within subdomains IX and X, respectively, are also likely to affect kinase activity, although this has not been confirmed (Wang et al. 1997a). Nerlich et al. (2003) used laser capture microdissection, followed by PCR-SSCP analysis of 105 tissue specimens from 12 patients with invasive SCC of the larynx. A total of 23 cancer-associated missense- or frameshift mutations were identified (Table 3.1). There were no significant morphologic differences between mutant and nonmutant tumor cells. Tumor cell proliferation rates, as measured by Ki-67 immunostaining, was greater at the tumor periphery than in the tumor center and seemed to correlate with areas containing TGFBR2 gene mutant cells (Nerlich et al. 2003). A missense mutation at codon 526 causing a glutamic acid to glutamine substitution (E526Q) in the TbR-II serine/threonine kinase domain was identified in 1 of 26 esophageal SCC (Tanaka et al. 2000). This mutation is identical to the one we identified in the SqCC/Y1 HNSCC cell line and is associated with loss of serine/threonine kinase activity (Garrigue-Antar et al. 1995). Finally, Chen et al. (1999) identified a G:T transversion in exon 3 of TGFBR2 that introduces a premature stop codon (E142Stop) and presumably results in the synthesis of a truncated soluble exoreceptor in a case of SCC of the uterine cervix. In contrast to these three studies, no

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W. Xie and M. Reiss

mutations were identified in other series of esophageal SCC (Garrigue-Antar et al. 1996), HNSCC (Chen et al. 2001b) or cervical SCC (Chu et al. 1999; Herzog et al. 2001) (Table 3.1). Overall, somatic mutations of the TGFBR2 gene do occur in human SCC, but the frequency of these events is low at approximately 10%. In contrast to the relatively low frequency of TGFBR2 mutations, reductions in TbR-II expression appear to be much more common in human SCC (Eisma et al. 1996; Fukai et al. 2003; Garrigue-Antar et al. 1995; Wang et al. 1997a, 2009) (Table 3.1). Thus, we found cell surface TbR-II expression to be markedly reduced in each of seven human SCC cell lines examined (Reiss and Stash 1990). Consistent with this initial observation, other investigators have reported that TbR-II protein expression is often reduced in SCC cell lines, particularly in metastatic variants (Fukuchi et al. 2002a; Mincione et al. 2008; Wang et al. 2009). Similar trends have been observed in human SCC tissue specimens. For example, Garrigue-Antar et al. (1996) reported low to undetectable TGFBR2 mRNA levels in 6 of 21 human esophageal SCC. This included one case associated with hypermethylation of promoter sequences of the TGFBR2 gene (Garrigue-Antar et al. 1996). Similar reductions of TGFBR2 gene expression have been noted by other investigators in HNSCC and in cervical SCC (Herzog et al. 2001; Lu et al. 2006; Wang et al. 1997a). To determine whether TbR-II loss occurs mainly at the pre- or post-translational level, Lu et al. (2006) compared TGFBR2 transcript and protein levels in 32 paired samples of HNSCC and adjacent normal tissue. Close to 70% (22 of 32) HNSCC specimens exhibited a >50% decrease in TGFBR2 mRNA level compared with the average expression level in normal tissue samples. Immunohistochemical staining for TbR-II was of similar intensity in both normal oropharyngeal epithelium and the mucosa adjacent to the tumor, but was significantly reduced or lost in HNSCC specimens. Importantly, TbR-II loss observed by immunohistochemistry correlated with reduced mRNA levels, indicating that reduction or loss of TbR-II expression in human HNSCC occurs predominantly at the transcriptional level (Lu et al. 2006). Consistent with these findings, TbR-II protein expression is often markedly reduced in human SCC compared to normal mucosa (Table 3.1) (Andl et al. 2006; Eisma et al. 1996; Fukai et al. 2003; Mincione et al. 2008; Muro-Cacho et al. 1999; Paterson et al. 2001; Wang et al. 2009). Overall, approximately 60% of human SCC appear to be associated with significant reductions in TbR-II protein expression (Table 3.1). Several studies have suggested that loss of TbR-II receptor expression is associated with progression of HNSCC. While normal oral squamous epithelium homogenously expresses TbR-II, matched cases of dysplastic epithelium and carcinoma in situ showed a mild decrease in receptor expression. Well-differentiated to moderately differentiated carcinomas showed heterogeneous expression of variable intensity, and, in poorly differentiated carcinomas, TbR-II expression was significantly reduced or completely lost (Mincione et al. 2008; Muro-Cacho et al. 1999). Furthermore, in a small study of 13 oral HNSCC, TbR-II protein expression in lymph node metastases was lower than in the primary tumors, and lower in the primary tumors than in adjacent normal oral epithelium. Similar trends were found for TbR-I expression, but these differences were not statistically significant

3 Alterations of Transforming Growth Factor-b Signaling in SCCs

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(Paterson et al. 2001). The fact that reduced TbR-II expression has been associated with tumor grade, depth of invasion and metastasis suggests that it is a contributing factor involved in the progression of HNSCC. Finally, several studies have provided evidence for functional inactivation of TbR receptors in SCC based on loss of expression of the phosphorylated form of Smad2, pSmad2 (Fukuchi et al. 2002a; Muro-Cacho et al. 2001; Xie et al. submitted; Xie et al. 2003; Yan et al. 2000). Thus, Muro-Cacho et al. (2001) reported loss of pSmad2 in 5 of 13 HNSCC. Subsequently, we found that 14% (24 of 170) of HNSCC failed to express pSmad2 (Xie et al. 2003). In a more recent study of 792 human HNSCC, 18.5% failed to express pSmad2 (Xie et al. submitted). Moreover, among 198 patients with survival information, those with pSmad2 negative tumors had a better overall survival rate compared with those with pSmad2-positive SCC. Our results indicate that inactivation of TGF-b receptor and Smad signaling occurs in a subset of HNSCC and the defects of TGF-b receptor signaling may be associated with favorable clinical outcome (Xie et al. submitted). In contrast, Fukuchi et al. (2006) reported that, in patients with esophageal SCC, the absence of pSmad2 was predictive of poor survival (Fukuchi et al. 2006). One possible explanation for the discordant results between these two studies is the fact that we controlled for total Smad2 expression, a precaution that was not taken in the study by Fukuchi et al. (2006). In aggregate, complete inactivation of TGF-b receptor signaling, as evidence by intragenic mutation of one of the receptor genes or by absence of pSmad2, is a relatively infrequent event that occurs in approximately 15% of human SCC and appears to be associated with a relatively good clinical outcome. In contrast, a quantitative reduction in TGF-b receptor expression appears to occur in approximately 60% of SCC, and appears to be associated with tumor progression and, therefore, a worse clinical outcome. These conclusions are entirely consistent with our findings that TGF-b signal strength is a major determinant of the gene expression profiles and cellular phenotype of human keratinocytes (Kareddula et al. 2008). Furthermore, we might speculate that the two types of SCC-associated TGFBR2 mutants exemplify the two major altered TGF-b signaling phenotypes seen in this disease (Bharathy et al. 2008). In one class, exemplified by the E526Q mutant, TGF-b signaling is completely abrogated, giving rise to a low-grade verrucous carcinoma (Bharathy et al. 2008; De et al. 1998; Reiss et al. 1985). On the other hand, the R537P TGFBR2 mutant may be representative of the class of SCC in which TGF-b receptor signaling is attenuated but not completely abolished, resulting in a shift from Alk-5/Smad2/3 signaling to Alk-2/3/Smad1/5 signaling that results both in loss of cell cycle control and in a more aggressive tumor phenotype (Bharathy et al. 2008).

3.2.4 Smads Following activation by TGF-b receptors, Smad proteins serve as intracellular transducers of TGF-b signals to activate specific transcriptional programs and cellular

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responses. This raises the question whether SCC development and progression might be associated with structural or functional alterations of Smad signaling. 3.2.4.1 Receptor-Associated Smads (Smad2 and -3) Thus far, only three cancer-associated mutations of the SMAD2 gene have been reported in human SCC cell lines (Maliekal et al. 2003; Qiu et al. 2007) (Table 3.1). One of them is a missense mutation, located in exon 8 (MH2 domain) at codon 276 (TCG to TTG, Ser to Leu) that is associated with loss of the second SMAD2 allele. While the S276L mutation was not associated with a change of Smad2 mRNA or protein expression, it may affect protein–protein interactions or protein stability. However, no functional studies of this mutant were reported (Qiu et al. 2007). The other two reported SMAD2 gene mutations consisted of single nucleotide deletions or insertions, resulting in truncation of the Smad2 protein (Maliekal et al. 2003). Besides this small number of (presumed) inactivating mutations, significant reductions in Smad2 mRNA or protein expression have only been noted in approximately 2% of human SCC (Table 3.1) (Fukuchi et al. 2006; Kloth et al. 2008; Maliekal et al. 2003; Muro-Cacho et al. 2001; Xie et al. submitted; Xie et al. 2003). 3.2.4.2 Common Mediator Smad (Smad4) Yan et al. (2000) have reported on the status of the common mediator Smad, Smad4, in a panel of seven human SCC lines known to be refractory to TGF-bmediated cell cycle arrest. Three of these SCC lines, C4-1, CE-48 and FaDu, failed to express Smad4, two on the basis of transcriptional silencing and one by a posttranscriptional mechanism (Table 3.1). Similarly, Qiu et al. (2007) identified a homozygous nonsense mutation in exon 5 of the Smad4 gene (codon 245, CAG to TAG, Glut to stop) in another human HNSCC cell line. This mutation resulted in complete loss of Smad4 protein expression. The HNSCC cell line, Fadu, has undergone a homozygous deletion of SMAD4 (Qiu et al. 2007). Reiss et al. (1997) showed that restoration of the SMAD4 locus suppresses tumor progression. Microcell mediated transfer was used to introduce a wild-type copy of chromosome 18 into FaDu cells. Among the total 10 hybrid clones of chromosome 18, five developed into invasive carcinomas in nude mice at a significantly lower rate and with a longer latency compared to parental tumor cells, whereas the five remaining clones were tumorigenic. Each of the hybrid clones that were either completely or partly suppressed carried an intact copy of SMAD4, whereas this gene was deleted in the two most tumorigenic clones. Furthermore, the presence of SMAD4 correlated with partial restoration of cellular responsiveness to TGF-b. These results provide further evidence for tumor suppression by SMAD4 in the HNSCC (Reiss et al. 1997). In a subsequent study, Hummer et al. (2003) reexpressed the Smad4 gene in FaDu cells, both by microcell-mediated chromosome transfer and by retroviral infection. This study confirmed our findings that the reexpression of the

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Smad4 gene by either method partially restored TGF-b responsiveness in FaDu cells with respect to growth inhibition. However, only the microcell hybrids showed growth retardation in organotypic raft culture and an enhanced ability to upregulate fibronectin. The results suggest that, in addition to a homozygous deletion of Smad4, FaDu cells may carry additional defects within the TGF-b signaling pathway, thereby limiting the extent of TGF-b responsiveness upon Smad4 reexpression. Alternatively, the region of chromosomal deletion might include additional genes that cooperate with Smad4 in suppressing tumor growth. Kim et al. (1996) examined 20 primary HNSCC tumors and 16 HNSCC cell lines for potential defects of the SMAD4 gene. One nonsense mutation at codon 526 was identified in two cell lines (UMSCC22A and UMSCC22B) derived from the primary tumor and a lymph node metastasis of the same patient, respectively. In addition, loss of heterozygosity (LOH) at l8q21 was observed in 47% (7 of 15) of informative tumors (Kim et al. 1996). In a second study, LOH of 18q21 was found in 28% (14 of 50) of HNSCC. However, no intragenic mutations or deletions of SMAD4 were identified in any of the 5 HNSCC cases for which full sequencing of all 11 exons of the SMAD4 gene was carried out (Schutte et al. 1996). Similarly, Osawa et al. (2000) failed to detect any deleterious mutations in the SMAD4 gene in a series of 20 esophageal SCC (Table 3.1). In human SCC, decreased expression of Smad4 appears to occur more commonly than genomic alterations of the SMAD4 gene (Table 3.1). For example, among 170 HNSCC specimens assembled in a tissue array, we found that 38 cases (22%) failed to express Smad4 protein (Xie et al. 2003). We have recently extended these findings in a study of 792 HNSCC specimens, 96 (12%) of which failed to express Smad4 (Table 3.1). These results indicate that loss of Smad4 expression is relatively common in human HNSCC, suggesting that this plays a role in the tumorigenesis of a significant subset of HNSCC (Xie et al. submitted). Interestingly, in contrast to loss of receptor signaling, no significant associations between Smad4 status and clinical outcome of patients were observed (Xie et al. submitted). Similarly, loss of Smad4 expression is relatively common in esophageal SCC (51%). However, even though Smad4 loss was associated with depth of invasion (p = 0.0008) and TNM stage (p = 0.008), it did not appear to have a negative impact on clinical outcome (Fukuchi et al. 2002b). In aggregate, approximately 25% of SCC cell lines and tumor specimens appear to be associated with loss of Smad4 protein expression as a consequence of genetic inactivation of the SMAD4 gene (Table 3.1). However, the biological or clinical significance of these events remain to be determined. 3.2.4.3 Inhibitor Smads (Smad6 and -7) The primary role of the so-called inhibitory Smads, Smad6 and -7, is to provide negative feedback to the TGF-b signaling pathway. Therefore, one might predict that overexpression of either of these I-Smads might be associated with SCC development. However, a small number of studies suggest that the opposite may be the

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case (Table 3.1). Thus, in one study, Smad7 expression was markedly reduced in one of five esophageal SCC cell lines examined (Fukuchi et al. 2002a). Along the same lines, Osawa et al. (2004) found Smad6 and -7 expression to be decreased as a function of depth of invasion in the case of esophageal SCC. Whether these findings can be validated in other types of SCC, and what their biological and clinical significance is, remains to be determined.

3.2.5 Summary and Conclusion In summary, genetic mouse models have clearly demonstrated that either activation or attenuation of the TGF-b and the TGF-b signaling pathway can have a major impact on either the genesis and/or the progression of SCC in the epidermis as well as in the head and neck region. In general, inactivation of the TGF-b signaling pathway in stratified squamous epithelium promotes the de novo emergence of benign papillomas that have the potential to progress to invasive SCC. On the other hand, activation of TGF-b signaling in established SCC clearly favors their progression to highly invasive and metastatic SCC. Moreover, a large number of reports of structural and functional alterations in TGF-b pathway components in human SCC cell lines as well as tumor specimens strongly support the idea that this pathway plays an important role in human SCC as well. However, whether and to what extent the genetic mouse models phenocopy the human disease remains to be clarified. For example, even though the mouse models might predict that a reduction in ambient TGF-b levels might promote SCC development, studies of TGF-b expression in human SCC have yielded conflicting results and no clear pattern has emerged. On the other hand, the parallels between genetic mouse models and the human disease are much stronger with regard to the role of TGF-b receptors. In this case, attenuation of either TbR-I or -II signaling promotes SCC development in mice, and mutation and/or loss of expression of TbR-I or -II receptors are commonly seen in human SCC. Somatic mutations of either the TGFBR1 or TGFBR2 gene have been detected in a small fraction (<10%) of human SCC. In addition, approximately 40–60% of human SCCs are associated with a significant decrease in either TbR-I or -II expression. Perhaps most interesting is the emerging realization that these two types of alterations have very different biological and clinical implications (Bharathy et al. 2008; Kareddula et al. 2008). Thus, approximately 10–15% of HNSCCs display evidence of functional inactivation of TbR receptor signaling, as defined by the absence of pSmad2 and -3 or the presence of an inactivating TGFBR gene mutation (Table 3.1). Patients with this tumor type appear to have a particularly favorable clinical outcome (Xie et al. submitted) (Table 3.1). In contrast, those whose tumors continue to express pSmad2 and -3 presumably include the 40–60% of cases in which TbR-I or -II expression is reduced but not eliminated. In this context, exposure of the tumor cells to high levels of bioactive TGF-b activates a proinvasive and -metastatic gene expression program (Kareddula et al. 2008). In vivo, this scenario appears to be associated with a worse clinical outcome. In fact, genetic mouse

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models appear to phenocopy the two HNSCC subtypes in which TbR receptor signaling is abrogated versus those in which signaling is partly retained (Bian et al. 2009; Han et al. 2005; Lu et al. 2006). Thus, in mice in which the Tgfbr-2 gene is deleted in stratified squamous epithelia, only 35% of mice with DMBA-initiated SCC developed regional lymph node metastases (Lu et al. 2006). In contrast, in TGF-b1/dominant-negative Tgfbr-2 bitransgenic mice in which TGF-b receptor signaling is attenuated but not eliminated entirely, 60% of the animals developed metastases to lymph nodes and lungs (Han et al. 2005). Thus, even though tumorassociated TGF-b1 expression was significantly increased in both models, the metastatic potential of the arising tumors was highly dependent on the specific type of alteration the Tgfbr-2 gene had undergone. We might speculate that the two types of SCC-associated TGFBR2 mutants exemplify the two major altered TGF-b signaling phenotypes seen in this disease (Bharathy et al. 2008). In one class, exemplified by the E526Q mutant, TGF-b signaling is completely abrogated, giving rise to a lowgrade verrucous carcinoma (Bharathy et al. 2008; De et al. 1998; Reiss et al. 1985). On the other hand, the R537P TGFBR2 mutant may be representative of the class of SCC in which TGF-b receptor signaling is attenuated but not completely abolished, resulting in a shift from Alk-5/Smad2/3 signaling to Alk-2/3/Smad1/5 signaling that results both in loss of cell cycle control and in a more aggressive tumor phenotype (Bharathy et al. 2008). Therefore, we would like to propose that a structural and functional analysis of the TbR receptors represents a powerful prognostic tool for the management of patients with SCC.

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Chapter 4

Aberrant Activation of HGF/c-MET Signaling and Targeted Therapy in Squamous Cancer Zhong Chen

Abstract Aberrant activation of the c-MET signaling pathway has been identified in multiple carcinomas, including bladder, renal, cervical, colon, breast, ovary, lung, esophagus, gastric, and head and neck cancers. The underlying genetic and functional abnormalities include gene amplification of c-MET/hepatocyte growth factor (HGF) gene loci, c-MET mutation, overexpression of c-MET and HGF due to transcriptional dysregulation or promoter polymorphisms, and autocrine or paracrine activation mediated by cytokines and growth factors. HGF/c-MET signaling promotes an aggressive malignant phenotype, including increased cell proliferation and survival, epithelial-mesenchymal transition (EMT), invasion, angiogenesis and inflammation, and metastasis. HGF and its coregulated proinflammatory and proangiogenic growth factors in serum, as well as c-MET mutation, amplification and phosphorylation in tumor specimens, have been identified as important indicators for patient prognosis and responses to therapies targeting HGF/c-MET. Targeting HGF/c-MET and related signaling pathways are important strategies in the development of anticancer drugs, and a panel of small molecule inhibitors and biological antagonists have been tested in clinical trials.

4.1 Background and Overview Carcinoma progression involves a series of pathologic and phenotypic changes, including overexpressed cytokines and growth factors, activated receptors and intermediate kinases, uncontrolled DNA replication and cellular growth, and invasion and metastatic spread. Most carcinomas actively produce a repertoire of proinflammatory and proangiogenic cytokines and growth factors, which promote

Z. Chen (*) Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 10 Center Drive, Building 10, 5D55, Bethesda, MD 20892, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_4, © Springer Science+Business Media, LLC 2011

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changes in tumor cell phenotype and the formation a new stroma, with influx of host inflammatory and mesenchymal cells, neoangiogenesis, and lymphangiogenesis. Subsequent proteolysis leads to penetration and breakdown of the basement membrane and interstitial connective tissue, and promotes the spread of metastatic cells via lymphatics and blood vessels to distant sites. In addition, stromal cells in the tumor microenvironment, such as activated fibroblasts, endothelial cells, smooth muscle cells, and inflammatory infiltrating cells, are commonly found adjacent to the tumor counterpart, where paracrine interactions between cancer and stromal cells occur in response to dysregulated signaling mediated by growth factors and cytokines. In such autocrine and paracrine interactions, the HGF/c-MET pathway is an essential regulatory node between carcinoma cells and surrounding stroma microenvironment.

4.1.1 Hepatocyte Growth Factor/Scatter Factor HGF/SF, hereafter called HGF, was originally identified from sera of partially hepatectomized rats, as a potent mediator of primary hepatocyte mitogenesis and cell scattering in culture (Nakamura et al. 1984; Mizuno and Nakamura 1993). HGF is produced as a pro-HGF of 728 a.a. (amino acid), coding for both a and b subunits, and cleaved during cytoplasmic translocation. The mature HGF, composed of one 69 kDa (kilodalton) a subunit and one 34 kDa b subunit, connects through a single disulfide bond between cysteine 487 of the a subunit and cysteine 604 of the b subunit (Nakamura et al. 1987). The a subunit contains four kringle domains, which play an important role in protein interaction and are involved in the regulation of proteolytic activity (Atkinson and Williams 1990). Deletion of the kringle domains significantly abolished HGF-induced hepatocyte proliferation and scattering (Nakamura 1991). In addition, HGF protein has ~40% structural similarity to plasminogen (Mizuno and Nakamura 1993). The “scatter factor” and “fibroblast-derived morphogenic factor” were also identified to be HGF (Mizuno and Nakamura 1993).

4.1.2 c-MET c-MET is the receptor for HGF, which was cloned as a transforming gene from a chemically transformed human osteosarcoma-derived cell line (Cooper et al. 1984). Subsequently, c-MET was identified as an oncogene with tyrosine kinase activity (Dean et al. 1985), with a protein structure closely related to RON (also called macrophage stimulating 1 receptor, MST1R; Benvenuti and Comoglio 2007). c-MET mRNA is coded by single gene and translated as a protein of single a.a. chain. The protein is cleaved to generate a disulphide-linked heterodimer formed by an extracellular alpha chain (50 kDa) and a beta chain (145 kDa, Fig. 4.1). The beta subunit of c-MET, which contains extracellular, transmembrane, tyrosine kinase and phosphorylation domains, is

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able to bind the beta subunit of HGF (Bottaro et al. 1991). HGF binding to c-MET initiates receptor dimerization and phosphorylation of the major phosphorylation sites (tyrosine residues pY1234, pY1235, pY1349, and pY1356) of the tyrosine kinase domain, which are essential for c-MET activation (Fig. 4.1). HGF-mediated c-MET activation alters cellular adhesion, migration, and extracellular matrix

Fibroblast

Endothelium HGF VEGF, FGF IL-6, IL-8/Gro

pS985 pY1003

Integrins EGFR PlexinB CD44

c-Met GRB2 SOS

RAS RAF

pY1234 pY1235 STAT pY1349 PLCγ GAB1 SHC pY1356 GRB2 PI3K SRC SHP2

RAC1

AKT

MEK MAPK

Proliferation Cell cycle

PAK

FAK

Invasion, Cell motility Adhesion, Junction formation, Cytoskeleton

mTOR

Growth, Survival, Angiogenesis

Fig. 4.1 HGF binding results in c-Met autophosphorylation of tyrosinesY1234 andY1235 within the activation loop of the kinase domain and subsequent phosphorylation of tyrosinesY1349 and Y1356 near the –COOH terminus. Important adapter proteins and direct kinase substrates activated downstream in the c-Met pathway include growth factor receptor-bound protein 2 (GRB2), Grb2-associated adaptor protein 1 (GAB1), phosphatidylinositol 3-kinase (PI3K), son of sevenless (SOS), rat sarcoma oncogene homolog (RAS), mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3/5 (STAT 3/5), SRC, SRC homology protein tyrosine phosphatase 2 (SHP2), SRC homology domain c-terminal adaptor homolog (SHC), phospholipase c-g (PLC), Ras-related C3 botulinum toxin substrate 1 (RAC1), p21-activated kinase (PAK), focal adhesion kinase (FAK), AKT, and mammalian target of rapamycin (mTOR). Crosstalk between c-Met and various membrane protein partners, includes the epidermal growth factor receptor (EGFR), the plexin B family, a6b4 integrin, and CD44, results in additional signaling response modulation.

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integrity, ultimately leading to tumor invasion and metastasis (Birchmeier et al. 2003; Jiang et al. 2005; Matsumoto and Nakamura 2006).

4.2 Genetic Defects and Oncogenic Activation Contribute to HGF/c-MET Signaling The human HGF gene was mapped to chromosome 7q11.2-q21.1 (Weidner et al. 1991; Saccone et al. 1992), which spans about 70 kB (kilobase) and consists of 18 exons and 17 introns. The gene coding for c-MET, the cellular receptor of HGF, is located on chromosome 7q31, in close proximity to the HGF gene. Thus, cells with amplification of chromosome 7q may simultaneously overproduce both the factor and its receptor to acquire invasive properties through an autocrine mechanism, as discussed below. An increase in the copy number of chromosome 7q is one of the most common chromosome abnormalities observed in human squamous cell carcinoma (SCC) of the head and neck and esophagus (Du Plessis et al. 1999; Hannen et al. 2004; van Dekken et al. 2006, 2008; Martin et al. 2008). Aberrant c-MET signaling through c-MET gene amplification, mutations, and autocrine or paracrine activation of c-MET receptor has been reported in a variety of human carcinomas including breast, prostate, lung, pancreatic, bladder, and head and neck cancers (Birchmeier et al. 2003). Genetic alterations, such as c-MET mutations (germ-line and somatic), gene amplification and gene rearrangement, causally contribute to the aberrant activation of c-MET signaling pathway in a broad spectrum of epithelial malignancies. c-MET mutations have been identified in head and neck squamous carcinoma (HNSCC), and childhood hepatocellular carcinoma, as well as gastric, lung, ovarian and thyroid cancers (http://www.vai.org/met). Missense c-MET mutations are found in all patients with hereditary papillary renal cell carcinomas (PRCC) and in a subset of sporadic PRCC (Schmidt et al. 1999). Activating mutations alter sequences within the kinase or juxtamembrane domain, leading to ligand-independent activation of receptor tyrosine kinases (Liang et al. 1996). Mutated c-MET is associated with increased aggressiveness and extensive metastases in various carcinomas (Park et al. 1999; Lorenzato et al. 2002). In HNSCC, c-MET mutation is associated with higher potential for lymphatic invasion and metastasis (Di Renzo et al. 2000). More frequently in carcinomas, overexpression of c-MET has been observed due to gene amplification (gastric, esophagus, HNSCC, PRCC, lung and colon cancers) or transcriptional activation resulting from mutated oncogenes and constitutively activated transcription factors. For example, in a portion of hereditary PRCC patients, chromosome 7 trisomy harbors nonrandom duplication of the mutant c-MET allele. Because both HGF and c-MET genes reside in chromosome 7q, such chromosome amplification or gene duplication leads to profound activation of HGF/c-MET signaling pathway and an aggressive phenotype (Kovacs 1993; Zhuang et al. 1998). In addition, mutated or activated oncogenes, such as RAS (rat sarcoma oncogene homolog), can induce c-MET overexpression through transcriptional mechanisms (Ivan et al. 1997; Furge et al. 2001; Boccaccio and Comoglio 2006).

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Fewer genetic alterations have been observed in HGF. Although some carcinomas do produce HGF, which activates c-MET signaling pathway through autocrine regulation, stromal cells are the major source of HGF production in the tumor microenvironment. In HNSCC, HGF is abundantly detected in tumor stroma derived fibroblasts, tumor stroma, and serum, but not in HNSCC tumor cells (Dong et al. 2001a, 2001b; Knowles et al. 2009). By contrast, overexpression of HGF gene is often observed in breast cancer, but it is transcriptionally silenced in normal differentiated breast epithelia (Tuck et al. 1996; Kang et al. 2003). A truncated deletion of mononucleotide repeat of 30 deoxyadenosines (30As) has been identified in a transcriptional repressor region located 750 bp upstream from the transcription start site in human HGF promoter (Ma et al. 2009). Functional studies revealed that the truncation mutation modulates chromatin structure and DNA–protein interactions, leading to constitutive activation of the HGF promoter in human breast carcinoma cell lines (Ma et al. 2009). Interestingly, 51% of African Americans and 15% of Caucasians harbor a truncated variant in their breast tumors. The truncated allele is associated with aberrant HGF expression and early breast cancer incidence, supporting the importance of overexpressed HGF as a risk factor of developing breast cancer (Ma et al. 2009). In addition, the positive feedback loop between c-MET and HGF has been observed in invasive human breast carcinomas, where the two molecules are frequently overexpressed together. The upstream activation signals, including c-MET activation, induce HGF transcription through c-SRC activation (the cellular homolog of the transforming sequence of Rous sarcoma virus) and STAT3 binding (signal transducer and activator of transcription 3) to the HGF promoter. Coexpression of STAT3 and activated c-SRC increased HGF expression, which significantly enhanced cell scattering in breast epithelial cells (Wojcik et al. 2006). Other studies confirmed the conclusion that breast tissue is a uniquely permissive environment for HGF transactivation by c-SRC and STAT3, which may allow for the aberrant activation of HGF transcription during the early stages of breast transformation (Sam et al. 2007). Thus far, the role of these mechanisms identified in breast cancer have not been examined or defined in SCC.

4.3 HGF/c-MET Signaling, Transcriptional Regulation and Cross-talk with Other Pathways HGF binds to the extracellular domain of c-MET receptor and induces the activation of multiple signaling cascades (Fig. 4.1). The intracellular portion of the c-MET receptor is comprised of a juxtamembrane domain, kinase domain and a C-terminal regulatory tail, which contains docking and direct interacting sites with downstream signaling molecules (Birchmeier et al. 2003). The juxtamembrane domain contains serine 985 (pS985), phosphorylation of which leads to inhibition of the receptor kinase activity (Trusolino and Comoglio 2002). Tyrosine1003 (pY1003) is another phosphorylation site in this region, which is capable of binding CBL (Cas-Br-M murine ecotropic retroviral transforming sequence homolog) to

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mediate ubiquitin ligase activity and promote c-MET receptor polyubiquitination and degradation (Peschard et al. 2001; Abella et al. 2005). The kinase domain contains two tyrosine residues (pY1234 and pY1235), which are responsible for regulating c-MET enzyme catalytic activity. Two additional tyrosine residues (pY1349 and pY1356) exist in the C-terminal tail, where their phosphorylations provide multifunctional docking sites for various intracellular adaptors and signaling molecules (Ponzetto et al. 1994) (Fig. 4.1). c-MET tyrosine phosphorylation at pY1349 and pY1356 residues are critical to execute c-MET function in biological and malignant processes (Bardelli et al. 1998). The phosphorylation is responsible for mediating high-affinity interactions with multiple cellular components, such as GAB1 (GRB2-associated binder 1), GRB2 (growth-factor-receptor bound-protein 2), mitogen-activated protein kinase (MAPK), PI3K (phosphatidylinositol 3-kinase), PLC (phospholipase C), RAS, SRC, SHC (Src-homology-2-domain-containing), SHP2 (SH2-domain containing protein tyrosine) phosphatase 2, and STAT3 to regulate downstream biological events (Birchmeier et al. 2003; Benvenuti and Comoglio 2007). Among the signal molecules downstream from c-MET receptor, GAB1 is a crucial scaffolding adaptor protein in responding to extracellular stimuli, through direct interaction with tyrosine phosphorylation sites (mainly pY1349) (Maroun et al. 1999; Sachs et al. 2000). The binding leads to a prolonged phosphorylation of GAB1, which mediates additional interactions and phosphorylation of multiple molecules through their SH2 domains (SRC-homology-2 domain), such as SHP2, PI3K, PLC, and CRK (V-CRK avian sarcoma virus CT10 oncogene homolog; Gu and Neel 2003; Rosario and Birchmeier 2003). The activation cascades of the intermediate kinases and phosphatases lead to activation of RAS, RAF (V-RAF-1 murine leukemia viral oncogene homolog), and ERK (extracellular signal-regulated kinase)/MAPK pathways. The GAB1-SHP2-ERK/MAPK cascade regulates ETS (V-ETS avian erythroblastosis virus E26 oncogene homolog)/AP-1 (activator protein 1) transcription factors and adhesion molecules, which control cell proliferation, junction formation, and cell migration (Ridley et al. 1995; Potempa and Ridley 1998; Maroun et al. 1999; Birchmeier et al. 2003). Both ERK/MAPK and the PI3K pathways contribute to c-MET signals that mediate adherence, cell spreading and motility (Potempa and Ridley 1998); while AKT activation mainly mediated cell survival (Fan et al. 2001; Xiao et al. 2001). Another phosphorylation site (pY1356) is also capable of binding to these molecules with additional GRB2 and SHIP interaction (Birchmeier et al. 2003). Other downstream molecules from c-MET receptor signaling, such as RAS, RAC1 (RAS-related C3 Botulinum toxin substrate 1) and PAK (p21-activated kinase), control cytoskeletal rearrangement and cell adhesion (Ridley et al. 1995; Royal et al. 2000), and RAP1 (RAS-related protein 1) regulate cell motility (Lamorte et al. 2000; Sakkab et al. 2000). In addition, JNK (c-Jun N-terminal kinase), STAT3, and NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) directly or indirectly participate in c-MET signaling pathways, which lead to cell transformation, anchorage-independent growth, and branched endothelial tubule formation (Schaper and Kubin 1997; Boccaccio et al. 1998; Muller et al. 2002; Zhang et al. 2002). We have shown that HGF/c-MET signaling

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induces VEGF (vascular endothelial growth factor), and PDGFa and b (plateletderived growth factor a and b) through ERK1/2 and PKC (protein kinase C) pathways (Dong et al. 2001a, b; Worden et al. 2005). The regulation of VEGF and PDGF gene expression is through the induction of EGR-1 (early growth response-1) transcription factor expression (Worden et al. 2005), which belongs to a family of related zinc finger transcription factors (Sukhatme et al. 1988; Mages et al. 1993). HGF/c-MET signaling promotes vascular endothelial cell migration and angiogenesis in carcinomas, such as HNSCC, skin SCC, papillary carcinoma of the thyroid, breast and prostate cancers (Di Renzo et al. 1991; Grant et al. 1993; Dong et al. 2001a, b; Davies et al. 2003; Scarpino et al. 2003). The mechanism of action has been shown to directly activate PI3K/AKT and ERK/MAPK pathways in endothelial cells (Sengupta et al. 2003), or via induction of VEGF or interleukin-8 (IL-8) (Dong et al. 2001a) and inhibition of thrombospondin-1 (Zhang et al. 2003). Activation of the HGF/c-MET signaling cascade also results in the phosphorylation, ubiquitylation, and degradation of the E-cadherin and cadherin-associated proteins (Matteucci et al. 2006). The loss of E-cadherins disrupts tight junctions leading to detachment of malignant cells from the primary site of tumor formation (Hiscox and Jiang 1997). In addition, HGF has been shown to induce phosphorylation of FAK (Beviglia and Kramer 1999; Lai et al. 2000), which is involved in integrin mediated signal transduction and aids in cell motility. HGF/c-MET signaling stimulates detachment and increases mobility of tumor cells, which is coupled with the induction of tumor cell’s binding to and degradation of the basement membrane and extracellular matrix. HGF/c-MET signaling has also been found to increase the localization of mechanical linkers of the cytoskeleton protein paxillins to integrins, to promote the expression of a2 and a3 integrins (Beviglia and Kramer 1999; Lai et al. 2000). In addition, HGF/cMET signaling has been reported to stimulate production of the urokinase-type plasminogen activator (uPA)-dependant proteolytic network, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) (Gong et al. 2003; Nishimura et al. 2003). Once the tumor cell has broken the barrier formed by the basement membrane and the extracellular matrix, HGF/c-MET signaling promotes invasion of distant tissue by increasing the expression of adhesion molecules such as CD44 on the endothelium and the tumor cells (Hiscox and Jiang 1997; Mine et al. 2003). The increased interaction of the tumor cells with the endothelium of blood vessels ultimately enables tumor cell invasion and the resulting metastasis to distant tissues.

4.4 HGF/c-MET Mediated Paracrine and Autocrine Regulatory Networks As mentioned above, in the tumor microenvironment, HGF is expressed predominately by stromal cells, while the c-MET receptor is mainly expressed by cancer cells (Kolatsi-Joannou et al. 1997). Activation of c-MET in SCC occurs often

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through HGF-dependent paracrine mechanisms (Dong et al. 2001a, 2004; Knowles et al. 2009). HGF has been implicated as an important factor produced by stroma (Matsumoto et al. 1996; Matsumoto and Nakamura 1997) in responding to tumor-produced proinflammatory and angiogenic factors, such as IL-1, IL-6, EGF, fibroblast growth factor (FGF), PDGF, and tumor transforming growth factor-a (TGF-a) in vivo (Gohda et al. 1994; Seslar et al. 1995, Nakamura et al. 1997). Overexpression of activated c-MET and increased responsiveness to HGF has been observed in human HNSCC, which lead to increased angiogenesis and metastasis. These effects can be direct through promotion of scattering and migration of tumor cells, or indirectly through induction of proinflammatory and proangiogeneic factors, such as IL-8, VEGF and PDGF (Dong et al. 2001a; Worden et al. 2005). The invasion and metastasis mediated by HGF/c-MET signaling is demonstrated by in vitro models where invasion of collagen gel by oral SCC is enhanced with stromal cells or stromal-derived conditioned media (Matsumoto et al. 1989) and clinical investigations demonstrating that elevated HGF levels found in HNSCC patients’ serum is correlated with increased serum levels of IL-8 and VEGF, as well as a poor prognosis (Dong et al. 2001a; Allen et al. 2007). PDGF and VEGF involved in angiogenesis in HNSCC have been shown to be among the important targets of the HGF/c-MET mediated EGR-1 signaling (Worden et al. 2005). Recently, studies of HGF/c-MET expression and signaling were conducted in HNSCC cell lines, primary tissues and xenograft animal models (Zou et al. 2007; Knowles et al. 2009; Seiwert et al. 2009). Consistent with our previous findings, c-MET was over-expressed and functionally activated in HNSCC cells, and HGF was secreted by HNSCC tumorderived fibroblasts, but not by tumor cells. c-MET signaling promoted AKT and MAPK activation, and as well as release of the inflammatory cytokine IL-8. Cell growth and wound healing were also stimulated by HGF. These results show that HGF acts mainly as a paracrine factor in HNSCC cells, where the activation of HGF/c-MET pathway are through frequently up-regulated downstream components and increased signal responsiveness. HGF/c-MET mediated paracrine regulatory mechanisms between tumor and stroma cells have been also demonstrated in animal models. The interaction of transformimg growth factor-b (TGF-b) signaling with HGF/c-MET pathway has been observed in a knockout animal model, where conditional deletion of the type II TGF-b receptor (TGFbRII KO) in fibroblasts promotes mammary tumor metastasis and correlates with increased expression of HGF (Cheng et al. 2008). TGFbRII KO fibroblasts exhibited enhanced HGF/c-MET/Ron signaling, which is mediated by STAT3 and ERK1/2 MAPK activation, to promote scattering and invasion of mammary carcinoma cells. The study characterized the negative TGF-b and positive HGF signaling in mediating tumor-stromal interactions to promote mammary tumor cell scattering and invasion, which are important steps in the metastatic process (Cheng et al. 2008).

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4.5 Abnormality of HGF and c-MET in Carcinoma Patients Previously, we identified c-MET overexpression in a multistage murine epidermal SCC metastatic model as a critical signaling receptor to promote cell proliferation, induce proangiogenic and proinflammatory cytokine production, and enhance migration (Dong et al. 2004). In examination of a panel of human HNSCC cell lines, we observed overexpression and activation of c-MET (Dong et al. 2001a), as well as activated downstream signaling involved in MAPK/ERK, PI3K/AKT, and PKC pathways (Dong et al. 2001a; Worden et al. 2005). In a recent study of 121 HNSCC patient specimens and 20 HNSCC cell lines, a more complete picture of multiple abnormalities of c-MET signaling has been revealed. Seiwert et al. showed that 84% of HNSCC tissues and 90% of cell lines exhibited c-MET overexpression, and increased c-MET gene copy number was observed in 13% tumor specimens. Novel c-MET mutations have been identified in 13.5% of HNSCC patients (Seiwert et al. 2009). Recently, c-MET overexpression and mutation have been identified as one of the molecular mechanisms for drug resistance in targeting EGFR in lung cancer, HNSCC and other carcinomas. In searching for the molecular mechanisms of acquired drug resistance to EGFR kinase inhibitors in lung cancers with EGFR mutations, Engelman et al. described a gefitinib-sensitive carcinoma line that developed resistance as a result of focal amplification of the c-MET gene (Engelman et al. 2007). Inhibition of c-MET signaling in these cells restored their sensitivity to gefitinib. c-MET amplification was detected in 4 of 18 (22%) lung cancer specimens that had developed resistance to gefitinib or erlotinib. They found that amplification of c-MET induced gefitinib resistance through HER3/ERBB3 (v-erb-B2 avian erythroblastic leukemia viral oncogene homolog 3) mediated activation of PI3K pathway (Engelman et al. 2007). This finding has been confirmed in a larger study with lung adenocarcinomas harboring EGFR mutations, where analysis of tumor samples from multiple independent patient cohorts revealed that c-MET was amplified in tumors from 9 of 43 patients (21%) with acquired resistance, but in only two tumors from 62 untreated patients (3%). These cells with c-MET amplification are resistant to erlotinib and an irreversible EGFR inhibitor (CL-387), but sensitive to a multikinase inhibitor (XL880) against c-MET (Bean et al. 2007). More studies confirmed that HGF induced c-MET activation restored PI3K/AKT signaling pathway, and increased gefitinib resistance of lung adenocarcinoma cells with EGFR-activating mutations (Yano et al. 2008). Unlike previously reported mechanisms involving c-MET mutations and gene amplifications, in this study, strong immunoreactivity for HGF in cancer cells was detected, indicating that HGF-mediated c-MET activation is another mechanism of gefitinib resistance in lung cancer. We previously reported increased HGF level in serum from patients with HNSCC (Dong et al. 2001a), which is consistent with the recent survey of 121 HNSCC patients where HGF overexpression was present in 45% of cases (Seiwert

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et al. 2009). We showed that increased HGF was correlated with higher levels of angiogenic factors IL-8 and VEGF in serum of patients with HNSCC as compared with that in normal volunteers (Dong et al. 2001a). Experimentally, we demonstrated that HGF significantly induced IL-8 and VEGF cytokine production in eight HNSCC lines tested, which corresponded with an increase in phosphorylation of c-MET in HNSCC (Dong et al. 2001a). We further investigated HGF induced proinflammatory and proangiogenic factors in a separate clinical trial of 29 patients with HNSCC, where most patients had stage III and IV disease, and were treated with paclitaxel concomitantly with radiation or surgery, with a median of 37 months follow-up (NIH Protocol 95-C-0162). We confirmed that the serum level of HGF, along with other cytokines and growth factors, including IL-6, IL-8, VEGF, and GRO-1, was increased in HNSCC patients. Longitudinal changes in serum HGF, IL-6, IL-8, and VEGF were detected with treatment response, relapse, or complications in individual patients, where HGF showed the strongest relationship with survival (Druzgal et al. 2005; Van Waes et al. 2005; Allen et al. 2007). In an independent study with 3-year follow-up, we tested serum factors of 30 patients with locally advanced (stage III/IV) oropharyngeal SCC who underwent cisplatin based chemoradiation therapy. Longitudinal increases in individual serum levels of HGF, IL-6, IL-8, VEGF and GRO-1 predicted decreased cause-specific survival when adjusted for smoking history. For a given individual, a significant increase in any three or more factors predicted poorer cause-specific survival (Allen et al. 2007). In addition, in a larger set of patients with 78 primary HNSCC, 8 with recurrent HNSCC and 71 healthy control subjects, an increased HGF level was significantly correlated with tumor stage and progression. Curative resection of the tumor decreased HGF serum to normal level, and significantly increased HGF was observed in recurrent HNSCC patients. Taken together these data support the idea that serum HGF is significantly correlated with tumor progression and could be a strong predictor of recurrence in HNSCC (Kim et al. 2007). Serum HGF levels in patients with esophageal squamous cell carcinoma (ESCC) have been evaluated in 149 patients, where basal serum HGF was found to be significantly higher in patients with ESCC than in control subjects, and correlated significantly with advanced tumor metastasis stage and survival. Consistent with our findings in HNSCC, Ren et al. found increased HGF serum levels correlated positively with serum levels of VEGF and IL-8 (Ren et al. 2005). Their data indicate that serum HGF could be a useful biomarker of tumor progression and a valuable independent prognostic factor in patients with ESCC, as an autocrine/paracrine factor via enhancing angiogenesis and tumor cell invasion and migration. HGF serum levels were also investigated in a clinical trial of 45 patients with clear cell renal cell carcinoma (RCC), when compared with 45 normal healthy controls. Patients with RCC had significantly higher serum HGF, which significantly correlated with clinical stage, tumor grade and prognosis. Again data support the hypothesis that serum HGF might be used as a prognostic indicator for RCC (Tanimoto et al. 2008). In addition, HGF serum was investigated in 124 patients with invasive breast cancer undergoing surgery; the control group consisting of 35 patients with benign breast tumor. The serum soluble HGF in patients with invasive breast cancer was

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significantly higher than that of the control group. High HGF levels are associated with patients having negative estrogen receptor, poorer differentiated tumor, advanced primary and TNM tumor staging, advanced lymph node status, and distant metastases. Thus, preoperative serum HGF levels might reflect the severity of invasive breast cancer (Sheen-Chen et al. 2005).

4.6 Therapeutic Targeting of HGF/c-MET Pathway in Carcinomas Due to the broad biological impacts of HGF/c-MET signaling in cancer pathogenesis, extensive efforts have been directed toward developing agents to inhibit HGF/c-MET pathway signaling, including small molecule inhibitors or humanized antibodies. Small molecule inhibitors specifically block the catalytic activity of c-MET, targeting c-MET downstream signaling pathways, or inhibit other receptor or intermediate kinases that exhibit crosstalk with the c-MET pathway. Small molecule inhibitors have the advantage of easier delivery, with relatively higher penetration into tumor tissues and are more cost effective. In contrast, the strategy using humanized monoclonal antibodies allows highly specific targeting of HGF or c-MET, sustained serum concentrations due to the prolonged half-life of antibodies, and potential to induce host immune responses against tumors, such as antibodydependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. However, antibodies may have less effective penetration into tumor tissues. The high manufacturing cost of antibody production is an additional factor.

4.6.1 Humanized Antibodies Monoclonal antibody antagonists against c-MET or HGF are being developed to interrupt HGF and c-MET interaction (Burgess et al. 2006; Kim et al. 2006; Martens et al. 2006). A monovalent humanized anti-c-MET antibody has been generated from the monoclonal antibody 5D5, designated as one armed 5D5 (OA5D5) or MetMAb (Genentech) (Martens et al. 2006; Jin et al. 2008). This antibody exhibits antagonistic characteristics with high affinity binding to c-MET receptor, and prevents HGF binding, leading to the inhibition of c-MET phosphorylation and activation of downstream signaling pathways. In a preclinical study of a mouse model bearing glioblastomas containing a c-MET-activating mutation, local administration of OA5D5 exhibited significant and near complete inhibition of tumor growth (Martens et al. 2006). OA5D5 exhibits enhanced anti-tumor activity when used in combination with EGFR and VEGF inhibitors (Martens et al. 2006; Jin et al. 2008; Merchant et al. 2008a, b). In addition, results from an initial Phase I clinical trial suggested that OA5D5 is safe and well tolerated as a single agent (Salgia et al. 2008).

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To prevent HGF binding to c-MET and subsequently activation of downstream signaling pathways, a fully human IgG2 monoclonal antibody, AMG102, was identified that selectively binds and neutralizes HGF (Burgess et al. 2006; Kakkar et al. 2007). In preclinical tumor models, AMG102 shows potent inhibition of HGF/cMET-dependent tumor growth (Burgess et al. 2006; Gao et al. 2006). In a phase I clinical trial, 31 patients with solid tumors were treated with AMG102, and where 25 patients were available for tumor assessment, 12 of these patients had stable disease for 3 months (Gordon et al. 2007). AMG102 significantly enhanced the therapeutic effects of other agents in combination therapies. In a phase I clinical trial, where AMG102 was combined with the antiangiogenesis agents bevacizumab or motesanib, stable disease with suppression of tumor growth was observed in 8 of 10 evaluable patients without significant toxicity (Rosen et al. 2008).

4.6.2 Small Molecule Inhibitors Several small molecule inhibitors targeting c-MET kinase domains have been developed recently and evaluated in clinical trials, including XL880, XL184, ARQ197, PF2341066, and MP470. These small molecule inhibitors can be classified as c-MET specific or with a broader targeting capacity to other receptors or kinases. XL880 and XL184 (Exelixis) are orally active, ATP-competitive inhibitors that primarily target c-MET and VEGFR. XL880 (GSK1363089) also exhibits potent activity against PDGFR, KIT, FLT3and TIE-2, and suppresses cancer cell growth (Zillhardt et al. 2008). XL880 was generally tolerated, the side effects were treatable and reversible, and has been tested in Phase I and II clinical trials (http://www. exelixis.com/pipeline_xl880.shtml). In a Phase I study, 39 of 45 patients had either tumor regression or stable disease. Histological analyses of tumor samples from four treated patients showed significant decreases in c-MET phosphorylation, accompanied by markedly reduced phospho-AKT levels and increased tumor cell death (LoRusso et al. 2005; LoRusso et al. 2006; Eder et al. 2007; Shapiro et al. 2007). Recently, interim data was collected from an ongoing Phase II trial of XL880 in sporadic PRCC patients, a total of 21 patients are enrolled. Those patients were genotyped for c-MET mutations: 5 had activating c-MET mutations and 16 had wild type c-MET. Of 19 patients with measurable disease evaluable for tumor responses, 15 patients (79%) had a decrease in tumor size, including one patient with a partial response. All 19 evaluable patients had stable disease for 3 months, including 12 patients with stable disease for 6–15 months. Tumor biopsies from one patient demonstrated growth inhibition and induction of apoptosis following treatment (Ross et al. 2007a, b). Multiple phase II trials of XL880 have been started in patients with advanced hereditary PRCC, gastric, and HNSCC (http://www.exelixis.com/pipeline_xl880.shtml). In addition to the primary targets, XL184 (BMS-90735) also effectively inhibits RET, KIT, FLT3, and TIE-2 (http://www.exelixis.com/pipeline_x1880.shtml, http://

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www.exelixis.com/pipeline_xl184.shtml; Kurzrock et al. 2007; Ross et al. 2007a; Salgia et al. 2007; Shapiro et al. 2007). A phase III clinical trial has been initiated in medullary thyroid cancer (MTC) based on encouraging data from an ongoing phase I trial of XL184 in 69 patients with various solid tumors, including 17 MTC patients evaluable for response. These data showed a disease control rate (partial responses or prolonged stable disease for more than 3 months) of all evaluable MTC patients, with 53% of those patients (9 of 17) experiencing partial responses. Most of the MTC patients in the trial had previously failed other standard and targeted treatments, including tyrosine kinase inhibitors, chemotherapeutic agents, immunotherapy, and radiotherapy. This phase III trial is a single-agent therapy in 315 patients with unresectable, locally advanced, or metastatic medullary thyroid cancer (MTC). Additional Phase I/II trials in patients with non-small cell lung cancer (NSCLC) were initiated in early 2008 (http://www.cancer.gov/search/ ViewClinicalTrials.aspx?cdrid=586641&version=HealthProfessional&Protocolsea rchid=4281891, http://www.exelixis.com/pipeline_xl184.shtml; Salgia et al. 2007; Zillhardt et al. 2008). The trials are to evaluate the safety, tolerability, and highest safe dose of XL184 in combination with the EGFR inhibitor erlotinib, and to further evaluate the objective response rate of daily oral administration of XL184, with or without erlotinib, in NSCLC patients who have progressed after erlotinib treatment. ARQ197 (ArQule) is a highly selective non-ATP-competitive c-MET inhibitor (http://www.arQule.com). ARQ197 has been tested in 60 patients with multiple metastatic solid tumors and 49 patients were evaluated. Three patients with prostate, neuroendocrine, and testicular cancers had partial responses. Thirty-one patients (63.2%) had stable disease, of which 18 (36.7%) had prolonged stable diseases for 16–74 weeks, suggesting a possible antimetastatic effect (Garcia et al. 2007). Future clinical evaluation of the antitumor and antimetastatic activity as the single agent, or in combination with other drugs, will be tested in Phase II clinical trials of pancreatic cancer, NSCLC, prostate and gastric cancers (http:// phx.corporate-ir.net/phoenix.zhtml?=82991&p=irol-presentations, http://www. arQule.com). PF-2341066 (Pfizer) is a highly selective small molecule inhibitor targeting c-MET kinase with less potent inhibition of anaplastic lymphoma kinase (ALK), as well as with selective suppression of a large group of kinases (Zou et al. 2007). In several xenograft mouse tumor models, including HNSCC, gastric, glioblastoma, lung, renal, and prostate cancers, PF-2341066 has demonstrated potent antitumor activity (Zou et al. 2007; Knowles et al. 2009; Seiwert et al. 2009). Multiple Phase I/II trials of PF-2341066 have been conducted in patients with advanced solid tumors, including lung, colon, breast, gastric, head and neck, and renal cancers (http://clinicaltrials.gov/ct2/show/nct00585195?cond=%22lmyphoma%2c+largecell%2C+ki-1%22&rank=13, http://content.ll-0.com/dnl/devthx-july07.pdf?i07167193724, http://www.pfizer.com/investors/presentations/presentations.jsp). MP470 (SuperGen) is an orally active, multitargeted tyrosine kinase inhibitor of wild type c-MET and RET, as well as mutant KIT, PDGFR and FLT3 (http://ir. supergen.com/phoenix.zhtml?c=105560&p=irol-news&nyo=1, http://www.supergen.

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com/subpages/prod-dev.asp; Qi et al. 2009). Recently, the efficacy of MP470 or MP470 plus Erlotinib was evaluated in vitro using three prostate cancer cell lines in culture and in a mouse xenograft model. MP470 exhibits low IC50 in prostate cancer cell lines and in the tumor model treated with MP470 alone or in combination with Erlotinib. This combination treatment completely inhibited phosphorylation of the HER family members (HER1, 2, 3), PI3K and Akt activities (Qi et al. 2009). Several phase I trials of MP470 as a single agent in metastatic solid tumors are underway, and dose-escalation studies to evaluate MP470 in combination with carboplatin/paclitaxel, carboplatin/etoposide, docetaxel, topotecan, and EGFR inhibitor erlotinib have been started (http://www.clinicaltrials.gov/ct2/results?term=mp470).

4.7 Summary and Future Directions Aberrant activation of HGF and c-MET signaling has been identified in a broad panel of SCC and other carcinomas due to genetic mutations, gene amplifications, transcriptional activations, or paracrine and autocrine regulations. Activation of HGF/c-MET signaling is one of most critical growth factor and receptor mediated pathways that contribute to aggressive malignant phenotypes, such as resistance to therapy, increased angiogenesis, invasion, and metastasis. However, while dependency on HGF/c-MET signaling has been identified in certain subsets of carcinomas, the precise mechanisms of HGF/c-MET mediated activation and cross-talk with other signaling pathways must be further investigated to fully understand the significance in cancer prognosis and therapeutic responses. The expression and mutation status of HGF/c-MET could serve as the biomarkers for the molecular classification of carcinomas and the selection of personalized medication. The efficacy of novel drugs targeting HGF/c-MET pathway should be tested under the guidance of the biomarkers related to molecules involved in HGF/c-MET signaling pathway. Acknowledgements This work is supported by NIDCD/NIH Intramural project Z01-DC-00016 and Z01-DC-00073. The author would like to express appreciation to Dr. Gang Dong (NIAID/ NIH), for his effort in reference management and graphic drawing.

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Chapter 5

The Epidermal Growth Factor Receptor in Normal and Neoplastic Epithelia Susan K. Repertinger, Justin G. Madson, Kyle J. Bichsel, and Laura A. Hansen

Abstract The epidermal growth factor receptor (EGFR) regulates a plethora of cellular and tissue functions in epithelia including cell division, survival, differentiation, and migration. EGFR signaling is up-regulated in pathologies involving aberrant growth like squamous cancer, and facilitates neoplastic progression. Many mechanisms through which the effects of EGFR are modulated in normal and pathological processes have been identified. In particular, recent research has yielded important and surprising insights into the effects of EGFR-dependent signaling on cutaneous biology and carcinogenesis. This review focuses on EGFR signaling in normal biology and squamous cancer, with emphasis on the skin as a model organ to illustrate the biological significance of EGFR signaling.

5.1 Introduction The epidermal growth factor receptor (EGFR) has been implicated in the development and progression of neoplasms arising in virtually all epithelial cell types (Hynes and Macdonald 2009). Elucidation of the biological significance of EGFR signaling under normal physiologic conditions informs our understanding of how dysregulation of EGFR facilitates carcinogenesis. Investigation of EGFR has revealed a surprising diversity of functions for the receptor that vary depending on both the tissue and the cellular environment within the organ. The effects of EGFR signaling in normal and neoplastic cells are discussed, with examples provided by investigations of the skin and other epithelia.

L.A. Hansen (*) Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA e-mail: [email protected]

A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_5, © Springer Science+Business Media, LLC 2011

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5.2 EGFR, its Ligands, and Family Members The erythroblastic leukemia viral (v-Erb-b) oncogene homolog family of ERBB receptors is comprised of four transmembrane proteins: EGFR (ERBB1/HER1), ERBB2 (HER2/Neu), ERBB3 (HER3) and ERBB4 (HER4). ERBB family members are expressed in all epithelia, but only EGFR, ERBB2 and ERBB3 are expressed in the skin (Stoll et al. 2001). EGFR, ERBB2 and ERBB4 are receptor tyrosine kinases with a myriad of downstream actions, while ERBB3 has a defective tyrosine kinase domain (Guy et al. 1994). Ligand binding of epidermal growth factor (EGF) family members to EGFR, ERBB3 or ERBB4 triggers ERBB receptor dimerization and activation (Klapper et al. 2000). Of note, there is no known ligand for ERBB2 (Klapper et al. 2000). EGF-motifs are also found elsewhere such as on poxviruses and extracellular matrix molecules and have mitogenic effects (Tzahar et al. 1998; Swindle et al. 2001); however, the significance is unknown. ERBB ligands include EGF, heparinbinding EGF (HB-EGF) (Hashimoto et al. 1994), transforming growth factor (TGFa) (Coffey et al. 1987), epiregulin (Shirakata et al. 2000), amphiregulin (AR) (Cook et al. 1991), betacellulin (Watanabe et al. 1994), and epigen (Strachan et al. 2001), many of which are synthesized by keratinocytes (Hashimoto 2000) (Fig. 5.1). Another ligand group that binds only ERBB3 and ERBB4 is the neuregulins (Falls 2003). Ligand precursors are trafficked to the plasmalemma of various cells including fibroblasts and keratinocytes where they are cleaved to become active (Freeman 2004). Active EGF family members preferentially bind certain ERBB family members; for example, EGF, TGFa, and amphiregulin favor EGFR over other ERBB receptors (Klapper et al. 2000; Stoll et al. 2001). EGF family members have diverse actions and receptor affinities according to the tissue in which they are expressed (Stoll et al. 2009). EGFR and ERBB2 can also be activated through ligand-independent mechanisms including UV-induced reactive oxygen species generation (Ley et al. 1992; Huang et al. 1996; Bender et al. 1997; Madson et al. 2006; Xu et al. 2006; El Abaseri et al. 2005; Han et al. 2008), G-protein-coupled receptors (Bhola and Grandis 2008), other receptor tyrosine kinases, and cell adhesion molecules (Fischer et al. 2003) (Fig. 5.1). Upon activation by ligand or otherwise, the ERBB receptors undergo conformational change and homo- or heterodimerize with other family members (reviewed in Citri and Yarden 2006). While ERBB2 does not have a known ligand, it is the preferential dimerization partner for other family members (reviewed in Citri and Yarden 2006). Upon dimerization, the receptors undergo transphosphorylation at multiple sites where various docking molecules and adapter proteins are recruited (Schlessinger 2000; Yarden and Shilo 2007). There is a correlation of specific signal-transduction pathway activation according to the site of phosphorylation (Yarden and Shilo 2007). Signaling pathways activated by ERBB receptors include (1) phospholipase C (PLC); (2) Janus kinase (JAK)/Signal transducer and activators of transcription (STAT); (3) phosphotidylinositol-3-kinase (PI3K)/AKT; (4) SRC and SRC family members; and (5) mitogen activated protein

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Ligands (EGF, TGFα, HB-EGF, AR) Receptor tyrosine kinases

EGFR

UV-induced reactive oxygen species

P

Erbb Dimer

Cell adhesion molecules G-protein-coupled receptors

P

Src

PI3K/AKT PLC

Jak/STAT MAPK

Apoptosis, Survival, Proliferation, Differentiation, Migration Fig. 5.1 EGFR family members are activated by multiple mechanisms to regulate diverse biological functions.

kinase (MAPK) superfamily members including extracellular signal-regulated kinases (ERK1/2), p38 kinase, and Jun NH2-terminal kinases (JNK1/2) (Olayioye et al. 1999; Ren et al. 2002; Schlessinger 2000) (Fig. 5.1). These ERBB-activated signaling pathways have been implicated in the regulation of cell cycle progression, cell proliferation, apoptosis, differentiation, and migration. Following activation, ERBB receptor dimers bound to ligand are rapidly internalized and degraded or are recycled (Yarden and Shilo 2007). The ERBB receptors are also influenced by various positive and negative feedback loops involving autocrine and paracrine mechanisms. EGFR signaling is implicated in carcinogenesis at many sites, including hepatocellular, head and neck, renal cell, skin, and papillary thyroid carcinomas (Liu et al. 2007; Andl et al. 2003). Increased EGFR signaling in epithelial cancers occurs through several mechanisms. These include overexpression of EGFR and/or its ligands, EGFR transactivation, genetic alterations of EGFR (reviewed in Normanno et al. 2006) and a change in cellular localization of the receptor (Hyatt et al. 2008). EGFR transactivation occurs through a wide variety of molecules, including other receptors (Donnini et al. 2007), metalloproteinases (Singh et al. 2009), and cytokines

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(Mascia et al. 2003). Genetic alterations in the receptor include amplifications, deletions, duplications, or small in-frame deletions or point mutations in the kinase domain (reviewed in Normanno et al. 2006). EGFR-activating mutations are seen in human squamous cell carcinoma (SCC) of multiple sites, including the lung (reviewed in Linardou et al. 2009), head and neck (Hama et al. 2009; Dassonville et al. 1993), and oral cavity (Sheu et al. 2009). The most common mutated variant of EGFR, EGFRvIII, allows for ligand-independent receptor activation and occurs primarily in breast, lung and head and neck carcinomas (Pedersen et al. 2001). EGFR signaling is involved in a plethora of actions in both normal physiologic conditions and pathologies such as cancer. The biological significance of EGFR in cell division, cell death, cell migration and invasion, and differentiation in normal and neoplastic epithelia is the focus of the remainder of this review. Given the extensive characterization of EGFR signaling and function in the skin, and the surprising roles of EGFR in the various cell types of the skin, this mouse model provides important insight into the biological role of EGFR signaling in many squamous epithelia.

5.3 Biological Significance of EGFR Signaling Revealed by Mouse Skin Models The complexity of cutaneous keratinocyte organization and function has allowed for the identification of many non-overlapping and disparate functions of EGFR that vary both spatially and temporally. The development of mouse models with altered EGFR activity, manifesting a surprising variety of EGFR functions during development and carcinogenesis, has facilitated this research. Thus, this review will draw upon the complexity of EGFR-dependent functions in the skin to illustrate the potential variety of EGFR effects in epithelial biology and carcinogenesis. Our research and the results of others using mouse models with altered EGFR activity shows that the role of EGFR in cutaneous development and carcinogenesis depends specifically on the cell’s localization within the skin (Murillas et al. 1995; Sibilia et al. 1995; Threadgill et al. 1995; Hansen et al. 1996, 1997, 2000; Casanova et al. 2002; Repertinger et al. 2004; Maklad et al. 2009; Richardson et al. 2009). Down-modulation of EGFR expression in the developing hair placode is necessary for specification of follicular rather than epidermal cell fate (Richardson et al. 2009). EGFR also regulates patterning of sensory innervation, axonal outgrowth and axonal branching during development of the skin (Maklad et al. 2009). Postnatally, EGFR stimulates proliferation in the epidermis, and differentiation rather than proliferation in the hair follicle (Hansen et al. 1997; Murillas et al. 1995). EGFR is necessary for normal cell cycle arrest and apoptosis in hair follicles at catagen during hair follicle cycling (Hansen et al. 1997; Murillas et al. 1995) and our unpublished observations. Squamous cancer cells and cell lines more closely mimic the behavior of epidermal keratinocytes rather than follicular cells in terms of their response to EGFR (Dlugosz

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et al. 1997; Andl et al. 2003; Fischer et al. 2003; Modjtahedi et al. 1998; Woodworth et al. 2005). In fact, cutaneous epithelial cells, even those of follicular origin, acquire an epidermal-like pattern of response to EGFR signaling when cultured as dispersed cells in monolayer (unpublished observations). Reprogramming the cellular response following EGFR activation to a follicular pattern is a potential and unexplored mechanism for cancer cell differentiation, cell cycle arrest, and apoptosis.

5.4 EGFR and Cell Proliferation Epithelial proliferation is required for normal physiologic processes, including embryologic development, wound healing, and various adaptive responses. The various roles of EGFR in the developing follicular and interfollicular epithelia are discussed in the previous section. In other developing epithelia, not only is EGFR activation important in driving cellular proliferation, but also is necessary in inhibiting epithelial proliferation at precise time points in development. This fine balance is necessary for normal development of saccules within the distal lung. In Hbegf null newborn mice, a significant increase in cell proliferation occurs within the developing lung, resulting in thickened saccular walls that reduce the terminal saccular space area (Sibilia and Wagner 1995; Minami et al. 2008; Miettinen et al. 1995). Furthermore, crosses between HB-EGF mutant mice and hypomorphic Egfr Waved 2 mice suggest that HB-EGF and EGFR cooperate in this process (Minami et al. 2008). EGFR signaling also regulates early embryonic mouse gut development in chemically defined organ culture. In control mice, cellular proliferation initially occurs throughout the small intestinal epithelium, but later localizes to intervillous crypt regions (Duh et al. 2000). This localization increases with EGF treatment when compared with organ culture treated by EGFR inhibitors. This finding may account for the gut hypoplasia seen in Egfr null mice (Threadgill et al. 1995). Additionally, differentiation of endoderm into intestinal epithelium occurs when undifferentiated stratified epithelium is converted into a mature epithelial monolayer. This monolayer is formed through selective apoptosis of superficial cells lining the lumen, while cell proliferation is restricted to basally-located epithelial cells. In a three-dimensional embryonic gut culture system, EGFR inhibition reduces proliferation and survival of cells within cultured gut explants (Abud et al. 2005). During the wound healing process, EGFR signaling stimulates keratinocyte proliferation at the wound margins behind migrating keratinocyte tongues (Singer and Clark 1999; Kusewitt et al. 2009). This increased EGFR signaling is due in part to a transient elevation of EGFR and EGFR-dependent induction of its ligands HB-EGF and AR, which occurs in skin and other epithelia following injury (reviewed in Werner and Grose 2003). Inhibition or genetic deletion of EGFR decreases proliferation and epidermal hyperplasia adjacent to the wound (Repertinger et al. 2004). While EGFR appears to accelerate wound healing, other signaling pathways must also be activated during the healing process (Repertinger et al.

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2004). Similarly, the systemic administration of the EGFR inhibitor gefitinib in human cancer patients decreases epithelial proliferation in response to corneal injury, although the drug does not retard repair following full-thickness wounding (Nakamura et al. 2001). EGFR signaling is involved in several physiologic processes which allow organisms to adapt and survive following structural insults. For example, following partial resection, many organs show cellular hyperplasia and/or hypertrophy within the remaining tissue. Following massive small bowel resection, enterocytes proliferate within the remaining mucosa, producing taller villi and deeper crypts, increasing mucosal absorptive surface area. EGFR activation or inhibition after massive small bowel resection results in an amplified or attenuated enterocyte proliferative response, respectively (Sheng et al. 2006). Similarly, goblet cell density increases following massive small bowel resection, while EGFR inhibition results in diminished goblet cell density (Jarboe et al. 2005). EGFR activation increases cellular hyperplasia within the parathyroid gland secondary to chronic kidney disease, with requirement of TGF-a for its development; pharmacologic inhibition of EGFR activation with erlotinib prevents this up-regulation of parathyroid TGF-a and the progression of growth (Arcidiacono et al. 2008). Dysregulated EGFR signaling is implicated in a number of benign hyperproliferative disease states of the skin, such as psoriasis vulgaris, a common disorder in humans (Piepkorn et al. 2003; Yoshida et al. 2008). Histologically, psoriasis vulgaris in well-developed plaques is characterized by elongated rete ridges of even length, suprabasal mitoses, parakeratosis containing neutrophilic abscesses, and an absent granular layer. Overexpression of EGFR ligands and abnormal localization of EGFR itself within the epidermis is one component of the disease phenotype, as TGF-a, HB-EGF, and AR are overexpressed in psoriatic epidermis when compared to normal keratinocytes (Yoshida et al. 2008; Powell et al. 1999). Similarly, psoriatic keratinocytes incubated with a monoclonal antibody against EGFR or with tyrosine kinase inhibitors show inhibited EGFR phosphorylation and regression of the psoriatic phenotype (Powell et al. 1999; Varani et al. 1998). However, there are case reports showing exacerbation of human psoriasis after treatment with EGFR tyrosine kinase inhibitors (Zorzou et al. 2004). Benign and malignant cutaneous neoplasms are commonly occurring tumors in humans and animals. Ultraviolet (UV) light has long been known to activate EGFR in human and murine skin (Knebel et al. 1996; Huang et al. 1996; Ley and Ellem 1992; Xu et al. 2006; El Abaseri et al. 2005, 2006). With repeated UV exposure in animal models, a continuum of disease severity is seen, with the initial development of benign epithelial hyperplasia, followed by the development of the benign squamous papilloma, some of which progress to malignant SCC. A similar sequence occurs in human skin, with the development of the UV-induced actinic keratosis, a lesion which shows varying severities of squamous dysplasia and hyperkeratosis. Some actinic keratoses progress to SCC, with full-thickness dysplasia and possible invasion. As with benign epidermal hyperplasia, EGFR appears necessary to maintain the proliferative population in the basal cell compartment in papillomas of murine

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skin (Hansen et al. 2000). Primary keratinocytes from EGFR-deficient mice can be transformed in vitro by a replication-defective v-RasHa retrovirus and grafted to a skin site in vivo. Squamous papillomas form at the graft site, but are small, achieving an average size of 20% of similarly treated wild-type keratinocytes (Dlugosz et al. 1997). Similarly, in genetically initiated v-RasHa transgenic Tg.AC mice, topical treatment or intraperitoneal injection of EGFR inhibitors before UV exposure significantly decreases the number and size of squamous papillomas (El Abaseri et al. 2005). Moreover, in the absence of EGFR, cycling tumor cells migrate into the suprabasal compartment of the epidermis and initiate differentiation programs, followed by cell cycle arrest (Kolev et al. 2008). Therefore, the function of EGFR in squamous tumors may be to maintain a proliferative pool of basal cells and prevent premature terminal differentiation. Similarly, EGFR is commonly expressed in human skin cancers where it is associated with poor prognosis when coexpressed with other family members (Krahn et al. 2001). Increased EGFR signaling contributing to cellular proliferation in epithelial cancers occurs through several mechanisms. These include overexpression of EGFR and/or its ligands, EGFR transactivation, genetic alterations of EGFR (reviewed in Normanno et al. 2006) and a change in cellular localization of the receptor (Hyatt and Ceresa 2008). Cellular localization of activated EGFR appears to determine whether proliferation or apoptotic pathways are activated. For example, in a breast cancer cell line over-expressing EGFR, only endocytosed, activated EGFR stimulates CASPASE-3 and induces cell death, while activated EGFR retained at the cell surface inhibits CASPASE-3 and promotes cellular proliferation (Hyatt and Ceresa 2008). A recent study examined the roles of the Nf2 tumor suppressor gene Merlin and EGFR in kidney tumorigenesis by generating a mouse model with a targeted deletion of Nf2 in the proximal convoluted epithelium of the kidney (Morris et al. 2009). Nf2 mutant kidneys were hyperproliferative and rapidly developed invasive carcinoma resembling human clear cell renal cell carcinoma. Proliferation in these lesions was dependent on EGF (Morris and McClatchey 2009). EGFR also appears to interact with oncogenic RET/PTC in the development of human papillary thyroid carcinoma. Rearrangements of the RET protooncogene that generate RET/PTC (papillary thyroid cancer) are a genetic hallmark of papillary thyroid carcinomas (Fagin 2004). Conditional activation of RET/PTC in a thyroid carcinoma cell line increased both the expression and activation of EGFR, while inhibition of EGFR decreased cell proliferation of these carcinoma cells (Croyle et al. 2008).

5.5 EGFR and Apoptosis and Cell Survival Apoptosis is a form of programmed cell death that occurs following withdrawal of trophic stimuli or various cytotoxic agents. Apoptosis is important to many physiologic processes, including embryological development and prevention of damaged

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cells from entering the proliferative pool. EGFR signaling pathways resulting in apoptosis may be dysregulated in benign skin diseases, such as pemphigus vulgaris. Pemphigus vulgaris in humans is an autoimmune disease in which circulating antibodies bind and destroy components of the desmosome (reviewed in Schmidt and Waschke 2009). Histologically, pemphigus vulgaris is characterized by increased keratinocyte apoptosis and by acantholysis of suprabasal keratinocyte layers. The pemphigus vulgaris immunoglobulin (PV-IgG,) which targets desmosomal cadherin desmoglein 3, activates EGFR in cultured keratinocytes, which is followed by phosphorylation of its downstream substrates MAPK/ERK and the transcription factor c-JUN, resulting in EGFR internalization (Frusic-Zlotkin et al. 2006). Substantial evidence also documents apoptotic cell death in the epidermis and in cultured keratinocytes upon abrogation of EGFR signaling (Lewis et al. 2003; Canguilhem et al. 2005). Inhibition of EGFR prior to UV exposure also increases apoptosis and suppresses skin tumorigenesis (El Abaseri et al. 2005). EGFR suppresses UV-induced apoptosis through the activation of PI3K/AKT signaling (Wan et al. 2001). Evidence of the importance of apoptosis in suppression of EGFRdependent tumorigenicity is provided by experiments in which expression of the anti-apoptotic Bcl-2 gene restores tumor formation in hypomorphic Egfr and Egfr null backgrounds (Sibilia et al. 2000). While abrogation of EGFR signaling usually increases apoptosis, EGFR activation, paradoxically, can trigger cell death in cancer cells over-expressing the receptor (Chin et al. 1997). In a breast cancer cell line overexpressing EGFR, application of EGF triggers anoikis (Kottke et al. 1999). As mentioned above, the cellular localization of the receptor affects the choice between apoptosis and proliferation (Hyatt and Ceresa 2008). Finally, the effects of EGFR inhibitors on epithelial tumors are diverse, and include down-regulation of MAPK and PI3K/AKT-dependent signaling cascade with decreased BCL2 expression and increased apoptosis (reviewed in Zahorowska et al. 2009).

5.6 EGFR Stimulation of Cell Migration and Invasion During Wound Healing and in SCC Cell migration is required for reepithelialization during wound healing. Neoplastic cells are also highly migratory and are invasive, a key property that distinguishes them from normal cells. EGFR is implicated in both normal and neoplastic migration and invasion through multiple mechanisms. EGFR is involved in many aspects of wound repair and plays a pivotal role in the keratinocye migration necessary for reepithelialization in the skin such that wound healing is delayed in the absence of EGFR signaling (Repertinger et al. 2004; Pastore et al. 2008; Forsberg et al. 2008). EGFR acts through SLUG and GSK3 (Koivisto et al. 2006; Kusewitt et al. 2009) to stimulate JAK-STAT-induced migration, potentially through increased MMP-1 expression (Andl et al. 2004).

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EGFR is also implicated in SCC progression and is associated with poorer p rognosis (reviewed in Rogers et al. 2005). Administration of EGFR inhibitors reduces the metastasis of carcinomas, additional evidence of their importance in invasion (Oliveira et al. 2006; Zahorowska et al. 2009). To become invasive, epithelial cells must break their anchoring connections to each other and to basement membrane components, and also must express proteolytic enzymes in order to degrade extracellular matrix components. Oncogenic EGFR facilitates downmodulation of cellular adhesions and up-regulates protease expression (reviewed in Rogers et al. 2005). EGFR activation internalizes the cell adhesion molecule E-cadherin via b-catenin phosphorylation and through phosphorylation of a6b4 integrins, freeing keratinocytes from basement membrane and cell–cell connections (Mariotti et al. 2001; Yasmeen et al. 2006). GTP-binding proteins of the Rho family, including CDC42 and Rac1 are activated by EGFR, resulting in cytoskeletal rearrangement and cell migration (Rogers et al. 2005; Yang et al. 2004). EGFR works through phospholipase C, PI3K and MAPK to facilitate these interactions (Chen et al. 1994; Dise et al. 2008; Xie et al. 1998; Li et al. 2009). EGFR signaling also plays a role in extracellular matrix degradation. For example, EGFR activation of MAP kinases up-regulates MMPs; including MMP-1, 3, 7, 9, 10, 11, and 13; and disintegrin and matrix metalloproteases (ADAMs), which are highly expressed in some squamous cancers (Rogers et al. 2005; Sahin et al. 2004; Takamune et al. 2007). MMPs and ADAMs; especially ADAM-10, 12, and 17; are implicated in ectodomain shedding of EGF-ligands and auto/paracrine activation of EGFR (Ohtsu et al. 2006; Edwards et al. 2008). Thus, integration of multiple signaling pathways downstream from EGFR lead to increased cell migration and invasion of the extracellular matrix associated with malignant progression.

5.7 EGFR and Differentiation in the Skin and Other Epithelia Two types of epithelia exist in the body and perform multiple functions including secretion, absorption, and protection. Simple epithelium is a one cell-thick layer of cells, with all cells making contact with the basement membrane. Stratified epithelium is made up of multiple cell layers that undergo a process of terminal differentiation, where the basal layers contain more undifferentiated progenitors and the cells differentiate as they move to the apical surface. Expression of intermediate filament proteins facilitates the formation of each layer along with alterations of junctional contacts (reviewed in Fuchs 1990). Differentiation in the epithelium results from a cell fate decision that reduces the proliferative capacity of the cell and initiates the terminal differentiation program. The mechanisms for this cell fate specification are not fully defined; however, multiple mechanisms have been described (reviewed in Vincent et al. 2009; Koch and Nusrat 2009; Koster and Roop 2007). These cell fate decisions are important not only in normal tissue, but in epithelial cancers as well.

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Numerous studies show that EGFR suppresses differentiation in both stratified and simple epithelia (Getsios et al. 2009; Hallman et al. 2008; Visco et al. 2004; Brandt et al. 2000). In epidermal keratinocytes, differentiation occurs when proliferating cells in the basal layer migrate apically and initiate the terminal differentiation program. This program includes the down-regulation of EGFR (Sibilia and Wagner 1995; Nanney et al. 1990) and may involve asymmetric distribution of EGFR in daughter cells (Lechler et al. 2005). EGFR expression promotes proliferation in the epidermis; daughter cells that contain greater amounts of receptor retain their proliferative potential while cells with fewer receptors undergo terminal differentiation (Lechler and Fuchs 2005). A more active role for EGFR in differentiation results from the interaction of EGFR and NOTCH family members. In both C. elegans and D. melanogaster, EGFR and Notch can either work together or oppose each other during differentiation (Sundaram 2005). In keratinocytes, increased expression of EGFR causes higher proliferative potential and increased cellular migration while increased NOTCH signaling results in progression to the spinous layer and differentiation (Fuchs 2008). The loss of EGFR results in reduced proliferation and increased differentiation while decreased NOTCH causes the opposite (Fuchs 2008). EGFR and NOTCH act as negative regulators of each other in the differentiating epidermis, with EGFR-meditated signaling antagonizing NOTCH signaling, while upregulation of NOTCH results in transcription of EGFR/Ras inhibitors. MAPK phosphorylation of Groucho inactivates this corepressor, resulting in inhibition of NOTCH responses through Enhancer of split (E(SPL)). The up-regulation of NOTCH homolog LIN-12 in C. elegans drives the transcription of LIP-1 (ERK phosphatase), ARK-1 (EGFR inhibitor) and other proteins that cause EGFR endocytosis and degradation (Sundaram 2005; Shilo 2005). Interestingly, evidence exists for a role of NOTCH as both a tumor suppressor and oncogene in carcinogenesis (Radtke and Raj 2003). In two human model cancer cell lines, NOTCH signaling is activated by oncogenic RAS, and NOTCH-1 is necessary to maintain the neoplastic phenotype (Weijzen et al. 2002), suggesting an interaction between these signaling pathways in human carcinoma. EGFR activation can also regulate cell fate specification during the formation of ectodermal appendages. In the developing epidermis EGFR activation by EGF specifies epidermal rather than follicular cell fate (Kashiwagi et al. 1997). Downmodulation of EGFR in the developing hair placode is necessary for hair follicle development (Richardson et al. 2009). As these examples demonstrate, the role of EGFR in differentiation and cell fate specification may be active, through interaction with other pathways, or passive, through a loss of mitogenic stimulation and subsequent cell cycle exit following down-modulation of the receptor. It has become clear that multiple mechanisms may be responsible for epithelial differentiation and cell fate determination. Such mechanisms may be not only cell type-dependent but also context-dependent. This cell type-dependence has clinical significance as illustrated by the role of EGFR in promoting tumorigenesis in different organs. Because of its overexpression in various tumors it has recently become a key target for treating certain solid tumors, including, renal cell, prostate, colorectal, and SCC (Ciardiello et al. 2008). Although the efficacy of EGFR

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i nhibitors to treat cancer largely results from slowing mitogenic signaling, increasing apoptosis and reducing angiogenesis (Ciardiello and Tortora 2008), these inhibitors may also promote the differentiation of cancer cells, a possibility that remains largely uninvestigated.

5.8 Conclusions Review of the literature reveals a daunting multiplicity of roles for EGFR in squamous epithelia and the malignancies arising from them. As mechanisms of EGFR action are identified, the specificity of EGFR signaling pathways for normal versus neoplastic cells and for one cell type compared to another is also apparent. Thus, much investigation is required to fully understand the influence of EGFR signaling and to effectively utilize EGFR-targeting strategies for the treatment of squamous carcinoma.

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Chapter 6

Cyclooxygenase-2 Signaling in Squamous Cell Carcinomas Joyce E. Rundhaug and Susan M. Fischer

Abstract Cyclooxygenase-2 (COX-2) is the inducible isoform of the enzymes that initiate prostaglandin synthesis from arachidonic acid. While COX-2 is generally not expressed in most unperturbed adult tissues, it can be induced by multiple stimuli, including growth factors, cytokines, ultraviolet (UV) irradiation, tumor promoters and other stressors. Induction most often involves transcriptional activation of the COX-2 gene via transcription factor binding to cis-acting elements in its promoter. COX-2 is overexpressed in many epithelial cancers including most human and mouse squamous cell carcinomas (SCCs). Mouse skin carcinogenesis models have been extensively used to study the molecular events involved in SCC development. Inhibition of COX-2 activity with pharmacological agents, as well as genetic manipulation of COX-2 expression levels with transgenic and knockout mice, have demonstrated that up-regulated COX-2 expression/activity during tumor promotion is critically important for the development of mouse skin tumors and SCCs using both chemical and UV carcinogenesis protocols. PGE2, a major product of COX-2 in epithelial tissues, signals through four G protein-coupled receptors, EP1-EP4, which show differential affinities for PGE2 and couple to different G proteins and downstream signaling pathways. EP1, EP2, and/or EP4 have been shown to be involved in UV and chemical induction of mouse skin tumors, keratinocyte proliferation, epidermal hyperplasia, inflammation and angiogenesis.

6.1 Introduction Cyclooxygenase (COX), also called prostaglandin endoperoxidase H or prostaglandin H synthase, catalyzes the first and rate-limiting step in the conversion of arachidonic acid to prostaglandins (PGs), prostacyclins and thromboxanes S.M. Fischer (*) The University of Texas M.D. Anderson Cancer Center, Science Park – Research Division, Smithville, TX 78957, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_6, © Springer Science+Business Media, LLC 2011

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Fig. 6.1 Conversion of arachidonic acid to prostaglandins (PGs), prostacyclins and throm boxanes is catalyzed by cyclooxygenases (COXs). Arachidonic acid is released from membrane phospholipids by phospholipase A2. In a two-step reaction, COX-1, which is constitutively expressed, and COX-2, which is highly inducible by multiple stimuli, initially insert two molecules of O2 into arachidonic acid to form PGG2 followed by reduction of the hydroperoxide moiety to a hydroxyl group to form PGH2. PGH2 is then converted to bioactive eicosanoids via specific synthases.

(Fig. 6.1). COX initially incorporates two molecules of oxygen into arachidonic acid, released from membrane phospholipids by phospholipase A2, to form PGG2 and then via a peroxidase reaction reduces PGG2 to PGH2 (Smith et al. 2000). Subsequently, specific PG, prostacyclin and thromboxane synthases convert the unstable PGH2 intermediate to bioactive eicosanoids. There are two major COX isoforms, which are encoded by separate genes on different chromosomes (Smith et al. 2000). COX-1 is constitutively expressed in most tissues and its PG products are involved in normal physiological functions, such as maintenance of the gastric mucosa and regulation of renal blood flow (Subbaramaiah and Dannenberg 2003). On the other hand, COX-2 is generally not expressed in normal, quiescent tissues, but is highly inducible by mitogenic and inflammatory stimuli, such as growth factors, cytokines, hormones, ultraviolet (UV) radiation, carcinogens, tumor promoters, wounding, and other stressors (Smith et al. 2000; Trifan and Hla 2003). PGs derived from COX-2 are involved in pathophysiological functions such as cell proliferation, apoptosis, angiogenesis, inflammation and fever (Funk 2001; Trifan and Hla 2003). A third isoform, COX-3, has been identified, which is a splice variant of COX-1 and is highly expressed in the brain, kidney, and heart (Kis et al. 2006). However, because the alternative splicing causes a shift in the reading frame, COX-3 does not have cyclooxygenase enzyme activity (Kis et al. 2006).

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6.2 COX-2 Expression in Human Squamous Cell Carcinomas (SCCs) While COX-2 expression is generally undetectable in unperturbed adult epithelial tissues, it is overexpressed in a variety of human and rodent epithelial cancers, including SCCs. In human head and neck SCCs (HNSCCs), COX-2 mRNA levels were found to be increased almost 150-fold over levels detected in normal oral mucosa from healthy volunteers, while levels in normal-appearing epithelia adjacent to HNSCCs were increased approximately 50-fold (Chan et al. 1999). Likewise, COX-2 protein was undetectable in the normal oral mucosa of healthy volunteers, but present in HNSCCs (Chan et al. 1999). These data correlate with high levels of PGs found in HNSCCs (Karmali et al. 1984). In another study of HNSCCs, COX-2 immunohistochemical staining was detected in the majority of tumors, particularly in tumors of higher pathological stage, and was significantly higher in tumors with histologically confirmed lymph node metastasis as compared with those without metastasis (Gallo et al. 2002). PGE2 levels were also elevated in HNSCC tumors compared with matched unaffected mucosa and were highest at the invasive edge of the tumors (Gallo et al. 2002). COX-2 overexpression and higher PGE2 tumor levels were associated with poorer overall survival rates (Gallo et al. 2002). On the other hand, in laryngeal SCCs, strong COX-2 immunostaining was found in well-differentiated areas of tumors and expression was lost in poorly- differentiated, more malignant tumors (Ranelletti et al. 2001). Low levels of COX-2 were associated with poorer overall survival compared with high levels of expression in laryngeal SCCs (Ranelletti et al. 2001). The difference in high versus low COX-2 expression correlating with poorer survival in HNSCC compared with better survival in laryngeal SCC is presumably because high COX-2 expression is associated with more malignant, aggressive disease in HNSCC, but with more differentiated, less malignant tumors in laryngeal SCC. In the esophagus, COX-2 protein levels determined by immunohistochemistry and Western blotting were found to be elevated in SCCs compared with normal esophageal squamous epithelia, while COX-1 levels were similar in normal and cancerous tissues (Zimmermann et al. 1999). Similarly, overexpression of COX-2 mRNA and protein levels were found in both esophageal squamous dysplasia and in SCCs compared with normal epithelia in a study done in China (Yu et al. 2003). In this latter study, high COX-2 expression was correlated with a higher proliferation index and high p53 levels in dysplasias and SCCs, but did not correlate with histological grade, pathological stage or lymph node metastasis. Another group also found the majority of esophageal SCCs had high COX-2 immunostaining primarily in the well-differentiated portions of the tumors (Heeren et al. 2005). However, there was no significant difference in overall survival of patients with high versus low intensity of COX-2 immunostaining, although the number of patients with SCCs was small (Heeren et al. 2005). Although overexpression of COX-2 has been demonstrated in the tumor cells of only some (28–51%) SCCs of the uterine cervix (Ferrandina et al. 2002a, b;

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Kim et al. 2002), it was shown to be associated with poorer overall survival, shorter interval to recurrence and to higher treatment failure rates compared to low or negative COX-2 expression (Kim et al. 2002; Pyo et al. 2005). In human skin SCCs, COX-2 was found to be expressed in the vast majority of tumors, with positive immunostaining generally in both the tumor cells and adjacent stroma, while there was little or no staining in normal human skin (Buckman et al. 1998; An et al. 2002). In addition, positive COX-2 immunostaining was also detected in greater than 80% of actinic keratoses and in Bowen disease, which are premalignant lesions and essentially SCC in situ, respectively, although staining was less intense than in SCCs (An et al. 2002). Within the lesions in all cases, COX-2 staining was cytoplasmic and limited to the suprabasal (differentiated) layers. Similarly, COX-2 expression and PGE2 production was significantly greater in human SCC cell lines compared to the nontumorigenic human keratinocyte HaCaT cell line (Higashi et al. 2000). Thus, COX-2 is frequently overexpressed in various human SCCs compared with little or no expression in normal epithelial tissues. In some tissues, up- regulated expression appears to be an early event in that premalignant lesions, carcinoma in situ and/or normal-appearing tissue adjacent to SCCs also show elevated COX-2 expression. In some SCCs, COX-2 overexpression is associated with poorer overall survival or response to therapy, while in other SCCs, COX-2 expression is associated with the more differentiated regions of the tumor and signifies a less malignant or aggressive phenotype.

6.3 Mouse Skin Carcinogenesis Models of SCC Mouse skin carcinogenesis models have been widely used to study the various stages and molecular events associated with the development of SCCs. The multistage chemical induction of skin tumors is accomplished by topical application of a subcarcinogenic dose of a carcinogen, such as 7,12-dimethylbenz[a]anthracene (DMBA), to the shaved backs of mice, followed by repeated applications of a tumor promoter, most often 12-O-tetradecanoylphorbol-13-acetate (TPA). The carcinogen treatment results in genetic mutations (in the proto-oncogene H-Ras in the case of DMBA) giving rise to initiated cells in the epidermis and hair follicles, which remain morphologically normal unless further treated (DiGiovanni 1994). Subsequent treatments with a tumor promoter induce epidermal proliferation, hyperplasia and inflammation resulting in the selective clonal expansion of initiated cells to form initially premalignant tumors called papillomas, some of which eventually progress to become invasive SCCs (DiGiovanni 1994). A more physiologically relevant model for human skin cancer utilizes repetitive UV irradiation treatments to induce skin tumors in mice. Most often SKH hairless mice are irradiated three times/week with sub- or minimally-erythemic UV (predominantly UVB) doses, which result in the formation of papillomas, keratoacanthomas and eventually SCCs (Kligman and Kligman 1981; Gallagher et al. 1984). In this model, the

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early initiating event is thought to be UV-induced mutations in the tumor suppressor gene, Tp53, which have also been shown to be present in human actinic keratoses, Bowen disease, and SCCs (Boukamp 2005). UV irradiation also acts a tumor promoter by inducing inflammation, keratinocyte proliferation and epidermal hyperplasia (Lu et al. 1999; Bowden 2004). Constitutive overexpression of COX-2 mRNA and protein as well as elevated levels of PGE2 and PGF2a have been shown in chemically-induced mouse skin papillomas and SCCs (Müller-Decker et al. 1995). Immunostaining for COX-2 showed strong staining in the basal layer of papillomas and in follicular keratinocytes in SCCs (Müller-Decker et al. 1998). In UV-induced mouse skin tumors, COX-2-positive staining was seen in some, but not all papillomas, while intense COX-2 staining was found in the granular layer and in the tumor stroma of SCCs, compared with no staining in normal mouse epidermis (An et al. 2002). Thus, upregulated expression of COX-2 is present in mouse skin SCCs, as well as in premalignant lesions, similar to that seen in human skin and other SCCs.

6.4 Regulation of COX-2 Gene Expression As noted earlier, COX-2 expression can be induced by multiple stimuli. In mouse skin keratinocytes various tumor promoters, such as TPA, anthralin, benzoyl peroxide, and okadaic acid, as well as epidermal growth factor (EGF), induced COX-2 mRNA expression with varying kinetics (Maldve and Fischer 1996). The COX-2 product PGE2 has also been shown to induce COX-2 mRNA expression and promoter activity in keratinocytes via a cyclic AMP (cAMP)-linked pathway, which provides a positive feedback loop (Maldve et al. 2000). Acute exposure of human and mouse skin to UVB irradiation strongly induced COX-2 mRNA and protein expression (Buckman et al. 1998; Fischer et al. 1999; Athar et al. 2001; An et al. 2002; Tripp et al. 2003). Using the HaCaT human keratinocyte cell line, up-regulation of COX-2 expression by UVB was shown to be mediated via UVB activation of p38 mitogen-activated protein kinase (MAPK) that results in the phosphorylation of the cAMP-response element (CRE) binding protein (CREB) and activating transcription factor-1 (ATF-1), which then bind to the CRE site in the COX-2 gene promoter, and activate COX-2 transcription (Chen et al. 2001; Tang et al. 2001). In addition, absorption of UVB energy by tryptophan converts it to a ligand for the arylhydrocarbon receptor (AhR) causing AhR to release its associated proteins including Src (Fritsche et al. 2007). Src then activates the EGF receptor (EGFR) and the downstream MAPK cascade to induce COX-2 transcription (Fritsche et al. 2007). While most studies have focused on UVB induction of COX-2 and skin cancer, it has been estimated that UVA wavelengths are responsible for 70% of the total induction of COX-2 and PGE2 by solar light (Mahns et al. 2004). UVA irradiation produces reactive oxygen species, which leads to activation of phospholipase A2, as well as p38 and JNK MAPKs and ultimately in the up-regulation of COX-2

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expressionand PG production (Bachelor and Bowden 2004). UVA activation of p38 causes stabilization of COX-2 mRNA and thus increased protein expression (Bachelor et al. 2002). Two cis-acting elements in the mouse Cox-2 gene promoter, a nuclear factor-IL6 (NF-IL6) site at −138/−131 and an E-box site at −53/−48 are important for constitutively elevated expression of COX-2 in a mouse skin SCC cell line (Kim and Fischer 1998). CCAAT/enhancer-binding proteins (C/EBPs) and upstream stimulatory factors (USFs), respectively, were identified as the nuclear factors that bound to these two sites and transcriptionally activated the Cox-2 promoter in SCC cells (Kim and Fischer 1998). The differential expression of the C/EBP isoforms between normal skin and SCC cells suggested that these factors were responsible for COX-2 overexpression in SCCs (Kim and Fischer 1998). In addition, the COX-2 gene promoter contains nuclear factor-kB (NF-kB) binding sites (Yamamoto et al. 1995) and COX-2 has been identified as a direct transcriptional target of NF-kB (Pahl 1999). Parallel up-regulated expression of NF-kB and COX-2 has been demonstrated in oral premalignant lesions and oral SCCs relative to normal oral mucosa (Santhi et al. 2006; Sawhney et al. 2007). Cigarette smoke condensate and smokeless tobacco extract, as well as the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been shown to activate NF-kB and induce COX-2 expression in HNSCC and oral precancer and cancer cell lines (Anto et al. 2002; Sawhney et al. 2007). The COX-2-selective inhibitor celecoxib suppressed cigarette smoke condensate-induced activation of NF-kB and COX-2 expression (Shishodia and Aggarwal 2004). Likewise, the skin tumor promoter TPA was shown to activate NF-kB via phosphorylation of inhibitor of kBa (IkBa) by extracellular signaling-regulated kinase 1/2 (ERK1/2) in mouse skin, which led to the induction of COX-2 expression (Chun et al. 2003). Both curcumin and cocoa polyphenols were able to inhibit TPA-induced COX-2 expression in mouse skin by blocking TPA activation of NF-kB and inhibiting ERK activity (Chun et al. 2003; Lee et al. 2006). Thus, COX-2 expression can be transcriptionally regulated by NF-kB and several potential chemopreventive agents appear to block COX-2 expression by inhibiting NF-kB activation.

6.5 Role of COX-2 Overexpression in SCC Development While COX-2 overexpression has been demonstrated in numerous SCCs and various tumor promoters and mitogenic stimuli can induce COX-2 expression, COX inhibitors have been used to directly demonstrate that this increased COX-2 level plays a causal role in SCC development. Early studies demonstrated that topical treatment with the nonsteroidal anti-inflammatory drug (NSAID), indomethacin, which is a general COX inhibitor, could effectively inhibit DMBA/TPA-induced mouse skin tumor development (Verma et al. 1980). Concurrent application of PGE2 was able to reverse indomethacin’s inhibition of the tumor promoting effects of TPA (Verma et al. 1980). Similarly, a COX-2-selective inhibitor, SC-58125, blocked TPA-induced

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PGE2 production in mouse skin, as well as DMBA/TPA-induced skin tumor formation. Thus, TPA induction of COX-2 expression is important for its tumor promoting effects (Müller-Decker et al. 1998). In another study, a different COX2-selective inhibitor, celecoxib, administered in the diet throughout the promotion phase reduced tumor yield by approximately 50% and feeding celecoxib during DMBA initiation had no greater effect (Müller-Decker et al. 2002). Again, these results suggest that COX-2 activity is involved in tumor promotion. Likewise, in the UV-induced skin tumorigenesis model, feeding mice with either indomethacin or celecoxib or topical treatment with celecoxib after each UV exposure dramatically reduced tumor development, as well as UV-induced PGE2 synthesis (Fischer et al. 1999; Wilgus et al. 2003a; Tober et al. 2006). Topical treatment with celecoxib was also shown to inhibit UV-induced vascular permeability, infiltration of inflammatory cells and oxidative DNA damage (Wilgus et al. 2003a). In another study, oral administration of either indomethacin or the COX-2-selective inhibitor, SC-791, decreased UV-induced epidermal proliferation and increased UV-induced apoptosis (Tripp et al. 2003). Together, these results suggest that COX-2 induction is necessary for keratinocyte survival and proliferation after UV irradiation and that COX-2-mediated PGE2 production is critical for UV-induced inflammation, all of which contribute to skin tumorigenesis. When celecoxib treatment (either oral or topical) was begun after UV-induced tumors were established, new tumor formation was prevented, but there was little or no effect on the regression of the tumors that were already there (Fischer et al. 2003; Wilgus et al. 2003b). Thus, COX-2 overexpression seems to be more important in the early development of skin tumors in these models and may not be as necessary for the maintenance of established tumors. On the other hand, inhibition of COX-2 activity with NS-398 or reduction of COX-2 expression with antisense oligos inhibited the growth of two human skin SCC cell lines (Higashi et al. 2000), which suggests that COX-2 overexpression contributes to SCC proliferation. Evidence for the relevance of COX-2 expression on human skin SCC development comes from an epidemiological study, which found that regular users of NSAIDs had lower risk of developing actinic keratoses and SCC (Butler et al. 2005). Because COX-2-selective inhibitors can have COX-independent effects (Grösch et al. 2006; Niederberger et al. 2006), genetic approaches have also been used to demonstrate a direct role of COX-2 in mouse skin tumorigenesis models. Chemically induced skin carcinogenesis was reduced by 75% in both COX-1 and COX-2 knockout mice compared to wild-type mice, which correlated with premature keratinocyte terminal differentiation in the knockout mice (Tiano et al. 2002). However, in the UV carcinogenesis model, COX-1 deficiency had no effect on tumor development even though it enhanced UV-induced apoptosis (Pentland et al. 2004). Furthermore, deficiency of just one allele of COX-2 significantly delayed the onset of UV-induced skin tumorigenesis and reduced multiplicity by 50–65% (Fischer et al. 2007). Similar to that seen with COX-2-selective inhibitors, UV-induced proliferation was reduced and apoptosis increased in heterozygous as well as homozygous COX-2 knockout mice compared to wild-type mice (Chun and Langenbach 2007; Fischer et al. 2007). Together these results suggest that both

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COX-1 and COX-2 contribute to chemically-induced carcinogenesis, but COX-2 expression is critical for UV induction of skin tumors. To complement the studies done with COX-2 knockout mice, transgenic mice that overexpress COX-2 in the basal layer of the epidermis via either keratin-14 (K14) or keratin-5 (K5) promoters have been generated. Experiments with these mice, which have elevated levels of PGE2 in their epidermis (Bol et al. 2002; Müller-Decker et al. 2002), have shown that they are more sensitive than wild-type mice to developing skin tumors after carcinogen initiation without further treatment with a tumor promoter (Müller-Decker et al. 2002; Rundhaug et al. 2007). In the UV carcinogenesis model, the K14.COX-2 transgenic mice developed skin tumors earlier and developed more tumors/mouse than wild-type mice (Fischer et al. 2007). Together these data demonstrate that elevated levels of COX-2 activity and/ or PGE2 in the skin can act as an endogenous tumor promoter.

6.6 Signaling Downstream of PGE2 EP Receptors All of the PG and thromboxane products of COX-2 bind to and signal through a family of seven-transmembrane G protein-coupled receptors designated DP, EP, FP, IP and TP, which are specific for PGD2, PGE2, PGF2a, prostacyclin (PGI2) and thromboxane A2 (TXA2), respectively (Breyer et al. 2001) (Fig. 6.2). Ligand-binding specificity of the receptors is determined by the extracellular and transmembrane

Fig. 6.2 PGs, prostacyclin (PGI2) and thromboxane A2 (TXA2) signal by binding to specific seven-transmembrane G protein-coupled receptors. PGE2 signals through four EP receptors, which are coupled to different G proteins leading to the generation of various second messengers. The EP3 receptor is expressed as multiple splice variants, which due to different C-terminal tails couple to different G proteins and activation of different signaling pathways. IP3, inositol 1,4,5-trisphosphate; cAMP, cyclic AMP; EGFR, epidermal growth factor receptor, PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase.

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domains, while the cytoplasmic C-terminal domain regulates coupling to specific G proteins, constitutive activity, signal transduction, receptor phosphorylation and desensitization/internalization (Breyer et al. 2001). Because PGE2 is a major COX product in the skin and other epithelial tissues, most studies have focused on PGE2 signaling through the EP receptors. There are four EP receptors, EP1-EP4, which show differences in PGE2 affinity and signal through different pathways (Fig. 6.2). EP3 and EP4 are high-affinity receptors and bind PGE2 in the subnanomolar range, while EP1 and EP2 are low-affinity receptors, binding PGE2 at low nanomolar concentrations (Konger et al. 2005a). The EP1 receptor is coupled to the Gq protein and activates phospholipase C-b, which leads to generation of inositol 1,4,5-trisphosphate (IP3), increases in intracellular calcium and activation of protein kinase C (Breyer et al. 2001; Tober et al. 2006; Zhang et al. 2007). The EP2 and EP4 receptors are coupled to Gs, which activates adenylate cyclase resulting in elevation of cAMP levels (Hata and Breyer 2004). EP2mediated increases in cAMP lead to activation of protein kinase A (PKA) and subsequent phosphorylation of CREB and glycogen synthase kinase-3 (GSK-3) (Regan 2003; Hata and Breyer 2004; Chun et al. 2007). On the other hand, EP4 signaling can activate phosphatidylinositol 3-kinase (PI3K), Akt and ERK1/2 via a PKA-independent pathway (Regan 2003; Hata and Breyer 2004; Chun et al. 2007). Additional differences between EP2 and EP4 signaling are due to differences in their C-terminal tails, with EP4 having a longer tail resulting in agonist-induced desensitization and internalization, which does not occur with EP2 (Hata and Breyer 2004). EP3 exists as multiple alternatively spliced variants, which have unique C-terminal tails and are coupled to different signaling pathways (Narumiya et al. 1999; Breyer et al. 2001). In general, EP3 has been reported to be coupled to Gi, which inhibits adenylate cyclase and lowers cAMP levels (Narumiya et al. 1999; Breyer et al. 2001; Hata and Breyer 2004). However, individual EP3 splice variants have also been shown to stimulate increases in cAMP, intracellular calcium and IP3 via coupling to Gs, Gq and/or Go (Narumiya et al. 1999; Breyer et al. 2001; Hata and Breyer 2004).

6.7 Role of EP Receptors in Skin Carcinogenesis Primary cultures of human keratinocytes have been shown to express EP2-4 mRNA (Konger et al. 1998). Based on immunohistochemical analysis, EP1 protein was expressed predominantly in the cytoplasm of the granular layer as well as in the plasma membrane and perinuclear/nuclear areas of the basal and spinous layers of human epidermis (Konger et al. 2005a). EP2 was localized primarily in the perinuclear/nuclear areas and plasma membrane of the basal and spinous layers with scattered and less intense cytoplasmic staining in the granular layer (Konger et al. 2005a). Three splice variants of EP3 were also expressed in human skin, predominantly in the basal and lower spinous layers (Konger et al. 2005b). In normal mouse skin there was a low level of EP1 immunostaining, mainly in the granular layer;

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EP2 showed patchy expression throughout the epidermis; EP3 was moderately expressed and EP4 was largely undetectable (Lee et al. 2005). Acute UV irradiation of mouse skin resulted in up-regulation of EP1, down-regulation of EP3 and no change in EP2 or EP4 expression as determined by immunohistochemical staining (Lee et al. 2005). Another group found that UV exposure led to up-regulation of EP1, EP2, and EP4 protein expression by Western blotting (Chun et al. 2007). In UV-induced mouse skin SCCs, EP1 showed intense immunostaining, EP2 was patchy, EP3 was very low and EP4 was moderately expressed (Lee et al. 2005). In human skin SCCs, all four EP receptors were expressed by immunostaining and SCCs had significantly elevated mRNA expression of EP1, EP2, and EP4 compared to adjacent normal skin (Lee et al. 2005). Thus, elevated expression of EP1, EP2, and/or EP4 in mouse and human SCCs may be contributing to the development of these tumors. Knockout and transgenic EP mouse models, as well as treatment with EP-specific agonists and antagonists, have been used in skin carcinogenesis protocols to determine the contributions of each of the EP receptors to skin tumor development. Fewer EP2 knockout mice developed tumors using the DMBA/TPA protocol and the mice that did had fewer tumors/mouse compared to wild-type mice (Sung et al. 2005). This correlated with a reduced epidermal proliferative and hyperplastic response to TPA, as well as reduced TPA induction of interleukin-1a and dermal macrophage infiltration in the knockout mice (Sung et al. 2005). Overexpression of EP2 in the skin of K5.EP2 transgenic mice resulted in increased benign and malignant tumor development relative to wild-type mice (Sung et al. 2006). TPA-induced proliferation, hyperplasia, angiogenesis and macrophage infiltration were all greater in EP2 transgenic mice compared to wild-type mice (Sung et al. 2006). In contrast, DMBA/TPA induction of skin tumors was similar in EP3 knockout and wild-type mice (Sung et al. 2005). However, another group found a delayed appearance and reduced tumor number at 11 weeks of tumor promotion in EP3 knockout mice relative to wild-type mice, although tumor incidence and multiplicity were not significantly different by 25 weeks (Shoji et al. 2005). These data suggest that EP2 mediates, at least in part, the inflammation, proliferative and tumor promoting effects of TPA. In the UV skin carcinogenesis model, as in the chemical carcinogenesis model, EP2 knockout mice developed fewer tumors and had reduced UV-induced epidermal proliferation and hyperplasia compared to wild-type mice (Brouxhon et al. 2007a). However, the tumors that did develop in the EP2 knockout mice tended to be larger, less differentiated and more aggressive than tumors from wild-type mice (Brouxhon et al. 2007a). UV-induced inflammation (ear swelling, blood flow, and infiltration of inflammatory cells) was reduced in EP2 knockout mice, as well as in wild-type mice treated with an EP4 antagonist compared to untreated wild-type mice (Kabashima et al. 2007). Expression of E-cadherin, which contributes to cell– cell adhesion, has been shown to be down-regulated by chronic and acute UV exposure and this was abrogated in EP2 knockout mice (Brouxhon et al. 2007b). The excessive UV-induced apoptosis seen in COX-2 knockout versus wild-type mice was reduced by treatment of the knockout mice with either an EP2 or EP4

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agonist (Chun et al. 2007). Topical treatment of mice with an EP1 antagonist reduced UV induction of skin edema, inflammatory cell infiltration and skin tumor development (Tober et al. 2006). Together these studies suggest that UV induction of PGE2 synthesis mediates keratinocyte proliferation, inflammation, loss of cell– cell contacts, cell survival, and skin tumorigenesis via EP1, EP2, and/or EP4 signaling.

6.8 PGE2 Regulation of Keratinocyte and SCC Proliferation One of the ways in which COX-2-derived PGE2 promotes SCC development is through stimulation of keratinocyte and SCC cell proliferation. The mechanisms and signaling pathways involved in PGE2 stimulation of cell proliferation have generally been studied in cell culture models. PGE2-induced proliferation of primary keratinocytes in culture was significantly reduced in cells from EP2 knockout mice and increased in K5.EP2 transgenic cells relative to wild-type cells (Ansari et al. 2007). PGE2 induction of keratinocyte proliferation, as well as COX-2 expression, in wild-type and K5.EP2 transgenic keratinocytes was blocked by pretreatment with an adenylate cyclase inhibitor (Ansari et al. 2007), which suggests that proliferation induced by PGE2 involves EP2 stimulation of cAMP production and positive feedback to further induce COX-2 expression and more PGE2 production. Through the use of pathway-specific inhibitors, PGE2-induced proliferation of primary mouse keratinocytes was also shown to involve activation of Protein Kinase A (PKA), Epidermal Growth Factor Receptor (EGFR), PI3K and ERK1/2 (Ansari et al. 2008). Inhibiting these pathways attenuated PGE2-induced binding of NF-kB, activator protein-1 (AP-1) and CREB to the promoters of cyclin D1 and vascular endothelial growth factor (VEGF) genes and expression of cyclin D1 and VEGF (Ansari et al. 2008). To relate this to COX-2 expression in vivo, K14.COX-2 transgenic mice were found to have greater keratinocyte proliferation, expression of cyclin D1 and VEGF, and activation of EGFR, NF-kB, AP-1 and CREB than wildtype mice and these effects were similar to that seen in PGE2-treated wild-type mice (Ansari et al. 2008). Indomethacin inhibition of PGE2 production inhibited the growth of primary cultures of adult human keratinocytes and this was reversed by treatment with either an EP2 or EP4 agonist or with dibutyryl cAMP (Konger et al. 1998). On the other hand, treatment with an EP3 agonist further inhibited DNA synthesis in indomethacin-treated human keratinocytes (Konger et al. 1998, 2005b). Inhibition of keratinocyte growth by EP3 activation correlated with production of ceramide and sn-1,2-diacylglycerol second messengers and induction of the cell cycle inhibitor, p21 (Konger et al. 2005b). Thus, PGE2 activation of EP2 and/or EP4 stimulates human keratinocyte growth, while EP3 activation inhibits growth. Similar to primary keratinocytes, indomethacin treatment inhibited growth of a malignant mouse keratinocyte cell line that produces high levels of PGE 2 (Thompson et al. 2001). In this case, addition of an EP1 agonist or PGE2 was able

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to reverse indomethacin growth inhibition (Thompson et al. 2001). PGE2 stimulation of cell proliferation and invasion of two human SCC cell lines was blocked by treatment with either inducible nitric oxide synthase (iNOS) silencing RNA or iNOS/guanylate cyclase inhibitors (Donnini et al. 2007). The signaling pathway mediating PGE2 stimulation of proliferation was shown to involve activation of EP2, PKA and c-Src, leading to EGFR, iNOS and subsequently ERK1/2 activation (Donnini et al. 2007). Taken together, most of these studies implicate EP2 induction of elevated cAMP levels, leading to activation of PKA signaling as the major pathway by which PGE2 induces proliferation of keratinocytes and their SCC counterparts. EGFR and ERK1/2 activation also seem to be involved downstream of EP2. On the other hand, EP3 activation inhibits keratinocyte proliferation. Since EP3 is a highaffinity receptor, low concentrations of PGE2 would favor growth inhibition and differentiation, while higher concentrations, as seen with tumor promoter and UV treatments, during inflammation and in SCCs, would favor activation of the lowaffinity EP2 receptor and stimulation of proliferation (Konger et al. 2005b). Reduced expression of EP3 in SCCs would further contribute to the imbalance of EP2/EP3 proliferation signals.

6.9 Clinical Studies with COX Inhibitors There have been a few clinical trials using COX, and in particular COX-2 selective, inhibitors for either therapy or chemoprevention of human SCC. In an oral cancer chemoprevention study, a non-selective COX inhibitor, ketorolac, was administered as an oral rinse twice a day for 90 days to patients with oropharyngeal leukoplakia with the rationale that local administration of the COX inhibitor would reduce gastrointestinal side effects (Mulshine et al. 2004). Although the COX inhibitor was well-tolerated, there was no significant change in leukoplakia histology between those using ketorolac compared to a placebo rinse. In a more recent small pilot study, patients with oral premalignant lesions were treated with the COX-2 selective inhibitor, celecoxib. Comparing baseline and posttreatment biopsies, 12 of 18 (67%) showed improvement in the degree of dysplasia after 12 weeks and 8 of 11 (73%) showed continued improvement after 12 months (Wirth et al. 2008). This study also looked at potential biomarkers and found a reduction in PGE2 levels and trends towards reduced COX-2 and Ki67 (a proliferation marker) expression with celecoxib treatment (Wirth et al. 2008). Similarly in a pilot study with 14 cervical cancer patients, 10 days of celecoxib treatment significantly reduced tumor biopsy COX-2 and Ki67 immunostaining and microvessel density, as well as a trend towards lower PGE2 levels (Ferrandina et al. 2003). On the other hand, in an esophageal SCC chemoprevention trial in patients with premalignant squamous dysplasia, celecoxib treatment for 10 months did not significantly influence changes in dysplasia grade compared to placebo treatment (Limburg et al. 2005). Treatment of patients with recurrent or metastatic HNSCC with a combination of an EGFR inhibitor, gefitinib, and celecoxib resulted in partial responses in 4 of 18

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patients (Wirth et al. 2005). Another case report showed that treatment of a patient with locoregional metastatic cutaneous SCC with a combination of an EGFR antibody, cetuximab, and celecoxib resulted in complete or partial regression of several metastases (Jalili et al. 2008). Thus, treatment or chemoprevention of SCCs with COX inhibitors shows promise, but more studies are needed to determine which tumors/patients will benefit the most and what doses and routes of administration will yield the most efficacious results with the least side effects. Additional clinical trials using celecoxib or other COX-2-selective inhibitors for the chemoprevention or treatment of non-SCCs have also been conducted (see e.g., Higuchi et al. 2003; Mazhar et al. 2006; Yona and Arber 2006).

6.10 Summary and Conclusions COX-2 overexpression is frequently seen in numerous human and mouse SCCs relative to little or no expression in normal epithelial tissues. This leads to elevated PG levels in SCCs, particularly PGE2, which is often the major COX-2 product. COX-2 expression is highly inducible by various inflammatory and mitogenic stimuli, including UV irradiation and tumor promoters, which is accomplished by transcriptional activation of the COX-2 gene, as well as stabilization of COX-2 mRNA. Pharmacological and genetic manipulation of COX-2 activity and expression has demonstrated unequivocally that COX-2 plays an important role in the development of mouse skin tumors and SCCs by both chemical and UV carcinogenesis protocols. PGE2 appears to mediate most of the effects of COX-2 overexpression via signaling through its EP1, EP2, and/or EP4 receptors, whose expression is also up-regulated in human and mouse skin SCCs. Activation of these receptors leads to increased levels of intracellular calcium and cAMP and activation of PKA, EGFR, PI3K, and MAPK pathways to stimulate proliferation, cell survival, inflammation and angiogenesis, thereby promoting SCC development. The signaling pathways activated by PGE2 are the same as those activated by TPA, which suggests that this is how PGE2 has endogenous tumor promoting activity. Thus, pharmacological targeting of the EP receptors, in particular EP2, could potentially be used for the chemoprevention of SCCs, which may circumvent some of the unwanted cardiovascular side effects of COX-2 inhibitors (Yona and Arber 2006). Acknowledgments We apologize to all those researchers whose important work might not have been acknowledged due to editorial constraints. This work was supported by the following grants from NIH: CA100140, ES07784 and CA16672.

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Chapter 7

Interacting Signaling Pathways in Mouse Skin Tumor Initiation and Progression Christophe Cataisson and Stuart H. Yuspa

Abstract The multistage induction of squamous cell cancer (SCC) on mouse skin as a consequence of chemical exposures has remarkable phenotypic and genotypic homology to human SCC development. Genetically altered mouse models have been instrumental in defining the respective contribution of signaling pathways on the development of SCC in vivo. Central to the skin carcinogenesis process is the activation of the EGFR-Ras-MAPK pathway. While hyperactivation of the pathway can often be attributed to activating mutations in ras genes, it appears that for the majority of cases the pathway is activated by alterations in upstream modulators as well as downstream effectors that are integral to the altered phenotype of the initiated keratinocytes. This chapter will review data from experimental inductions of cutaneous SCC with a special emphasis on mouse models. Particularly, the role of the EGFRRas-MAPK pathway, protein kinase C, nuclear factor kappa B and the expression of proinflammatory factors by transformed keratinocyte will be covered in more detail.

7.1 Introduction Nonmelanoma skin cancer is the most common cancer in the Caucasian population in the USA. Approximately 80% of nonmelanoma skin cancers are basal-cell carcinomas and 20% are squamous cell carcinomas (SCC). Unlike almost all basalcell carcinomas, cutaneous SCC are associated with substantial risk of metastasis and share many of the features of lethal solid tumors of the internal organs. Exposure to ultraviolet radiation is the most common cause of cutaneous SCC. Ionizing radiation, human papillomavirus infection, chemical agents (polycyclic aromatic hydrocarbons for example) have been described as causal factors for the development of SCC. Patients who suffer from hereditary defects in DNA repair (e.g., xeroderma pigmentosum) or blistering disorders (e.g., recessive dystrophic S.H. Yuspa (*) Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH, Bethesda, MD 20892, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_7, © Springer Science+Business Media, LLC 2011

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epidermolysis bullosa) are extremely sensitive to development of SCC. Similarly, immunocompromised organ transplant recipients are at greater risk of developing SCC. Finally, SCCs are more likely to develop in injured or chronically diseased skin (for review Mueller 2006). Genetic analyses of squamous tumors at these sites have revealed certain common alterations. Among these are mutations or inactivation of p53, activating mutations or enhanced signaling through Ras, up-regulation of the EGFR by mutation, amplification or transcription, inactivation of cell cycle inhibitors, upregulation of cell cycle drivers, reduction in TGFb signaling and often complex changes in expression of AP-1 regulated genes. In order to identify underlying mechanisms by which these changes influence human disease, molecular information obtained from human tumor studies must be integrated with biological processes. This is often achieved best by studying relevant animal models. For cancer research, relevancy implies a target tissue representative of the common human cancer, a pattern of pathogenesis consistent with the multistage nature of cancer development in that target site, and molecular events identical or parallel to those that occur in the human tumor. The model becomes more valuable if a predictable sequence of phenotypic changes is coincident with reproducible genetic and epigenetic events. The multistage induction of SCCs on mouse skin as a consequence of chemical exposures fulfills many of these criteria, and this model has shown remarkable phenotypic homology to human SCC development. This chapter will review data from experimental models of cutaneous SCC with a special emphasis on mouse models. Particularly, the role of the EGFR-RasMAPK pathway, protein kinase C, nuclear factor kappa B (NF-kB) and the expression of proinflammatory factors by transformed keratinocyte will be covered in more detail.

7.2 EGFR-Ras-MAPK Pathway Ras (H-, K-, and N-Ras) belongs to a family of small guanosine triphosphatases (GTPases) that normally couple receptor activation to downstream effector pathways that control diverse cellular response including proliferation, differentiation and survival. Ras proteins cycle between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound conformations. The activation state of Ras is controlled by the cycle of hydrolysis of bound GTP, which is catalyzed by GTPase activating proteins (GAPs; e.g., p120GAP, NF1 neuro fibromin), and the replacement of bound GDP with fresh GTP, which is catalyzed by guanine nucleotide exchange factors (GEFs, e.g., SOS1-2) and a family of guanine nucleotide-releasing factors (RASGRP1-4) Ebinu et al. 1998; Stone, 2006. Activating RAS mutations occur in approximately 15% of human cancers and mutations have been found at residues 12, 13, 59, and 61 with positions 12 (glycine) and 61 (glutamine) being the most common (Bos 1989). Recently germline mutations in HRAS and KRAS have been implicated in developmental disorders in humans (for example, the Costello syndrome for HRAS Aoki et al. 2005).

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Mutations identified in Costello syndrome substitute amino acid at position 12 and 13 common to somatic mutations in tumors. Those mutations impair the intrinsic GTPase activity and confer resistance to GAPs, thereby causing mutant RAS to accumulate in its GTP-bound active form. There is a loss or silencing of the wild type HRAS allele reported in malignancies of Costello syndrome patients including bladder cancer and rhabdomyosarcoma (Estep et al. 2006). While pancreas (60% with KRAS mutations), colon (32% with KRAS mutations), thyroid (7% with NRAS and 5% with KRAS mutations), lung (19% with KRAS mutations), melanoma (18% with NRAS mutations) and cervix (9% with HRAS and 9% with KRAS mutations) have the highest prevalence of ras mutation (Schubbert et al. 2007), a smaller fraction of human skin and head and neck SCC harbors activating RAS mutations (Pierceall et al. 1991; Kreimer-Erlacher et al. 2001; Rumsby et al. 1990; Campbell et al. 1993; Spencer et al. 1995). However, transduction of primary human keratinocytes with an oncogenic HRAS in combination with CDK4 or blockade of nuclear factor-kB (NF-kB) is sufficient to cause SCC formation when grafted on SCID mice (Lazarov et al. 2002; Dajee et al. 2003). Similarly, animal model studies indicate that heterozygous activating Ras gene mutations in keratinocytes are sufficient to induce benign squamous lesions. The most studied model is the two-stage skin carcinogenesis protocol where initiation occurs by a single application of a carcinogen like 7, 12-dimethylbenz[a]-anthracene (DMBA) followed by repeated application of a tumor promoting agent typically 12-O-tetradecanoylphorbol-13acetate (TPA) (Abel et al. 2009). The majority of skin cancers initiated with DMBA contain Hras1 activating transversion mutations (A to T) at the second base of codon 61 (Balmain et al. 1984). Accordingly, mice deficient for the Hras1 gene develop much fewer papillomas in the DMBA/TPA tumor model (Ise et al. 2000). Similarly, retroviral introduction of the v-rasHa oncogene in primary mouse keratinocytes reproduces the phenotypic alterations observed in DMBA-initiated cells (Roop et al. 1986). These observations point to Ras signaling as a critical element in epidermal neoplasia. GTP-bound Ras or constitutively active mutant Ras leads to persistent activation of downstream effectors. The most intensively studied is the protein serine/threonine kinase RAF which relocates to the membrane upon Rasmediated autophosphorylation and activation. Raf then phosphorylates and activates mitogen-activated protein kinase kinase 1 and 2 (MEK1 and MEK2). In turn those kinases are capable of phosphorylating and activating the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (extracellular signal-regulated kinases 1 and 2). ERK1 and 2 have multiple cytoplasmic and nuclear targets among those cytoskeletal proteins and transcription factors (Kyriakis and Avruch 2001). Mice deficient for ERK1 show resistance to DMBA/TPA skin carcinogenesis by developing fewer and smaller tumors compared to their WT controls (Bourcier et al. 2006). Using the same DMBA/TPA model, mice deficient for epidermal MEK1 develop fewer tumors than WT animals, while tumor incidence and burden was unchanged in MEK2 deficient animals (Scholl et al. 2009b). It therefore appears that the role of MEK1 during skin carcinogenesis is isoform specific. This result contrasts with another study showing redundancy among MEK1 and MEK2 and gene dosage strongly influencing Ras induced epidermal hyperplasia (Scholl et al. 2009a).

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While the contribution of Raf to carcinogenesis through the activation of ERK is well documented, a recent study shows that Raf can also contribute to skin tumorigenesis through the inhibition of the RhoGTPase target Rok-a (Ehrenreiter et al. 2009). Although those studies provide considerable evidence that the Raf-MEKERK pathway is a critical mediator of Ras-induced epidermal carcinogenesis, studies have also demonstrated that Ras activates other effectors to promote tumorigenesis. Ras can interact directly with the catalytic domain (p110 subunit) of type I phosphatidylinositol 3-kinases (PI3Ks) leading to activation of the lipid kinase as a result of its translocation to the membrane and conformational changes. Among the Ras isoforms, HRAS was described as being the most potent activator of PI3K in comparison to KRAS (Yan et al. 1998). Introducing a missense mutation in the sequence of p110a (the mutant being still active but cannot interact with Ras), Gupta and colleagues were able to demonstrate a drastic reduction in tumor formation in mutant mice subjected to DMBA-TPA induced carcinogenesis (Gupta et al. 2007). A third effector pathway comprises the Ral (RAS-Like) small GTPasespecific GEFs members. These exchange factors activate Ral, which can stimulate phospholipase D. Among these exchange factors, deletion of RalGDS (RAL guanosine nucleotide dissociation stimulator) reduces tumor incidence in a chemically induced skin carcinogenesis study (Gonzalez-Garcia et al. 2005). A fourth effector pathway consists of the GEF Tiam1 (T-cell lymphoma invasion and metastasis 1) that activates the Rac small GTPase. Tiam1-deficient mice are resistant to the development of Ras-induced skin tumors initiated with 7, 12-dimethylbenzanthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate but a greater proportion progressed to malignancy, suggesting that Tiam1 deficiency promotes malignant conversion (Malliri et al. 2002). RAS-GTP can also bind directly to phospholipase C epsilon (PLCe) (Kelley et al. 2001). PLC catalyzes the hydrolysis of phosphatidylinositol 4, 5-bisphosphate into two important second messengers, diacylglycerol (DAG), and inositol 1, 4, 5-trisphosphate (IP3). DAG binds to a number of target proteins, among those, protein kinase C (PKC) isozymes (their respective function will be reviewed in more detail in the next section), and regulates their activities. IP3 opens calcium channels on the surface of intracellular stores, increasing cytosolic free calcium concentration. PLCe−/−mice showed a delayed onset and markedly reduced incidence of skin carcinoma after DMBA/TPA (Bai et al. 2004). The same group also demonstrated that TPA induced cutaneous response is much reduced in PLCe−/−mice while the in vivo proliferative response of keratinocytes was not affected. This observation points toward a critical role of PLCe during tumor promotion and progression. While activating ras mutations are present in some SCC, activation of the Ras pathway is more common as 75% of cutaneous SCC contain a GTP-bound active Ras (Dajee et al. 2003). To produce this state of activation, Ras genes can be amplified, or upstream activators can be activated or amplified. Interestingly overexpression of RASGRP1 in basal keratinocytes leads to spontaneous skin tumor formation (in the absence of DMBA mediated Hras1 mutation) associated with increased GTP bound RAS levels (Oki-Idouchi and Lorenzo 2007). The elevation of RASGRP expression in human SCC has not been documented. However, the most

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studied and functionally implicated upstream activator of Ras is the epidermal growth factor receptor (EGFR). Numerous studies have viewed the EGFR as a regulator of epithelial cell growth and a mediator of enhanced proliferation in tumors by virtue of activating mutations, amplification or transcription. In fact, the EGFR is one of the most frequently altered proteins in cancer (Harari 2004). EGFR is constitutively activated in epidermal papillomas and frequently amplified in SCC (Kiguchi et al. 1998). Furthermore overexpression of TGFa by transgenic targeting to mouse epidermis followed by topical promotion by TPA or wounding is sufficient to produce squamous cell papillomas on mouse skin (Vassar et al. 1992; Dominey et al. 1993) in the absence of Ras mutations. Keratinocytes express four members of the EGFR ligand family (TGFa, amphiregulin, betacellulin and HB-EGF) (Glick et al. 1991; Dlugosz et al. 1997; Dlugosz et al. 1995), and the expression of all four ligands is increased in neoplastic keratinocytes. These ligands stimulate the EGFR therefore creating an autocrine growth loop whereby oncogenic ras transformed cells synthesize growth factor to which they then respond. Genetic deletion of the EGFR in mice results in early postnatal death and hypoplastic epidermis with low BrdU labeling index (Threadgill et al. 1995), suggesting this receptor is a positive growth regulator for normal epidermis. The EGFR null mouse on a CD1 background survives the gestational period and several days of postnatal life. Cultured keratinocytes and skin grafts onto nude mouse hosts have been used to catalogue the consequences of EGFR ablation in skin (Threadgill et al. 1995; Dlugosz et al. 1997; Hansen et al. 1997). After transduction with an oncogenic ras retrovirus, EGFR null keratinocytes are capable of forming squamous papillomas upon in vivo grafting, but the tumors are very small. Unexpectedly, the proliferating pool of cells from these tumors is as large as that of similarly treated and grafted control CD1 keratinocytes. However, in the absence of the EGFR, proliferating cells migrate prematurely from the basal cell compartment into the suprabasal cell pool, where they undergo growth arrest and terminal differentiation. This suggests that in tumors, alternate pathways exist to stimulate proliferation in the absence of the EGFR, and a previously unrecognized function of the EGFR is to maintain the proliferative pool of tumor cell in the basal cell compartment (Hansen et al. 2000). Wound closure is delayed in grafts of EGFR null skin (Repertinger et al. 2004). However, the number of proliferating keratinocytes in the healing wound is very high, particularly after a slight delay. This emphasizes the existence of alternative pathways for stimulation of keratinocyte proliferation. Other unexpected findings in the wound experiments are an increase in neutrophilic infiltration and a reduction in angiogenesis in EGFR null skin wounds, both qualities that could influence tumor development. Pioneering studies in human primary keratinocytes treated with EGFR inhibitors have revealed that EGFR blockade (and presumably genetically mediated deletion) alters chemokine/cytokine expression by keratinocytes in an inflammatory environment (Mascia et al. 2003; Pastore et al. 2005, 2008; Pivarcsi et al. 2007). These observations have functional relevance to the observation made in the clinic where patients treated with EGFR inhibitors develop severe skin rashes. Whether that altered chemokine/cytokine production upon EGFR blockade takes place in the tumor microenvironment remains to be determined, but such

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Fig. 7.1 Upstream activators and downstream effectors of ras-mediated carcinogenesis in mouse skin.

modulation of the immune/inflammatory response could be a part of the beneficial therapeutic response to EGFR inhibition. Finally, defining alternative pathways for growth stimulation in the absence of EGFR is of great interest since EGFR inhibitors are now used in the clinical treatment of many cancers (Ciardiello and Tortora 2008). Figure 7.1 depicts the pathways contributing to ras-induced skin carcinogenesis as they are presented in that section.

7.3 Role of Individual Protein Kinase C Isoforms in Mouse Skin Carcinogenesis The protein kinase C (PKC) family of calcium and/or lipid-activated serine-threonine kinases function downstream of nearly all membrane-associated signal transduction pathways. The seminal discovery of PKC as the major intracellular receptor for the tumor promoting phorbol esters, suggested a critical role of PKCs in tumor formation (Castagna et al. 1982; Leach et al. 1983). A number of studies have indicated that PKC isoforms contribute to the regulation of skin homeostasis, and alterations in PKC signaling are fundamental to the pathogenesis of cutaneous diseases, including hyperproliferative, inflammatory and neoplastic lesions (for review Breitkreutz et al. 2007; Griner and Kazanietz 2007). Six different PKC isozymes are expressed in mouse and human keratinocytes; they are broadly classified by their activation characteristics. The conventional PKC isozyme (PKCa) is activated by calcium and diacylglycerol (DAG) or phorbol ester, whereas the novel isozymes (deh) and atypical isozymes (zm) are calcium independent but

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a ctivated by distinct lipids. Studying the contribution of PKC to tumorigenesis is complicated by the fact that each individual PKC has a distinct role in that process and that role might be cell-type dependent within the different cell populations present in the tumor microenvironment. An additional level of complexity is added by the fact that the DAG signal can be transduced by non-PKC-phorbol ester effectors with some being implicated in cancer progression (Griner and Kazanietz 2007). The absence of PKC isoform specific agonists or antagonists has hindered the determination of individual isoform function. Several laboratories have addressed the contribution of PKC isoforms to skin carcinogenesis using genetically modified mouse models demonstrating sometimes opposite effects for different isozymes (for review Breitkreutz et al. 2007). Earlier studies in our models had indicated that oncogenic Ras increases the activity of PKCa, and this activity was an important factor in the altered expression of differentiation markers detected in papillomas (Dlugosz et al. 1994). In order to study these relationships further, we targeted PKCa to the epidermis and hair follicles of transgenic mice with a keratin 5 promoter cassette (K5-PKCa mice) (Cataisson et al. 2003). In the absence of stimulation, the skin or cultured keratinocytes from multiple transgenic lines was normal. However, when stimulated by TPA at doses too low to significantly activate endogenous PKC, a severe intraepidermal neutrophilic inflammation was evoked, and the epidermis and upper hair follicles were disrupted (Wang and Smart 1999; Cataisson et al. 2003). This phenotype was unique to PKCa skin targeted transgenics and not seen in mice targeting other PKC isoforms to the epidermis (Reddig et al. 2000; Reddig et al. 1999; Jansen et al. 2001). K5-PKCa mice and their wild-type littermates (WT) were compared for susceptibility to 100 mg DMBA initiation followed by 1 mg (1.6 nmol) TPA twice weekly for 20 weeks of promotion. By weeks 21, 58% of the K5-PKCa mice developed tumors (with an average tumor multiplicity of 4.6 tumors per tumor-bearing animal) while all WT mice were tumorfree (Cataisson et al. 2009). Sixty-eight percent of papillomas converted to carcinomas by week 50 reflecting the genetic predisposition for malignant skin tumor development on an FVB/N background (Hennings et al. 1993). Our results demonstrated that PKCa overexpression in basal keratinocytes and potentially the consequent acute inflammation sensitize mice to skin carcinogenesis using a limiting DMBA initiation-TPA promotion protocol. Previous studies have reported the sensitivity to tumor promotion of mice overexpressing PKCa in the epidermis on a C57/Bl6 background (Wang and Smart 1999) or FVB/N background (Jansen et al. 2001) under the control of keratin 5 or keratin 14 respectively. Both studies reported no differences for tumor susceptibility comparing transgenic mice with their wild-type counterpart. While those results appear contradictory to our study, they can be explained by the TPA regiment used for promotion. We have use a much lower dose (2 nmol TPA twice a week) while the study on FVB/N mice used 5 nmol TPA twice a week (Jansen et al. 2001) and 3 nmol TPA three times a week for the other study (Wang and Smart 1999), conditions that were favorable for tumor development in wildtype mice. Chemically induced skin carcinogenesis represents a classical inflammationassociated tumor model. Landmark studies have demonstrated the importance of proinflammatory signaling pathways in tumor promotion by showing that mice

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d eficient for TNFa or its receptors are resistant to DMBA/TPA-induced skin carcinogenesis (Moore et al. 1999; Arnott et al. 2004). The persistent hyperplasia and infiltration of inflammatory cells observed during tumor promotion in K5-PKCa mice suggest that those two phenomena could facilitate carcinogenesis. PKCa activation induces keratinocyte apoptosis via an AP-1 dependent pathway (Cataisson et al. 2003) and causes the intraepidermal recruitment of neutrophils through NF-kB mediated expression of CXCR2 ligands (Cataisson et al. 2006). Furthermore we have observed that PKCa overexpression in basal keratinocytes renders TNFa dispensable during TPA induced acute cutaneous inflammation in the K5-PKCa mouse model. Indeed, TNFa deficiency is associated with reduced PKCa activity and altered AP-1 gene regulation during tumor promotion in the epidermis suggesting that PKCa is a downstream target of TNFa (Arnott et al. 2002). Whether PKCa overexpression in basal keratinocytes would compensate for TNFa deficiency and restore susceptibility to tumor promotion remains an open question. Transgenic mice overexpressing PKCd in basal keratinocytes are resistant to skin carcinogenesis (Reddig et al. 1999) likely because of an increased apoptotic response during promotion. Studies from our laboratory have implicated several PKC isoforms as regulators of keratinocyte differentiation and, in particular, PKCd as a mediator of a mitochondrial death pathway associated with the terminal phases of that process (Li et al. 1999). Following the transduction of keratinocytes with oncogenic ras, PKCd is inactivated by src-kinase mediated tyrosine phosphorylation at tyrosine residues 64 and 565 (Denning et al. 1996; Joseloff et al. 2002), rendering initiated cells resistant to the differentiation-inducing affects of phorbol ester tumor promoters (Denning et al. 1993) while normal keratinocytes remain vulnerable, migrate prematurely into the suprabasal compartment and are eliminated from the tissue. Elimination of PKCd mediated cell death is a requirement for tumor formation in mouse skin carcinogenesis and a similar requirement appears to exist for human skin tumor development (D’Costa et al. 2006). These results strongly support a tumor suppressor function for PKCd in the skin. In contrast mice overexpressing PKCe when subjected to two-stage skin carcinogenesis had reduced papilloma incidence while progression to invasive and metastatic carcinoma was greatly increased (Reddig et al. 2000), with some carcinomas arising independently of precursor papillomas lesions. PKCe can phosphorylate the serine residue 727 of the Signal Transducers and Activators of Transcription 3 (Stat3) and increase its activity (Aziz et al. 2007), while independent studies have established the critical contribution of Stat3 during progression of skin carcinogenesis (Chan et al. 2004). These data highlight the critical function of PKC during tumor promotion and progression and their sometime antagonistic or opposite functions depending on the target organ, for example the tumor suppressive activity of PKCa in the adenomatous polyposis coli (Apc) mouse model (Oster and Leitges 2006). The complexity and intricacy of the PKC signal is certainly contributing to the disappointing efficacy of PKC inhibitors in the clinic (Mackay and Twelves 2007). However, a better understanding of PKC downstream effector targets might offer new therapeutic venues.

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7.4 SCC and the NF-kB Connection The transcription factor NF-kB encompasses a family of five members: p50/p105, p52/p100, c-Rel, p65/RelA and Rel-B. NF-kB homo- and heterodimers are usually located in the cytoplasm because of their association with inhibitor of kB (IkB) proteins. Activation of the IkB kinase (IKK) complex (IKKa, IKKb and IKKg subunits) causes the phosphorylation of IkB at Ser32 and Ser36, targeting for ubiquitilation, proteasomal degradation and release of the association with NF-kB. This exposes the nuclear localization signal of the NF-kB dimers and allows for their translocation to the nucleus and transactivation of gene expression. Because NF-kB has both antiapoptotic activities and can promote cell cycle progression, its constitutive activation is advantageous to cancer cells. Indeed the loss of the tumor suppressor CYLD (a deubiquitinating enzyme that limits NF-kB signaling) causes multiple skin tumors in patients suffering from familial cylindromatosis (Bignell et al. 2000). Mice deficient for Cyld are highly susceptible to chemically induced skin carcinogenesis. In keratinocytes, CYLD normally limits the nuclear interaction of Bcl-3 with p50/52 and therefore the induction of cyclinD1 by TPA (Massoumi et al. 2006). Studies of human SCC and their respective mouse models indicate that NF-kB as well as its transcriptional targets are frequently activated (Budunova et al. 1999; Loercher et al. 2004), and this activation promotes carcinogenesis (Martin-Oliva et al. 2004; Loercher et al. 2004; Loukinova et al. 2000). Recent studies have demonstrated that pro-inflammatory factors (like cytokines and chemokines) are among the protumorigenic targets of Ras signaling (Sparmann and Bar-Sagi 2004; Ancrile et al. 2007). Our own studies in mouse keratinocytes have demonstrated that transduction of keratinocytes with oncogenic ras induced CXCR2 ligands in the absence of TPA stimulation. Ras induction of CXCR2 ligands was mediated by autocrine activation of EGFR and NF-kB and potentiated by PKCa. Furthermore, the receptor CXCR2 itself is expressed by keratinocytes suggesting a potential autocrine activity. Indeed, oncogenic ras transduced CXCR2 null keratinocytes formed only small skin tumors in orthotopic skin grafts to CXCR2 intact hosts, whereas transformed wildtype keratinocytes produced large tumors. In vitro, CXCR2 was essential for CXCR2 ligand-stimulated migration of ras-keratinocytes and for ligand activation of ERK and Akt pathways. Both migration and activation of ERK and Akt were restored by CXCR2 reconstitution of CXCR2 null keratinocytes. Thus, activation of CXCR2 on ras-transformed keratinocytes has both promigratory and protumorigenic functions. The upregulation of CXCR2 ligands after initiation by oncogenic ras and promotion with TPA in the mouse skin model provides a mechanism to stimulate migration by both autocrine and paracrine pathways (Cataisson et al. 2006, 2009). Our data demonstrate that autocrine CXCR2 signaling contributes to keratinocyte tumorigenic behavior but paracrine activities of CXCR2 ligands have been proposed to have tumor promoting effects as well through the maintenance of inflammation in the tumor microenvironment as well as directly causing neovascularization (Loukinova et al. 2000; Sparmann and Bar-Sagi 2004; Dhawan and Richmond 2002; Waugh and Wilson 2008; Vandercappellen et al. 2008). Therefore it appears that ras alters the

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EGFR v-rasHa RASHa

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Fig. 7.2 Oncogenic ras alters the keratinocyte phenotype via a series of feedback autocrine and paracrine signals.

p henotype of murine keratinocytes through a series of autocrine/paracrine loops (Fig. 7.2). Furthermore, recent studies have reported that CXCL1 paracrine activity was causing senescence in tumor-associated fibroblasts (Yang et al. 2006; Acosta et al. 2008) facilitating tumor growth. Finally, CXCR1 and CXCR2 genes were expressed in the majority of head and neck SCC cells derived from primary tumors or matching lymph node metastases (Muller et al. 2006). Studies addressing the role of individual chemokine receptors in the chemically induced skin tumor model are lacking, only one elegant study is indirectly suggesting a facilitating role for a subset of CC chemokines acting through CCR 1–5. Nibbs and associates have reported that skin targeted overexpression of the atypical chemokine receptor D6 (a nonsignaling receptor that can sequester chemokines that normally bind CCR1–5) can confer protection from tumor formation, and conversely D6 deficient mice show increased sensitivity to tumor formation (Nibbs et al. 2007). Because individual chemokine receptor expression is not restricted to cells from the hematopoietic lineage, defining their function during carcinogenesis will require the use of mouse models with tissue targeted deletion. Paradoxically, NF-kB blockade in the skin (through transgenic expression of non degradable dominant negative form of IkBa (IaBaDN), or keratinocyte specific deletion of IKKb causes massive inflammation and hyperplasia (van Hogerlinden et al. 1999; Pasparakis et al. 2002) that leads to SCC development in the K5-IkBaDN mouse model. Of note, SCC that form in this model do not harbor ras mutations suggesting that persistent inflammation can compensate for Ras signaling when NF-kB is blocked (van Hogerlinden et al. 2002). Interestingly the

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skin phenotype is rescued by ablation of TNFR1 on non immune cells (Pasparakis et al. 2002; Lind et al. 2004). Inhibition of NF-kB is required together with oncogenic Ras to transform human keratinocytes to SCC in a grafting model (Dajee et al. 2003). In this model of human SCC, blockade of NF-kB allows for TNFR1 and JNK mediated upregulation of CDK4 to overcome Ras induced growth arrest (Zhang et al. 2005). Caution should be applied when extrapolating those observations made in experimental models to human SCC as clinical evidence for a role of NF-kB blockade in the development of SCC is still missing. Nevertheless, those studies suggest that upon certain conditions NF-kB blockade can promote SCC development and therefore more research is needed to understand that duality of NF-kB in the skin (Pikarsky and Ben-Neriah 2006; Sur et al. 2008). Finally, in the DMBA/TPA chemical carcinogenesis model, reduction in IKKa expression promotes the development of papillomas and carcinomas (Park et al. 2007) while overexpression of IKKa confers protection (Liu et al. 2006). Epidermal targeted deletion of IKKa has allowed for the molecular dissection of the tumor suppressor activity of IKKa. IKKa counteracts the EGFR autocrine loop initiated by oncogenic ras in keratinocytes by physically repressing the transcription of EGFR ligands and ADAMS responsible for their shedding (Liu et al. 2008).

7.5 Conclusions Nonmelanoma skin cancer incidence has been escalating in the last decades making this disease a major public health issue. While SCC has a higher propensity for invasion and metastasis, therapeutic strategies are limited and patients have to resort to surgery. Understanding genetic and epigenetic changes associated with the pathogenesis of SCC will help define new targets for treatment. The mouse model of chemically induced skin carcinogenesis has been instrumental in helping define those changes. While the EGFR-Ras-Raf-MEK-ERK pathways plays a central role, the mouse models have clearly demonstrated that others effectors are critical for the maintenance of SCC. While more inhibitors are moving to the clinic, results obtained from the mouse model suggest that a successful therapeutic strategy might require concurrent inhibition of multiple effectors in SCC.

References Abel EL, Angel JM, Kiguchi K, DiGiovanni J (2009) Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications. Nat Protoc 4:1350–1362 Acosta JC, O’Loghlen A, Banito A et al. (2008) Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell 133:1006–1018 Ancrile B, Lim KH, Counter CM (2007) Oncogenic Ras-induced secretion of IL6 is required for tumorigenesis. Genes Dev 21:1714–1719 Aoki Y, Niihori T, Kawame H, Kurosawa K et al. (2005) Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat Genet 37:1038–1040

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Chapter 8

Protein Kinase C and the Development of Squamous Cell Carcinoma Mitchell F. Denning

Abstract An important role for protein kinase C (PKC) signaling in squamous cell carcinoma (SCC) of the skin has been recognized since the early 1980s, when PKC was discovered to be the major receptor for phorbol ester tumor promoters commonly used in mouse skin chemical carcinogenesis studies. Since then, we have gained a tremendous understanding of the unique roles for different PKC isoforms in keratinocyte, KC, proliferation, differentiation, and apoptosis, as well as how PKC regulation and signaling integrate into the etiology of chemically induced SCC skin. In addition, the role of PKC in ultraviolet (UV)-induced cutaneous SCC is becoming appreciated. This is a significant development as human skin cancers are very common and are caused primarily by exposure to UV radiation. This chapter will summarize our current understanding of PKC function and regulation in normal KCs, as well as the etiology of both chemical and UV-induced cutaneous SCCs.

8.1 Protein Kinase C Signaling in Normal Keratinocytes Significant insight into the function and signaling of protein kinase C (PKC) isoforms in cutaneous squamous cell carcinoma (SCC) has in fact come from studies of normal KCs. Treatment of mouse skin with the general PKC agonist 12-O-tetradeconylphorbol-13-acetate (TPA) results in a complex and massive transient hyperplasia. Both increased DNA synthesis and increased differentiation accompany this TPA-induced hyperplastic response (Astrup and Iversen 1983; Aldaz et al. 1985). In culture, TPA is primarily a potent differentiation stimulus for both mouse and human KCs, although increased DNA synthesis does occur with delayed kinetics (Yuspa et al. 1976). Multiple PKC isoforms are responsible for the differen tiation response of KCs to TPA (Prowse et al. 1999; Brugarolas et al. 1995; Huitfeldt et al. 1991; Efimova and Eckert 2000; Deucher et al. 2002; Tibudan et al. 2002). M.F. Denning (*) Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60153, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_8, © Springer Science+Business Media, LLC 2011

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TPA predominantly induces a differentiation response of cultured KCs, as compared with the hyperplastic response in vivo, possibly due to the lack of immune and other systemic/stromal components in vitro. For example, TPA induces extensive release of proinflammatory cytokines, such as tumor necrosis factor a (TNFa) GM-CSF, G-CSF, and MIP-2, and these have both direct and indirect (i.e. recruiting inflammatory cells) effects on epidermal KCs (Moore et al. 1999; Aziz et al. 2006; Wang and Smart 1999; Cataisson et al. 2003, 2006, 2009).

8.2 Keratinocyte PKC Isoform Heterogeneity KCs express five PKC isoforms (a, d, e, h, and z) which have distinct tissue distribution and functions within the epidermis (Dlugosz et al. 1992; Denning et al. 1995a; Denning 2004). PKCa is the only calcium-dependent PKC isoform in KC, and it is also activated by phorbol esters or diacylglycerol via its two C1 domains. PKCa is localized to the plasma membrane and cell–cell junctions in suprabasal, differentiating KC within the epidermis, and it accumulates to a high level in the granular layer (Tibudan et al. 2002; Denning 2004; Jerome-Morais et al. 2009; Cataisson et al. 2006; Jansen et al. 2001a). PKCd, PKCe, and PKCh are calciumindependent isoforms but are also activated by TPA/DAG. PKCd is expressed at high levels throughout the epidermis, while PKCh is localized exclusively to the granular layer, consistent with its role in terminal differentiation. PKCe is expressed throughout the mouse epidermis, but stronger staining is detected in the proliferative basal layer of human epidermis (Reddig et al. 2000; Denning 2004). This is consistent with the reduced PKCe levels during calcium-induced differentiation of human KCs (Yang et al. 2003). PKCz is the only atypical PKC isoform in KC, and thus lacks both calcium and TPA/DAG responsiveness. PKCz can be activated by a variety of lipid second messengers, however the role of PKCz signaling in normal KC function and SCC development is not clear. TPA-induced KC differentiation involves multiple PKC isoforms and is associated with enhanced granular layer differentiation, and suppressed spinous layer differentiation (Dlugosz and Yuspa 1993; Deucher et al. 2002; Efimova and Eckert 2000; Ueda et al. 1996a). PKCd and PKCh are consistently reported to induce differentiation marker expression, while PKCa and PKCe have been variably implicated in KC differentiation induction (Takahashi et al. 1998; Efimova and Eckert 2000; Ueda et al. 1996a; Deucher et al. 2002; Seo et al. 2004; Ohba et al. 1998; Cabodi et al. 2000; Denning et al. 1995a). Extracellular calcium is a potent inducer of KC differentiation and a calcium gradient exists in the epidermis which is postulated to be important for KC differentiation. Calcium-induced KC differentiation involves increased phosphatidylinositol turnover and elevation of cellular diacylglycerol (DAG) levels (Lee and Yuspa 1991). Multiple PKC isoforms become activated during calcium-induced KC differentiation, although these isoforms are likely to play redundant roles in epidermal homeostasis in vivo as individual PKC isoform knockout mice are not reported to have any alterations in epidermal

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d ifferentiation (Denning et al. 1995a; Chida et al. 2003; Leitges et al. 2001a, b; Lessmann et al. 2006; Miyamoto et al. 2002). Extracellular calcium induces KC differentiation in part by binding to a cell surface calcium-sensing receptor, CaR, and activating E-cadherin (Tu et al. 2001). E-cadherin engagement activates phosphatidylinositol-4-phosphate-5-kinase and phosphatidylinositol-3-kinase, promoting phosphatidylinositol turnover and leading to the activation of phospholipase C-g and generation of DAG and inositol triphosphate (Tu et al. 2008; Xie and Bikle 2007; Xie et al. 2009; Lee and Yuspa 1991). While DAG can directly activate multiple PKC isoforms in KCs (PKCa, d, e, h), the elevation of intracellular calcium due to inositol triphosphate-mediated release from intracellular stores is also required for KC differentiation (Li et al. 1995a, b). Notably, PKCa is the only calcium responsive PKC isoform in KCs, and is activated more rapidly than other PKC isoforms in response to phospholipase C-coupled signaling events (Denning et al. 1995a; Lenz et al. 2002).

8.3 PKC Effector Pathways The targets of PKC isoforms responsible for KC differentiation and growth arrest are not completely understood. PKCs are potent activators of multiple mitogenactivated protein kinase (MAPK) pathways, and ERK and p38 are linked to PKCmediated KC differentiation marker expression (Efimova et al. 2002; Seo et al. 2004). These MAPK pathways impinge upon differentiation genes via AP-1 and C/ EBPa transcription factors (Efimova et al. 2002; Balasubramanian et al. 2002). Calcium induces the expression of the AP-1 proteins FRA2, JUNB, and JUND in a PKC-dependent manner, and PKCa down-regulation with Bryostatin abolishes calcium-induced AP-1 complexes binding to DNA, implicating PKCa in calciuminduced AP-1 activation (Rutberg et al. 1996). PKCs are also activators of NF-kB, functioning as IkB kinase kinases (Ueda et al. 1996b; Schonwasser et al. 1998; Seo et al. 2004; Efimova et al. 2002, 2004). IkB kinase a and NF-kB play an essential role in KC differentiation and the associated growth arrest, suggesting that the PKC-mediated activation of NF-kB may also contribute to the prodifferentiation of PKC (Hu et al. 2001; Takeda et al. 1999; Seitz et al. 1998, 2000). Perhaps more relevant to carcinogenesis than differentiation marker expression is the induction of KC growth arrest by PKC activation. This is because well- differentiated SCCs typically express an array of squamous differentiation markers, but still have increased numbers of basal and suprabasal proliferating cells which are primarily responsible for tumor formation (Tennenbaum et al. 1993). Thus, differentiation marker expression per se is not a barrier to aberrant proliferation in neoplastic KCs. PKCa is membrane localized and activated in the first suprabasal layer in normal epidermis where irreversible growth arrest occurs (Denning 2004; Jerome-Morais et al. 2009) (Fig. 8.1). Using overexpression and shRNA approaches, PKCa has been shown to be both necessary and sufficient for normal KC differentiation growth arrest (Jerome-Morais et al. 2009). PKCa

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Fig. 8.1 Function of PKC isoforms in normal epidermis. PKCs are activated during KC differentiation by extracellular calcium via the plasma membrane calcium-sensing receptor (CaR) and E-cadherin, which is coupled to phospholipase C and results in the generation of diacylglycerol (DAG) and inositol triphosphate (IP3) second messengers. PKCs can also be activated directly by TPA. PKCd promotes KC differentiation marker expression via p38, AP-1 and C/EBPa. PKCh induces KC growth arrest via Fyn and the induction of cyclin-dependent kinase inhibitors p21 and p27. PKCa, which is preferentially activated by calcium, can promote KC growth arrest and differentiation, or hyperplasia via ERK and possibly cytokine release when overexpressed.

s ignaling, leading to KC growth arrest, involves PKC activation in response to calcium-induced activation of PLCg. PKCa phosphorylates S100C which triggers the redistribution into the nucleus with nucleolin to activate Sp-1 and activate the CDKN1A (p21) promoter (Sakaguchi et al. 2004, 2005). The induction of CDKN1A is required for PKCa-induced growth arrest (Jerome-Morais et al. 2009). PKCd and PKCh have also been implicated in phorbol ester-induced growth arrest in cultured KC (Ohba et al. 1998; Ishino et al. 1998). PKCh, which is expressed exclusively in the granular layer of the epidermis, is also capable of inducing KC growth arrest. PKCh-induced growth arrest involves induction of the Src family kinase FYN, which can induce CDKN1A and CDKN1B (p27) resulting in a G1 arrest (Cabodi et al. 2000). PKCh has also been demonstrated to bind directly to the cyclin E/CDK2/CDKN1A complex and inhibit CDK2 activity in KCs (Kashiwagi et al. 2000). PKCh null mice had no alterations in baseline proliferation, but did have prolonged epidermal hyperplasia in response to TPA, indicating that PKCh plays a role in resolution of the hyperplastic response to TPA (Chida et al. 2003).

8.4 Chemical Carcinogenesis in Mouse Skin The classic two-stage mouse skin chemical carcinogenesis regimen, involving initiation with the carcinogen DMBA and promotion with the PKC agonist TPA has been an exceptionally powerful model for dissecting the molecular, cellular, and

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genetic mechanisms of SCC development. The critical mutagenic effect for DMBA initiation is activating mutation in the HRAS gene at codon 61 (Quintanilla et al. 1986; Roop et al. 1986). Hras activation is sufficient to permit the selective outgrowth of “initiated” KCs by chronic TPA treatment, which develop initially into benign papillomas and can progress into invasive SCCs. Mouse skin chemical carcinogenesis has also focused the attention of cancer researchers on the role of PKC in SCC etiology as a result of the PKC agonist activity of TPA and other phorbol ester tumor promoters. Despite this focus, a comprehensive understanding of how TPA promotes squamous papillomas is still lacking. Acute PKC activation by TPA induces many immediate early genes involved in cell proliferation which are involved in SCC development, including Odc1, Cox2, Fos, and Myc (Huppi et al. 1994; Gilmour et al. 1987; Aziz et al. 2006; Skouv et al. 1987; Wang et al. 2001; Kennard et al. 1995). PKC activation also induces the expression of proinflammatory cytokines, such as TNFa, supporting the link between inflammation and cancer. Indeed, mice lacking TNFa or its receptors are resistant to chemical carcinogenesis (Moore et al. 1999; Arnott et al. 2004). Further complicating the tumor promoting effects of TPA is the existence of multiple PKC isoforms with unique biological activities. Thus, one reason why the mechanisms of tumor promotion by TPA remains elusive is largely due to multiple PKC isoforms with activities capable of both promoting (hyperplasia, inflammation) and inhibiting (differentiation, apoptosis) SCC development (Fig. 8.2). Transgenic mice, overexpressing PKCa or PKCe in their epidermis, have dramatically enhanced hyperplastic response to TPA, which is accompanied by a massive neutrophil infiltration (Jansen et al. 2001a; Wang and Smart 1999; Cataisson et al. 2003, 2005, 2006). This infiltration induces extensive KC cell death in the interfollicular Chemical Carcinogenesis DMBA PKC d

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Fig. 8.2 Function of PKC isoforms in chemical carcinogenesis. Initiation with DMBA induces an activating Hras mutation, which stimulates the production of autocrine epidermal growth factor receptor (EGFR) ligands. Activation of the EGFR suppresses differentiation and activates Src family kinases (SRC/FYN), which can tyrosine phosphorylate and inhibit the pro-apoptotic PKCd. Activation of PKCa results in NF-kB activation and production of cytokines which lead to inflammation and subsequent regenerative hyperplasia. PKCa-induced CXCR2 ligands promote KC migration. Activation of PKCe by TPA can lead to TNFa production, resulting in massive inflammation, and STAT3, providing a potent proliferation and survival signal. PKCh activation is involved in limiting the extent of TPA-induced hyperplasia.

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e pidermis, and the ensuing regenerative hyperplasia, predominately from the hair follicles, is postulated to be a contributing factor to SCC development. However, depleting mice of neutrophils did not reduce the TPA-induced hyperplasia in K5/ PKCa mice, indicating that neutrophil infiltration was not responsible for the hyperplastic response to TPA (Cataisson et al. 2009). After the acute activation of PKC by TPA treatment, PKC isoforms become down-regulated and are transiently lost from the epidermis (Hansen et al. 1990). There is evidence that this PKC down-regulation, not the acute activation of PKC, is critical for tumor promotion (Hansen et al. 1990; Kischel et al. 1989; Mills et al. 1992). This is consistent with the growth inhibitory and prodifferentiation activities of multiple KC PKC isoforms. Because chronic, repeated PKC activation/down-regulation by TPA in the absence of DMBA initiation is not sufficient for tumor formation in naïve skin, alterations in PKC signaling elicited by Ras activation need to be considered to explain the selective growth advantage of initiated KCs harboring activating Hras mutations. Hras-transformed KCs are resistant to the differentiating-inducing effects of both calcium and TPA, and in fact TPA stimulates DNA synthesis in KCs expressing active Hras (Dlugosz and Yuspa 1991; Yuspa et al. 1985; Hennings et al. 1987). Ras activation induces differential alterations in PKC isoform activation which may explain the altered response of Ras-transformed KCs to TPA (Dlugosz et al. 1994; Denning et al. 1993). In cultured mouse KCs, Hras activation induces an inhibitory tyrosine phosphorylation on PKCd (Denning et al. 1993, 1996). Since PKCd is a prodifferentiation and proapoptotic PKC isoform, this inhibitory phosphorylation would shift the response of KCs treated with TPA towards proliferation and survival. Members of the Src tyrosine kinase family are responsible for this inhibitory phosphorylation, and can be activated by autocrine EGFR ligands secreted by Ras-transformed KC (Denning et al. 1996, 2000). PKCd can be tyrosine phosphorylated on multiple sites by Src family kinases, but tyrosines 64 and 565 are required to block differentiation in Ras-transformed KCs, implicating these residues as inhibitory phosphorylation sites (Szallasi et al. 1995; Joseloff et al. 2002). PKCd has a “C2-like” domain which, although is not calcium-responsive, is able to bind phosphotyrosine, consistent with the interactions between PKCd and Src family kinases (Sano et al. 2005a). Note that the effects of tyrosine phosphorylation on PKCd are cell type and context dependent, and other tyrosine phosphorylation residues (Y311) are correlated with increased PKCd activity (Zhu et al. 2008).

8.4.1 Role of PKCd In mouse skin, PKCd protein levels and enzymatic activity are markedly reduced in mouse squamous papillomas (Reddig et al. 1999; Wheeler et al. 2002). This loss of PKCd protein may be related to tyrosine phosphorylation since phosporylation of PKCd by SRC can also trigger PKCd degradation (Zang et al. 1997; Blake et al. 1999).

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Human KCs transformed with active HRAS lose expression of PKCd at both the protein and mRNA level, suggesting that transcriptional repression may be an additional mechanism to suppress PKCd signaling (Geiges et al. 1995; D’Costa et al. 2006). Thus, there are at least three general mechanisms that may inhibit PKCd in Ras-transformed mouse KCs: (1) tyrosine phosphorylation to inhibit its enzymatic activity, (2) degradation of the protein, and (3) transcriptional repression of the PRKCD gene. Transgenic mice overexpressing PKCd in the epidermis via the keratin 14 promoter are highly resistant to chemical carcinogenesis, resulting in lower papilloma and carcinoma incidence (Reddig et al. 1999; Jansen et al. 2001a; Wheeler et al. 2002; Aziz et al. 2006). The mechanism of tumor suppression by PKCd is independent of Odc1 induction since Odc1 is induced even higher in PKCd transgenic mice (Wheeler et al. 2002). Other potential mechanisms for tumor suppression by PKCd include enhanced apoptosis, enhanced differentiation (hyperkeratosis), and decreased proliferation as measured by PCNA expression (Aziz et al. 2006; Reddig et al. 1999). Multiple TPA-induced signaling events were decreased in PKCd transgenic mice, including Akt, p38, COX2, and the expression of the proinflammatory cytokines TNFa, GM-CSF, and G-CSF (Urtreger et al. 2005). Thus, the loss of PKCd in the chemical carcinogenesis model appears to be important for papillomas and SCC development, supporting its role as a tumor suppressor for SCC development.

8.4.2 Role of PKCa Ras activation elevates the activation of PKCa, and this is linked to the autocrine production of EGFR ligands and elevation of cellular DAG levels (Dlugosz et al. 1994; Lee et al. 1992; Cheng et al. 1993). Ras activation alters the KC differentiation program toward a simple epithelium due to EGFR ligand production, especially TGFa, and PKCa activity has been linked to this altered differentiation program (Cheng et al. 1993; Dlugosz et al. 1994). Three independent groups have evaluated PKCa transgenic mice for sensitivity to skin chemical carcinogenesis (Wang and Smart 1999; Jansen et al. 2001a; Cataisson et al. 2009). Two studies found no effect of PKCa over-expression using standard tumor promoting dose of TPA (3 and 5 nmol twice/week) (Wang and Smart 1999; Jansen et al. 2001a), while the other study used a lower subcarcinogenic dose of TPA and found enhanced sensitivity in the PKCa transgenic mice (Cataisson et al. 2009). All groups found that PKCa overexpression resulted in a massive TPA-induced inflammatory cytokine release, neutrophil infiltration, and hyperplasia. Surprisingly neutrophil infiltration was not required for epidermal hyperplasia, but the production and release of CXCR2 ligands was linked to enhanced KC migration and activation of ERK and Akt pathways (Cataisson et al. 2009). The PKCa-mediated CXCR2 ligand production was necessary for neutrophil infiltration and required NF-kB activation (Cataisson et al. 2003, 2005, 2006,

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2009). TPA treatment of PKCa transgenic KCs also induced enhanced apoptosis and inhibited DNA synthesis, and the enhanced apoptotic effect required AP-1 (Cataisson et al. 2003). Chemical carcinogenesis experiments in PKCa null mice found enhanced tumor incidence in the knockout mice, supporting a tumor suppressive role for PKCa, but contradicting the oncogenic effects of PKCa found in the low-dose TPA studies (Hara et al. 2005; Cataisson et al. 2009). The PKCa null mice had no differences in basal BrdU incorporation or inflammatory cytokine (TNFa, interleukin-1b) production, but did have reduced TPA-induced hyperplasia, wound healing hyperplasia, and EGFR ligand release. To summarize, PKCa plays multiple roles in KC proliferation, differentiation, migration, and cytokine release, and the overall role in SCC development is difficult to establish based on the current over-expression and loss-of-function studies. Additional loss-of-function studies and analysis of PKCa expression/activation in mouse papillomas and SCCs will be needed to clarify the function of PKCa in SCC etiology.

8.4.3 Role of PKCe The ability of PKCe overexpression to promote metastatic SCC in the chemical carcinogenesis model has been firmly established by Dr. Verma and colleagues (Verma et al. 2006). Transgenic mice expressing PKCe from the powerful keratin 14 promoter display spontaneous epidermal hyperplasia and enhanced carcinoma development despite reduced papilloma incidence in response to chemical carcinogenesis protocols (Reddig et al. 2000). Interestingly, the carcinomas that developed in PKCe transgenic mice were moderately differentiated, TPA-independent, and metastatic, which is quite rare for chemically induced mouse tumors (Reddig et al. 2000; Jansen et al. 2001a; Jansen et al. 2001b). Two mechanisms have been postulated for the oncogenic nature of PKCe. The first involves enhanced TPA-induced TNFa shedding, which is a key mediator of tumor promotion in mouse skin (Wheeler et al. 2003, 2004, 2005). TPA-treated PKCe mice also produce other proinflammatory cytokines, massive neutrophil infiltration, and destruction of the interfollicular epidermis (Li et al. 2005). The ensuing regenerative and prolonged TPA-induced hyperplasia from hair follicles may explain the etiology of the aggressive carcinomas in these mice. The other mechanism involves STAT3 phosphorylation (Ser727) and activation (Aziz et al. 2007). STAT3 is a critical mediator of skin carcinogenesis, and is important for both the survival and proliferation of KCs in response to DNA damage (Chan et al. 2004; Sano et al. 2005b; Aziz et al. 2007). Despite these oncogenic effects of PKCe, the induction and/or activation of PKCe is not observed and is often decreased in chemically induced murine SCCs. Interestingly, however, as in PKCe transgenic mice, PKCe activation has also been implicated in invasion and motility of human head and neck SCC (HNSCCs), which are tobacco carcinogen-related (Pan et al. 2006). In HNSCCs, PKCe is also a mediator of Hepatocyte Growth Factor/c-MET receptor signal mediated activation

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of transcription factor Egr-1 and angiogenesis factor expression (Worden et al. 2005). Thus additional studies are needed to clarify the role of endogenous PKCe in murine and human SCC development (Reddig et al. 2000; Wheeler et al. 2005).

8.4.4 Role of PKCh The strong association between PKCh with KC squamous differentiation suggested that this PKC could function to limit the proliferation and transformation of KC by inducing growth arrest and differentiation (Kashiwagi et al. 2000, 2002; Koizumi et al. 1993; Cabodi et al. 2000). In fact, chemical carcinogenesis studies on PKCh null mice indicated that they were more sensitive to chemical carcinogenesis and had prolonged TPA-induced hyperplasia (Chida et al. 2003). PKCh is also activated by cholesterol sulfate, a cholesterol metabolite generated during the late stages of KC differentiation (Ikuta et al. 1994; Denning et al. 1995b). Interestingly, cholesterol sulfate induced granular layer KC differentiation and inhibited chemical skin carcinogenesis (Chida et al. 1995; Denning et al. 1995b). Thus, the granular layer expression and activation of PKCh may function to limit the proliferation of KCs in the suprabasal layers, as well as in response to TPA treatment.

8.5 UV Carcinogenesis There is strong epidemiological and experimental evidence that exposure to UV radiation, especially high cumulative exposure to UVB (280–320 nm), is a major etiological factor for the development of cutaneous SCC in humans (Vitasa et al. 1990; Soehnge et al. 1997). The molecular genetics of UV carcinogenesis differ from chemical skin carcinogenesis. Unlike chemical carcinogenesis, which results in almost universal activating Ha-Ras mutations during initiation, UV carcinogenesis frequently causes mutations in the TP53 tumor suppressor gene, and these are associated with reduced UV apoptosis and altered cell cycle kinetics in response to UV (Zhang et al. 2005; Ziegler et al. 1994). HRAS mutations are found in UV-induced SCCs at a lower frequency (<50%) than in chemically induced papillomas and SCCs, and occur later in the natural history of the disease (Pierceall et al. 1991; Spencer et al. 1995). In addition, UV-induced SCCs frequently progress through an actinic keratoses pre-malignant stage rather than benign papillomas. UV signal transduction pathways are very pleiotropic and have important similarities with TPA-induced signaling pathways. UV activates similar immediate early genes, including COX2, ODC1, and FOS and induces epidermal inflammation (Chen et al. 1999b; Einspahr et al. 2008). However, UV also causes both direct and indirect DNA damage, resulting in growth arrest and either repair, apoptosis, or mutation. UV activates many growth factor receptors in a growth factor-independent manner dependent on reactive oxygen species (Huang et al. 1996; Ashida et al. 2003; Aragane et al. 1998; Rosette and Karin 1996; Sheikh et al. 1998). UV also elevates DAG levels in

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ROS

Mcl-1

EGFR

PKC e

Caspase

Positive Feedback Loop

Ras STAT3

TNFα

PKC d

DNA Damage

p53

Proliferation Hyperplasia Survival

Apoptosis

Fig. 8.3 Function of PKC isoforms in UV carcinogenesis. UV radiation induces DNA damage, which can activate TP53 to induce apoptosis. UV-induced apoptosis also involves reduction of the anti-apoptotic MCL1, which leads to increase caspase activation and cleavage/activation of PKCd. PKCd phosphorylates and increases the degradation of MCL1. This sets up a positive feedback loop involving increased turn-over of MCL1, activation of caspases, and further activation of PKCd. However, UV-induced reactive oxygen species (ROS) activate multiple growth factor receptors, including the epidermal growth factor receptor (EGFR), which is coupled to Ras. UV can also activate HRAS by mutation. HRAS activation suppresses PKCd levels, and thus inhibits UV apoptosis. PKCd may also contribute to the activation of p53, and thus functions as a tumor suppressor. PKCe can promote UV carcinogenesis by activating STAT3, leading to enhanced proliferation/survival and inhibition of UV apoptosis, and by inducing TNFa which promotes hyperplasia in the epidermis.

KCs, but with delayed kinetics (24 h) (Punnonen and Yuspa 1992). Despite this growth factor receptor activation and DAG elevation by UV irradiation, the global activation of PKCs by UV is not well-established, and conflicting reports exits regarding the membrane translocation (i.e., activation) of PKC isoforms in KCs (Denning et al. 1998; Matsui et al. 1996; Chen et al. 1999a) (Fig. 8.3). UV radiation acts as both an initiator and promoter for skin carcinogenesis, and both effects must be considered in the context of PKC signaling. Although no mutations in PKC isoforms have been described in either chemically or UV induces SCC, PKC signaling and the expression of specific isoforms is altered in SCC. Human SCC cell lines have defective PKCa up-regulation and membrane translocation (i.e., activation) in response to extracellular calcium (Yang et al. 2003). Because PKCa has a role in KC growth arrest during differentiation, the lack of PKCa activation in human SCC lines may contribute to defect in growth arrest in the suprabasal compartment of SCCs (Tibudan et al. 2002; Jerome-Morais et al. 2009).

8.5.1 Role of PKCd in UV Response and Carcinogenesis UV irradiation of KCs does cause a robust activation of PKCd by triggering proteolysis in the hinge domain, resulting in a constitutively active catalytic fragment (Denning et al. 1998). The cleavage-dependent activation of PKCd is specific as

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other PKC isoforms were not cleaved in response to UV. Caspases, especially caspase-3, are responsible for the cleavage and activation of PKCd, and the catalytic fragment generated redistributes to both mitochondria and nucleus where it participates in apoptosis induction (Denning et al. 2002; Sitailo et al. 2004; DeVries-Seimon et al. 2007; DeVries et al. 2002; Li et al. 1999). Inhibition of PKCd cleavage by multiple approaches, including mutation of the caspase cleavage site, protects KCs from apoptosis, indicating that PKCd cleavage is necessary for UV-induced apoptosis (D’Costa and Denning 2005). In addition, ectopic expression of the PKCd catalytic fragment is sufficient for apoptosis induction (Denning et al. 2002; Sitailo et al. 2004). Multiple PKCd substrates involved in apoptosis have been identified, including the anti-apoptotic Bcl-2 family member MCL1, phospholipid scramblase-3 (PLSCR3), and DNA-dependent protein kinase (PRKDC) (Sitailo et al. 2006; Liu et al. 2003; Bharti et al. 1998). For example, the PKCd catalytic fragment phosphorylates and increases the turn-over of MCL1, thus sensitizing KC to further caspase activation. This establishes a positive feedback loop for PKCd cleavage/activation and mitochondrial apoptosis (Sitailo et al. 2006). UV-induced apoptosis requires loss of MCL1 from the cell, and thus PKCd activation may augment that process (Nijhawan et al. 2003). PKCd-induced apoptosis also involved production of death receptor ligands in some cell types (Gonzalez-Guerrico and Kazanietz 2005). Note that PKCd cleavage, activation, and apoptosis induction is not restricted to UV, but is involved in a wide range of apoptotic processes (Yoshida 2007; Humphries et al. 2006). PKCd levels are reduced in approximately 30% of human SCCs, and in all UV-induced mouse tumors examined (D’Costa et al. 2006; Aziz et al. 2006). Given the important role of PKCd in UV-apoptosis and the tumor suppressor function of apoptosis in UV carcinogenesis, the loss of PKCd expression in SCC cells could provide a selective survival advantage and thus promote expansion of KCs with low PKCd levels. This was tested by reexpressing full length PKCd in Ras-transformed HaCaT cells which have low PKCd levels as a result of transduction with active HRAS. PKCd reexpression induced elevated apoptosis and dramatically inhibited growth of these cells in nude mice (D’Costa et al. 2006). Thus, the loss of PKCd expression in Rastransformed human KC is necessary for their survival and tumorigenicity. The mechanism of PKCd loss/down-regulation in KCs is unclear, but Rasactivation is sufficient to reduce PKCd protein and RNA levels in the human transformed epidermal KC line HaCaT (Geiges et al. 1995; D’Costa et al. 2006). Note that the percentage of human SCCs with low PKCd is approximately equal to the percentage with Ras activation (30–50%). In addition, the majority of human SCCs with low PKCd were found to have elevated EGFR activation, suggesting activation of this growth factor receptor pathway which includes Ras (D’Costa et al. 2006). PKCd transgenic mice were not resistant to UV-induced tumor formation, suggesting that PKCd is not a tumor suppressor for UV skin carcinogenesis (Verma et al. 2005). However, the tumors that developed had low PKCd protein levels indicating that even the K14-driven PKCd transgene was down-regulated by some mechanism. The adjacent epidermis still expressed high levels of PKCd, suggesting that loss of PKCd expression was required for tumors to form in this UV model.

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Furthermore, PKCd levels are not rate limiting for UV apoptosis as overexpression of PKCd in human KCs does not enhance sensitivity to UV apoptosis (unpublished observation). Thus, the lack of protection from UV carcinogenesis by PKCd overexpression does not exclude a tumor suppressor function as KCs already express sufficient PKCd for an efficient apoptotic response to UV.

8.5.2 Role of Other PKCs in UV Response PKCe over-expression sensitizes epidermal KCs to the overall damaging effects of UV radiation, but inhibits apoptosis (sunburn cell formation), and enhances UV carcinogenesis (Wheeler et al. 2004). The elevated UV damage partially involved enhanced TNFa production, and was associated with enhanced hyperplasia. The molecular mechanism for these effects may involve activation of STAT3 by direct binding and activating phosphorylation on Ser727 (Aziz et al. 2007). The role of the prodifferentiation isoform PKCh in UV carcinogenesis has not been evaluated, however PKCh is able to block UV apoptosis in KCs (Matsumura et al. 2003). This effect may be due to the antiapoptotic effects of growth inhibition and KC differentiation (Chaturvedi et al. 1999). Further studies are needed to determine if the prodifferentiation or antiapoptotic activities of PKCh would favor tumor suppression or tumor formation, respectively.

8.6 Summary and Future Directions p53 mutations are common (>90%) in UV-induced SCCs, and are even detected in asymptomatic, sun-exposed skin and actinic keratoses (Ziegler et al. 1994). Several studies have found functional and regulatory interactions between p53 and PKCs. The majority of these studies support the activation of TP53 by multiple PKC isoforms, and this suggest that PKCs which are lost or inactivated in SCCs (i.e., PKCa, PKCd) may impede the activation or expression of TP53 (Abbas et al. 2004; Lee et al. 2006; Liu et al. 2007; Niwa et al. 2002; Ryer et al. 2005; Yamaguchi et al. 2007; Yoshida et al. 2006; Youmell et al. 1998). This would contribute to the tumor suppressor function of these PKC isoforms. In conclusion, specific tumor promoting or inhibiting functions for PKCs in the development of SCCs induced by either chemical carcinogens or UV radiation are becoming clear. The complexities of PKC isoform heterogeneity and the central integration of PKC into multiple major signaling pathways have complicated the elucidation of clear-cut functions in skin SCC etiology. However, potent inhibitory effects of PKC antagonists have been demonstrated in human HNSCC in vitro (Pan et al. 2006; Worden et al. 2005), suggesting that selective antagonists or agonists warrant investigation for the development of novel targeted therapeutics for the treatment of SCC.

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Chapter 9

The Transcription Factor AP-1 in Squamous Cell Carcinogenesis: Lessons from Mouse Models of Skin Carcinogenesis Jochen Hess and Peter Angel

Abstract A large body of experimental and clinical data provides strong evidence for an important role of the activator protein-1 (AP-1) in normal epithelial development and homeostasis, and that deregulated AP-1 activity contributes to pathophysiological processes, including neoplastic transformation of keratinocytes and malignant progression of tumor cells. AP-1 is a dimeric transcription factor mainly composed of members of the Jun and Fos families, which are key nuclear targets of several signal transduction pathways, particularly those involving the activation of mitogen-activated protein kinases. The analysis of genetically modified mouse models and cell lines derived thereof has provided important new insights how Jun and Fos proteins regulate cellular processes of keratinocyte differentiation, proliferation, survival and neoplastic transformation. Although some direct AP-1 target genes such as matrix metalloproteinases have been identified, further genome-wide and functional approaches will be required to comprehensively understand the causal connectivity between altered AP-1 function and squamous cell carcinogenesis.

9.1 The AP-1 Transcription Factor Much of our current knowledge on the characteristics of transcription factors comes from the discovery and study of the activator protein (AP-1). AP-1 describes an activity that controls both basal and inducible transcription of target genes containing AP-1-binding sites (consensus sequence TGAG/CTCA), also known as TPA-responsive elements (TREs) (Angel and Karin 1991). AP-1 is a dimeric transcription factor that is mainly composed of Jun (Jun, JunB, and JunD)

P. Angel (*) DKFZ-ZMBH Alliance, Division of Signal Transduction and Growth Control (A100), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_9, © Springer Science+Business Media, LLC 2011

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and Fos (Fos, FosB, Fosl1, and Fosl2) family members (Angel and Karin 1991). Each of these proteins is differentially expressed and regulated, and consequently each cell type has a complex mixture of AP-1 dimers with subtly different functions (Wagner 2001). The founding members of both families, Jun and Fos, were originally cloned as a result of efforts to identify the cellular counterparts of the viral oncogenes vJun and vFos that were described as the transforming activity of the avian sarcoma virus 17 (Bos et al. 1988) and FBJ/FBR murine sarcoma virus (Curran et al. 1983; Van Beveren et al. 1983), respectively. When the cellular counterparts of the viral oncoproteins were discovered, their overexpression or activation by well-known oncogenes, such as oncogenic Ras, was found to efficiently transform cells in culture or in mouse model systems (Eferl and Wagner 2003). According to their function in controlling gene transcription AP-1 subunits are composed of a region responsible for binding to a specific DNA recognition sequence (DNA binding domain) and a second region that is required for transcriptional activation (transactivation domain, TAD). The DNA binding domain, also known as the bZip region, can be subdivided in two evolutionary conserved, independently acting domains (Fig. 9.1): the basic domain (b), which is rich in basic amino acids and responsible for contacting the DNA, and the leucine zipper (Zip) region characterized by heptad repeats of leucines forming a coiled-coil structure that enables protein dimerization, which is a prerequisite for DNA binding mediated by the basic domain. The composition of the Zip domain is also responsible for the specificity and the stability of homo- and heterodimers that are formed by various Jun and Fos proteins (Eferl and Wagner 2003). While Jun proteins can form homodimers and heterodimers with Fos family members, Fos proteins can only heterodimerise with members of the Jun family. It is worthwhile to note that although Jun and Fos proteins share a high degree of structural homology, the individual AP-1 dimers exert obvious differences in their DNAbinding affinity and their capability to regulate gene transcription (Angel and Karin 1991; Shaulian and Karin 2002). This is, at least in part, due to the fact that individual Jun and Fos proteins have significantly different transactivation potential. Whereas, Jun, Fos, and FosB are considered strong transactivators, JunB, JunD, Fosl1 and Fosl2 exhibit only weak transactivation potential. Under some circumstances, the latter might even act as repressors of AP-1 activity by competing for binding to TRE motifs or by forming less active heterodimers with Jun, Fos, or FosB. A plethora of stimuli, including growth factors, cytokines, stress signals, bacterial and viral infection, as well as oncogenic signals, have been shown to enhance AP-1 function in a given cell (Fig. 9.2). Accordingly, AP-1 has been implicated in the control of a diverse range of cellular processes, such as cell proliferation, differentiation, apoptosis, and oncogenic transformation (Eferl and Wagner 2003; Hess et al. 2004). Regulation of the net AP-1 activity can be achieved through changes in transcription of genes encoding AP-1 subunits, control of their mRNA stability, post-translational modifications and turnover of pre-existing or newly synthesized AP-1 subunits, as well as specific interaction between Jun or Fos proteins and other

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Fig. 9.1 Schematic representation of cellular Jun and Fos proto-oncoproteins and their viral counterparts. Both proteins exhibit several domains, including the bZIP domain, transactivation domains, and docking sites for distinct kinases, such as Jnk and Erk. These kinases phosphorylate serine and threonine residues and thereby modulate the activity and stability of Jun and Fos proteins. Differences between cellular protooncoproteins and viral counterparts are indicated.

transcription factors or cofactors (Eferl and Wagner 2003; Hess et al. 2004). In this context, the mechanism of posttranslational control by phosphorylation has been most extensively studied with a focus on evolutionary conserved mitogen and cellular stress induced signal transduction pathways, such as the MAP kinase pathway (Figs. 9.1 and 9.2; Chang and Karin 2001).

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Fig. 9.2 The functional integration of AP-1 signaling. Numerous extra- and intracellular stimuli induce intracellular signaling cascades, including MAPK signaling, and thereby regulate expression and/or activity of the AP-1 transcription factor. AP-1 is a dimeric transcription factor that is mainly composed of Jun (Jun, JunB, and JunD) and Fos (Fos, FosB, Fosl1, and Fosl2) family members. Upon activation, AP-1 regulates the expression of downstream target genes implicated in the regulation of various cellular processes, regulating normal physiological as well as pathological conditions, including cancer.

9.2 AP-1 Function In vivo: Genetically Modified Mouse Models The establishment of mouse models with genetic disruption and/or transgenic overexpression as well as the availability of mutant cell lines derived from these animals represented a major breakthrough in our understanding of the regulatory functions of AP-1 subunits. The specific phenotypes of the individual mouse models, induced by defects in cells or tissues in which the subunits were particularly important or where their absence became first limiting, support the notion that AP-1 dimers exhibit unique and independent functions in vivo. As a general rule derived from

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all studies, the AP-1 subunits must be present in a complementary and coordinated manner in order to ensure proper development or physiology of the organism (Eferl and Wagner 2003; Hess et al. 2004). Here we will focus on the models that specifically addressed the role of Jun and Fos proteins in keratinocyte physiology and during epithelial malignancy.

9.2.1 AP-1 Function in Normal Skin Development and Homeostasis Conventional knockout approaches demonstrated that the expression of JunD, Fos and FosB is dispensable for normal embryogenesis and with regard to skin biology revealed no obvious defect in keratinocyte differentiation or function. In contrast, loss of Jun, JunB, Fosl1 and Fosl2 resulted in embryonic lethality or postnatal death, hampering functional studies of tissue function or homeostasis in adult mice (Eferl and Wagner 2003; Hess et al. 2004). To circumvent this problem, conditional tissue and cell type specific ablation of these AP-1 subunits using the Cre/loxP system has become the major experimental tool to study their function in physiological and pathological conditions in vivo. So far, unique functions in skin development and/or homeostasis was demonstrated for Jun and JunB. In order to address the function of Jun during skin development, mice were generated with conditional Jun ablation in basal keratinocytes (JunDep). JunDep animals displayed a normal epidermal architecture suggesting that Jun is not essential for skin development and other Jun family members may functionally compensate for its loss (Li et al. 2003; Zenz et al. 2003). However, JunDep mice exhibited a dramatic failure of eyelid fusion during embryogenesis, resulting in an open eye phenotype at birth, which is most likely a result of insufficient expression and function of the epidermal growth factor receptor (Egfr) (Li et al. 2003; Zenz et al. 2003). Therefore, it is not surprising that hypomorphic Egfr mutant strains or genetically modified animals with impaired Egfr signaling phenocopied the defect of JunDep mice (see references in Zenz et al. [2003]). In addition, Jnk-deficient mice showed an eyelid closure defect associated with markedly reduced Egfr function and loss of expression of the ligand Egf, supporting the assumption that regulation of Jun function by phosphorylation might be crucial for its function in keratinocytes (Weston et al. 2004). Reduced levels of Egfr and its ligand, heparin-binding EGF (Hbegf), also seem to be responsible for a severe proliferation and differentiation defect in keratinocytes lacking Jun function. However, this phenotype was only observed in primary keratinocyte cultures in vitro but not in normal skin homeostasis in vivo. This observation suggested that Jun determines the fate of keratinocyte physiology by an intrinsic genetic program, including transcription of genes implicated in Egfr signaling, which could be compensated by paracrine-acting factors derived from adjacent dermal fibroblasts. Indeed, impaired keratinocyte proliferation and differentiation could be rescued not only by the addition of Egfr ligands, but also by Fgf7 (originally described as KGF) and Csf2 (originally described as GM-CSF)

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(Zenz et al. 2003). Intriguingly, in an organotypic in vitro model system composed of fibroblasts and keratinocytes Jun and JunB antagonistically control the expression of Fgf7 and Csf2 in fibroblasts and thereby contribute to the tightly balanced cytokine-regulated mesenchymal-epidermal interaction in the regulation of keratinocyte proliferation and differentiation (Szabowski et al. 2000). Meanwhile, global gene expression profiling with control, Jun-deficient, and JunB-deficient fibroblasts highlighted differential expression of further cytokines and chemokines, such as Lcn2, Ptn, and Cxcl12, and support the concept that AP-1 activity regulates complex genetic programs of cell proliferation and differentiation in epithelial tissues in a cell autonomous manner and through induction of paracrine effectors (Fig. 9.3) (Florin et al. 2004, 2005). In line with this assumption, ablation of JunB in skin revealed pronounced epidermal hyperproliferation, disturbed differentiation, and prolonged inflammation upon wounding or treatment with the phorbol esters 12-O-tetradecanoylphorbol13-acetate (TPA) (Florin et al. 2006). Finally, an inducible epidermal deletion of Jun and JunB in adult mice resulted in a phenotype resembling histological and molecular hallmarks of psoriasis, including arthritis-like lesions (Zenz et al. 2005). Both phenotypes are closely linked with an aberrant expression of cytokines and chemoattractants, such as S100a8 and S100a9, that impact keratinocyte proliferation and differentiation, as well as activation and recruitment of immune cells implicated in acute and chronic inflammation (Gebhardt et al. 2006).

Fig. 9.3 AP-1−regulated processes and target genes in cancer. AP-1 is a central regulator of tumor-associated target genes during carcinogenesis and is critically involved in cellular processes, such as cell cycle regulation, cell invasion, cell signaling, cytokine and chemokine signaling, and inflammation.

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9.2.2 Regulation and Function of AP-1 in Squamous Cell Carcinogenesis The initial identification of Jun and Fos as cellular homologs of retroviral oncoproteins, together with the finding that growth factors, cytokines and tumor promoters augment their activity immediately linked AP-1 with cellular growth control, cell survival and neoplastic transformation (Eferl and Wagner 2003; Hess et al. 2004). Additionally, the analysis of well-established cell culture and mouse model systems demonstrated that AP-1 activity is essential for neoplastic transformation of keratinocytes and tumor progression (Durchdewald et al. 2009; Eferl and Wagner 2003; Young et al. 2003).

9.2.2.1 Jun Function in Mouse Models of Skin Carcinogenesis Again, mouse genetics was used to address the role of Jun proteins in neoplastic transformation and malignant progression of keratinocytes in vivo (Table 9.1). As an example, transgene expression of a dominant-negative deletion mutant of Jun (K14-TAM67) in basal keratinocytes inhibited two-stage skin carcinogenesis in a mouse model with ectopic expression of the human papilloma virus oncogene E7 (K14-HPV16-E7) (Young et al. 1999). However, TAM67 affected tumorigenesis without blocking hyperproliferation or cell survival of keratinocytes suggesting an important role of AP-1 in late stages of tumor promotion and progression (Young et al. 2002). Accordingly, TPA-induced epidermal hyperplasia was indistinguishable between JunDep and control mice, although JunDep animals developed smaller papillomas after crossbreeding with K5-SOS-F transgenic animals, a tumor-prone model system with a constitutive active Ras signaling pathway in keratinocytes (Zenz et al. 2003). Similarly to the situation in eye-lid closure, tumors of JunDep mice showed drastically reduced Egfr expression compared with control tumors. Intriguingly, Jun is not only a potent transcriptional inducer of Egfr and its ligands, but is also an important target of the Egfr signaling pathway, suggesting the existence of a positive feed-back-loop during squamous cell carcinogenesis. Recently, Kolev and colleagues identified EGFR signaling as a key negative regulator of NOTCH1 gene expression in primary human keratinocytes, intact epidermis and skin SCCs (Kolev et al. 2008 Nat Cell Biol). The underlying mechanism for negative control of the NOTCH1 gene in human cells, as well as in a mouse model of Egfr-dependent skin carcinogenesis, involves transcriptional suppression of p53 by Jun (Kolev et al. 2008). This finding was in line with previous work using mouse embryo fibroblasts in which Jun was identified as a direct negative regulator of Tp53 gene expression and function (Schreiber et al. 1999), as well as the genetic interaction of Jun and p53 that apparently controls tumor formation in a mouse model of hepatocellular carcinogenesis (Eferl et al. 2003).

192 Table 9.1 Summary of genetically modified mouse models carcinogenesis Mouse strain Phenotype Impaired tumor formation in mice with K14-TAM67 keratinocyte-specific expression of the human papilloma virus oncogene E7 JunDep Impaired tumor formation in mice with keratinocyte-specific expression of the oncogenic human SOS-F transgene JunAA Delayed tumor formation in mice with keratinocyte-specific expression of the oncogenic human SOS-F transgene hK1-vFos Spontaneous epidermal hyperplasia and papilloma formation after long latency Fos−/− Resistance against malignant progression in the two-stage skin carcinogenesis model Impaired tumor formation in mice with FosDep keratinocyte-specific expression of the oncogenic human SOS-F transgene K5-AFos Impaired tumor formation in the two-stage skin carcinogenesis model and transdifferentiation of squamous cell tumors into sebaceous adenomas Jnk1−/− Accelerated tumor growth and malignant progression in the two-stage skin carcinogenesis model Jnk2−/− Impaired tumor formation in the two-stage skin carcinogenesis model Impaired tumor formation in the two-stage Erk1−/− skin carcinogenesis model

J. Hess and P. Angel that affect mouse skin References Young et al. (2002) and Young et al. (1999)

Zenz et al. (2003)

Behrens et al. (2000)

Greenhalgh et al. (1993b) Saez et al. (1995) Durchdewald et al. (2009)

Gerdes et al. (2006)

She et al. (2002)

Chen et al. (2001)

9.2.2.2 Fos Protein Functions in Malignant Progression In the past, many experimental approaches focused on the oncogenic function of Fos proteins and highlighted their key role in neoplastic transformation and malignant progression of epithelial cells (Durchdewald et al. 2009). Originally, Greenhalgh and colleagues generated transgenic mice that express vFos under the control of the human Keratin-1 promoter (hK1-vFos) to investigate the role of activated Fos protein in the transformation of keratinocytes in vivo (Greenhalgh et al. 1993b). Adult transgenic mice displayed hyperplasia and hyperkeratosis and developed papillomas after long latency. Meanwhile, hK1-vFos animals were crossbred with numerous mouse strains to unravel the synergism of vFos with other oncogenes and/or tumor suppressor genes, such as Ras, Tgfa, Tp53, and Pten, in neoplastic transformation of epidermal keratinocytes (Greenhalgh et al. 1993a; Wang et al. 2000; Wang et al. 1995; Yao et al. 2008). The crucial role of Fos-containing protein complexes for multistage skin carcinogenesis was further supported with Fos-deficent mice (Fos−/−) and animals that specifically lack Fos expression in keratinocyes

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(FosDep). Both showed reduced tumor formation in mouse models of skin carcinogenesis and supported an essential function of Fos-regulated genetic programs during tumor formation and malignant conversion (Durchdewald et al. 2009; Saez et al. 1995). Recently, Gerdes and colleagues generated another transgenic mouse model in which A-Fos, a dominant-negative mutant that inhibits AP-1 DNA binding, was conditionally expressed in keratinocytes (K5-AFos). Whereas, inhibition of Fos/ AP-1 activity during chemically induced skin carcinogenesis dramatically reduced the number of benign and malignant squamous lesions, expression of A-Fos after tumor formation caused squamous tumors to trans-differentiate into sebaceous adenomas (Gerdes et al. 2006). Vice versa, reactivating AP-1 in sebaceous tumors results in a reciprocal trans-differentiation into squamous tumors, supporting the notion that Fos/AP-1 activity in keratinocytes regulates tumor cell lineages and is essential to maintain the squamous tumor cell identity. But how does Fos affect malignant progression of epithelial tumor cells? Reichmann and colleagues, who showed that over-expression of a conditional FosER fusion protein in mammary epithelial cells caused loss of epithelial polarity and augmented invasive growth (Reichmann et al. 1992), found one possible answer. Intriguingly, Fos induced characteristic molecular features of epithelial-mesenchymaltransition (Eger et al. 2000, 2004; Mejlvang et al. 2007). This is a critical step in tumor progression providing tumor cells with the capacity to escape from the primary tumor and to invade the surrounding tissue (Thiery and Sleeman 2006). 9.2.2.3 Regulation of AP-1 Function in Skin Cancer Development by MAPKs As mentioned above, an important mechanism to regulate AP-1 function is posttranslational modification of distinct family members, including Jun N-terminal phosphorylation (JNP) at the serine residues 63 and 73 by the Jun N-terminal kinases (JNKs) (Fig. 9.1). The JNK signal transduction pathway plays an important role in coordinating the cellular stress response, including apoptosis. However, several lines of evidence also support an important role of the JNK pathway in major cellular functions, such as cell proliferation, and transformation (Weston and Davis 2007). Knockin mouse models and cell lines harboring a mutant Jun allele, which has the JNK phosphoacceptor serines changed to alanines (JunAA), were used to determine the function of JNP during oncogenic transformation in vitro and tumor formation in vivo (Behrens et al. 2000). Indeed, skin tumor formation was significantly delayed in K5-SOS-F mice expressing the mutant JunAA allele, suggesting a function of JNP as a target for the oncogenic Ras pathway in keratinocytes. In line with this assumption, JNP was also required for efficient transformation of immortalized fibroblasts by vRas in vitro as well as bone tumor formation in vFos transgenic mice in vivo (Behrens et al. 2000). More recently, the generation of Jnk1- and Jnk2-deficient mice (Jnk−/− and Jnk2−/−) provided the opportunity to address the role of specific Jnk isoforms during

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neoplastic transformation of keratinocytes and skin carcinogenesis in vivo. While Jnk2−/− mice exhibited significant reduction in papilloma burden compared to wildtype controls, Jnk1−/− animals were more susceptible to chemically induced skin tumor growth and malignant progression (Chen et al. 2001; She et al. 2002). Collectively, these data demonstrated for the first time that Jnk1 and Jnk2 are important but antagonistic regulators of neoplastic transformation and tumor development. The molecular mechanism by which both kinases function in chemically induced tumor formation seems to be complex and correlated with altered DNAbinding activity of AP-1. However, no difference in Jun phosphorylation was detected among wild-type, Jnk1−/−, and Jnk2−/− mouse skin after TPA treatment (She et al. 2002), implying that neither Jnk1 nor Jnk2 deficiency affect AP-1 activity upon TPA administration by N-terminal Jun phosphorylation. Accordingly, both studies provided experimental evidence that differential modulation of Erk and Akt signaling pathways, and subsequent AP-1 DNA-binding activity in Jnk1−/− and Jnk2−/− mouse skin may, at least in part, account for the distinct roles of both kinases in skin carcinogenesis (She et al. 2002). It is well established that activation of Erk and Akt kinases induces expression of Jun and Fos proteins, leading to an increased AP-1 DNA-binding activity and transcriptional activation. Furthermore, increased activity of Erk and Akt kinases was found during mouse skin carcinoge nesis, supporting their relevance for keratinocyte transformation and malignant progression (Segrelles et al. 2002). In line with this assumption, Bourcier and colleagues could demonstrate that Erk1-deficient mice (Erk1−/−) were almost resistant against chemically induced skin papilloma development accompanied by a drastic reduction of TPA-induced Fos expression (Bourcier et al. 2006).

9.3 AP-1 Target Genes in Squamous Cell Carcinogenesis The identification of key regulators of distinct molecular processes in epithelial cancer cells that are under the direct control of AP-1 is essential to understand its function during tumor development and to find therapeutic targets for novel strategies of translational cancer research. Despite the well-documented function of AP-1 in neoplastic transformation and malignant progression only a few unambiguously identified tumor-associated target genes have been described so far. Thus, genome-wide approaches with material from informative tumor models have been accomplished in the past to elucidate the role of AP-1-dependent gene regulatory networks during squamous cell carcinogenesis and to highlight critical tumorassociated target genes. As an example, we performed global gene expression profiling with samples of the extensively used mouse model of two-stage skin carcinogenesis for which AP-1 was shown to be a key player. Our studies revealed a comprehensive list of tumorassociated genes some of which represent well-known AP-1 target genes in other in vitro and in vivo model system (e.g, Ccnd1, Cd44, Fosl1, Lcn2, Mmp9, Mmp13, S100a8, S100a9), or share at least one putative AP-1 binding site in their proximal

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promoter region (Hummerich et al. 2006; Schlingemann et al. 2003). In another study, Matthews and colleagues examined the expression profile of K14-HPV16-E7 transgenic and control skin samples in the presence or absence of TAM67 expression to identify target genes under conditions known to inhibit skin carcinogenesis (Matthews et al. 2007). This approach revealed a limited number of genes implicated in inflammation, tumor invasion and metastasis, including Ptgs2, Spp1, Cxcl1, Alox5ap, Mmp10, and Plaur. More recently, we analyzed skin tumor samples from control and FosDep mice that developed in the genetic background of K5-SOS-F transgenic mice (Durchdewald et al. 2008). Expression profiling revealed a comprehensive list of differentially expressed genes some of which represent previously identified candidate genes in epithelial carcinogenesis. Gene clustering according to their functional annotation showed that most of these genes are implicated in tumor-relevant processes, such as cellular movement and morphology, cell cycle progression, cell death and cell signaling (Durchdewald et al. 2008). Interestingly, one candidate gene whose expression strictly depends on Fos activity in cell lines and tumor tissue encoded the small mucin-like glycoprotein Podoplanin (Pdpn). Pdpn has attracted major interest in the field of cancer research since its expression closely correlated with malignant progression and metastasis of epithelial cancer and could promote tumor cell invasion in vitro and in vivo by EMT-dependent and independent mechanisms (Wicki and Christofori 2007).

9.4 Targeting of AP-1 or Its Upstream Signaling Pathways as Anticancer Therapy for Squamous Cell Carcinoma (SCC) There is a controversial discussion on the question whether oncogenic transcription factors themselves, such as AP-1, are suitable drug targets for clinical application. Some general doubts are that (1) AP-1 subunits are ubiquitously expressed and essential for normal cell physiology, (2) AP-1 consist of several subunits showing redundant functions during cellular transformation and tumor development, (3) frequent mutations in AP-1 subunits have not been found so far that could serve as putative target sites for drug interference. However, Zhang and colleagues demonstrated that specific targeting of Jun expression using a specific DNA enzyme (DNAzyme) strategy impairs expression of AP-1 target genes in cell culture and inhibits solid tumor growth in several mouse model of SCC and melanoma (Zhang et al. 2004, 2006). DNAzymes are synthetic, single-stranded DNA catalysts that can be engineered to bind to their complementary sequences in a target mRNA and cleave the transcript at predetermined phosphodiester linkages (Schubert et al. 2003). Meanwhile, DNAzyme-dependent targeting of Jun was also applied on mouse tumor models of liposarcoma and osteosarcoma and revealed beneficial outcomes (Dass et al. 2008a, b). Another strategy has been the generation of high-affinity peptides or small molecule inhibitors that act as stable drugs by interference with homo- and

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h eterodimerization of AP-1 family members and DNA-binding activity (Mason et al. 2006; Aikawa et al. 2008). More recently, Aikawa and colleagues succeeded in de novo design of a specific small molecule inhibitor for Jun/Fos activity using three-dimensional (3D) pharmacophore modeling and confirmed its activity in a preclinical mouse model of arthritis (Aikawa et al. 2008). The pharmacophore modeling was based on the 3D structure of the bZIP domain of the AP-1-DNA complex. Yet, more than 50 human bZIP proteins that make up 20 families have been identified and participate in a wide range of important biological processes. One has to consider that a peptide or small molecule inhibitor designed to bind optimally to the bZIP domain of Jun or Fos may also bind strongly to one or more undesired bZIP-containing targets, sharing strong sequence and structural similarities. Hence, Grigoryan and colleagues developed a strategy for addressing specificity in protein-design calculations that rests on the trade-off between maximizing affinity and introducing specificity (Grigoryan et al. 2009). Indeed, this computational method revealed anti-bZIP peptides according to the prediction of high target binding affinity and minimal interaction with themselves and with members of the 19 nontarget bZIP families. Two of the best designs targeted Jun and Fos proteins, and using anti-Fos peptides provided a way to disrupt Jun-Fos, but neither Jun-Jun nor Jun-ATF dimers. Although further experimental studies are urgently needed to determine and improve the therapeutic efficacy of AP-1 specific inhibitors, these approaches might represent promising new strategy for the treatment of patients with SCCs. In the past, tremendous efforts also have been made to develop small molecules or other blocking reagents that interfere with oncogenic signaling pathways upstream of AP-1 activity. In particular, the RAS-MAPK pathway has been the subject of intense preclinical and clinical studies leading to the development of pharmacologic inhibitors for the treatment of human cancer (Roberts and Der 2007; Friday and Adjei 2008). As mentioned above, the RAS-MAPK signaling cascade and members of the EGFR family are well known to induce AP-1 activity in normal and pathophysiological conditions, including cancer. Although most inhibitors that specifically target EGFR, RAS, MEK or ERK interfere with expression of AP-1 family members or well known target genes in cell culture models, only a limited number of studies addressed their impact on AP-1 in preclinical mouse tumor models or cancer patient. As an example, Jimeno and colleagues studied the consequence of gefitinib and erlotinib treatment on Fos expression. Gefitinib and erlotinib are two EGFR small molecule kinase inhibitors that demonstrated promising results in preclinical trial and cancer patients, including patients with SCC. They found Fos expression as a reliable marker of anti-EGFR effects applying gefitinib and erlotinib treatment on human cancer cell lines in culture or in xenograft mouse models. Furthermore, patient tumor samples from a clinical trial of gefitinib in patients with solid tumors confirmed that expression of Fos can be used as biomarker in clinical biopsies (Jimeno et al. 2006).

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9.5 Conclusion and Perspectives A large body of experimental and clinical data provide strong evidence for an important role of distinct AP-1 subunits in normal epithelial development and homeostasis, and that deregulated AP-1 activity critically contributes to pathophysiological processes, including neoplastic keratinocytes transformation and epithelial malignancy. Although, some direct AP-1 target genes have been identified in mouse models of skin carcinogenesis further genome-wide and functional approaches will be required to comprehensively understand the causal connectivity between altered AP-1 function and squamous cell carcinogenesis. Acknowledgments Our work was supported by the German Ministry for Education and Research (National Genome Research Network NGFN-2), the Deutsche Krebshilfe e.V., the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Systems Biology, and by the Studienstiftung des deutschen Volkes.

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Eferl R, Wagner EF (2003) AP-1: a double-edged sword in tumorigenesis. Nat Rev Cancer 3:859–868 Eger A, Stockinger A, Park J et al. (2004) beta-Catenin and TGFbeta signalling cooperate to maintain a mesenchymal phenotype after FosER-induced epithelial to mesenchymal transition. Oncogene 23:2672–2680 Eger A, Stockinger A, Schaffhauser B et al. (2000) Epithelial mesenchymal transition by c-Fos estrogen receptor activation involves nuclear translocation of beta-catenin and upregulation of beta-catenin/lymphoid enhancer binding factor-1 transcriptional activity. J Cell Biol 148:173–188 Florin L, Hummerich L, Dittrich BT et al. (2004) Identification of novel AP-1 target genes in fibroblasts regulated during cutaneous wound healing. Oncogene 23:7005–7017 Florin L, Knebel J, Zigrino P et al. (2006) Delayed wound healing and epidermal hyperproliferation in mice lacking JunB in the skin. J Invest Dermatol 126:902–911 Florin L, Maas-Szabowski N, Werner S et al. (2005) Increased keratinocyte proliferation by JUNdependent expression of PTN and SDF-1 in fibroblasts. J Cell Sci 118:1981–1989 Friday BB, Adjei AA (2008) Advances in targeting the Ras/Raf/MEK/Erk mitogen-activated protein kinase cascade with MEK inhibitors for cancer therapy. Clin Cancer Res 14:342–346 Gebhardt C, Nemeth J, Angel P et al. (2006) S100A8 and S100A9 in inflammation and cancer. Biochem Pharmacol 72:1622–1631 Gerdes MJ, Myakishev M, Frost NA et al. (2006) Activator protein-1 activity regulates epithelial tumor cell identity. Cancer Res 66:7578–7588 Greenhalgh DA, Quintanilla MI, Orengo CC et al. (1993a) Cooperation between v-fos and v-rasHA induces autonomous papillomas in transgenic epidermis but not malignant conversion. Cancer Res 53:5071–5075 Greenhalgh DA, Rothnagel JA, Wang XJ et al. (1993b) Hyperplasia, hyperkeratosis and benign tumor production in transgenic mice by a targeted v-fos oncogene suggest a role for fos in epidermal differentiation and neoplasia. Oncogene 8:2145–2157 Grigoryan G, Reinke AW, Keating AE (2009) Design of protein-interaction specificity gives selective bZIP-binding peptides. Nature 458:859–864 Hess J, Angel P, Schorpp-Kistner M (2004) AP-1 subunits: quarrel and harmony among siblings. J Cell Sci 117:5965–5973 Hummerich L, Muller R, Hess J et al. (2006) Identification of novel tumour-associated genes differentially expressed in the process of squamous cell cancer development. Oncogene 25:111–121 Jimeno A, Kulesza P, Kincaid E et al. (2006) C-fos assessment as a marker of anti-epidermal growth factor receptor effect. Cancer Res 66:2385–2390 Kolev V, Mandinova A, Guinea-Viniegra J et al. (2008) EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer. Nat Cell Biol 10:902–911 Li G, Gustafson-Brown C, Hanks SK et al. (2003) c-Jun is essential for organization of the epidermal leading edge. Dev Cell 4:865–877 Mason JM, Schmitz MA, Müller KM et al. (2006) Semirational design of Jun-Fos coiled coils with increased affinity: Universal implications for leucine zipper prediction and design. Proc Natl Acad Sci USA 103:8989–8994 Matthews CP, Birkholz AM, Baker AR et al. (2007) Dominant-negative activator protein 1 (TAM67) targets cyclooxygenase-2 and osteopontin under conditions in which it specifically inhibits tumorigenesis. Cancer Res 67:2430–2438 Mejlvang J, Kriajevska M, Berditchevski F et al. (2007) Characterization of E-cadherin-dependent and -independent events in a new model of c-Fos-mediated epithelial-mesenchymal transition. Exp Cell Res 313:380–393 Reichmann E, Schwarz H, Deiner EM et al. (1992) Activation of an inducible c-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion. Cell 71:1103–1116 Roberts PJ, Der CJ (2007) Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for treatment of cancer. Oncogene 26:3291–3310

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Saez E, Rutberg SE, Mueller E et al. (1995) c-fos is required for malignant progression of skin tumors. Cell 82:721–732 Schlingemann J, Hess J, Wrobel G et al. (2003) Profile of gene expression induced by the tumour promotor TPA in murine epithelial cells. Int J Cancer 104:699–708 Schreiber M, Kolbus A, Piu F et al. (1999) Control of cell cycle progression by c-Jun is p53 dependent. Genes Dev 13:607–619 Schubert S, Gül DC, Grunert HP et al. (2003) RNA cleaving ‘10-23’ DNAzymes with enhanced stability and activity. Nucleic Acid Res 31:5982–5992 Segrelles C, Ruiz S, Perez P et al. (2002) Functional roles of Akt signaling in mouse skin tumorigenesis. Oncogene 21:53–64 Shaulian E, Karin M (2002) AP-1 as a regulator of cell life and death. Nat Cell Biol 4:E131–E136 She QB, Chen N, Bode AM et al. (2002) Deficiency of c-Jun-NH(2)-terminal kinase-1 in mice enhances skin tumor development by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res 62:1343–1348 Szabowski A, Maas-Szabowski N, Andrecht S et al. (2000) c-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin. Cell 103:745–755 Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7:131–142 Van Beveren C, van Straaten F, Curran T et al. (1983) Analysis of FBJ-MuSV provirus and c-fos (mouse) gene reveals that viral and cellular fos gene products have different carboxy termini. Cell 32:1241–1255 Wagner EF (2001) AP-1 reviews. Oncogene 20:2333–2497 Wang XJ, Greenhalgh DA, Donehower LA et al. (2000) Cooperation between Ha-ras and fos or transforming growth factor alpha overcomes a paradoxic tumor-inhibitory effect of p53 loss in transgenic mouse epidermis. Mol Carcinog 29:67–75 Wang XJ, Greenhalgh DA, Lu XR et al. (1995) TGF alpha and v-fos cooperation in transgenic mouse epidermis induces aberrant keratinocyte differentiation and stable, autonomous papillomas. Oncogene 10:279–289 Weston CR, Davis RJ (2007) The JNK signal transduction pathway. Curr Opin Cell Biol 19:142–149 Weston CR, Wong A, Hall JP et al. (2004) The c-Jun NH2-terminal kinase is essential for epidermal growth factor expression during epidermal morphogenesis. Proc Natl Acad Sci USA 101:14114–14119 Wicki A, Christofori G (2007) The potential role of podoplanin in tumour invasion. Br J Cancer 96:1–5 Yao D, Alexander CL, Quinn JA et al. (2008) Fos cooperation with PTEN loss elicits keratoacanthoma not carcinoma, owing to p53/p21 WAF-induced differentiation triggered by GSK3beta inactivation and reduced AKT activity. J Cell Sci 121:1758–1769 Young MR, Farrell L, Lambert P et al. (2002) Protection against human papillomavirus type 16-E7 oncogene-induced tumorigenesis by in vivo expression of dominant-negative c-jun. Mol Carcinog 34:72–77 Young MR, Li JJ, Rincon M et al. (1999) Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion. Proc Natl Acad Sci USA 96:9827–9832 Young MR, Yang HS, Colburn NH (2003) Promising molecular targets for cancer prevention: AP-1, NF-kappa B and Pdcd4. Trends Mol Med 9:36–41 Zenz R, Eferl R, Kenner L et al. (2005) Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins. Nature 437:369–375 Zenz R, Scheuch H, Martin P et al. (2003) c-Jun regulates eyelid closure and skin tumor development through EGFR signaling. Dev Cell 4:879–889 Zhang G, Dass CR, Sumithran E et al. (2004) Effect of deoxyribozymes targeting c-Jun on solid tumor growth and angiogenesis in rodents. J Natl Cancer Inst 96:683–696 Zhang G, Luo X, Sumithran E et al. (2006) Squamous cell carcinoma growth in mice and in culture is regulated by c-Jun and its control of matrix metalloproteinase-2 and -9 expression. Oncogene 25:7260–7266

Chapter 10

NF-kB, IkB Kinase and Interacting Signal Networks in Squamous Cell Carcinomas Antonio Costanzo, Giulia Spallone, and Michael Karin

Abstract Nuclear factor-kB (NF-kB) transcription factors and the IkB kinases (IKKs) that activate them are central coordinators of innate and adaptive immune responses. More recently, it has become clear that NF-kB signaling also has a critical role in cancer development and progression. The canonical NF-kB pathway contributes to squamous cell carcinoma (SCC) development by interacting with other signaling pathways including tumor suppressive and oncogenic pathways in a tissue specific manner. In this chapter, we will summarize recent advances in the understanding of interconnections between NF-kB and IKKs, including IKKa which has NF-kB independent functions, in the context of SCC development and progression and as potential target for novel chemotherapy approaches.

10.1 Introduction In 1986, Nuclear factor-kB (NF-kB) was first identified as a nuclear factor that binds to an enhancer element of the immunoglobulin k light-chain gene and was thought to be restricted to B cells (Sen and Baltimore 1986). In its active DNAbinding form, NF-kB is a heterogeneous collection of dimers, composed of different combinations of members of the NF-kB/Rel family (Karin and Ben-Neriah 2000). NF-kB transcription factors play an important role in integrating multiple stress stimuli and regulating innate and adaptive immune responses seen in states of inflammation, infection and injury (Bonizzi and Karin 2004). By now, the name “NF-kB” for these transcription factors is no longer accurate, as NF-kB proteins reside in the cytoplasm of all resting cells, and upon stimulation they enter the nucleus and bind to a large array of enhancer sequences (Naugler and Karin 2008).

M. Karin (*) Laboratory of Gene Regulation and Signal Transduction, School of Medicine, University of California, San Diego, 9500 Gilman Drive MC 0723, La Jolla, CA 92093-0723, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_10, © Springer Science+Business Media, LLC 2011

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Furthermore, the NF-kB binding site in kappa light chain enhancer is not critical for kappa gene expression or rearrangement (Xu et al. 1996). The aim of this chapter is to focus on the role of the NF-kB signaling pathway and the IkB kinases, in linking the pathophysiological processes of inflammation and cancer in squamous cell carcinoma (SCC). SCC is one of the most common invasive cancers in the world, with an annual incidence of 250,000 in the USA (Chen et al. 2001a, b). Although the molecular mechanisms leading to SCC development and progression are likely to be heterogeneous, it is widely accepted that the NF-kB pathway plays an important role in both initial transformation steps and in progression of SCC.

10.2 Composition and Regulation of NF-kB NF-kB dimers can be made up of five homologous subunits (RelA/p65, c-Rel, RelB, p50/NF-kB1, and p52/NF-kB2). They are held in the cytoplasm of nonstimulated cells by specific proteins, the inhibitors of NF-kB(IkBs) which are characterized by presence of multiple ankirin repeats (Fig. 10.1a). Acting immediately upstream to the IkB-bound NF-kB dimers is the IKK complex, composed of two catalytic (IKKa and IKKb) and one regulatory (IKKg/NEMO) subunits (Naugler and Karin 2008; Häcker and Karin 2006). A wide range of agonists, including tumor necrosis factor alpha (TNFa), lipopolysaccharide (LPS), interleukin-1 (IL-1), ligands for Toll-Like Receptors (TLRs), and various stressors lead to activation of the IKK complex, which then phosphorylates the two amino-terminal serine residues of IkBs proteins, targeting them for ubiquitination and degradation by the 26 S proteosome (Häcker and Karin 2006). Two other IKK-related kinases that phosphorylate amino-terminal serine residues 32 and 36 in IkBa and 19 and 23 in IkBb are IKKe/ TBK1 and IKKi (Häcker and Karin 2006). The liberated NFkB dimers travel to the nucleus and engage a variety of transcriptional programs. Though there is a broadening complexity to NF-kB signaling, the two most recognized pathways are the so-called “classical” and “alternative.”

10.2.1 The Classical Pathway of NF-kB Signaling The classical pathway includes IKKg/NEMO, IKKa, and IKKb subunits, and depends on IKKb kinase activity for activation, nuclear localization of p50:RelA dimers, and is related to inflammation (Fig. 10.1b)(Naugler and Karin 2008). The alternative pathway depends on IKKa activation via the upstream NF-kB-inducing kinase NIK, which activates IKKa homodimers, independently of either IKKb or IKKg, leading to the phosphorylation and processing of p100 and nuclear trans location of p52:RelB dimers (Fig. 10.1b) (Naugler and Karin 2008; Bonizzi and Karin 2004; Hayden and Ghosh 2004). The alternative pathway is activated by a subset of TNF family members and is important in lymphoid organogenesis

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Fig. 10.1 (a) Structure of principal NF-kB signaling pathway components. Transcription factors RelA(p65), cRel and RelB possess an N-terminal Rel Homology Domain and C-terminal transcriptional activation domain. p105 and p100 contain C-terminal IkB-like domains composed of multiple ankyrin repeats that are proteolytically removed to yeld p50 and p52, respectively. IkB proteins contain several C-terminal ankyrin repeats and a variable N-terminal region. Components of IkB Kinase complex include the IKKa and IKKb catalytic subunits, an N-terminal protein kinase domain and C-terminal Leucin Zipper (LZ), and Helix-Loop Helix domain (HLH). IKKg/ NEMO is the regulatory subunit and it contains two Coiled Coil domains (CC), a Leucin Zipper and a C-terminal Ring Finger (RF) domain. (b), NF-kB is activated through two main signaling pathways. The classical pathway (left) is activated by a wide range of extracellular stimuli including TNFa, IL-1, and toll-like receptor ligands which lead to activation of TAK1. Receptor engagement also results in recruitment of the IKK complex and its phosphorylation by TAK1. The IKK complex in turn phosphorylates N-terminal residues on IkB molecules which target them to K48-linked ubiquitination and proteasome-mediated degradation. This leads to the release of NF-kB dimers that translocate to the nucleus to induce transcription of specific genes. The alternative pathway (right) is activated by a subset of TNF receptor family members which lead to stabilization of NF-kB-inducing kinase (NIK). NIK activates IKKa which phosphorylates the p100 NF-kB2 precursor to induce the proteasomal-mediated degradation of its IkB-like C-terminus. This results in the release of p52/RelB dimers which activate a different set of NF-kB-dependent genes.

(Bonizzi and Karin 2004). Recent studies indicate that both pathways of NF-kB activation are implicated in tumorigenesis (Naugler and Karin 2008) but an NF-kBindependent role for IKKa as a tumor suppressor was also identified (Marinari et al. 2008; Maeda et al. 2007). The NF-kB proteins belong to two groups: those that do not require proteolytic processing and those that do require proteolytic processing (Karin et al. 2002). The first group includes RelA/p65, c-Rel and RelB.

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NF-kB1/p105 and NF-kB2/p100 belong to the second group, whose proteolytic processing gives rise to the mature p50 and p52 subunits, respectively. Members of both groups dimerize, the common dimer being p50:RelA. Due to the presence of a strong transcriptional activation domain, RelA is responsible for most of NF-kB transcriptional activity. p50:c-Rel dimers are less abundant and seem to be activated with slower kinetics. Both p50:RelA and p50:c-Rel dimers are regulated by IkB proteins, whereas RelB is mostly bound to p100 in the cytoplasm of non-stimulated cells. Proteolytic processing of p100 results in the release of p52:RelB dimers, which translocate to the nucleus where RelB, unlike RelA and c-Rel, can have both activating and repressing functions (Karin et al. 2002). Different NF-kB dimers exhibit different binding affinities for kB sites bearing the consensus sequence GGGRNNYYCC, where R is purine, Y is pyrimidine, and N is any base (Karin and Ben-Neriah 2000; Miyamoto and Verma 1995). To better understand the physiological function of these proteins targeted disruption of individual Rel and Nfkb loci has been carried out in mice (Karin and Ben-Neriah 2000; Gerondakis et al. 2006). These “knockout” studies reveal overlapping as well as specific activities for each NF-kB protein. For instance, RelA/p65 was found to be critical for survival mainly through suppression of hepatocyte apoptosis (Beg et al. 1995 ). 10.2.1.1 Post-translational Modifications Regulate REL Protein Activity Not only the IkB but also the Rel proteins themselves, are subject to signal-induced post-translational modifications that alter their DNA binding and their transactivating function (Viatour et al. 2005; Perkins 2006). These modifications alter various physiological functions of the NF-kB factors. Most important amongst these modifications is phosphorylation and the basal and signal-induced phosphorylation of RelA are by far the best-characterized. Site-specific phosphorylation is often a prerequisite for other modifications regulating RelA activity. Such subsequent posttranslational modifications include acetylation, ubiquitinylation, and isomerization of specific amino acid residues. Today, nine putative sites of RelA phosphorylation are known: six serine and three threonine residues. Three of these sites (Ser-276, Ser-311, and Thr-254) are located within the N-terminal RHD, whereas six acceptor sites (Ser-468, Ser-529, Ser-535, Ser-536, Thr-435, and Thr-505) are found within the C-terminal transactivation domain. The effects of RelA phosphorylation at these specific sites vary dramatically, ranging from transcriptional activation to the complete repression of certain genes. The pattern of RelA phosphorylation is most likely stimulus- and cell type-specific. It becomes more and more apparent that RelA phosphorylation at different sites serves as an integrator for multiple incoming signals, which could control both the kinetics and strength of the transactivating potency of RelA. Functionally, RelA ubiquitinylation is a potential mechanism regulating the termination of NF-kB–mediated inflammatory responses (Lawrence et al. 2005) and may also contribute to the oscillation of nuclear NF-kB activity (Nelson et al. 2004). The phosphorylation status of Rel proteins, especially of RelA, a decisive event controlling their association with

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either activating Histone Acetyl Transferases (e.g., CBP/p300) or suppressing Histone Deacetylases, was a hallmark discovery in the field of NF-kB regulation (Zhong et al. 2002). Upon TNFa stimulation, RelA is acetylated at five specific lysine residues: Lys-122, -123, -218, -221, and -310 (Liu et al. 2006; Hoberg et al. 2006). RelB is phosphorylated at Thr-84 and Ser-552 (Marienfeld et al. 2001). These phosphorylation events have functional consequences that are contrary to the effects of RelA phosphorylation. RelB phosphorylation is induced by specific signals and is a prerequisite for subsequent proteasomal degradation (Marienfeld et al. 2001). Regulation of c-Rel activity is also achieved to a large extent by its signalinduced phosphorylation. An initial hint stressing the importance of c-Rel phosphorylation came from a study reporting that tyrosine phosphorylation can be rapidly induced by G-CSF in human neutrophils (Druker et al. 1994). However, neither the protein tyrosine kinase mediating the G-CSF effect nor the target tyrosine residue (or residues) has been identified. Subsequently, a complex of a 65 kDa serine/threonine protein kinase with murine c-Rel was reported (Fognani et al. 2000). This kinase apparently has two binding sites that mediate C-terminal phosphorylation of c-Rel, but the specific phospho-acceptor sites within c-Rel and the identity of the protein kinase have not been identified. Similar to the IkBs, p105, the precursor of the Rel protein p50, is site-specifically phosphorylated, then polyubiquitinylated and proteolytically degraded in the 26S proteasome. Although it has been known for many years that p50 as well as its p105 precursor are inducibly phosphorylated upon stimulation of various cell types (Naumann and Scheidereit 1994), reports about site-specific phosphorylation of the mature p50 and the functional consequences of such a posttranslational modification are rare. So far, the finding that the catalytic subunit of PKA (PKAc) can inducibly phosphorylate Ser-337, which is located within the RHD of p50, stands alone. Since p50 has no transactivation domains, the only functional consequence appears to be an enhanced p50 DNA binding (Guan et al. 2005).

10.2.2 Atypical Activation Pathways The mechanisms of activation described above appear to be generally applicable to all potent NF-kB activators, but two additional activation pathways were also reported. One is observed as a result of hypoxia or pervanadate treatment and is believed to require phosphorylation of IkBa at Tyr-42 (Imbert et al. 1996; Beraud et al. 1999). The exact protein tyrosine kinases involved in this pathway are not known, but certain members of the Src family were proposed to be responsible for IkB phosphorylation. The subsequent dissociation of tyrosine-phosphorylated IkBa from NF-kB was suggested to be mediated by interaction with phosphoinositide-3 (PI3) kinase and not by degradation by the 26S proteasome (Beraud et al. 1999). Because Tyr-42 is not conserved in other IkB family members, this pathway is specific for IkBa. The second atypical activation pathway is observed in cells exposed to short-wavelength ultraviolet (UV) radiation (254 nm). UV radiation

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induces IkBa degradation via the 26S proteasome, but this process is not mediated by phosphorylation of Ser-32 and Ser-36 or Tyr-42 (Bender et al. 1998; Li and Karin 1998). The mechanism of IkBa degradation in response to UV radiation is thought to be dependent on Casein Kinase 2 (CK2), which phosphorylates the C-terminal PEST domain of Ik-B (Kato et al. 2003). In both of these alternative pathways, NF-kB activation is considerably slower and weaker than the response to the prototypical NF-kB activators of the classical pathway, TNF-a, IL-1, or LPS (Karin and Ben-Neriah 2000).

10.3 NF-kB, Inflammation and Cancer With the recognition that inflammatory conditions are often associated with or preceed cancer, it was natural to associate NF-kB with cancer, as was first suggested several years ago (Karin and Ben-Neriah 2000). Rudolf Virchow, one of the fathers of modern pathology, first postulated a link between inflammation and cancer from observing leukocytes in neoplastic tissue and suspected that inflammation might support or promote cancer (Balkwill and Mantovani 2001). This notion has reemerged in the last decade in part because of the recognition that many chronic infections are associated with development of cancer. Approximately 15% of the global cancer deaths or 1.2 million cases of infection-related malignancies per year have been attributed to chronic infections and the accompanying inflammatory reaction (Parkin et al. 2001), and 15–20% of cancer deaths arise from preventable infections (Kuper et al. 2000). Likewise, many chronic inflammatory conditions of noninfectious origin increase the risk and accelerate the progression of diverse cancers (Karin 2006). The common denominator in these conditions is the presence of chronic inflammatory infiltrate invariably associated with activation of NF-kB and its effector pathways, an elevated secretion of cytokines and chemokines (Naugler and Karin 2008). Chronic inflammation is often caused by the pathogenic action of bacterial products and proteins encoded by viruses such as human papillomaviruses (HPV), Epstein–Barr virus (EBV), and hepatitis B virus (HBV). HPV and EBV are the significant risk factors for malignancies such as cervical and head and neck SCC (HNSCC), while HBV is associated with hepatocellular carcinoma. Such viruses operate through inflammation-related mechanisms, in addition to inhibition of tumor suppressors (Coussens and Werb 2002). Bacterial products such as LPS are also implicated in chronic inflammation associated cancer. Besides such products, Helicobacter pylori, one of the main contributors to gastric cancer, has not been shown to contain any oncogenes such as those identified in viruses (Roder 2002). Noninfectious causes of chronic inflammation also include external agents such as cigarette smoke, asbestos, silica and diseases such as inflammatory bowel diseases and ulcerative colitis that are thought to increase the risk of colorectal cancer by approximately 1% per year (Warzocha et al. 1998). The organs that are most susceptible to chronic inflammation with consequent tumor development are the lungs, bladder, esophagus, pancreas and, most commonly, the gastrointestinal

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tract (Balkwill and Mantovani 2001; Karin 2006). In addition to epidemiological data which link inflammation with cancer, polymorphisms in the TNF locus that lead to increased levels of TNF production are associated with poor prognosis and disease severity in patients with non-Hodgkin’s lymphoma (Warzocha et al. 1998). Gene-cluster polymorphisms in the IL1 locus, which encodes the pro-inflammatory IL-1 cytokine are found in patients with stomach cancer (El-Omar et al. 2000). TNF-a and IL-1a are expressed and enhance activation of NF-kB in HNSCC (Duffey et al. 2000; Wolf et al. 2001; Jackson-Bernitsas et al. 2007). Additional polymorphisms that are associated with increased risk of prostate cancer were recently identified in a gene cluster that encodes TLRs (Sun et al. 2005). Recently, bacterial LPS and toll receptors have also been implicated in activation of NF-kB in HNSCC (Szczepanski et al. 2009). Conversely, long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, as well as natural compounds, such as ginseng and green-tea extracts, resveratrol and curcumin (all of which attenuate activation of NF-kB), reduces the incidence of cancers of the colon, lungs, stomach, esophagus and ovaries, as well as Hodgkin’s lymphoma (Wang et al. 2003).

10.4 NF-kB and SCC SCCs arise from stratified epithelia and represent the most common kind of cancer worldwide (Alam and Ratner 2001). Most common SCCs that arise from the skin, are clinically evident at the premalignant stage (actinic keratoses) and therefore, are readily diagnosed and treated. By contrast, HNSCC, lung SCC, esophageal SCC, urothelial SCC, and bladder SCC are often diagnosed at later stages and cause significant mortality. In particular, HNSCC represents the fifth most common cancer worldwide and 7,300 deaths occur annually in the US (Jemal et al. 2009). Although advances have been made in understanding the molecular pathogenesis of SCC, the role of NF-kB signaling pathways in this context has not been fully elucidated. The current model proposes that the canonical NF-kB pathway contributes to SCC development by interacting with other signaling pathways including tumor suppressive and oncogenic pathways in a tissue specific manner. We will summarize recent advances in the understanding of interconnections between NF-kB and IKK in the context of SCC development and progression.

10.4.1 Interaction of NF-kB and MAPK Pathways in SCC Contrary to what is expected from the assumed general function of NF-kB in cancer (antiapoptotic, proproliferative), down-regulation of NF-kB activity seems to be an important step of keratinocyte transformation into SCC, through regulation of the activity and oncogenic potential of Ras and MAPK pathways (Dajee et al. 2003).

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Considerable experimental evidence confirms that NF-kB and oncogenic Ras can alter proliferation in epidermis, the most common site of SCC (Miller and Weinstock 1994; Alam and Ratner 2001). Both Ras and NF-kB were implicated in epidermal SCC in mice (Yuspa 1994; Pazzaglia et al. 2001; van Hogerlinden et al. 1999). Whereas inhibition of NF-kB enhances apoptosis in certain tumors (Yamamoto and Gaynor 2001), blockade of NF-kB predisposes the murine skin to development of SCC (van Hogerlinden et al. 1999; Seitz et al. 1998). This correlates with the observation that the majority of human SCCs display reduced NF-kB function, and experimentally induced NF-kB blockade with IkB promotes SCC in both murine and human epidermal tissue (van Hogerlinden et al. 1999; Dajee et al. 2003; Lind et al. 2004). In addition, clinical data support the concept that chronic skin inflammations may confer protection against keratinocyte transformation. Thus, the incidence of SCC forming in psoriasis plaques, which are chronically inflammed skin lesions expressing high levels of TNFa, IL-6, and other cytokines known to promote cancer in other tissues, is very rare. This is even more suprising, given that psoriasis patients are treated with immunosuppressive therapies such as cyclosporine or UVB irradiation, and in the past were treated topically with the tumor promoter coal-tar. These carcinogenic agents, together with chronic inflammation should induce high frequency of skin cancer on psoriatic plaques, but that is never observed in clinical practice (Nickoloff 2001). One possible explanation is that high levels of activated NF-kB in psoriatic keratinocytes (Lizzul et al. 2005) may induce growth arrest and facilitate the acquisition of a senescent phenotype that inhibits tumor formation. These clinical observations were mechanistically explained by Khavari’s group who showed that in non-neoplastic human epidermal keratinocytes both NF-kB and oncogenic Ras induce cell-cycle arrest (Dajee et al. 2003). Growth arrest triggered by oncogenic Ras can be bypassed by IkBamediated inhibition of NF-kB, generating malignant human epidermal tissue resembling SCC. As previously mentioned, SCC progression is thought to be induced by the aberrant activity of a network of interrelated signaling pathways, and it was found that NF-kB influences JNK activity through regulation of reactive oxygen species (ROS) production (Kamata et al. 2005). Indeed, the main target of NF-kB “tumor suppressive” activity in keratinocyte transformation seems to be the JNK pathway. NF-kBinduced antioxidant enzymes (MnSOD and thioredoxin) decrease ROS levels in the cell, thereby preventing inhibition of JNK-inactivating dual-specifity phosphatases and ensuring transient JNK activation in response to tumor promoting cytokines such as TNFa. In fact, inhibition of TNF receptor 1 (TNFR1) signaling components like MKK7, JNK, and AP1 as well as ablation of TNFR1 and TNFa using genetic, pharmacologic, or antibody mediated approaches abolished the development of SCC in mice and inhibited invasive human epidermal neoplasia induced by Ras and NF-kB blockade in a tumor cell autonomous fashion (Fig. 10.2a) (Zhang et al. 2007). The TNFR1/MKK7/JNK/AP1 cascade thus promotes human neoplasia and represents a potential therapeutic target for human epithelial cancers. Inflammation mediated by TNFa and TNFR1, is important in autoimmunity and has recently been implicated in neoplasia (Arnott et al. 2004; Balkwill 2002). In

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Fig. 10.2 (a) Interaction between the NF-kB and JNK pathways in development of skin SCC. TNFR1 engagement activates both the NF-kB and JNK pathways that have opposite activities on cyclin dependent kinase 4 (CDK4). expression. NF-kB down-regulates JNK activity through induction of antioxidant enzymes (e.g., MnSOD). which repress ROS-mediated JNK activation. NF-kB downregulation in keratinocyte leads to prolonged and enhanced activation of the JNK pathway resulting in upregulation of CDK4, increased proliferation and transformation. (b) Crosstalk between the NF-kB pathway and JAK-STAT pathway in HNSCC development. Inflammation induces NF-kB-mediated trancription of IL-6, another inflammatory cytokine that in turn acts on the gp130 receptor subunit in an autocrine fashion leading to the activation of STAT3. NF-kB and STAT3 cooperate in transcriptional activation of antiapoptotic molecules such as Bcl-XL which promote HNSCC progression.

fact TNF and TNFR1 were first shown to be involved in tumorigenesis in a mouse model of epidermal SCC. Both AP1 and NF-kB family members are concomitantly activated by signals transmitted through TNFR1, via JNK and IKK, respectively (Baud and Karin 2001; Chen and Goeddel 2002). Activation of the JNK cascade rescues Ras-induced epidermal cell cycle arrest in primary human keratinocytes (Dajee et al. 2003; Lazarov et al. 2002). Therefore one possible role for JNK cascade in Ras-induced skin tumorigenesis might be to promote escape from G1 growth restraints. Furthermore, the critical transition through the G1 phase of the cell cycle into the S1 phase and DNA replication is regulated by cyclin dependent kinases (CDKs), including CDK4/6 (Murray et al. 2004). In human keratinocytes, CDK4 down-regulation has been identified as a safeguard against neoplastic transformation by oncogenic Ras (Lazarov et al. 2002). In this context, RAS can trigger CDK4 protein degradation in a process that can be inhibited by IkB (Dajee et al. 2003), suggesting that NF-kB opposes epidermal tumorigenesis by altering the levels of a core cell cycle regulator (Fig. 10.2a). In agreement with this, NF-kB

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activation caused selective CDK4 down-regulation, which led to G1 arrest (Dajee et al. 2003; Hinata et al. 2003). Furthermore, Zhang et al. (2007) demonstrated that interfering with NF-kB function increases expression and tissue distribution of CDK4. This CDK4 up-regulation is dependent on both TNFR1 and JNK. The work of Zhang et al. ( 2007), together with data presented by others (Lind et al. 2004) support a model in which locally produced TNFa activates epidermal cell TNFR1, which then alters CDK4 protein levels in a NF-kB and JNK-regulated manner to control cell cycle progression in the epidermis (Fig. 10.2a). In contrast to many other cell types, epidermal keratinocytes display opposite responses to IKK/NF-kB and JNK/AP1 activation by inducing cell growth arrest and hyperproliferation, respectively. Unlike NF-kB, which does not affect differentiation (Dajee et al. 2003; Seitz et al. 1998), AP-1 proteins regulate epidermal differentiation (Mehic et al. 2005). The balance of these two signaling cascades and the combined cellular effects orchestrated by subtle differences in AP-1 heterodimerization may ultimately regulate epidermal homeostasis (Angel et al. 2001; Mehic et al. 2005). This possibility is further supported by the differential expression of AP-1 subunits in normal human epidermis as well as psoriatic tissues (Angel et al. 2001; Mehic et al. 2005; Haider et al. 2006). In addition, JNK isoforms have also been shown to induce differential cellular effects (Sabapathy et al. 2004; Chen et al. 2001a, b; She et al. 2002). Consistent with these notions, the role of the MKK7/JNK/AP-1 signaling cascade in epidermal differentiation appears to be complex and is context dependent. Furthermore it is reasonable to postulate that the blockade of this pathway may provide a promising approach for treating human epidermal cancers such as SCC. Indeed, combination of a JNK antagonist together with a proteasome antagonist of NF-kB activation appears to enhance cytotoxicity in HNSCC resistant to proteasome inhibitor bortezomib (Chen et al. 2008) Thus, the recent development of novel molecular-targeted therapies may now afford the rational selection of treatment modalities for SCC patients based on specific molecular mechanisms whose deregulated activity contributes to the initiation, development, and metastatic spread of this cancer type.

10.4.2 Interaction of NF-kB and the STAT Pathway As further confirmation of the emerging notion that SCC results from the aberrant activity of a network of interrelated signaling pathways rather than a dysfunction of a single biochemical cascade, a cross-talk between the NF-kB and the STAT3 signaling pathways was found to play an important role in the pathogenesis of HNSCC. The NF-kB inducible cytokine, IL-6, promotes STAT3 activation, and proliferation and survival of HNSCC (Hong et al. 2000; Squarize et al. 2006; Lee et al. 2006; Lee et al. 2008). HNSCC progression often involves the accumulation of a number of genetic and epigenetic alterations in tumor suppressor proteins, such as p53, p16, and RB, concomitant with aberrant activity of signaling molecules that drive unrestricted growth of HNSCC cells (Forastiere et al. 2001). Roughly, 90% of

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all HNSCC cases exhibit enhanced expression of epidermal growth factor receptor (EGFR) (Dassonville et al. 1993; Grandis et al. 1998a; Grandis and Tweardy 1993) and approximately 50% of all advanced HNSCC cases present an elevated activity of this polypeptide growth factor tyrosine kinase receptor (Kong et al. 2006). Many of the downstream targets for EGFR, including STAT3, the Ras–ERK, and the PI3K–Akt pathways are also often activated in HNSCC (Grandis et al. 1998b, 2000; Amornphimoltham et al. 2004). However, STAT3 activation in HNSCC cells, was found to be EGFR-independent and instead is mediated by the gp130 cytokine receptor (Squarize et al. 2006; Lee et al. 2006). Activation of gp130 is primarily driven by the inflammatory cytokine IL-6 which is produced by HNSCC cells themselves (Sriuranpong et al. 2003). Interestingly production of IL-6 by HNSCC cells requires NF-kB activity (Duffey et al. 1999) (Fig. 10.2b). Inhibition of NF-kB led to down-regulation of IL-6 expression, together with a decreased release of many other inflammatory cytokines, such as IL-8, IL-10, G-CSF, and GM-CSF (Duffey et al. 1999; Squarize et al. 2006). Contrarily to skin SCC, the majority of HNSCC display constitutive NF-kB activation (Ondrey et al. 1999) correlating with increased IL-6 expression in a statisticallty significant manner (Squarize et al. 2006). These results identify at least three levels for possible therapeutic intervention: inhibition of NF-kB activation, IL-6 blockade, and inhibition of STAT3 activation. For each of these, drugs have been developed and introduced in clinical trials (Nishimoto et al. 2000; Wu et al. 2009; Kandel 2009). The NF-kB-IL-6-STAT3 crosstalk was recently shown to play a critical role in the development of colitis associated cancer, although in that case IL-6 is produced by lamina propria macrophages rather than by colon adenocarcinoma cells (Grivennikov et al. 2009).

10.4.3 Interaction of NF-kB and TGFb Pathway The TGFb signaling pathway performs an essential regulatory role in maintaining normal epithelial homeostasis (Derynck et al. 2001) (Siegel and Massague 2003). TGFb signals through three TGFb receptor (TbR) subunits (TbRI, TbRII, and TbRIII), resulting in phosphorylation of Receptor-Smad (R-Smad) Smad2 and Smad3, which, with Smad4, enter the nucleus and regulate transcription. TGFb potently inhibits epithelial proliferation through induction of the cyclin-dependent kinase inhibitor genes p15INK4b, p21Cip1, and p57Kip2 and down-regulation of c-MYC and ID1 expression (Derynck et al. 2001). Thus, TGFb signaling shows potent tumor suppression activity, and transcriptional inactivation or mutations of TbRI and TbRII have been reported in human epithelial malignancies (Markowitz and Roberts 1996). Whereas mutation of TbRII is uncommon in HNSCC development, decreased TbRII expression occurs frequently and leads to a less differentiated, more aggressive phenotype (Huntley et al. 2004; Wang et al. 1997). TGFb was detected in both in situ and invasive SCCs at levels that correlate with malignancy (Oft et al. 1996; Oft et al. 1998; Han et al. 2005). Experimental evidence suggests that in SCCs TGFb is required for progression from carcinoma in situ to invasive

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cancer and for the epithelial to mesenchymal transition (EMT) that results in the genesis of spindle cell carcinomas and a metastatic phenotype (Oft et al. 1996; Oft et al. 1998; Han et al. 2005; Li et al. 2006). In addition, TGFb overproduced by epithelial cells acts on the tumor microenvironment to induce the release of inflammatory cytokines, metalloproteinases, and angiogenic factors that contribute to the progression and invasiveness of carcinoma cells, which become resistant to TGFb mediated growth arrest (Li et al. 2006; Gomis et al. 2006a; Gomis et al. 2006b). TGFb and NF-kB interact at multiple levels in both normal epithelia and in their transformed derivatives. The TGFb-induced signaling protein Smad7 bind to TAB2 and TAB3, which are adaptors that link the kinase TAK1 to “upstream” regulators in the TNFR1 signaling pathway (Hong et al. 2007). The formation of Smad7TAB2 and Smad7-TAB3 complexes results in suppression of TNFR1 signaling through TAK1. Furthermore, expression of a transgene encoding Smad7 in mouse skin suppressed inflammation and NF-kB nuclear translocation substantially and disrupted the formation of endogenous TRAF2-TAK1-TAB2 and TRAF2-TAK1TAB3 complexes. Thus, Smad7 is a critical mediator of TGFb signals that block proinflammatory TNF signals (Hong et al. 2007). Deficient expression of TGFb receptor II (TbRII) is common in human HNSCC and in combination with carcinogen or oncogenic ras promotes HNSCC in TbRII-/- mice (Lu et al. 2006). Recently, deficiency in expression of TbRII was found to contribute to enhanced responsiveness to TNF-a and NF-kB activation of human HNSCC lines, and NF-kB activation, proinflammatory cytokine expression and HNSCC in squamous epithelia in these mice (Cohen et al. 2009). A second mode of interaction between TGFb and NF-kB signaling pathways occurs at the IKKa level. IKKa is one of the components of the IKK complex, although, IKKa also functions as a molecular switch that controls epidermal differentiation (Hu et al. 2001). This unexpected function requires IKKa nuclear translocation but does not depend on its kinase activity, and is independent of NF-kB signaling. Ikka–/– mice present with a hyperproliferative and undifferentiated epidermis characterized by complete absence of a granular layer and stratum corneum (Hu et al. 2001). The function of IKKa in keratinocytes is strictly linked to regulation of the TGFb pathway. A critical role for IKKa in TGFb signaling in stratified epithelia was recently identified during normal keratinocyte differen tiation. IKKa directly interacts with R-SMADs and promotes transcription of anti-proliferative TGFb target genes such as Mad1 and Ovol1 (Myc inhibitors), independently of SMAD4 (Fig. 10.3b), thus promoting definitive exit from cell cycle and terminal differentiation (Descargues et al. 2008). This newly identified function of IKKa is also important in SCC development. Indeed, IKKa also acts as a tumor suppressor in stratified epithelia in a NF-kB independent manner, and its expression and nuclear localization are progressively down-regulated during malignant progression of SCC and acquisition of an invasive phenotype (Maeda et al. 2007; Marinari et al. 2008). The interaction between IKKa and the TGFb signaling pathway is defective in a subset of SCCs. SCC cell lines displaying reduced IKKa expression or that are unable to shuttle IKKa to the nucleus. These cells also display defective TGFb-induced growth arrest due to inability to activate

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Fig. 10.3 (a) Interaction between NF-kB and the p53 family of tumor suppressors in SCC. Mutant p53 potentiates the activation of anti-apoptotic and proliferative NF-kB-dependent genes by repressing TGFb receptor expression thus impairing TGFb-mediated inhibition of TNF signaling. DNp63, a p53 paralogue overexpressed in SCC binds to NF-kB subunit cRel and represses transcription of the CDK inhibitor p21 thereby promoting cancer cell proliferation. (b) Interaction between IKKa and the TGFb signaling pathway in SCC progression. IKKa, which is downregulated in invasive SCC, also acts as a component of the TGFb signaling pathway. IKKa binds to phosphorylated R-SMADs after receptor engagement and translocates to the nucleus where it is involved in the activation of a subset of TGFb anti-proliferative genes such as Mad1 and Ovol1. Downregulation of IKKa and inability to shuttle it to the nucleus impairs anti-proliferative signals and promotes cancer growth and invasivity.

TGFb-dependent antiproliferative genes such as CDKi and Myc inhibitors. Sensitivity to TGFb together with activation of TGFb antiproliferative target genes such as Mad1 and Ovol1 was rescued in these cells by introduction of a constitutively nuclear IKKa variant. SCC cell lines overexpressing nuclear IKKa are unable to grow when xenografted in SCID mice, further confirmig an important tumor suppressive role for IKKa (Marinari et al. 2008). These results suggest that the tumor-suppressive activity of IKKa in stratified epithelia may be exerted in part via the TGFb signaling pathway and that components of NF-kB pathway can regulate SCC progression in NF-kB independent manner.

10.4.4 NF-k B and the p53 Pathway Another pathway with which NF-kB interacts is the p53 pathway, the most frequently altered pathway in human cancers. Mutations in the p53 tumor suppressor gene often lead to the constitutive overproduction of mutant p53 proteins, which may exert a cancer-promoting activity (Strano et al. 2007; Weisz et al. 2007a).

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Recently a link between such activated p53 proteins and NF-kB signaling was discovered (Weisz et al. 2007b). Activated mut-p53 can promote NF-kB activation in response to cytokine stimulation in cultured cancer cells and also within real tumors (Weisz et al. 2007b). However, it remains to be determined whether all, or only some, tumor-associated p53 mutations exert a similar effect. Mutant p53 expression correlates positively with NF-kB activity in cultured cancer cells even without external triggers (Gulati et al. 2006; Scian et al. 2005). Thus, mutant p53 may maintain higher basal NF-kB activity, which is further elevated when a proper activation signal such as TNFa is delivered. In particular, basal NF-kB activity can be augmented by the transactivation potential of mutant p53 resulting in enhanced expression of NF-kB target genes in human premalignant and malignant HNSCC lesions (Weisz et al. 2007). The molecular mechanism whereby this is achieved awaits further elucidation, although the data suggest that mutant p53 may affect both the strength and duration of NF-kB activation. Recently, the Van Waes group demonstrated that inhibition of the TGFb signaling pathway may constitute an important link between p53 mutational status and overexpression of NF-kB target genes in HNSCC (Fig. 10.3a)(Cohen et al. 2009). HNSCC lines overexpressing mutant (mt) TP53 and human tumor specimens with positive TP53 nuclear staining exhibited reduced TGFb Receptor II (TbRII) expression and knocking down mtTP53 induced TbRII, increasing TGFb downstream gene expression while inhibiting proinflammatory NF-kB target gene expression. Transfection of ectopic TbRII directly restored TGFb signaling while inhibiting IkBa degradation and suppressing serine-536 phosphorylation of NF-kB p65 and NF-kB transcriptional activation, linking these alterations. Finally, experiments with TbRII conditional knockout mice show that abrogation of TGFb signaling promotes the sustained induction of NF-kB and its proinflammatory target genes during HNSCC tumorigenesis and progression. These findings elucidate a regulatory framework in which attenuated TGFb signaling promotes NF-kB activation and squamous epithelial malignancy in the setting of altered TP53 status. The p53 family of trascription factors contains two more genes p63 and p73, which are only rarely mutated in human cancers (Deyoung and Ellisen 2007; Levrero et al. 2000). Recently, a link between DNp63, the isoform most abundantly expressed in basal layer of stratified epithelia and in SCCs, and NF-kB activation was identified (King et al. 2008). In normal keratinocytes overexpressing DNp63a and in human SCC, cells DNp63a physically associates with phosphorylated, transcriptionally active nuclear c-Rel, resulting in increased c-Rel nuclear accumulation. Importantly, enhanced cell proliferation driven by elevated DNp63a are attenuated by silencing of c-Rel or overexpression of non degradable IkBa, indicating that c-Rel containing complex formation is critical to the ability of elevated DNp63a to maintain proliferation in the presence of growth arresting signals (King et al. 2008). Consistent with a role in growth regulation, DNp63a-c-Rel complexes bind a promoter motif and repress expression of the cyclin-dependent kinase inhibitor p21WAF1 in human SCC cells or keratinocytes that overexpress DNp63a (Fig. 10.3a). The relationship between DNp63a and activated c-Rel is reflected in their strong nuclear staining in the proliferating compartment of HNSCC (King et al. 2008).

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10.5 Therapeutic Strategies Targeting NF-kB in Cancer Molecular studies demonstrated that widely used drugs such as NSAIDs and glucocorticoids, known for their chemopreventive or therapeutic activities against human cancers, inhibit NF-kB, among other biological effects (Roman-Blas and Jimenez 2008). It is therefore possible that some of the anti-cancer activities of NSAIDs and glucocorticoids could be exerted through inhibition of NF-kB. These findings resulted to the initiation of new clinical trials with old compounds such as sulfasalazine that inhibits NF-kB signaling (Sebens et al. 2008). However, novel agents were developed as deliberate NF-kB or IKK inhibitors (Lee and Hung 2008) whereas other agents target generally important processes that also affect NF-kB activation, such as the proteasome (Teicher et al. 1999). A comparison between the two types was conducted on prostate cancer cell lines, demonstrating that proteasome inhibitors generally require lower concentrations to preclude NF-kB activation of by either TPA or TNFa (Gasparian et al. 2009). This observation by itself does not invalidate the strategy of IKK inhibition, as the disparity may reflect different affinity of the inhibitors to their targets, different rates of accumulation, efflux and degradation within a cell (Gasparian et al. 2009). Today, the most impressive clinical data have been obtained with bortezomib, a proteasome inhibitor, for the treatment of multiple myeloma. Bortezomib ([Velcade®], formerly known as PS-341) is a boronic acid derivative that blocks the active site of the proteasome with a high degree of selectivity against other proteases (Chang et al. 2004; Teicher et al. 2004) and is currently Food and Drug Administration approved for the treatment of recurrent or refractory multiple myeloma (Saunders 2005). Proteasome inhibition has been shown to attenuate NF-kB activation, as well as increase expression of cell cycle inhibitors and tumor suppressors such as p53 and p21 (Adams et al. 1999). In HNSCC cells, treatment with bortezomib downmodulates expression of NF-kB -regulated genes cyclin D1, Bcl-XL, IAP-1, IL-6 and VEGF; inhibits cellular proliferation and angiogenesis, and promotes apoptosis (Sunwoo et al. 2001; Van Waes et al. 2005). Consistent with NF-kB target gene products inducing drug or radiation resistance, inhibition of NF-kB with bortezomib was shown to have radiosensitizing effects in several SCC preclinical models (Van Waes et al. 2004). A phase I clinical trial is under way examining the effects of bortezomib with concurrent irradiation in patients with recurrent or metastatic HNSCC (Van Waes 2007). Although only a relatively low starting dose level has been investigated, preliminary results indicate that HNSCC tumors from a subset of patients appear to be sensitive to proteasome inhibition, demonstrating nuclear p65/RelA and increased apoptosis, along with changes in NF-kB target gene expression. Dose-limiting toxicities of hypotension and hyponatremia were observed in early cohorts of this phase I trial; however, grade 3 or 4 mucositis anticipated in a trial with re-irradiation or radiosensitization was not observed, suggesting a possible antiinflammatory and mucoprotective effect of NF-kB inhibition on normal tissue (Van Waes 2007). In addition to its potential radiosensitizing effects, administration of proteasome inhibitors with

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systemic chemotherapy agents such as camptothecin, cisplatin, and paclitaxel may result in additive or synergistic activity. ECOG 1304, a study examining the utility of combining bortezomib with irinotecan, is currently under way (Van Waes 2007). Potentially, combining targeted therapies with systemic chemoterapeutic agents may allow for greater cytotoxicity at lesser doses in vivo, creating HNSCC treatment regimens with greater efficacy and less acute and long-term toxicities (Van Waes 2007). These data support continued investigation into combination therapy involving these agents in both the preclinical and clinical setting.

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Chapter 11

Regulation of Squamous Cell Carcinoma Carcinogenesis by Peroxisome ProliferatorActivated Receptors Jeffrey M. Peters and Frank J. Gonzalez

Abstract Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that respond to endogenous and exogenous signaling molecules and modulate cellular functions. PPARs regulate many target genes that in turn influence cell growth, differentiation, and inflammatory signaling. These changes can occur through direct transcriptional up-regulation of target genes, through secondary changes subsequent to direct transcriptional up-regulation of target genes, and through protein-protein interactions. The focus of this chapter is to summarize PPAR-dependent regulation of cellular signaling in squamous epithelium, and how these changes influence cancer.

11.1 Introduction 11.1.1 Transcriptional Regulation by PPARs Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily. Three PPARs have been identified including PPARa, PPARb/d (also referred to as PPARb or PPARd), and PPARg. Structurally, PPARs have important features, some of which are shared with other members of the nuclear receptor superfamily including a ligand binding domain, a region important for heterodimerization of retinoid X receptor (RXR) and coactivator recruitment, and a DNA-binding domain that is important for binding to specific target gene regulatory elements. The ligand binding domain and DNA-binding domain of PPARs are similar to other nuclear receptors, but coding differences in the ligandbinding domain allow for discrimination between ligands and target gene activation specificities. PPARs modulate gene expression through a classic mechanism. J.M. Peters (*) Department of Veterinary and Biomedical Sciences, The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_11, © Springer Science+Business Media, LLC 2011

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Following ligand binding, PPARs heterodimerize with another nuclear receptor, RXR, and recruit coactivators with histone acetylase activity (Fig. 11.1). The activated transcriptional complexes then bind to direct repeat 1 elements, also called peroxisome proliferator response elements (PPREs), leading to recruitment of other transcription factors including RNA polymerase leading to up-regulation of gene expression. Through this mechanism, PPARs modulate many biological processes and regulate homeostasis. PPARs can modulate cellular responses by interfering with other transcription factors such as the p65 subunit of nuclear factor kappa B (NF-kB) (Fig. 11.1). Thus, PPARs can regulate cellular and physiological function by directly modulating target gene expression and indirectly modulating other transcription factors. PPARs are activated by specific ligands and it is thought that endogenous fatty acids and fatty acid derivatives are natural ligands for PPARs. For example, linoleic acid can activate PPARa and PPARb/d while 15-deoxy-D12, 14-prostaglandin J2 (PGJ2) specifically activates PPARg (Forman et al. 1997). Additionally, synthetic high affinity agonists of PPARs have been developed including the PPARg ligands rosiglitazone and pioglitazone (thiazolidinedione class of type 2 diabetic drugs), the PPARa ligands gemfibrozil and fenofibrate (fibrate class of hypolipidemic drugs), and the PPARb/d ligands GW501516 and GW0742 (reviewed in Peraza et al. 2006). In addition to regulation based on the presence of intracellular ligands

Fig. 11.1 Regulation of transcription by peroxisome proliferator-activated receptors (PPARs). Acting as ligand-activated transcription factors, PPARs can directly modulate target gene expression by facilitating recruitment of RNA polymerase. This is the classic mechanism of PPAR action. PPARs can also interact with other proteins such as the p65 subunit of NF-kB, AP1, STAT3, etc. and interfere with this signaling. This is one mechanism by which PPARs can inhibit expression of genes. This is particularly important for the anti-inflammatory activities associated with PPARs.

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(e.g., they become activated when endogenous or exogenous agonists are present in sufficient concentration), the expression patterns of the PPARs is another level of regulation. PPARs are expressed at varying levels in many tissues, with higher expression found in tissues where PPARs function in the control of lipid homeostasis. For example, expression of PPARa is high in hepatocytes where it functions to regulate expression of fatty acid catabolizing enzymes (as reviewed in Peters et al. 2005). PPARg is expressed at high levels in adipose tissue where it functions to mediate adipogenesis and lipid storage (Spiegelman et al. 1997). Expression of PPARb/d is notably higher in intestinal epithelium and keratinocytes (Girroir et al. 2008b) where it functions to mediate the induction of terminal differentiation (Burdick et al. 2006; Peters et al. 2008). The role of PPARs in squamous epithelium such as skin is just beginning to emerge. All three PPARs are expressed in skin, skin cancer models, and squamous cell carcinomas (SCCs), and despite some differences in expression, functional roles for all three PPARs have been described in squamous epithelium, which is the focus of the remainder of this chapter.

11.2 Modulation of Skin Cancer by PPARa The limited numbers of studies that have examined the functional role of PPARa in SCC are focused on skin. Expression of PPARa is lower in normal epidermis as compared to SCC based on immunohistochemical analysis of fixed tissue sections from 35 human patients (Nijsten et al. 2005). However, these data are based solely on immunohistochemistry (IHC), which has proved to be unsuitable in some instances due to nonspecific immunoreactivity of PPAR antibodies and the lack of appropriate controls (Foreman et al. 2009). Thus, in the absence of supportive data demonstrating more quantitative measures of protein expression that correlate with functional differences in PPAR-dependent target gene expression, data obtained for PPAR expression based solely on immunohistochemistry must be viewed with caution. However, there is one report that more specifically examined the role of PPARa in skin SCC. Thuillier et al. determined the effect of PPARa ligands on skin tumorigenesis in a two-stage chemical carcinogenesis bioassay (Thuillier et al. 2000). Topical application of Wy-14,643 or conjugated linoleic acid (CLA) inhibited skin tumor multiplicity, whereas topical application of either bezafibrate or troglitazone had little effect at inhibiting skin tumor multiplicity (Thuillier et al. 2000). Similar to results described above for human SCC, expression of PPARa as determined by IHC suggested lower levels in mouse SCC, but no quantification of the receptor was reported (Thuillier et al. 2000). In contrast, expression of PPARa was reported to be higher in the 308 or JWF2 and CH72 cell lines that represent papilloma and carcinoma stages of tumor progression, respectively, but quantification or statistical comparisons of PPAR expression were not done. Although these data are consistent with the idea that activation of PPARa inhibits tumor formation, the fact that both CLA and Wy-14,643 can function through other mechanisms besides PPARa makes this conclusion less clearcut.

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11.2.1 PPARa-Dependent Regulation of Differentiation and Inflammation in Skin One of the first observations linking PPARa with epidermal function was the observation that epidermal barrier development was increased by PPARa ligands (Hanley et al. 1997). Because the stratum corneum, the outermost layer of epi dermis, provides the layer between the environment and organisms with squamous epidermis, this study suggested that PPARa ligands could function to protect against exposure to xenobiotics, ultraviolet (UV) light, and other stressors by accelerating barrier development. Shortly after this report, the same group went on to demonstrate that the improvement in barrier formation by these ligands was likely mediated by PPARa-dependent regulation of terminal differentiation. Indeed, activating PPARa in human keratinocytes with clofibrate causes an increase in expression of involucrin (IVL) and transglutaminase-1 (TGM1) with an increase in cornified envelopes and a decrease in cell proliferation, which are all associated with induction of keratinocyte terminal differentiation (Hanley et al. 1998). Similarly, increased expression of filaggrin and loricrin were also noted in fetal rat epidermis following treatment with a PPARa ligand (Komuves et al. 1998). The increase in terminal differentiation marker expression did not occur in Ppara-null mice demonstrating the receptor dependence of this response. The mechanism by which PPARa induces terminal differentiation in keratinocytes remains uncertain. There is no evidence to date showing that the mechanism of action of PPARa is through binding to a functional PPRE in the promoter of genes important for keratinocyte terminal differentiation. While clofibrate and Wy-14,643 caused activation of an Involucrin promoter-luciferase construct (Komuves et al. 2000), subsequent mutational analysis of the AP-1 binding site in the Involucrin promoter suggested that PPAR responsiveness required AP-1. Thus in this context, activating PPARa causes an increase in AP1-dependent signaling in contrast to reports showing that PPARa can interact and inhibit AP-1 signaling in other models such as vascular smooth muscle cells (Delerive et al. 1999). More studies are necessary to determine how PPARa specifically regulates terminal differentiation in keratinocytes as this could occur through both indirect and direct transcriptional mediated mechanisms (Fig. 11.2). Because inhibition of inflammation is known to be associated with suppression of tumorigenesis, the inhibitory effect of PPARa ligands observed in a chemicallyinduced skin cancer model (Thuillier et al. 2000) could also be mediated in part by PPARa-dependent inhibition of inflammation. Consistent with this idea, ligands for PPARa inhibit UV-induced inflammation in human keratinocytes and topical application of a PPARa ligand increases the minimal erythema dose in UV-irradiated human skin (Kippenberger et al. 2001). Whether this effect was due to a sunscreen effect was not controlled for because topical application of the PPARa ligand preceded UV exposure. Interestingly, UV exposure also caused a decrease in the expression of PPARa, PPARb/d, and PPARg in human HaCaT keratinocytes (Kippenberger et al. 2001), suggesting that the observed increase in inflammation

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Fig. 11.2 Modulation of skin tumorigenesis by PPARa. PPARa agonists have been shown to inhibit chemically induced skin tumorigenesis. The mechanisms underlying this effect could involve direct modulation of target genes that might include those encoding proteins required for terminal differentiation that have not been identified to date. There is some evidence suggesting that this may be due to PPARa modulation of AP-1 signaling. Anti-inflammatory activities of PPARa and its ligands could also participate.

observed in response to UV exposure may be due in part to reduced expression of PPARs, which all possess potent anti-inflammatory activities (Hong and Tontonoz 2008). PPARa-dependent anti-inflammatory activities in skin have also been noted in atopic dermatitis models (Staumont-Salle et al. 2008). Ligand activation of PPARa can also inhibit inflammation by repressing other transcription factors including NF-kB, STAT, and AP-1 (Hong and Tontonoz 2008). The precise mechanism of the anti-inflammatory effects of PPARa and its ligands include both direct protein-protein interactions and receptor-independent effects (e.g., inhibition of proinflammatory enzymes) (Hong and Tontonoz 2008). Combined, there is some evidence suggesting that the inhibition of skin tumorigenesis observed with CLA and Wy-14,643 in a two-stage chemical carcinogenesis bioassay (Thuillier et al. 2000) could be due to the induction of terminal differentiation and/or through anti-inflammatory activities mediated by PPARa (Fig. 11.2). More studies are necessary to more definitively determine how PPARa influences SCC. It is of interest to note that PPARa regulates fatty acid catabolism in most tissues, but PPARa ligands are associated with lipid accumulation in the epidermis. The reason for this paradoxical difference is uncertain.

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11.3 Modulation of SCC by PPARb/d While there are more reports examining the potential role of PPARb/d in SCC as compared with PPARa, the data are still limited. These analyses have included description of PPARb/d expression and examining the effect of ligand activation of PPARb/d in skin SCC models coupled with knockout mouse approaches. Expression of PPARb/d is higher in skin SCC (Nijsten et al. 2005) and head and neck SCC (Jaeckel et al. 2001). However, these data were based in part on IHC and/ or unquantified analysis and therefore should be confirmed by direct quantitation of PPARb/d mRNA and protein. This is particularly true because nonspecific immunoreactivity of anti-PPARb/d antibodies can be a major problem (Foreman et al. 2009). For example, while one study using IHC had suggested increased expression of PPARb/d in colon in an Apcmin mutant mouse model (Ouyang et al. 2006), subsequent analysis using quantified western blotting with the same samples showed no increase in expression of PPARb/d (Foreman et al. 2009). Thus, while there is some evidence that PPARb/d may be increased in SCC, these data are limited. The effect of PPARb/d in SCC has also been examined more directly using high affinity ligands and knockout mouse models. Skin tumor formation induced by a 7,12-dimethylbenz[a]anthracene (DBMA)-12-O-tetradecanoylphorbol-13acetate (TPA) protocol is exacerbated in Pparb/d-null mice (Kim et al. 2004) suggesting that ligand activation of PPARb/d could be chemopreventive for chemically induced skin cancer. Consistent with this hypothesis, ligand activation of PPARb/d during the promotion phase of chemically induced skin cancer inhibited the onset of papilloma formation, the incidence of papillomas, and papilloma multiplicity (Bility et al. 2008). The average number of keratocanthomas was significantly reduced by ligand activation of PPARb/d in the wildtype mice and this effect was not found in Pparb/d-null mice (Bility et al. 2008). While keratocanthomas are benign lesions in humans, they can progress to malignant carcinomas in mice (Knutsen et al. 1986). This suggests that ligand activation of PPARb/d could potentially inhibit malignant conversion but was not examined due to the lack of SCC in wild-type mice (Bility et al. 2008). However, SCCs were only found in Pparb/d-null mice, and administration of a highly specific PPARb/d ligand had no influence on this endpoint (Bility et al. 2008). In contrast to these studies showing exacerbated skin tumorigenesis in Pparb/d-null mice where PPARb/d expression is globally deleted, selective deletion of PPARb/d in basal keratinocytes had no influence on chemically induced skin cancer (Indra et al. 2007). This suggests that exacerbated skin tumorigenesis observed in global Pparb/d-null mice could be mediated by mechanisms not associated with basal keratinocytes. Ligand activation of PPARb/d is also reported to inhibit cell proliferation of a 308, Sp1 and Pam212 skin cancer lines (Bility et al. 2008) suggesting that PPARb/d might be suitable for targeting for chemotherapy.

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11.3.1 PPARb /d -Dependent Regulation of Differentiation and Inflammation in Skin The chemopreventive effects of PPARb/d on skin cancer are likely mediated by modulation of cellular differentiation and inflammation (Fig. 11.3). One of the first roles described for PPARb/d in regulating cell growth was suggested by the observation that phorbol ester-induced hyperplasia is exacerbated in the absence of PPARb/d expression (Peters et al. 2000). This observation was confirmed using a different Pparb/d-null mouse (Michalik et al. 2001), suggesting that PPARb/d can attenuate cell proliferation in keratinocytes. One possible mechanism for attenuation of cell proliferation is that PPARb/d can induce terminal differentiation. Expression of PPARb/d precedes induced terminal differentiation in skin (Matsuura et al. 1999). Similarly, four independent laboratories have shown that ligand activation of PPARb/d mediates terminal differentiation in keratinocytes (Kim et al.

Fig. 11.3 Modulation of skin tumorigenesis by PPARb/d. In the absence of PPARb/d expression, chemically induced skin tumorigenesis is exacerbated. Ligand activation of PPARb/d can inhibit chemically induced skin tumorigenesis and this effect is not found in Pparb/d-null mice. The mechanisms underlying this chemopreventive effect could involve direct modulation of PPARb/d target genes that may include proteins essential for the induction of keratinocyte terminal differentiation and/or apoptotic signaling. Anti-inflammatory activities of PPARb/d and its ligands could also contribute the mechanisms of this chemopreventive effect. PPARb/d ligands can also function through mechanisms that are receptor-independent (e.g., inhibition of myeloperoxidase activity) that might also lead to inhibition of tumorigenesis.

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2006; Schmuth et al. 2004; Tan et al. 2001; Westergaard et al. 2001) and induction of terminal differentiation by PPARb/d does not occur in the absence of PPARb/d expression (Kim et al. 2006). Ligand activation of PPARb/d also increases expression of terminal differentiation markers in 308, Sp1 and Pam212 skin cancer lines (Bility et al. 2008), which is of interest because these cell lines are resistant to calcium-induced differentiation. The specific mechanism by which ligand activation of PPARb/d induces terminal differentiation is not clear, but one gene product associated with this process, adipocyte differentiation-related protein is a direct target gene of PPARb/d (Chawla et al. 2003). It is known that there is an increase in the expression of genes required for terminal differentiation in keratinocytes such as TGM1, several keratins, and small proline-rich proteins following ligand activation in keratinocytes (Kim et al. 2006; Schmuth et al. 2004; Tan et al. 2001; Westergaard et al. 2001). Whether PPARb/d directly regulates these genes due to PPREs in the promoter is unknown but should be evaluated. In addition to keratinocytes, there are many studies suggesting that PPARb/d also mediates terminal differentiation in other cell types (Aung et al. 2006; Burdick et al. 2007; Di Loreto et al. 2007; Hollingshead et al. 2008; Man et al. 2007; Marin et al. 2006; Matsuura et al. 1999; Nadra et al. 2006; Saluja et al. 2001; Varnat et al. 2006; Vosper et al. 2003; Werling et al. 2001). There is a good correlation between the induction of terminal differentiation and cell proliferation, and a large body of evidence exists showing that PPARb/d inhibits cell growth. This association was established in keratinocytes where the induction of terminal differentiation by PPARb/d was first identified (Borland et al. 2008; Burdick et al. 2007; Kim et al. 2004, 2005, 2006; Man et al. 2007; Michalik et al. 2001; Peters et al. 2000; Westergaard et al. 2001). Numerous studies have also demonstrated that PPARb/d inhibits cell proliferation in other cell types (Ali et al. 2005; Di Loreto et al. 2007; Fukumoto et al. 2005; Girroir et al. 2008a; Hollingshead et al. 2007a, 2008; Lim et al. 2009; Marin et al. 2006; Martinasso et al. 2006; Matthiessen et al. 2005; Müller-Brüsselbach et al. 2007; Otsuyama et al. 2007; Planavila et al. 2005; Sertznig et al. 2008; Teunissen et al. 2007; Yang et al. 2008). One related mechanism that may also contribute to the inhibition of cell growth found in response to ligand activation of PPARb/d is that cells undergoing terminal differentiation also exhibit an increase in apoptotic signaling. Indeed, there is evidence that PPARb/d can increase the presence of apoptosis markers including TUNEL-positive cells, caspase-3 activity, and annexin V staining (Borland et al. 2008; Kim et al. 2004; Marin et al. 2006). Convincing evidence shows that PPARb/d can inhibit keratinocyte cell growth and this effect is likely the due to induced terminal differentiation and/or apoptotic signaling. However, the specific target genes that mediate these effects are currently uncertain (Fig. 11.3). PPARb/d and ligands for PPARb/d also have potent anti-inflammatory activities in many tissue including skin and keratinocytes that could contribute to the mechanisms for the PPARb/d-dependent chemopreventive effects described above. Phorbol ester-induced inflammation (accumulation of neutrophils) in skin is greatly enhanced in the absence of PPARb/d expression (Peters et al. 2000). The observation that 1) experimentally induced colitis is exacerbated in Pparb/d-null mice (Hollingshead

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et al. 2007b), 2) cardiomyocytes from Pparb/d-null mice exhibit increased production of tumor necrosis factor-a (TNFa) in response to lipopolysacharide (LPS) (Ding et al. 2006), and 3) hepatotoxicity associated with increased NF-kB signaling and inflammation is enhanced in Pparb/d-null mice treated with carbon tetrachloride (Shan et al. 2008a, b), support the concept that PPARb/d can modulate inflammation in a variety of cell types. PPARb/d may mediate these effects by protein-protein interactions because anti-inflammatory signaling is found in the absence of exogenous ligands. However, the anti-inflammatory effects of PPARb/d could also be a result of modulation of gene expression following ligand activation. LPS-induced expression of TNFa is inhibited by GW0742 in rat cardiomyocytes (Ding et al. 2006). Additionally, PPARb/d ligands inhibit both LPS-stimulated inducible nitric oxide synthase (iNos) mRNA expression in macrophages (Welch et al. 2003) and LPS-induced expression of iNOS and monocyte chemoattractant protein-1 (MCP1) in cardiomyocytes (Planavila et al. 2005). This effect may be mediated by a direct interaction between PPARb/d and NF-kB. Similar findings of anti-inflammatory activities by PPARb/d and its ligands are found in a variety of cell models including inhibition of pro-inflammatory cytokines, inhibition of cell adhesion molecules and others (Arsenijevic et al. 2006; Barish et al. 2008; Bassaganya-Riera et al. 2004; Dyroy et al. 2007; Fan et al. 2008; Graham et al. 2005; Haskova et al. 2008; Jakobsen et al. 2006; Kang et al. 2008; Kilgore and Billin 2008; Kim et al. 2006, 2008; Man et al. 2007; Marathe et al. 2008; Nagasawa et al. 2006; Odegaard et al. 2008; Polak et al. 2005; Ravaux et al. 2007; Riserus et al. 2008; Rival et al. 2002, 2009; Rodriguez-Calvo et al. 2008; Schmuth et al. 2004; Sheng et al. 2008; Smeets et al. 2008; Takata et al. 2008). While many of the anti-inflammatory effects reported for PPARb/d and its ligands are associated with inhibition of NF-kB signaling (Fig. 11.3), it was also suggested that PPARb/d agonists may exert anti-inflammatory activities by directly interacting with ERK5 (Woo et al. 2006), or with STAT3 signaling (Kino et al. 2007). Finally, PPARb/d ligands can directly inhibit MPO (myeloperoxidase) activity, which is associated with neutrophil accumulation (Kim et al. 2006); an example of direct route for inhibition of inflammation by interfering with an enzyme. Thus, PPARb/d has potent anti-inflammatory activities. Whether these mechanisms contribute to the observed PPARb/d-dependent chemopreventive effects in skin tumorigenesis has not been critically examined. Collectively, there is evidence that PPARb/d may inhibit skin tumorigenesis by suppressing cell growth resulting from induction of keratinocyte terminal differentiation and modulating apoptosis and/or through modulation of inflammation. The specific mechanisms for these PPARb/d-dependent effects in SCC are uncertain.

11.4 Regulation of Squamous Cancers by PPARg The role of PPARg is SCC has been examined in greater detail as compared to PPARa and PPARb/d. Studies have examined expression patterns of PPARg, the effect of ligand activation on human SCC lines, the effect of ligand activation coupled

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with knockout mouse models and mechanistic studies. Interest in PPARg is greatest due to the fact that PPARg agonists are being examined as potential chemopreventive and chemotherapeutic candidates for a variety of cancers, including head and neck SCC. Based on IHC analysis of human skin SCC, expression of PPARg is not different between normal skin and SCC (Nijsten et al. 2005). In rat tongue SCC, expression of PPARg is increased as compared with normal rat tongue epithelium, but the semiquantitative analysis of these data indicate that this difference is not statistically significant (Yoshida et al. 2003). In contrast, expression of PPARg is reportedly lower in human esophageal SCC as compared with normal esophageal mucosa, as assessed by both mRNA expression and correlative IHC (Terashita et al. 2002). Similarly, exp ression of PPARg is lower in dysplastic urothelial squamous metaplasia as compared with normal urothelial epithelium (Varley et al. 2004). A number of studies have also demonstrated that SCC cell lines express PPARg but comparisons with “normal” cells is lacking. Most studies to date that have examined expression of PPARg focus on IHC and do not provide comparable markers of PPARg activity. In mice heterozygous for disruption of PPARg, DMBA-TPA-induced skin papillomas are significantly increased suggesting that constitutive expression of PPARg protects against chemically induced skin tumorigenesis (Nicol et al. 2004). Consistent with these studies, when PPARg is selectively disrupted in keratinocytes, chemically induced skin tumorigenesis is exacerbated including a significant increase in the incidence of SCC (Indra et al. 2007). Both in vivo and in vitro models have been used to extend these studies to prevention of squamous carcinogenesis through ligand activation of PPARg. Dietary administration of rosiglitazone or topical application of troglitazone did not prevent UV-induced and/or chemically-induced skin tumor development (He et al. 2005). This is the only in vivo study to date that has examined the effect of ligand activation of PPARg on skin cancer. Interestingly, expression of PPARg was not significant in normal mouse skin and essentially lacking in papillomas in two of these studies (He et al. 2005; Nicol et al. 2004), consistent with some studies suggesting that PPARg expression is diminished in tumors. Dietary administration of troglitazone significantly decreased the incidence of 4-nitroquinoline-induced rat tongue SCC (Yoshida et al. 2003), suggesting that ligand activation of PPARg can also target other squamous epithelium in addition to skin. Many studies have examined the effect of PPARg ligands on SCC cell growth using SCC cell lines, and all of these show inhibitory effects. Cell proliferation is inhibited by activating PPARg with PGJ2 in human oral SCC cell lines (SCC25, SCC9), but this effect is not found with either rosiglitazone or ciglitazone (Nikitakis et al. 2002). Cell proliferation of human oral SCC cell lines (BHY, HN, HNt) is inhibited by troglitazone and pioglitazone (Nakashiro et al. 2003). Since these effects were observed in cells that lacked significant expression of PPARg and measurable PPARg transcriptional activity, this inhibition was suggested to be a result of mechanisms that are independent of PPARg. Interestingly, the synthetic retinoid fenretidine (4-hydroxyphenylretinamide) can also cause increased activity of PPARg and significantly inhibit proliferation of CA9-22 and NA human oral SCC cell lines (Harris et al. 2005). Human esophageal SCC cell lines also exhibit inhibition of proliferation in response to PPARg ligands including T.T., T.Tn, EC-GI-10 cell lines (Rumi et al. 2002) and KYSE cell lines (Hashimoto

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Fig. 11.4 Modulation of squamous cancers by PPARg. Genetic deletion experiments indicate that skin tumorigenesis is exacerbated in the absence of PPARg expression. Studies examining the effect of ligand activation indicate that SCC is not altered by PPARg ligands in skin models, but other models of SCC (oral and esophageal) indicate some efficacy. The mechanisms underlying these effects include the induction of terminal differentiation through PPARg-dependent events, anti-inflammatory activities that may or may not be PPARg-dependent, and modulation of genes associated with apoptosis/cell cycle arrest.

et al. 2003). In contrast to these studies, an antagonist of PPARg inhibited proliferation of human oral SCC cell lines (Masuda et al. 2005). The reason why many reports suggest that activating PPARg will inhibit cell growth of SCC whereas one report suggests that inhibiting PPARg will do the same, remains to be determined. Collectively, there are compelling data from genetic deletion analysis and pharmacological activation of PPARg suggesting that targeting PPARg may prevent SCC growth, and a number of mechanisms have been postulated to explain these effects including the induction of terminal differentiation, induction of apoptosis and inhibition of inflammatory signaling (Fig. 11.4).

11.4.1 PPARg -Dependent Regulation of Differentiation and Inflammation in Skin Increased expression of PPARg is associated with the induction of terminal differentiation of human keratinocytes (Rivier et al. 1998) and activating PPARg with troglitazone inhibits cell proliferation of human keratinocytes (Ellis et al. 2000) and mouse

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keratinocytes (He et al. 2004). One mechanism that may contribute to this observed inhibition of cell growth is the induction of terminal differentiation as PPARg ligands can increase expression of TGM1 and IVL and improve barrier function in human keratinocytes (Mao-Qiang et al. 2004). However, these investigators also noted that PPARg ligands can inhibit phorbol ester-induced inflammation in epithelium, suggesting that anti-inflammatory activities associated with PPARg ligands could also contribute to the inhibition of cell growth observed in keratinocytes following stimulation with PPARg ligands. Indeed, anti-inflammatory activities of PPARg have also been delineated that are similar to other PPARs (e.g., inhibition of NF-kB signaling) while others are more unique (e.g., SUMOylated PPARg interacting with co-repressors of the NF-kB complex) (Pascual et al. 2005). Based on analysis of Pparg-null mice, the induction of terminal differentiation in keratinocytes requires PPARg while the antiinflammatory effects are independent of PPARg (Mao-Qiang et al. 2004). Increased expression of cytokeratins associated with terminal differentiation was also observed in urothelial cells in response to PPARg ligands (Varley et al. 2004). In hyperproliferative mouse skin, topical application of PPARg ligands attenuates BrdU incorporation (Demerjian et al. 2006). Cell cycle progression is blocked at the G1 phase of the cell cycle in human esophageal SCC cells following culture in PPARg ligands that is associated with increased expression of p27, p21 and p18 (Rumi et al. 2002). While there is clear evidence that PPARg is required to induce terminal differentiation in keratinocytes (Mao-Qiang et al. 2004), the inhibition of cell proliferation observed in some models appears to be mediated through mechanisms that do not exclusively require PPARg. Inhibition of PPARg activity using a PPARg antagonist or a dominant negative PPARg construct shows that decreased cell proliferation induced by troglitazone in C50 cells is independent of PPARg expression (He et al. 2004). This could be a result of downstream effects mediated by repression of inflammatory cytokines, which are thought to occur through PPARg-independent mechanisms (Mao-Qiang et al. 2004). Thus, these studies support the notion that the increased skin tumorigenesis observed when PPARg is disrupted could be due to reduced induction of terminal differentiation. These findings also support the idea that inhibition of cell proliferation observed in other SCC models could be due to PPARg-dependent and PPARgindependent mechanisms that cause induction of terminal differentiation, inhibition of cell proliferation and/or inhibition of inflammatory signaling. Indeed, in oral SCC, there is evidence that some PPARg agonists (e.g., PGJ2) can down-regulate STAT3, which could inhibit STAT3 signaling, but other PPARg ligands (e.g., rosiglitazone, ciglitazone) do not cause this effect (Nikitakis et al. 2002). Because not all PPARg agonists cause downregulation of STAT3, this supports the idea that this effect is likely not mediated by PPARg-dependent mechanisms.

11.5 Concluding Comments PPARs have the potential to be good targets for preventive and therapeutic inter vention in squamous cancer. The evidence linking PPARa with SCC carcinogenesis is limited, but there is strong evidence demonstrating that PPARa activation can

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induce terminal differentiation and inhibit cell proliferation in keratinocytes. The precise mechanisms underlying this effect remains uncertain but clearly require PPARa. There is also strong evidence showing that PPARb/d can protect against skin tumorigenesis based on studies using genetic deletion and ligand activation. Induced terminal differentiation and anti-inflammatory activities mediated through receptor-dependent and receptor-independent mechanisms appear to contribute to these effects. Loss of function analysis indicates that PPARg protects against chemically induced skin tumorigenesis including SCC, but the effects of ligand activation in this model do not support the idea that ligand activation will prevent skin SCC. Other models are more supportive of inhibitory effects in response to PPARg ligands, and these effects are likely mediated by the induction of terminal differentiation and inhibition of inflammation. As reviewed by Ondrey (2009), population-based evidence for potential role of PPARg agonists in chemoprevention of SCC emerged from analysis revealing a 40% decrease in head and neck SCC and 30% decrease in lung cancer in diabetics administered thiozolidinediones (Ondrey 2009). A recently completed NCI clinical trial with pioglitazone showed reversal of clinical lesions in patients with oral leukoplakia, which often precedes development of SCC (Ondrey 2009). Similar to PPARb/d, there is good evidence that the chemopreventive properties of PPARg ligands could be due in part to both receptordependent and receptor-independent mechanisms. In all cases, the specific target genes that mediate receptor-dependent mechanism for any of the PPARs is uncertain. Similarly, whether there is some redundancy in the target genes remains to be determined. The targeting of PPARs for the prevention and treatment of squamous carcinogenesis deserves further critical evaluation.

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Burdick AD, Bility MT, Girroir EE et al. (2007) Ligand activation of peroxisome proliferator- activated receptor-b/d (PPARb/d) inhibits cell growth of human N/TERT-1 keratinocytes. Cell Signal 19:1163–1171 Chawla A, Lee CH, Barak Y et al. (2003) PPARd is a very low-density lipoprotein sensor in macrophages. Proc Natl Acad Sci USA 100:1268–1273 Delerive P, De Bosscher K, Besnard S et al. (1999) Peroxisome proliferator-activated receptor a negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1. J Biol Chem 274:32048–32054 Demerjian M, Man MQ, Choi EH et al. (2006) Topical treatment with thiazolidinediones, activators of peroxisome proliferator-activated receptor-gamma, normalizes epidermal homeostasis in a murine hyperproliferative disease model. Exp Dermatol 15:154–160 Di Loreto S, D’Angelo B, D’Amico MA et al. (2007) PPARb agonists trigger neuronal differentiation in the human neuroblastoma cell line SH-SY5Y. J Cell Physiol 211:837–847 Ding G, Cheng L, Qin Q et al. (2006) PPARd modulates lipopolysaccharide-induced TNFa inflammation signaling in cultured cardiomyocytes. J Mol Cell Cardiol 40:821–828 Dyroy E, Rost TH, Pettersen RJ et al. (2007) Tetradecylselenoacetic acid, a PPAR ligand with antioxidant, antiinflammatory, and hypolipidemic properties. Arterioscler Thromb Vasc Biol 27:628–634 Ellis CN, Varani J, Fisher GJ et al. (2000) Troglitazone improves psoriasis and normalizes models of proliferative skin disease: ligands for peroxisome proliferator-activated receptor-gamma inhibit keratinocyte proliferation. Arch Dermatol 136:609–616 Fan Y, Wang Y, Tang Z et al. (2008) Suppression of pro-inflammatory adhesion molecules by PPAR-d in human vascular endothelial cells. Arterioscler Thromb Vasc Biol 28:315–321 Foreman JE, Sorg JM, McGinnis KS et al. (2009) Regulation of peroxisome proliferator-activated receptor-b/d by the APC/b-CATENIN pathway and nonsteroidal anti-inflammatory drugs. Mol Carcinog. doi:10.1002/mc.20546 Forman BM, Chen J, Evans RM (1997) Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors a and d. Proc Natl Acad Sci USA 94:4312–4317 Fukumoto K, Yano Y, Virgona N et al. (2005) Peroxisome proliferator-activated receptor delta as a molecular target to regulate lung cancer cell growth. FEBS Lett 579:3829–3836 Girroir EE, Hollingshead HE, Billin AN et al. (2008a) Peroxisome proliferator-activated receptorb/d (PPARb/d) ligands inhibit growth of UACC903 and MCF7 human cancer cell lines. Toxicology 243:236–243 Girroir EE, Hollingshead HE, He P et al. (2008b) Quantitative expression patterns of peroxisome proliferator-activated receptor-b/d (PPARb/d) protein in mice. Biochem Biophys Res Commun 371:456–461 Graham TL, Mookherjee C, Suckling KE et al. (2005) The PPARd agonist GW0742X reduces atherosclerosis in LDLR-/- mice. Atherosclerosis 181:29–37 Hanley K et al. (1997) Activators of the nuclear hormone receptors PPARalpha and FXR accelerate the development of the fetal epidermal permeability barrier. J Clin Invest 100:705–712 Hanley K, Jiang Y, He SS et al. (1998) Keratinocyte differentiation is stimulated by activators of the nuclear hormone receptor PPARa. J Invest Dermatol 110:368–375 Harris G, Ghazallah RA, Nascene D et al. (2005) PPAR activation and decreased proliferation in oral carcinoma cells with 4-HPR. Otolaryngol Head Neck Surg 133:695–701 Hashimoto Y, Shimada Y, Itami A et al. (2003) Growth inhibition through activation of peroxisome proliferator-activated receptor gamma in human oesophageal squamous cell carcinoma. Eur J Cancer 39:2239–2246 Haskova Z, Hoang B, Luo G et al. (2008) Modulation of LPS-induced pulmonary neutrophil infiltration and cytokine production by the selective PPARb/d ligand GW0742. Inflamm Res 57:314–321 He G, Thuillier P, Fischer SM (2004) Troglitazone inhibits cyclin D1 expression and cell cycling independently of PPARg in normal mouse skin keratinocytes. J Invest Dermatol 123:1110–1119

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Mao-Qiang M, Fowler AJ, Schmuth M et al. (2004) Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation. J Invest Dermatol 123:305–312 Marathe C, Bradley MN, Hong C et al. (2008) Preserved glucose tolerance in high fat diet-fed C57BL/6 mice transplanted with PPARg −/−, PPARd −/−, PPARg d −/− or LXRalpha b −/− bone marrow. J Lipid Res. doi:10.1194/jlr.M800189-JLR200 Marin HE, Peraza MA, Billin AN et al. (2006) Ligand activation of peroxisome proliferatoractivated receptor b/d (PPARb/d) inhibits colon carcinogenesis. Cancer Res 66:4394–4401 Martinasso G, Maggiora M, Trombetta A et al. (2006) Effects of di(2-ethylhexyl) phthalate, a widely used peroxisome proliferator and plasticizer, on cell growth in the human keratinocyte cell line NCTC 2544. J Toxicol Environ Health A 69:353–365 Masuda T, Wada K, Nakajima A et al. (2005) Critical role of peroxisome proliferator-activated receptor gamma on anoikis and invasion of squamous cell carcinoma. Clin Cancer Res 11:4012–4021 Matsuura H, Adachi H, Smart RC et al. (1999) Correlation between expression of peroxisome proliferator-activated receptor beta and squamous differentiation in epidermal and tracheobronchial epithelial cells. Mol Cell Endocrinol 147:85–92 Matthiessen MW, Pedersen G, Albrektsen T et al. (2005) Peroxisome proliferator-activated receptor expression and activation in normal human colonic epithelial cells and tubular adenomas. Scand J Gastroenterol 40:198–205 Michalik L, Desvergne B, Tan NS et al. (2001) Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)a and PPARb mutant mice. J Cell Biol 154:799–814 Müller-Brüsselbach S, Kömhoff M, Rieck M et al. (2007) Deregulation of tumor angiogenesis and blockade of tumor growth in PPARb-deficient mice. EMBO J 26:3686–3698 Nadra K, Anghel SI, Joye E et al. (2006) Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor b/d. Mol Cell Biol 26:3266–3281 Nagasawa T, Inada Y, Nakano S et al. (2006) Effects of bezafibrate, PPAR pan-agonist, and GW501516, PPARd agonist, on development of steatohepatitis in mice fed a methionine- and choline-deficient diet. Eur J Pharmacol 536:182–191 Nakashiro K, Begum NM, Uchida D et al. (2003) Thiazolidinediones inhibit cell growth of human oral squamous cell carcinoma in vitro independent of peroxisome proliferator-activated receptor gamma. Oral Oncol 39:855–861 Nicol CJ, Yoon M, Ward JM et al. (2004) PPARg influences susceptibility to DMBA-induced mammary, ovarian and skin carcinogenesis. Carcinogenesis 25:1747–1755 Nijsten T, Geluyckens E, Colpaert C et al. (2005) Peroxisome proliferator-activated receptors in squamous cell carcinoma and its precursors. J Cutan Pathol 32:340–347 Nikitakis NG, Siavash H, Hebert C et al. (2002) 15-PGJ2, but not thiazolidinediones, inhibits cell growth, induces apoptosis, and causes downregulation of Stat3 in human oral SCCa cells. Br J Cancer 87:1396–1403 Odegaard JI, Ricardo-Gonzalez RR, Red Eagle A et al. (2008) Alternative M2 activation of Kupffer cells by PPARd ameliorates obesity-induced insulin resistance. Cell Metab 7:496–507 Ondrey F (2009) Peroxisome proliferator-activated receptor gamma pathway targeting in carcinogenesis: implications for chemoprevention. Clin Cancer Res 15:2–8 Otsuyama KI, Ma Z, Abroun S et al. (2007) PPARb-mediated growth suppression of baicalein and dexamethasone in human myeloma cells. Leukemia 21:187–190 Ouyang N, Williams JL, Rigas B (2006) NO-donating aspirin isomers downregulate peroxisome proliferator-activated receptor (PPAR)d expression in APC(min/+) mice proportionally to their tumor inhibitory effect: Implications for the role of PPARd in carcinogenesis. Carcinogenesis 27:232–239 Pascual G, Fong AL, Ogawa S et al. (2005) A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma. Nature 437:759–763 Peraza MA, Burdick AD, Marin HE et al. (2006) The toxicology of ligands for peroxisome proliferator-activated receptors (PPAR). Toxicol Sci 90:269–295

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Chapter 12

p63 in Squamous Differentiation and Cancer Dennis R. Roop and Maranke I. Koster

Abstract Genes that are important for normal development and differentiation of tissues are often deregulated in cancers that originate from that tissue. An example of a gene that is both required for normal skin development and differentiation, and which is deregulated during tumorigenesis is the transcription factor p63. During epidermal development and in postnatal epidermis, p63 controls various processes including basement membrane formation, keratinocyte adhesion, terminal differentiation, and barrier formation. When p63 expression is deregulated, these processes do not occur normally, resulting in oncogenic transformation. In this chapter, we will review the role of p63 in normal epidermal development and differentiation, as well as the role of deregulated p63 expression in skin cancer.

12.1 Introduction Squamous cell carcinomas (SCCs) are tumors of epithelial origin that can occur on various body sites, including skin, head and neck, cervix, and lung. Of the various types of SCCs, skin SCCs are the most common, and they account for 20% of all skin cancers (Johnson et al. 1992). In the United States, over 250,000 new cases of skin SCCs are diagnosed each year leading to approximately 2,500 deaths (www.aad.org). The incidence of skin SCCs has increased in recent years, especially in areas with high ultraviolet (UV) exposure. In addition, cutaneous SCCs are particularly aggressive in immunosuppressed populations (e.g. organ transplant patients). Although they can be variable in appearance, most skin SCCs present as ulcerated lesions with hard, raised edges. Skin SCCs often lead to a local destruction of tissue, and surgical removal of skin SCCs may result in cosmetic deformities. Therefore, even though mortality from skin SCCs is lower than that of many other malignancies, cutaneous

M.I. Koster (*) Department of Dermatology, Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado Denver, Aurora, CO 80045, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_12, © Springer Science+Business Media, LLC 2011

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SCCs are associated with a relatively high morbidity. Epidemiological studies have linked the development of skin SCCs to chronic sun exposure, especially during childhood and adolescence (Leiter and Garbe 2008; MacKie 2006). In support of these findings, skin SCCs most frequently occur on sun-exposed areas of the body, such as the scalp, neck, and back of the hands. The link between chronic sun exposure and skin SCCs was further strengthened by the finding that chronic exposure of mice to ultraviolet B (UV-B) irradiation, the carcinogenic component of sunlight, results in the formation of skin SCCs (Forbes 1996). In the epidermis, most keratinocytes that have acquired UV-B induced DNA damage will be removed from the skin through apoptosis. However, if this process fails, the persistence of damaged keratinocytes with oncogenic mutations may ultimately lead to tumor formation. Oncogenic mutations that predispose to tumor formation often result in excessive proliferation signals, inhibiting differentiation, blocking apoptosis and/or inducing genomic instability (Renan 1993). Further, oncogenic mutations often result in the deregulation of genes that are required for normal skin development and differentiation. One gene that is required for normal skin development and differentiation, and is also deregulated during tumorigenesis is the transcription factor p63.

12.2 p53 Gene Family The p63 gene is a member of the p53 gene family which also includes p53 and p73. The p53 gene was first discovered in 1979, and although not initially appreciated, it is now known to function as a tumor suppressor gene that is frequently mutated in human cancers (Olivier et al. 2002; DeLeo et al. 1979). The two homologues of p53, p63 and p73, were identified almost two decades later (Kaghad et al. 1997; Yang et al. 1998). All three genes share a high degree of homology although p63 and p73 are more similar to each other than to p53 (Yang et al. 2002). The proteins encoded by all three genes contain the three typical domains of a transcription factor: a transactivation domain, a DNA binding domain, and an oligomerization domain (Yang et al. 2000). All three genes are also expressed as multiple isoforms generated by the use of different promoters, different translation start sites, and/or the use of alternative splicing (Murray-Zmijewski et al. 2006). However, despite their similarity in sequence, domain structure, and presence of multiple variants, the three members of the p53 gene family have strikingly different roles during development and tumorigenesis.

12.2.1 Structure 12.2.1.1 Transactivation Domain All members of the p53 gene family encode transcription factors with two different N-termini (Fig. 12.1). In all cases, these N-termini contain at least one transactivation domain and are generated through alternative promoter usage (Harms and Chen 2006). The most proximal promoter is located upstream of the first exon, and

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gives rise to full-length isoforms. For p63 and p73, these full-length isoforms contain a p53-like N-terminal transactivation domain (TA isoforms). The second promoter of p63 and p73 is located within intron 3, and gives rise to isoforms that lack the p53-like transactivation domain (DN isoforms). Because of the apparent lack of a transactivation domain, DNp63 and DNp73 proteins were initially believed to function primarily as dominant-negative molecules, and were believed to inhibit the function of the full-length TAp63 and TAp73 proteins (Serber et al. 2002; Yang et al. 1998). However, the finding that both DNp63 and DNp73 isoforms were able to activate gene expression, suggested that they may also contain a functional transactivation domain (Wu et al. 2003; Carroll et al. 2006; Dohn et al. 2001; King et al. 2003; Koster et al. 2007). Follow-up studies demonstrated that this internal transactivation domain is composed of exon 3’, the only unique exon for DNp63 and DNp73 isoforms, and the downstream located proline rich region (Liu et al. 2004; Helton et al. 2006). Because exon 3’ comprises part of this transactivation domain, this domain is unique for DNp63 and DNp73 proteins, and does not exist in TAp63 and TAp73 proteins. The presence of these different transactivation domains in DN and TA isoforms provides a plausible explanation for the finding that TA and DN isoforms can regulate the expression of different downstream target genes. Like p63 and p73, the p53 gene uses two promoters to generate full-length and truncated (DNp53) transcripts (Bourdon et al. 2005). However, unlike TAp63 and TAp73 proteins, full-length p53 proteins contain two transactivation domains, one of which is homologous to the transactivation domain that is present in TAp63 and TAp73. In contrast, the transactivation domain that is retained in DNp53 isoforms, is not homologous to the one found in DNp63 or DNp73 isoforms (Harms and Chen 2006).

12.2.1.2 DNA Binding Domain The core domains of p53, p63, and p73 all contain highly homologous, but not identical, DNA binding domains (Fig. 12.1). The DNA binding domain is responsible for the interaction of the p53, p63, and p73 proteins with recognition sites located in the regulatory regions of their target genes. The p53 binding site has been well-defined and consists of two copies of the 10-mer PuPuPuC(A/T)(T/A)

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GPyPyPy (where Pu is a purine [A or G] and Py is a pyrimidine [T or C]) separated by 2–8 nucleotides (el Deiry et al. 1992). Interestingly, p63 and p73 are also capable of interacting with p53 binding sites. In fact, in some cases, different p53 family members were found to interact with the same binding site within the promoter of target genes. For example, p53 and p63 interact with the same binding sites in the regulatory regions of the Perp and p21 (CDKN1A) genes (Ihrie et al. 2005; Westfall et al. 2003; Reczek et al. 2003). However, although there is some overlap in target genes for p53, p63, and p73, they have numerous unique target genes. For example, whereas p63 can activate expression of the epidermal-specific gene bullous pemphigoid antigen 1 (BPAG-1), p53 and p73 have no effect on the expression of this gene (Osada et al. 2005a). This differential regulation of target genes by p53, p63, and p73 is likely caused by different binding specificities for the consensus sequence. Indeed, p63 has a higher affinity for binding sites that contain a few mismatched nucleotides when compared to consensus p53 binding sites (Bian and Sun 1997; Ortt and Sinha 2006; Zeng et al. 1998; Osada et al. 2005b). 12.2.1.3 Oligomerization Domain It is well-accepted that a functional p53 transcription factor consists of a tetramer, formed by the interaction of four p53 monomers through their oligomerization domains (Chene 2001). However, despite the homologies between the oligomerization domains of p53 and p63, and p53 and p73, their oligomerization domains do not interact. One exception to this, as described in more detail below, is that some mutant forms of p53 are able to interact with p63 and p73. Furthermore, only weak interactions were observed between the oligomerization domains of p63 and p73, suggesting that these interactions may be of little biological relevance (Davison et al. 1999). However, it was demonstrated that p63 and p73 do physically interact in human head and neck squamous cell carcinomas (HNSCC), resulting in the promotion of cell survival (Rocco et al. 2006). 12.2.1.4 C-Terminal Sequences In addition to different N-terminal sequences, all members of the p53 gene family encode isoforms with different C-termini (Figs. 12.1 and 12.2). Of these, only the C-terminus of p53 contains a basic domain. This domain serves several functions, including allowing for efficient DNA binding, inhibiting transactivation of various target genes, and recognizing damaged DNA (Liu and Kulesz-Martin 2006). In contrast to p53, alternative splicing of p63 and p73 gives rise to various C-termini (three for p63 and six for p73), only the longest of which contains a SAM (sterile alpha motif) domain (Fig. 12.2) (Yang et al. 1998). SAM domains are evolutionary conserved protein–protein interaction domains that are primarily found in proteins involved in the regulation of developmental processes (Schultz et al. 1997). The

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Fig. 12.2 Structure of p63 isoforms. Alternative promoter usage gives rise to p63 isoforms containing a p53-like transactivation domain (TAp63) and isoforms lacking this domain (DNp63). DNp63 isoforms contain an internal transactivation domain which consists of exon 3¢ and the proline rich domain (PxxP). Three carboxy termini are generated by alternative splicing (a, b, g).

importance of the SAM domain for p63 protein function is highlighted by the finding that mutations in the p63 SAM domain underlie the developmental disorder Ankyloblepharon Ectodermal Dysplasia and Clefting (AEC) (McGrath et al. 2001). Patients with AEC suffer from severe skin fragility and have a combination of hair, nail, tooth, and limb abnormalities (Siegfried et al. 2005). However, the mechanism by which mutations in the p63 SAM domain cause AEC remains to be determined. Despite the critical role of the SAM domain for p63 function, few interacting proteins have currently been identified. One of these is c-Rel, a member of the nuclear factor-kB/reticuloendotheliosis family of transcription factors. c-Rel was found to interact exclusively with the SAM domain-containing DNp63a isoform, resulting in enhanced keratinocyte proliferation (King et al. 2008).

12.2.2 Expression Pattern In addition to their structural differences, another difference between the members of the p53 gene family is their expression pattern. Under homeostatic conditions, p53 protein expression is virtually undetectable. These low steady-state levels of p53 are caused by post-translational modifications which target p53 for proteasome-mediated degradation. However, this p53 degradation pathway is inhibited upon DNA damage, resulting in the accumulation of p53 protein. Under these conditions, p53 induces cell cycle arrest or apoptosis, thus preventing the persistence of cells with DNA damage (Hinds and Weinberg 1994). By removing cells with DNA damage, and thus potentially oncogenic mutations, p53 plays a critical role in tumor suppression. In vitro

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evidence suggests that, in certain cell types, such as liver and osteosarcoma cells, p63 and p73 may also be stabilized upon genotoxic stress and thus it has been suggested that these proteins may have an equivalent role in tumor suppression (Okada et al. 2002; Flores et al. 2002; Katoh et al. 2000). However, in contrast to p53, p63 and p73 are also constitutively expressed in a tissue-specific manner. p73 is expressed in a number of epithelial and neural structures, including nasal epithelium, the vomeronasal organ, the hippocampus, and the hypothalamus (Yang et al. 2000). Consistent with this expression pattern, p73 deficiency in mice leads to neurological, pheromonal, and inflammatory abnormalities (Yang et al. 2000). p63, on the other hand is primarily expressed in stratified epithelia, such as the epidermis, tongue, and esophagus (Yang et al. 1998). Loss of p63 in mice leads to aborted development of tissues that normally express this protein (Yang et al. 1999; Mills et al. 1999). Importantly, under homeostatic conditions, the three p53 family members do not have overlapping expression patterns, suggesting that they do not cooperate in gene regulation during normal development and differentiation.

12.3 p63 in Embryonic Development 12.3.1 Role of p63 in Epidermal Development Because p53 and p63 share such high homology, and even share some target genes, it was initially proposed that p63 would function much like p53. Thus, it was hypothesized that, like p53-deficient mice, mice lacking p63 would be developmentally normal and would be predisposed to develop various types of tumors. In contrast to these predictions, p63-deficient mice were found to display severe developmental defects (Yang et al. 1999; Mills et al. 1999). When p63-deficient mice are born, their skin appears translucent and the mice die shortly after birth due to excessive water loss. Upon histological examination, it was found that p63-deficient mice completely failed to develop an epidermis, and were instead covered by a single layer of epithelial cells. This single layer of epithelial cells represented cells of the surface ectoderm, the single-layered epithelium that initially covers the developing embryo and which normally develops into the epidermis (Koster et al. 2004). Thus, it was concluded that p63 plays a critical role in the earliest stage of skin development, namely epidermal specification. However, as described below, p63 plays additional roles after this initial stage of skin development has taken place.

12.3.2 p63 in Appendage Development In addition to the absence of an epidermis, p63-deficient mice display developmental abnormalities of skin appendages, such as teeth, hair follicles, and mammary glands (Yang et al. 1999; Mills et al. 1999). Furthermore, p63-deficient mice are

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born with truncated forelimbs and without hindlimbs. Interestingly, striking similarities were observed between p63-deficient mice and patients with ectodermal dysplasias, a group of disorders characterized by developmental abnormalities in appendages (Priolo et al. 2000). Indeed, it was found that several ectodermal dysplasias are caused by mutations in p63 (Rinne et al. 2007). These syndromes include, but are not limited to AEC, caused by mutations in the SAM domain, and Ectrodactyly Ectodermal Dysplasia and Clefting (EEC), caused by mutations in the DNA-binding domain (Celli et al. 1999; McGrath et al. 2001). Whereas both patients with AEC and EEC display tooth, hair, and nail abnormalities, only patients with AEC suffer from severe skin fragility (Rinne et al. 2007). In contrast, limb abnormalities are absent or mild in patients with AEC, whereas patients with EEC display severe syndactyly. Because appendage development requires extensive and continuous cross-talk between the developing epidermis and the underlying dermis (Chuong 1998), it was initially proposed that the abnormalities in appendage development in p63-deficient mice and ectodermal dysplasia patients were due to a failure of the single-layered ectoderm that covers the early embryo to develop into the normal multilayered epidermis that covers the body of newborn mice. In support of this hypothesis, it was documented that the crosstalk between the embryonic epidermis and the dermis, which is required for appendage development, does not occur normally in p63deficient mice (Mills et al. 1999; Laurikkala et al. 2006). However, in addition to this indirect role of p63 in appendage development, it was also found that p63 plays a direct instructive role in regulating the expression of genes that control appendage morphogenesis. For example, p63 directly induces expression of Dlx3, a gene which is required for the development of several skin appendages, including teeth and hair (Radoja et al. 2007). p63 is also required for the induction of P-cadherin during limb and hair follicle morphogenesis (Shimomura et al. 2008). Interestingly, inherited mutations in P-cadherin lead to limb abnormalities that are indistinguishable from those displayed by patients with EEC, suggesting that induction of P-cadherin by p63 is essential for normal limb development.

12.3.3 Role of TAp63 Isoforms in Skin Development 12.3.3.1 Commitment to Stratification While p63-deficient mice have been very useful in addressing the role this gene in normal development, the complete ablation of all isoforms of p63 in these mice prevented insight into the role of the different p63 isoforms in epidermal development and differentiation. To resolve this issue, subsequent in vitro and in vivo experiments were designed in which the role of one isoform, or one class of isoforms, was addressed (Koster et al. 2004; Koster et al. 2007; Truong et al. 2006). The role for TAp63 isoforms during epidermal development was initially addressed

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by the development of mice which inducibly express TAp63a. The goal of these experiments was to determine if TAp63a could direct the fate of single-layered epithelia into an epidermal fate. Because of technical limitations, it is not feasible to determine if expression of TAp63a in the surface ectoderm could induce an epidermal fate. Therefore, TAp63a was ectopically expressed in the single layered epithelia of the lung. Interestingly, under these conditions, TAp63a expression caused initiation of an epidermal program in lung epithelium, as evidenced by the induction of epidermal-specific genes (Koster et al. 2004). This is consistent with the finding that TAp63 isoforms are expressed when epidermal specification takes place, whereas their expression declines during later stages of epidermal development. These results suggest that TAp63 isoforms are required for the earliest stage of epidermal development, the commitment of cells of the surface ectoderm to an epidermal fate. 12.3.3.2 Terminal Differentiation In addition to the role of TAp63 isoforms during epidermal specification, it has been suggested that TAp63 isoforms may also play a role in epidermal terminal differentiation (Candi et al. 2006). Low levels of TAp63 isoforms have been reported in keratinocytes and in normal epidermis by some investigators (King et al. 2006; Gu et al. 2006). In support of a functional role for TAp63 isoforms in postnatal epidermis, it was found that down-regulating TAp63 isoforms in human skin equivalents resulted in mild defects in late stages of epidermal differentiation, namely the formation of the upper layers of the epidermis, the granular layer and the stratum corneum (Truong et al. 2006). Furthermore, it was found that TAp63 isoforms can induce the expression of genes involved in epidermal differentiation, including filaggrin and S100A2 (De Laurenzi et al. 2000; Candi et al. 2006; Kirschner et al. 2008). Collectively, these studies suggest that TAp63 isoforms may play roles in multiple steps of epidermal differentiation.

12.3.4 Role of D Np63 Isoforms in Skin Development Unlike TAp63 isoforms, DNp63 isoforms are expressed abundantly in developing and mature epidermis. The predominant DNp63 isoform that is expressed in late embryonic and postnatal epidermis is DNp63a, and its expression is primarily localized to the basal layer of the epidermis (Yang et al. 1998). However, lower levels of DNp63a protein are readily detectable in suprabasal cell layers and in differentiated keratinocytes in vitro (King et al. 2003). To further understand the role of DNp63 isoforms in the epidermis, two model systems have been employed. First, mice were generated in which DNp63 isoforms were specifically and inducibly down-regulated in the epidermis (Koster et al. 2007). Second, human skin equivalents were generated in which DNp63 expression was down-regulated using

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Terminal differentiation

Adhesion Basement membrane

Fig. 12.3 Role of DNp63 isoforms in postnatal epidermis. DNp63 isoforms are required for various processes that are essential for epidermal differentiation and epidermal integrity. Within the basal layer, DNp63a induces expression of genes that are required for basement membrane formation as well as genes required for keratinocyte adhesion. Further, DNp63a is required for the onset of epidermal terminal differentiation and for barrier formation.

s iRNAs (Truong et al. 2006). Although some differences were observed, terminal differentiation did not occur normally in either model, thus demonstrating a role for DNp63a in regulating epidermal terminal differentiation. In addition to its role in initiating an epidermal differentiation program in keratinocytes, DNp63a is also critical for cell adhesion, epidermal barrier formation, and basement membrane formation (Fig. 12.3) (Koster and Roop 2008). 12.3.4.1 Epidermal Differentiation One process that is controlled by DNp63a is the induction of epidermal terminal differentiation. Epidermal differentiation initiates when cells of the basal layer permanently withdraw from the cell cycle and move suprabasally. The terminal differentiation program ultimately results in the formation of a functional epidermal barrier. Studies using mouse models or human skin equivalents, demonstrated that a reduction in DNp63 expression led to the inability of keratinocytes to induce keratin 1 (K1), the earliest marker of epidermal differentiation (Koster et al. 2007; Truong et al. 2006). This failure to induce K1 expression was also observed in patients with AEC, a skin fragility disorder caused by p63 mutations (Koster et al. 2009). In addition, increased levels of DNp63a in primary keratinocytes led to a premature induction of K1 expression, further supporting a critical role for DNp63a in keratinocyte differentiation (Ogawa et al. 2008). Several target genes of DNp63a that mediate its ability to induce epidermal differentiation have been identified. First, DNp63a directly induces expression of IKKa, a gene required for cell cycle withdrawal during epidermal terminal differentiation (Koster et al. 2007; Marinari et al. 2008). Further, DNp63a induces the expression of genes required for cell cycle withdrawal, such as p57Kip2, a cyclin-dependent kinase inhibitor that is induced when keratinocytes undergo terminal differentiation (Beretta et al. 2005; Martinez et al. 1999). DNp63a also represses the expression of genes required for cell cycle progression, including

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cyclin B2 and cdc2 (Testoni and Mantovani 2006). Finally, DNp63a is required for the final stage of epidermal differentiation, the formation of the epidermal water barrier. At least two genes that are required for epidermal barrier formation, Alox12 and claudin 1, are directly induced by DNp63a (Lopardo et al. 2008; Kim et al. 2009).

12.3.4.2 Basement Membrane Formation The epidermis is separated from the underlying dermis by the basement membrane, which consists of extracellular matrix proteins secreted by epidermal keratinocytes and by dermal fibroblasts (McMillan et al. 2003). Keratinocytes are anchored to the basement membrane by integrins, heterodimeric transmembrane receptors that function both as cell-matrix adhesion molecules and as signaling centers for key biological processes such as migration (Larsen et al. 2006). Thus, the basement membrane is critical for maintaining the structural integrity and function of the skin. Interestingly, DNp63a is important both for the formation of the basement membrane as well as for anchoring keratinocytes to the basement membrane (Carroll et al. 2006; Kurata et al. 2004; Koster et al. 2007). First, DNp63a directly induces expression of Fras1, a major structural component of the embryonic basement membrane (Smyth and Scambler 2005). Interestingly, in mice and humans, Fras1-deficiency causes a severe embryonic blistering phenotype, demonstrating the importance for Fras1 in basement membrane integrity (Vrontou et al. 2003; McGregor et al. 2003). In addition, DNp63a induces expression of several integrin subunits, receptors for the basement membrane protein laminin (Kurata et al. 2004; Carroll et al. 2006).

12.3.4.3 Adhesion Another critical process for maintaining epidermal integrity is adhesion between keratinocytes. In the epidermis, cell adhesion between keratinocytes is mediated by multiprotein complexes called desmosomes (Cheng and Koch 2004). Interestingly, DNp63a plays a role in mediating cell adhesion by directly inducing the expression of the desmosomal component Perp (Ihrie et al. 2005). In the absence of Perp expression, mice display severe intraepidermal blistering further underscoring the importance of Perp in cell adhesion (Ihrie et al. 2005). In addition, cell adhesion defects have been reported in patients with AEC, a subset of who display aberrant Perp expression (Payne et al. 2005; Beaudry et al. 2009). Additional desmosomal components were found to be DNp63 targets in cultured mammary cells, however, it remains to be determined if DNp63 regulates the expression of these genes in the epidermis (Carroll et al. 2006).

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12.4 p63 in Squamous Cell Carcinomas 12.4.1 p63 in SCCs The p53 gene is mutated in over 50% of human tumors and in 44% of skin SCCs worldwide (Olivier et al. 2002; Petitjean et al. 2007). Most of these mutations are missense mutations leading to either a loss-of-function or a gain-of-function of the affected p53 proteins. p53-deficient mice are predisposed to develop various types of tumors, primarily lymphomas and sarcomas (Donehower et al. 1992). In contrast to p53, the two p53 homologues p63 and p73 are rarely mutated in human cancers (Mills 2006). Additionally, although germline mutations in p63 underlie a subset of ectodermal dysplasias, they are not associated with a cancer-prone phenotype (Rinne et al. 2007). This is in sharp contrast to inherited mutations in p53, which cause Li-Fraumeni syndrome, a tumor susceptibility syndrome in which patients generally develop several tumors at a young age (Malkin et al. 1990). In addition, unlike p53-deficient mice, mice heterozygous for p63 are not predisposed to develop spontaneous tumors, to develop tumors in response to a chemical carcinogenesis treatment, or to develop lymphomas upon gamma irradiation (Keyes et al. 2006; Perez-Losada et al. 2005). Although independently generated p63 heterozygous mice did show a tumor susceptibility phenotype, these mice also did not develop skin SCCs (Flores et al. 2005). Whereas mutations in p63 are rare in human cancers, amplification of the genomic region that harbors p63, 3q21-29, is frequent in human SCCs (Mills 2006). In fact, more than 50% of human head and neck SCCs (HNSCCs) display an amplification of this genomic region (Gebhart and Liehr 2000). Interestingly, both in HNSCC and lung SCC, amplification of the p63 gene was found to correlate with overexpression of p63 (Hibi et al. 2000). However, in follow-up studies, it was found that p63 overexpression is much more common than amplification of its genomic region, suggesting that mechanisms other than amplification result in increased p63 protein expression (Tonon et al. 2005; Geddert et al. 2003). In most cases, DNp63a is the p63 isoform that is overexpressed in SCCs, however, overexpression of TAp63 isoforms has also been documented in a smaller subset of SCCs (Mills 2006).

12.4.2 Role of TAp63 in SCCs 12.4.2.1 Tumor Progression As indicated above, increased TAp63 protein expression has been detected in a subset of human SCCs (Mills 2006). Although TAp63 proteins have been implicated in the induction of cell cycle arrest or apoptosis in some tissues (see below),

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increased TAp63 expression does not result in the induction of apoptosis in the epidermis (Candi et al. 2006; Koster et al. 2004). In contrast, overexpression of TAp63a in mouse epidermis led to increased proliferation and impaired differentiation (Koster et al. 2004). Further, when exposed to a skin chemical carcinogenesis protocol, mice overexpressing TAp63a develop skin SCCs at a faster rate than control mice (Koster et al. 2006). A high proportion of these skin SCCs undergo epithelial-mesenchymal transitions leading to metastasis. Therefore, whereas TAp63 induces cell cycle arrest and apoptosis in some cell types, it promotes tumorigenesis in the epidermis. 12.4.2.2 Stabilization Although some skin SCCs exhibit increased TAp63 expression, TAp63 protein expression is barely detectable in normal epidermis. In analogy with p53, a possible reason for the low TAp63 protein levels in the epidermis would be a rapid degradation of TAp63 isoforms under homeostatic conditions. Indeed, several E3 ubiquitin ligases which are responsible for ubiquitination and subsequent degradation of TAp63 proteins through the proteasome pathway have been identified (Li et al. 2008; Rossi et al. 2006). In further support of the importance of these E3 ubiquitin ligases for TAp63 degradation, it was found that down-regulation of these proteins results in increased TAp63 expression levels (Li et al. 2008; Hansen et al. 2007). Despite the low TAp63 protein levels under homeostatic conditions, it was found that, like p53, TAp63 proteins rapidly accumulate in response to genotoxic stress (Candi et al. 2007). Once stabilized, TAp63 proteins can induce cell cycle arrest or apoptosis. Like p53, in vitro evidence suggests that TAp63 isoforms can induce apoptosis both through death receptor and mitochondrial-mediated pathways (Candi et al. 2007). This is primarily accomplished by inducing the expression of classical p53 target genes, such as PIG3 and CD95 (Gressner et al. 2005; Helton et al. 2007). Interestingly, the proapoptotic role of TAp63 proteins requires both the N-terminal transactivation domain and the adjacent proline rich domain, providing an explanation for the inability of DNp63 isoforms to induce apoptosis (Helton et al. 2007; Sayan et al. 2007). Collectively, these findings suggest that stabilization of TAp63 may protect tissues from neoplastic transformation. However, whether increased TAp63 protein levels can, under certain circumstances, prevent SCC formation through this mechanism remains to be investigated. 12.4.2.3 Interaction with Mutant p53 As indicated above, p53 is one of the most frequently mutated genes in human cancers. Interestingly, a subset of mutant p53 proteins that are expressed in human cancers have acquired gain-of-function properties. One of these mutations is p53 R175H, a hot spot mutation which has been found in patients with Li-Fraumeni syndrome as well as in sporadic cancers (Petitjean et al. 2007). Mice

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that express p53 R172H (the mouse equivalent of human p53 R175H) display increased skin SCC formation and accelerated tumor progression when compared to mice that have lost p53 expression, supporting the idea that this specific p53 mutation represents a gain-of-function allele (Caulin et al. 2007). It has been postulated that the gain of function properties of mutant p53 proteins are caused, in part, by impairing the ability of TAp63 proteins to induce cell cycle arrest and/or apoptosis. Indeed, even though wild type p53 cannot interact with p63 or p73, it was found that p53 R175H can interact with both p63 and p73 (Gaiddon et al. 2001). Further, this interaction was also observed in vivo in osteosarcomas that develop in mice which express p53 R172H (Lang et al. 2004; Olive et al. 2004). The interaction between mutant p53 and TAp63 led to impaired TAp63 function as demonstrated by the inability of TAp63 to induce expression of its target genes in the presence of mutant p53 (Lang et al. 2004; Gaiddon et al. 2001; Adorno et al. 2009). Interestingly, despite the ability of the mutant p53 to interact with TAp63 in some cell types, this interaction does not occur in keratinocytes obtained from mice that express p53 R172H (Caulin et al. 2007). Thus, even though an interaction between mutant p53 and p63 may be of functional importance for a subset of human tumors, the relevance of these findings for human skin SCCs remains to be determined.

12.4.3 Role of D Np63 Isoforms in SCCs 12.4.3.1 Apoptosis During epidermal development, DNp63 isoforms have a very different role from TAp63 isoforms. Similarly, TAp63 and DNp63 isoforms play different roles during tumorigenesis. Unlike TAp63 isoforms, DNp63 isoforms are constitutively expressed at high levels in the epidermis, primarily in the basal layer. In addition to its roles in epidermal differentiation, keratinocyte adhesion, and basement membrane formation, DNp63 is involved in mediating the response to UV-B irradiation. Normally, in response to UV-B irradiation, epidermal keratinocytes undergo apoptosis, thus preventing damaged keratinocytes from persisting in the epidermis. Because keratinocytes with DNA damage could potentially undergo oncogenic transformation, UV-B-induced apoptosis is a critical mechanism for protecting the epidermis from carcinogenic events. Interestingly, when primary keratinocytes or mouse epidermis are exposed to UV-B irradiation, this results in a rapid decrease in DNp63a protein levels (Liefer et al. 2000; Westfall et al. 2005; Marchbank et al. 2003). This decrease is mediated both by a transcriptional down-regulation of DNp63a and by rapid degradation of DNp63a protein (Liefer et al. 2000; Westfall et al. 2005). These reduced levels of DNp63a allow primary keratinocytes to undergo apoptosis in response to UV-B irradiation (Ogawa et al. 2007; Lee et al. 2006). Consistent with these findings, forced expression of DNp63a in the epidermis prevents UV-B induced apoptosis (Liefer et al. 2000). This antiapoptotic role of DNp63a may be

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mediated in part by its ability to suppress the proapoptotic function of p73 (Rocco et al. 2006). Thus, down-regulating DNp63a is an important and necessary response to UV-B irradiation, allowing damaged keratinocytes to undergo apoptosis. 12.4.3.2 Tumor Progression Although increased DNp63a in the epidermis allows damaged keratinocytes to persist, thus providing a possible oncogenic stimulus, increased DNp63a expression during later stages of tumorigenesis is associated with a favorable outcome. In fact, several studies documented that SCCs that express DNp63a have a better prognosis than SCCs that do not express DNp63a (Massion et al. 2003; Zangen et al. 2005; Oliveira et al. 2007). This may be explained in part by the increased responsiveness of DNp63a-expressing SCCs to cisplatin, a chemotherapeutic agent that shows clinical activity in SCCs (Leong et al. 2007; Zangen et al. 2005). In support of a tumor-suppressive role for DNp63a, tumor progression was prevented by overexpressing DNp63a in SCC cell lines that normally metastasize (Adorno et al. 2009). Consistent with these findings, loss of DNp63 in cell lines promotes invasiveness and metastasis (Higashikawa et al. 2007; Adorno et al. 2009; Barbieri et al. 2006). In addition, loss of the DNp63a target gene Ikka from the epidermis, leads to spontaneous skin SCC formation in mice (Liu et al. 2008). Thus, even though reduced DNp63 levels in normal epidermis allow for the removal of damaged keratinocytes, loss of DNp63 at late stages of tumorigenesis appears to be a poor prognostic factor leading to increased aggressiveness of SCCs. A possible explanation for these findings would be that loss of DNp63a expression results in a loss of epithelial fate and an acquisition of a mesenchymal fate, thus resulting in the formation of aggressive spindle cell carcinomas. However, the precise mechanism underlying the poor prognosis of tumors with reduced DNp63 expression remains to be investigated.

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Harms KL, Chen X (2006) The functional domains in p53 family proteins exhibit both common and distinct properties. Cell Death Differ 13:890–897 Helton ES, Zhang J, Chen X (2007) The proline-rich domain in p63 is necessary for the transcriptional and apoptosis-inducing activities of TAp63. Oncogene 27:2843–2850 Helton ES, Zhu J, Chen X (2006) The unique NH2-terminally deleted (DN) residues, the PXXP motif, and the PPXY motif are required for the transcriptional activity of the DN variant of p63. J Biol Chem 281:2533–2542 Hibi K, Trink B, Patturajan M, Westra WH, Caballero OL, Hill DE, Ratovitski EA, Jen J, Sidransky D (2000) AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sci USA 97:5462–5467 Higashikawa K, Yoneda S, Tobiume K, Taki M, Shigeishi H, Kamata N (2007) Snail-induced down-regulation of DeltaNp63alpha acquires invasive phenotype of human squamous cell carcinoma. Cancer Res 67:9207–9213 Hinds PW, Weinberg RA (1994) Tumor suppressor genes. Curr Opin Genet Dev 4:135–141 Ihrie RA, Marques MR, Nguyen BT, Horner JS, Papazoglu C, Bronson RT, Mills AA, Attardi LD (2005) Perp is a p63-regulated gene essential for epithelial integrity. Cell 120:843–856 Johnson TM, Rowe DE, Nelson BR, Swanson NA (1992) Squamous cell carcinoma of the skin (excluding lip and oral mucosa). J Am Acad Dermatol 26:467–484 Kaghad M, Bonnet H, Yang A et al (1997) Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 90:809–819 Katoh I, Aisaki KI, Kurata SI, Ikawa S, Ikawa Y (2000) p51A (TAp63gamma), a p53 homolog, accumulates in response to DNA damage for cell regulation. Oncogene 19:3126–3130 Keyes WM, Vogel H, Koster MI, Guo X, Qi Y, Petherbridge KM, Roop DR, Bradley A, Mills AA (2006) p63 heterozygous mutant mice are not prone to spontaneous or chemically induced tumors. Proc Natl Acad Sci 103:8435–8440 Kim S, Choi IF, Quante JR, Zhang L, Roop DR, Koster MI (2009) p63 directly induces expression of Alox12, a regulator of epidermal barrier formation. Exp Dermatol 18(12):1016–1021 King KE, Ponnamperuma RM, Gerdes MJ, Tokino T, Yamashita T, Baker CC, Weinberg WC (2006) Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis. Carcinogenesis 27:53–63 King KE, Ponnamperuma RM, Yamashita T, Tokino T, Lee LA, Young MF, Weinberg WC (2003) DNp63a functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes. Oncogene 22:3635–3644 King KE, Ponnamperuma RM, Allen C, Lu H, Duggal P, Chen Z, Van Waes C, Weinberg WC (2008) The p53 Homologue DNp63a Interacts with the Nuclear Factor-kB Pathway to Modulate Epithelial Cell Growth. Cancer Res 68:5122–5131 Kirschner RD, Sanger K, Muller GA, Engeland K (2008) Transcriptional activation of the tumor suppressor and differentiation gene S100A2 by a novel p63-binding site. Nucleic Acids Res 36:2969–2980 Koster MI, Kim S, Mills AA, DeMayo FJ, Roop DR (2004) p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev 18:126–131 Koster MI, Marinari B, Payne AS, Kantaputra PN, Costanzo A, Roop DR (2009) DNp63 knockdown mice: a mouse model for AEC syndrome. Am J Med Genet 149A(9):1942–1947 Koster MI, Dai D, Marinari B, Sano Y, Costanzo A, Karin M, Roop DR (2007) p63 induces key target genes required for epidermal morphogenesis. Proc Natl Acad Sci USA 104:3255–3260 Koster MI, Lu SL, White LD, Wang XJ, Roop DR (2006) Reactivation of developmentally expressed p63 isoforms predisposes to tumor development and progression. Cancer Res 66:3981–3986 Koster MI, Roop DR (2008) Sorting out the p63 signaling network. J Invest Dermatol 128:1617–1619 Kurata Si, Okuyama T, Osada M, Watanabe T, Tomimori Y, Sato S, Iwai A, Tsuji T, Ikawa Y, Katoh I (2004) p51/p63 Controls subunit a3 of the major epidermis integrin anchoring the stem cells to the niche. J Biol Chem 279:50069–50077 Lang GA, Iwakuma T, Suh YA et al (2004) Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 119:861–872

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Larsen M, Artym VV, Green JA, Yamada KM (2006) The matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr Opin Cell Biol 18:463–471 Laurikkala J, Mikkola ML, James M, Tummers M, Mills AA, Thesleff I (2006) p63 regulates multiple signalling pathways required for ectodermal organogenesis and differentiation. Development 133:1553–1563 Lee Ho, Lee JH, Choi E, Seol JY, Yun Y, Lee H (2006) A dominant negative form of p63 inhibits apoptosis in a p53-independent manner. Biochem Biophys Res Commun 344:166–172 Leiter U, Garbe C (2008) Epidemiology of melanoma and nonmelanoma skin cancer–the role of sunlight. Adv Exp Med Biol 624:89–103 Leong CO, Vidnovic N, Deyoung MP, Sgroi D, Ellisen LW (2007) The p63/p73 network mediates chemosensitivity to cisplatin in a biologically defined subset of primary breast cancers. J Clin Invest 117:1370–1380 Li Y, Zhou Z, Chen C (2008) WW domain-containing E3 ubiquitin protein ligase 1 targets p63 transcription factor for ubiquitin-mediated proteasomal degradation and regulates apoptosis. Cell Death Differ 15:1941–1951 Liefer KM, Koster MI, Wang XJ, Yang A, McKeon F, Roop DR (2000) Down-regulation of p63 is required for epidermal UV-B-induced apoptosis. Cancer Res 60:4016–4020 Liu B, Xia X, Zhu F, Park E, Carbajal S, Kiguchi K, DiGiovanni J, Fischer SM, Hu Y (2008) IKKa is required to maintain skin homeostasis and prevent skin cancer. Cancer Cell 14:212–225 Liu G, Nozell S, Xiao H, Chen X (2004) DNp73b is active in transactivation and growth suppression. Mol Cell Biol 24:487–501 Liu Y, Kulesz-Martin MF (2006) Sliding into home: facilitated p53 search for targets by the basic DNA binding domain. Cell Death Differ 13:881–884 Lopardo T, Lo IN, Marinari B, Giustizieri ML, Cyr DG, Merlo G, Crosti F, Costanzo A, Guerrini L (2008) Claudin-1 is a p63 target gene with a crucial role in epithelial development. PLoS ONE 3:e2715 MacKie RM (2006) Long-term health risk to the skin of ultraviolet radiation. Prog Biophys Mol Biol 92:92–96 Malkin D, Li FP, Strong LC, Fraumeni JF Jr, Nelson CE, Kim DH, Kassel J, Gryka MA, Bischoff FZ, Tainsky MA (1990) Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250:1233–1238 Marchbank A, Su LJ, Walsh P, DeGregori J, Penheiter K, Grayson TB, Dellavalle RP, Lee LA (2003) The CUSP DNp63a isoform of human p63 is downregulated by solar-simulated ultraviolet radiation. J Dermatol Sci 32:71–74 Marinari B, Ballaro C, Koster MI, Giustizieri ML, Moretti F, Crosti F, Papoutsaki M, Karin M, Alema S, Chimenti S, Roop DR, Costanzo A (2008) IKK[alpha] Is a p63 Transcriptional Target Involved in the Pathogenesis of Ectodermal Dysplasias. J Invest Dermatol 129:60–69 Martinez LA, Chen Y, Fischer SM, Conti CJ (1999) Coordinated changes in cell cycle machinery occur during keratinocyte terminal differentiation. Oncogene 18:397–406 Massion PP, Taflan PM, Jamshedur Rahman SM et al (2003) Significance of p63 amplification and overexpression in lung cancer development and prognosis. Cancer Res 63:7113–7121 McGrath JA, Duijf PH, Doetsch V et al (2001) Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63. Hum Mol Genet 10:221–229 McGregor L, Makela V, Darling SM et al (2003) Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein. Nat Genet 34:203–208 McMillan JR, Akiyama M, Shimizu H (2003) Epidermal basement membrane zone components: ultrastructural distribution and molecular interactions. J Dermatol Sci 31:169–177 Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398:708–713 Mills AA (2006) p63: oncogene or tumor suppressor? Curr Opin Genet Dev 16:38–44 Murray-Zmijewski F, Lane DP, Bourdon JC (2006) p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress. Cell Death Differ 13:962–972

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Ogawa E, Okuyama R, Ikawa S, Nagoshi H, Egawa T, Kurihara A, Yabuki M, Tagami H, Obinata M, Aiba S (2007) p51/p63 inhibits ultraviolet B-induced apoptosis via Akt activation. Oncogene 27:848–856 Ogawa E, Okuyama R, Egawa T, Nagoshi H, Obinata M, Tagami H, Ikawa S, Aiba S (2008) p63/ p51-induced onset of keratinocyte differentiation via the c-Jun N-terminal kinase pathway is counteracted by keratinocyte growth factor. J Biol Chem 283:34241–34249 Okada Y, Osada M, Kurata S, Sato S, Aisaki K, Kageyama Y, Kihara K, Ikawa Y, Katoh I (2002) p53 gene family p51(p63)-encoded, secondary transactivator p51B(TAp63a) occurs without forming an immunoprecipitable complex with MDM2, but responds to genotoxic stress by accumulation. Exp Cell Res 276:194–200 Olive KP, Tuveson DA, Ruhe ZC, Yin B, Willis NA, Bronson RT, Crowley D, Jacks T (2004) Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell 119:847–860 Oliveira LR, Ribeiro-Silva A, Zucoloto S (2007) Prognostic significance of p53 and p63 immunolocalisation in primary and matched lymph node metastasis in oral squamous cell carcinoma. Acta Histochem 109:388–396, 2007 Olivier M, Eeles R, Hollstein M, Khan MA, Harris CC, Hainaut P (2002) The IARC TP53 database: new online mutation analysis and recommendations to users. Hum Mutat 19:607–614 Ortt K, Sinha S (2006) Derivation of the consensus DNA-binding sequence for p63 reveals unique requirements that are distinct from p53. FEBS Lett 580:4544–4550 Osada M, Nagakawa Y, Park HL et al (2005a) p63-Specific Activation of the BPAG-1e Promoter. J Invest Dermatol 125:52–60 Osada M, Park HL, Nagakawa Y et al (2005b) Differential recognition of response elements determines target gene specificity for p53 and p63. Mol Cell Biol 25:6077–6089 Payne AS, Yan AC, Ilyas E et al (2005) Two novel TP63 mutations associated with the ankyloblepharon, ectodermal defects, and cleft lip and palate syndrome: a skin fragility phenotype. Arch Dermatol 141:1567–1573 Perez-Losada J, Wu D, Delrosario R, Balmain A, Mao JH (2005) p63 and p73 do not contribute to p53-mediated lymphoma suppressor activity in vivo. Oncogene 24:5521–5524 Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M (2007) Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat 28:622–629 Priolo M, Silengo M, Lerone M, Ravazzolo R (2000) Ectodermal dysplasias: not only ‘skin’ deep. Clin Genet 58:415–430 Radoja N, Guerrini L, Lo Iacono N, Merlo GR, Costanzo A, Weinberg WC, La Mantia G, Calabro V, Morasso MI (2007) Homeobox gene Dlx3 is regulated by p63 during ectoderm development: relevance in the pathogenesis of ectodermal dysplasias. Development 134:13–18 Reczek EE, Flores ER, Tsay AS, Attardi LD, Jacks T (2003) Multiple response elements and differential p53 binding control Perp expression during apoptosis. Mol Cancer Res 1:1048–1057 Renan MJ (1993) How many mutations are required for tumorigenesis? Implications from human cancer data. Mol Carcinog 7:139–146 Rinne T, Brunner HG, van Bokhoven H (2007) p63-associated disorders. Cell Cycle 6:262–268 Rocco JW, Leong CO, Kuperwasser N, Deyoung MP, Ellisen LW (2006) p63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell 9:45–56 Rossi M, Aqeilan RI, Neale M, Candi E, Salomoni P, Knight RA, Croce CM, Melino G (2006) The E3 ubiquitin ligase Itch controls the protein stability of p63. Proc Natl Acad Sci 103:12753–12758 Sayan BS, Sayan AE, Yang AL, Aqeilan RI, Candi E, Cohen GM, Knight RA, Croce CM, Melino G (2007) Cleavage of the transactivation-inhibitory domain of p63 by caspases enhances apoptosis. Proc Natl Acad Sci 104:10871–10876 Schultz J, Ponting CP, Hofmann K, Bork P (1997) SAM as a protein interaction domain involved in developmental regulation. Protein Sci 6:249–253

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Serber Z, Lai HC, Yang A, Ou HD, Sigal MS, Kelly AE, Darimont BD, Duijf PH, van Bokhoven H, McKeon F, Dotsch V (2002) A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism. Mol Cell Biol 22:8601–8611 Shimomura Y, Wajid M, Shapiro L, Christiano AM (2008) P-cadherin is a p63 target gene with a crucial role in the developing human limb bud and hair follicle. Development 135:743–753 Siegfried E, Bree A, Fete M, Sybert VP (2005) Skin erosions and wound healing in ankyloblepharon-ectodermal defect-cleft lip and/or palate. Arch Dermatol 141:1591–1594 Smyth I, Scambler P (2005) The genetics of Fraser syndrome and the blebs mouse mutants. Hum Mol Genet 14(2):R269–R274 Testoni B, Mantovani R (2006) Mechanisms of transcriptional repression of cell-cycle G2/M promoters by p63. Nucleic Acids Res 34:928–938 Tonon G, Wong KK, Maulik G et al (2005) High-resolution genomic profiles of human lung cancer. Proc Natl Acad Sci U S A A102:9625–9630 Truong AB, Kretz M, Ridky TW, Kimmel R, Khavari PA (2006) p63 regulates proliferation and differentiation of developmentally mature keratinocytes. Genes Dev 20:3185–3197 Vrontou S, Petrou P, Meyer BI, Galanopoulos VK, Imai K, Yanagi M, Chowdhury K, Scambler PJ, Chalepakis G (2003) Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice. Nat Genet 34:209–214 Westfall MD, Joyner AS, Barbieri CE, Livingstone M, Pietenpol JA (2005) Ultraviolet radiation induces phosphorylation and ubiquitin-mediated degradation of DeltaNp63alpha. Cell Cycle 4:710–716 Westfall MD, Mays DJ, Sniezek JC, Pietenpol JA (2003) The DNp63a phosphoprotein binds the p21 and 14-3-3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay-Wells syndrome-derived mutations. Mol Cell Biol 23:2264–2276 Wu G, Nomoto S, Hoque MO et al (2003) aNp63a and TAp63a regulate transcription of genes with distinct biological functions in cancer and development. Cancer Res 63:2351–2357 Yang A, Kaghad M, Caput D, McKeon F (2002) On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet 18:90–95 Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, Andrews NC, Caput D, McKeon F (1998) p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, deathinducing, and dominant-negative activities. Mol Cell 2:305–316 Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398:714–718 Yang A, Walker N, Bronson R, Kaghad M, Oosterwegel M, Bonnin J, Vagner C, Bonnet H, Dikkes P, Sharpe A, McKeon F, Caput D (2000) p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 404:99–103 Zangen R, Ratovitski E, Sidransky D (2005) DNp63a levels correlate with clinical tumor response to cisplatin. Cell Cycle 4:1313–1315 Zeng X, Levine AJ, Lu H (1998) Non-p53 p53RE binding protein, a human transcription factor functionally analogous to P53. Proc Natl Acad Sci USA 95:6681–6686

Chapter 13

Effects of Natural and Synthetic Retinoids on the Differentiation and Growth of Squamous Cancers Humam Kadara and Reuben Lotan

Abstract A negative correlation between vitamin A intake and the incidence of epithelial tumors was observed about 80 years ago. This observation was followed by multiple studies, which have demonstrated that vitamin A and its metabolites as well as synthetic analogs (retinoids) could suppress carcinogenesis in a variety of epithelial tissues. In parallel, vitamin A and retinoids inhibit squamous cell differentiation in normal keratinocytes and in malignant squamous cell carcinomas (SCC) including those of the head and neck. Because the use of natural retinoids in the clinical setting was hampered by adverse side effects, synthetic retinoids were developed with the hope that they would exhibit potent anticancer chemopreventive and therapeutic properties but much lower toxicities than natural retinoids. In addition, synthetic retinoids and retinoid-related molecules, such as fenretinide [N-4-hydroxyphenyl)retinamide; 4HPR] and the adamantyl retinoid CD437, respectively, induce cancer cell apoptosis through retinoid receptor-dependent and more often independent mechanisms. This chapter reviews the major effects of retinoids on squamous cell differentiation, cancer cell growth and apoptosis, and aims to provide new insights into common and distinct mechanisms of action among the naturally occurring retinoic acid and the synthetic derivatives, 4HPR and CD437, in normal and malignant squamous head and neck cells.

13.1 Retinoids and Retinoid Receptors Vitamin A is a lipid soluble essential nutrient that cannot be synthesized in the body but rather must be metabolized from its ingested provitamin form, b-carotene, from dietary sources such as eggs, milk, and fish oil (Lotan 1980). Later, vitamin A was demonstrated to function as an essential growth factor in studies that monitored

R. Lotan (*) Department of Thoracic/Head and Neck Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_13, © Springer Science+Business Media, LLC 2011

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changes caused by vitamin A deficiency or its excess in experimental animals or organ cultures. Such studies established that vitamin A and its retinoid derivatives are crucial for vertebrate growth, night vision, differentiation of epithelial and mesenchymal cells and tissue as well as maintenance of reproductive function (Olson 1968; Wald 1968; De Luca 1977; Sporn 1977). Consequently, vitamin A deficiency manifests in various clinical conditions including night blindness, retardation, atrophy of the testes and immunodeficiency (De Luca 1991). Moreover, there is a strong link between vitamin A deficiency and the incidence of malignant epithelial lesions, first noted by Wolbach and Howe in the 1920s (Wolbach and Howe 1925). Conversely, the naturally occurring retinoids, retinoic acid and retinyl acetate, were found to inhibit the growth of transformed and malignant epithelial cells in vitro (Lotan and Nicolson 1977). More recently, it has been reported that cancer patients had lower serum retinol levels than patients not diagnosed with cancer but with similar clinical features (Kark et al. 1981). Furthermore, it was demonstrated that low intake of b-carotene increases the risk of breast cancer (Hislop et al. 1990; Moon 1994). Such important properties and phenotypes of vitamin A deficiency have stimulated many studies aimed at understanding the molecular mechanism by which retinoids regulate the growth and differentiation of normal and malignant epithelial cells. The major metabolites of vitamin A (retinol) are retinal and all-trans retinoic acid (ATRA). Retinol, the major transport form of the vitamin, is synthesized following reduction of retinal (formed by cleavage of b-carotene) by retinaldehyde reductases, or by hydrolysis of retinyl esters, another major dietary source and storage form of vitamin A, by retinyl ester hydrolases. ATRA is synthesized after irreversible oxidation of retinal by retinal dehydrogenases (Olson 1968; Blomhoff and Blomhoff 2006). When the body is in need of vitamin A, retinyl esters stored in the liver are hydrolyzed to retinol, which bound to retinol binding protein, is transported to target tissues (Blomhoff and Blomhoff 2006). In contrast, ATRA is formed in target tissues and cells (also indirectly from retinol). ATRA can be isomerized to 13-cis retinoic acid (13CRA) and 9-cis-retinoic acid (9CRA). The main mechanism by which retinoids regulate gene expression is via nuclear proteins related to the steroid and thyroid hormone receptor family, called retinoic acid receptors (RARs) and retinoid X receptors (RXRs). All three isomers of retinoic acid can bind with similar affinities and activate RARs. The RXRs are selectively activated by 9CRA. Different isotypes of RARs and RXRs (mainly a, b and g) form RAR/RXR heterodimers or to a lesser extent RXR/RXR homodimers and transduce the signals of the retinoic acids intracellularly by acting as ligand-activated transcription factor complexes that bind to consensus DNA sequences such as retinoic acid response elements (RAREs) (DR-2, DR-3 and DR-5) found in the promoter regions of target genes (Giguere et al. 1987; De Luca 1991; Kastner et al. 1995; Chambon 1996; Blomhoff and Blomhoff 2006). It is now well established that retinoid receptor signaling is regulated mainly by epigenetic mechanisms (Altucci and Gronemeyer 2001). In the absence of a ligand such as ATRA, histone deacetylases (HDACs) are recruited to the retinoid receptors by receptor-associated corepressors such as nuclear receptor corepressor or silencing mediator for retinoid and

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thyroid hormone receptors. HDACs in turn induce chromatin condensation and therefore inhibit the transactivational function of the retinoid receptors. Upon ligand binding, the association of the RAR/RXR heterodimers with corepressors is destabilized and allows for the interaction with coactivators that recruit histone acetyl transferases leading to chromatin decondensation and transactivation of the retinoic acid-regulated genes. This canonical retinoid receptor pathway through transactivation or repression of a variety of target genes is the major mechanism of retinoid signaling. The many retinoid-regulated genes include the retinoid receptors themselves such as, RARa and RARb, other transcription factors, major signaling molecules, growth factor receptors, insulin growth factor-binding proteins, tumor suppressors, cell cycle checkpoint regulators and metabolizing enzymes (Altucci and Gronemeyer 2001; Blomhoff and Blomhoff 2006). The vast range and large number of retinoic acid-regulated genes attests to the importance of retinoid signaling in many crucial physiological processes.

13.2 Retinoid-Induced Inhibition of Squamous Differentiation Several early observations illuminated the role of vitamin A on squamous differentiation. Vitamin A deficiency in experimental animal models leads to the development of squamous metaplasia in epithelia of the eye, nasal mucosa, respiratory tract and salivary glands (Hicks 1968; Beitch 1970; Hayes et al. 1970; Wong and Buck 1971; Harris et al. 1972; Wolbach and Howe 1925). In these early studies it was shown that vitamin A-deficiency caused replacement of mucus-secreting columnar epithelium with keratinizing squamous cells. Furthermore, removal of vitamin A in vitro induces squamous metaplasia in tracheal organ cultures (Marchok et al. 1975), and restoration of the vitamin to deprived animals was shown to inhibit squamous differentiation and reversed keratinization (Wong and Buck 1971; Harris et al. 1972; Lotan 1980). It is worthwhile to note that the epithelium of the oral cavity, including the mucosa lining the soft palate, tongue and floor of the mouth, is naturally nonkeratinizing (Ouhayoun et al. 1985) but undergoes abnormal squamous differentiation during pathological conditions such as vitamin A-deficiency or oral carcinogenesis (Shin et al. 1990; Lotan 1994). It is well established that premalignant oral lesions and SCCs express high levels of squamous differentiation markers compared to their normal counterparts (Lotan 1993). The squamous differentiation markers include but are not limited to K1 keratin (Poddar et al. 1991), involucrin (Poddar et al. 1991), transglutaminase I, cholesterol sulfotransferase (Jetten et al. 1990; Ta et al. 1990), filagrin, loricrin (Lotan 1993) and cornifin (Fujimoto et al. 1993). Furthermore, exposure of nonkeratinized buccal epithelial cells in vitro to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, causes an increase in expression of the late differentiation protein involucrin (Sundqvist et al. 1991), which is involved in formation of the cornified envelope in normal squamous epithelial cells.

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Retinoids have been studied extensively for their potential inhibitory effects on squamous differentiation in oral lesions and head and neck squamous cell carcinoma (HNSCC). HNSCC lesions and cell lines exhibit varying levels of squamous differentiation markers (Lotan 1994). For example, our laboratory has previously demonstrated that the HNSCC cell lines, 183, 886, 1,483, and SqCC/Y1, express varying levels of involucrin, translgutaminase I, and K1 keratin, with the latter two cells expressing all three markers, and the former two cells expressing only involucrin or none of the markers, respectively (Poddar et al. 1991). It has been suggested that the suppression of aberrant squamous differentiation by retinoids in premalignant and malignant head and neck tissues resembles the restoration of the normal differentiation process (Lotan 1994). Indeed, retinoid derivatives of vitamin A have been demonstrated to inhibit differentiation in premalignant and malignant squamous lesions. Retinoids inhibited squamous cell differentiation in normal keratinocytes in vitro (Rubin and Rice 1986; Eckert 1989; Jetten 1990) and in HNSCCs (Reiss et al. 1985; Jetten et al. 1990; Poddar et al. 1991). Retinoic acid treatment also restored the mucous phenotype of naturally nonsquamous human bronchial epithelial cells that undergo squamous differentiation when deprived of retinoids (Lee et al. 1996; Koo et al. 1999). It is also worthy to note that different concentrations of natural retinoid derivatives of vitamin A may exert distinct effects on cell differentiation as pharmacological but not physiological concentrations of ATRA induced differentiation of laryngeal epithelial cells and papilloma cells into columnar and ciliated epithelium (Mendelsohn et al. 1991). Retinoic acid treatment was also shown to suppress squamous differentiation in other squamous cancers such as cervical cancer. Treatment with ATRA reversed human endocervical cell metaplasia and HPV 18-immortalized endo- and ectocervical cell dysplastic epithelial differentiation (Yokoyama et al. 2001). Synthetic retinoid derivatives also inhibit squamous cell differentiation. At a low concentration, the synthetic retinoid related molecule 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) suppressed the expression of squamous differentiation markers and induced apoptosis in HNSCC cells through a retinoid receptor-dependent mechanism (Sun et al. 2000a). Treatment with ATRA and the synthetic retinoids N-(4-hydroxyphenyl)retinamide (4HPR) and CD437 of UMSCC-22B squamous HNSCC cells led to both common and distinct changes in protein expression assessed by high-throughput Western blotting (Kim and Lotan 2004). All three retinoids increased the protein levels of the Ets transcriptional factor family member, ELF3, which in had been previously shown to suppress the expression of several differentiation markers, such as keratin 4, in esophageal and cervical squamous cancers (Brembeck et al. 2000). However, the synthetic retinoids CD437 and 4HPR induced the differential expression of many proteins that were not modulated by treatment of ATRA. In particular, genes related to the proapoptotic retinoid receptor-independent function of both retinoids (Kim and Lotan 2004). The molecular basis of inhibition of squamous differentiation through signaling downstream of retinoid receptors is discussed in the section, retinoids and retinoid receptors, and discussed below in studies demonstrating loss of retinoid receptors during squamous carcinogenesis in humans and in animal models.

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In contrast to their inhibitory effects on squamous cell differentiation, retinoids are known to induce differentiation of various malignant cells including F9 embryonal carcinoma, leukemia, neuroblastoma, and melanoma cells (Lotan 1994). Additionally, the ability of retinoids to induce differentiation led to the rationale for their use in differentiation therapy most notably in the treatment of acute promyelocytic patients with ATRA (Huang et al. 1988; Castaigne et al. 1990). These contrasting findings shed light on the unique effects of retinoids on squamous differentiation and the distinctive role of vitamin A and retinoid signaling in the regulation of this process. The induction of squamous metaplasia due to vitamin A-deficiency and the reversal of the squamous phenotype by the vitamin A and its derivatives have led to the hypothesis that retinoid receptors are differentially expressed in normal, premalignant, and malignant tissues (Lotan 1994). Indeed, it was previously demonstrated that the retinoid receptor RARb was decreased in most cell lines isolated and cultured from oral leukoplakias and HNSCCs and was up-regulated following treatment with ATRA in cells expressing it at low levels (Crowe et al. 1991; Hu et al. 1991). Furthermore, overexpression of RARb is associated with a decrease in keratinization and in expression of squamous differentiation markers thus implicating RARb loss in aberrant squamous differentiation (Zou et al. 1999). In addition, overexpression of RARb in HNSCC cells increased their sensitivity to retinoid-induced inhibition of squamous differentiation (Wan et al. 1999). A progressive decrease in the expression of RARb, but not of RARa or RARg, has been demonstrated by in situ hybridization using tissue histological specimens of oral leukoplakias and HNSCCs compared with normal buccal epithelia (Xu et al. 1994). In addition, 13CRA treatment of patients with oral premalignant lesions that had decreased RARb expression increased the mRNA levels of this retinoid receptor and this increase was correlated with clinical response (Lotan et al. 1995). These findings demonstrated that RARb mediates the action of retinoids and can be valuable as an intermediate biomarker in retinoid-based oral carcinogenesis prevention trials. Furthermore, RARb expression level was later demonstrated to be downregulated in non-small cell lung cancer (NSCLC) tissues compared to their normal counterparts (Xu et al. 1997b) and decreased progressively during breast carcinogenesis (Xu et al. 1997a). Interestingly and in contrast, the mRNA levels of RARa, RARg, RXRa, RXRb and RXRg but not those of RARb decreased progressively during squamous skin carcinogenesis (Darwiche et al. 1995, 1996; Xu et al. 2001). The loss of retinoid receptor expression, specifically RARb, in squamous cancer, most notably HNSCC, further indicates the importance of retinoid signaling in maintenance of tissue differentiation and the value of retinoid derivatives in the prevention of squamous malignancies such as oral cancer.

13.3 Retinoids in Preclinical and Clinical Studies Retinoids have demonstrated considerable success in the prevention and treatment of cancer. In 1976, Lasnitzki reported the reversal of methylcholanthrene-induced hyperplasia, parakeratosis and metaplasia of mouse prostate epithelial cells in vitro by

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retinoic acid and retinol (Lasnitzki 1976). ATRA was found to inhibit transformation, assessed by colony formation, of rat tracheobronchial epithelial cells exposed to benzopyrene (Steele et al. 1990). In addition, the efficacy of retinoids in oral carcinogenesis inhibition was investigated in several models such as the hamster DMBA-induced hamster buccal pouch and tongue carcinogenesis models as well as the 4-nitroquinoline-1-oxide-induced mouse oral squamous cell carcinoma model (Goodwin et al. 1986; Inoue et al. 1993). It was shown that 13CRA was capable of inhibiting or delaying carcinogenesis in all three models. In the clinic, studies on retinoids focused on their chemopreventive potential by targeting individuals at an increased risk of developing cancer. This included patients who had premalignant lesions or those with early stage cancer but at high risk of developing second primary tumors (Lotan 1997). As mentioned before, 13CRA inhibited the development of oral leukoplakias in randomized placebo-controlled studies of patients with oral premalignant lesions and increased levels of the tumor suppressor RARb (Lotan et al. 1995). Treatment with vitamin A also decreased the incidence of esophageal cancer and increased survival in patients with esophageal dysplasias (Blot et al. 1993; Li et al. 1993). Furthermore, exposure to vitamin A decreased the incidence of second primary tumors in stage I NSCLC patients (Pastorino et al. 1993). In addition, 13CRA in adjuvant settings was shown to decrease recurrence in head and neck cancer patients inhibiting the incidence of second primary tumors; after a 32 months median follow-up, incidence of new second primary tumors was 4% and 24% in treated and control groups, respectively (Lotan 1997). Clinical evidence for the chemopreventive efficacy of retinoids has also been reported in breast cancer (Veronesi and Decensi 2001), renal-cell carcinoma (Miller et al. 2000) and cervical neoplasms (Meyskens et al. 1994). Retinoids also demonstrated chemotherapeutic activity and are now used in the clinic for treatment of skin disorders such as acne and psoriasis, Kaposi sarcoma, cutaneous T-cell lymphoma and more notably for the treatment of acute promyelocytic leukemia (APL) (Freemantle et al. 2003). APL patients present with a (15:17) chromosomal translocation (Rowley et al. 1977) causing the formation of a PML-RARa fusion protein that interferes with RAR/RXR heterodimer binding, increases recruitment of co-repressors and HDACs genes and thus represses the transcription of RARE containing target genes (Benedetti et al. 1997). Treatment with 13CRA induced APL differentiation in vitro and in vivo (Flynn et al. 1983). In addition, ATRA was effective in the complete remission of APL patients some of whom were resistant to first line therapies such as cytosine arabinoside indicating the usefulness of differentiation induction as a therapeutic strategy (Huang et al. 1988). Despite these successes there are several limitations on the use of retinoids in the clinic. Retinoic acid is readily catabolized as a result of the induction of the cytochrome P450, CYP26A1 (Muindi et al. 1992). Further, chronic administration of retinoic acid to patients results in undesirable side effects including skeletal abnormalities, mucocutaneous toxicity, hypertriglyceridemia, hypothyroidism, and teratogenesis (Collins and Mao 1999). In addition, it has been reported that long-term usage of retinoids causes adverse interactions with smoking in the prevention of lung cancer resulting in increased mortality especially among smokers (Lippman et al. 2001).

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It is worthy to note that in a randomized placebo-controlled chemoprevention trial, 13CRA failed to induce reversal of squamous metaplasia which was rather associated with smoking cessation (Lee et al. 1994). Such limitations of retinoids prompted the development of new strategies to improve retinoid efficacy such as the development and production of newer more effective retinoids with fewer or no side effects.

13.4 The Synthetic Retinoid 4HPR (Fenretinide) The synthetic analog of ATRA, 4HPR, was produced by the substitution of an amide-linked 4-(hydroxyphenyl) group for the carboxyl group of the retinoic acid precursor. This synthetic retinoid exhibited markedly reduced adverse side effects, such as liver toxicity, and was found to lack the ability to induce point mutations or chromosome aberrations and therefore was not genotoxic (Paulson et al. 1985). In preclinical studies, 4HPR was first shown to prevent breast cancer in rats (Moon et al. 1979). Interestingly, among all retinoids evaluated for effectiveness against chemically induced mammary carcinogenesis, retinyl acetate and 4HPR appeared to be the most efficacious. However, whereas retinyl acetate accumulates in the liver causing hepatotoxicity, 4HPR builds up in a dose-dependent manner in the mammary glands (Moon et al. 1979; Costa et al. 1995). In contrast to retinol and b-carotene, 4HPR was effective in inhibiting the development of lung adenocarcinomas in hamsters exposed to N-nitrosodiethylamine (Lotan 1997). In experimental animal models, 4HPR demonstrated chemopreventive efficacy against development of breast (Abou-Issa et al. 1995), prostate (Ohshima et al. 1985) and skin (McCormick and Moon 1986) cancers. In addition, 4HPR also exhibited chemotherapeutic properties in several in vitro and animal studies targeting neuroblastoma (Maurer et al. 1999), leukemia (Faderl et al. 2003) and lymphoma (Chan et al. 1997) cells. In the clinical setting, 4HPR was effective in chemoprevention of oral leukoplakia recurrence and new incidence (Chiesa et al. 2005) and in patients resistant to natural retinoids (Lippman et al. 2006). Moreover, the risk of second breast cancer in premenopausal women was significantly reduced by 4HPR, and this effect persisted for several years after treatment cessation in a 15-year long randomized phase III trial for breast cancer prevention (Veronesi et al. 2006). More recently, 4HPR therapy significantly delayed onset of ovarian cancer (Bast et al. 2007). The synthetic retinoid, 4HPR, has been shown to function though both retinoid receptor dependent and independent mechanisms. It is thought that 4HPR does not bind RARs due to the lack of a functional carboxyl group (Formelli et al. 1996). Nevertheless, transactivation studies have shown that 4HPR can activate RARa and RARb, as well as the RXRs (Fanjul et al. 1996; Formelli et al. 1996; Sun et al. 1999a). Moreover, cells with constitutively higher levels of RARs exhibited more sensitivity to 4HPR-induced cell death compared to cells with lower levels of the retinoid receptors (Liu et al. 1998; Sabichi et al. 1998). In addition, RARb/g antagonists were shown to suppress 4HPR-induced apoptosis, at least partly (Sun et al. 1999a; Sun et al. 1999b). On the other hand, there is substantially more

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evidence that the mechanism for induction of apoptosis by 4HPR is independent of the retinoid receptor, in most cell types (Dmitrovsky 2004). For example, cells depleted of the RAR gene are still sensitive to 4HPR-mediated apoptosis (Clifford et al. 1999). Moreover, RAR antagonists did not inhibit ceramide-induced 12-lipoxygenase (12-LOX)-mediated apoptosis in neuroblastoma cells (Lovat et al. 2000). Apoptosis induction by 4HPR is mediated by various mechanisms including ceramide induction (Maurer et al. 1999), triggering of the mitochondrial pathway and modulating mitochondrial membrane permeability and cytochrome c release (Hail and Lotan 2000), activation of 12-LOX predominantly in neuroblastoma cells (Lovat et al. 2002), nitric oxide production, for example in breast cancer cells (Simeone et al. 2002), and reactive oxygen species generation (ROS) (Delia et al. 1997; Oridate et al. 1997; Sun et al. 1999a; Hail and Lotan 2001a; Asumendi et al. 2002). It is now well established that the increase in ROS is an important mediator of apoptosis induction by 4HPR in many cell systems including transformed B-cells (Boya et al. 2003), adult T-cell leukemia (Darwiche et al. 2007), breast cancer (Hursting et al. 2002; Choi et al. 2004), cervical cancer (Oridate et al. 1997; Suzuki et al. 1999), HNSCC (Sun et al. 1999a), lung carcinoma (Sun et al. 1999a), neuroblastoma (Lovat et al. 2000), sarcoma (Batra et al. 2004; Myatt et al. 2005) and skin carcinoma cells (Hail and Lotan 2001a). The increase in ROS generation can cause sustained activation of the mitogen-activated protein kinases (MAPKs) Jun N-terminal kinase (JNK), p38 and ERK1/2 (Chen et al. 1999; Osone et al. 2004; Kim et al. 2006), triggering of the mitochondrial pathway (Hail and Lotan 2001a; Boya et al. 2003), 12-LOX activation in neuroblastoma cells (Lovat et al. 2004), activation of the p53 stress pathway and an increase in the Bax/Bcl2 ratio (Hail et al. 2006). More recent findings by our group and others highlighted novel downstream effects of ROS generation by 4HPR that culminate in the activation of the endoplasmic reticulum (ER) stress pathway (Tiwari et al. 2006; Corazzari et al. 2007; Kadara et al. 2007). Treatment of HNSCC cells with 4HPR induced hallmark features of ER stress activation including ER dilation, splicing and activation of the transcriptional factor X-box binding protein 1 (XBP1), increases in the mRNA and protein levels of growth arrest and DNA damage 153 (GADD153), as well as induction of the chaperones glucose-regulated protein 78/binding protein (GRP78/Bip) and heat shock proteins (HSPs) 70 and 90 (Kadara et al. 2007). Despite a documented prosurvival and antiapoptotic role for the inducible chaperone HSP70/HSPA1A (Sherman and Multhoff 2007), our studies using chemical inhibitors and small interfering RNA (siRNA) implicated a novel proapoptotic function for this chaperone in 4HPR-mediated cell death (Kadara et al. 2007). Other studies have shown, however, that 4HPR-mediated ROS generation can induce the prosurvival ER stress genes and chaperones, ERdj5 and ERp57, and that expression of these chaperones counteracts the pro-apoptotic action of this synthetic retinoid (Corazzari et al. 2007). In a more recent study, inhibition of ER stress-mediated protein disulfide isomerase activity increased apoptosis induction by 4HPR thus implicating the activation of a prosurvival arm of ER stress to counteract the anticancer effects of this synthetic retinoid (Lovat et al. 2008).

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While these studies highlight the pivotal role of the downstream effects of ROS, many gaps still exist in our knowledge of upstream mechanisms responsible for ROS generation by 4HPR. Because the enzyme, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), is implicated in ROS generation (Lambeth 2004), we hypothesized that it may be a target through which 4HPR increases ROS generation and induces apoptosis. Previously, we demonstrated that protein levels of the NADPH oxidase subunit, p67phox (De Leo et al. 1996), increased following 4HPR treatment in UMSCC-22B HNSCC cells (Kim and Lotan 2004). More recently, we demonstrated that the small GTPase, Rho-related C3 botulinum toxin 1 (RAC1), which acts as a catalytic subunit for NOX (Lambeth 2004; Cheng et al. 2006), was activated as early as 5 min following exposure to 4HPR. Inhibition of RAC1 activation with a small molecular weight inhibitor, NSC-23766 (Gao et al. 2004), or downregulation of RAC1 levels with siRNA significantly decreased 4HPR-induced ROS generation and subsequent apoptosis induction (Kadara et al. 2008b). Based on the known roles of RAC1 in cellular migration, invasion and metastasis (Chan et al. 2005), and elevated levels of endogenous ROS in metastatic cancer cells (Wu 2006), we compared isogenic pairs of primary and metastatic cell lines for sensitivity to 4HPR, activation of RAC1 and generation of ROS. We found that metastatic cancer cells displayed higher levels of RAC1 activation, ROS generation and cell death in response to 4HPR (Kadara et al. 2008b). Recently, it was demonstrated that the combination of 4HPR and the proteasome inhibitor, Bortezomib, was effective in cell growth inhibition and apoptosis of metastatic melanoma cancer cells (Hill et al. 2009). These important findings support the use of 4HPR in a chemotherapeutic setting and especially against advanced and metastatic tumors in contrast to natural retinoids which displayed mainly effectiveness in cancer prevention. In addition, these findings further support a retinoid receptorindependent function for the synthetic proapoptotic retinoid 4HPR.

13.5 The Synthetic Retinoid Related Molecule CD437 We previously screened many synthetic retinoid derivatives in search for effective agents with fewer undesired side effects than natural retinoids. The synthetic retinoid related molecule, CD437, emerged as a potent inducer of apoptosis and cell growth inhibition in several HNSCC and NSCLC cell lines (Sun et al. 1997, 2000b). CD437 was initially shown to be highly efficacious in inhibiting the growth of lung tumor xenografts in mice (Lu et al. 1997). CD437 was effective in apoptosis induction in a wide range of cancer cell types as reviewed earlier (Lotan 2003). Importantly, CD437 was capable of apoptosis induction in many cancer cell lines resistant to retinoic acid (Hsu et al. 1997; Sun et al. 1997; Gianni and de The 1999; Sun et al. 2000b). Moreover, CD437 induced apoptosis in primary cultures of APL blasts isolated from a newly diagnosed patient and from cases of relapse and resistance to ATRA (Mologni et al. 1999). In vivo, CD437 exhibited anti-cancer effects against human tumor xenografts in immunodeficient mice including melanoma

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(Schadendorf et al. 1996), APL (Ponzanelli et al. 2000), chronic lymphocytic leukemia (Zhang et al. 2002) and ovarian cancer (Langdon et al. 1998). CD437 was initially described to be selective for the retinoid receptor RARg (Bernard et al. 1992), and studies showing that a dominant negative pan retinoic acid receptor and a RARg antagonist could partially block the apoptogenic activity of CD437 (Holmes et al. 2000) supported a retinoid receptor-dependent mechanism of apoptosis induction. However, later studies support retinoid receptor independent mechanisms for the induction of apoptosis by this compound (Lotan 2003). Thus, retinoic acid resistant HL-60R cells, that lack both RARb and RARg and express a non-functional RARa mutant, and myeloma cells deficient in retinoic acid receptors, were sensitive to CD437-induced apoptosis (Hsu et al. 1997; Marchetti et al. 1999). Moreover, retinoic acid receptor antagonists failed to suppress apoptosis induction by CD437 (Sun et al. 1997). In contrast to these studies we have shown that CD437 induces retinoid receptor-dependent inhibition of squamous differentiation and receptor-independent induction of apoptosis in UMSCC-22B HNSCC cells (Sun et al. 2000a). More recently, CD437 was found to inhibit growth and induce apoptosis in F9 teratocarcinoma cells through RARg-dependent and receptor-independent pathways (Parrella et al. 2006). It appears that in most cases or cell systems, CD437 exerts its apoptogenic activities via retinoid receptor- independent mechanisms, but receptor dependent mechanisms cannot be excluded. It is plausible that differences in the mechanisms that dictate the antitumor effects of CD437 may be a result of variable doses of the synthetic retinoid used. As mentioned earlier, lower concentrations of 4HPR induced differentiation and growth arrest of F9 embryonal carcinoma cells whereas higher doses induced apoptosis (Clifford et al. 1999). Several mechanisms and pathways mediate the apoptogenic activity of CD437. The tumor suppressor p53 protein that induces apoptosis in response to several cellular stresses including chronic DNA damage (Vousden and Lu 2002) has been implicated in apoptosis induction by CD437, although p53 independent mechanisms for CD437’s apoptogenic activity were also reported. Chronic treatment with CD437 induces apoptosis in cancer cells with wild-type or mutant p53 or even cells lacking the tumor suppressor (Shao et al. 1995; Sun et al. 1999c). HaCaT immortalized human keratinocytes which express two mutant p53 alleles are susceptible to CD437-induced apoptosis (Wanner et al. 2002). In contrast, in NSCLC cells CD437 did increase the expression of p53 along with proapoptotic p53-regulated genes, such as BAX and death receptors (Sun et al. 1999c). Moreover, it was demonstrated using isogenic lung cancer cells, differing only in the expression of the E6 gene of the human papillomavirus (HPV) which compromises p53 tumor suppressor function, that CD437 was more effective in apoptosis induction in lung cancer cells with functional p53 whereas cells with the E6 gene were resistant to the synthetic retinoid and up-regulation of BAX or death receptors did not occur (Sun et al. 1999c). In addition, unlike lung cancer cells with functional p53, cells with the HPV-E6 gene did not exhibit any poly-ADP ribose polymerase (PARP) cleavage, up-regulation of DNA damage proteins such as replication protein A (RPA) or increase in expression

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of the cell growth inhibitor, growth and differentiation factor 15 (GDF15), in response to CD437 treatment (Kadara et al. 2006). Interestingly, CD437 was able to induce p53-target genes in melanoma cells expressing the HPV-E6 gene (Zhao et al. 2001). The role of the p53 tumor suppressor protein in CD437-mediated apoptosis may differ in various cell systems. CD437 was demonstrated in several studies to be a potent activator of the intrinsic or mitochondrial pathway of apoptosis. CD437 was documented earlier to cause release of cytochrome c from the mitochondria, for subsequent activation of caspase 9 and apoptosis induction, in various cancer cells (Lotan 2003). In an attempt to explore mechanisms of cytochrome c release from the mitochondria by CD437, it was demonstrated that this synthetic retinoid induces the expression of the transcriptional factor TR3, also known as NUR77, which in turn translocates to the mitochondria and inhibits the function of the anti-apoptotic BCL2 protein (Dawson et al. 2001). Moreover, CD437 was shown to cause an increase in mitochondrial membrane permeability which is required for the release of cytochrome c into the cytosol and preceded ROS generation and DNA fragmentation in the human myeloma cells and in cytoplasts of these cells that lack nuclei (Marchetti et al. 1999). To further support this mechanism of CD437’s apoptogenic activity, cotreatment of cells with the mitochondrial membrane transition inhibitor cyclosporine A blocked apoptosis induction by this synthetic retinoid. CD437 was also shown to induce ROS generation following the disruption of the mitochondrial membrane potential in several cells including cutaneous SCC cells (Hail and Lotan 2001b; Hail et al. 2001). Co-treatment of the same cells with the antioxidant vitamin C inhibited CD437-induced apoptosis. Moreover, CD437 caused a significant reduction in oxygen consumption in the SCC cells and co-treatment of the same cells with vitamin C blocked these effects and apoptosis, indicating the importance of the mitochondrial respiratory process for apoptosis induction by CD437 (Hail et al. 2001). In addition, several respiratory deficient clones of two SCC cell lines previously generated in our laboratory were resistant to CD437-induced apoptosis (Hail et al. 2001). Other mechanisms and cellular pathways have also been implicated in the apoptosis induction by CD437 including, increase in the BAX/BCL2 ratio, sustained activation of MAPKs such as JNK and p38, up-regulation and activation of death receptors and up-regulation of transcriptional factors such as MYC and E2F1 (Lotan 2003). CD437 was found to activate nuclear factor-kappa B for subsequent death receptor up-regulation and apoptosis induction in human prostate cancer cells (Jin et al. 2005). Moreover, CD437 caused DNA double-strand breaks leading to cytotoxicity in acute myeloid leukemia cells (Valli et al. 2008). Interestingly and similar to recent findings on novel mechanisms of 4HPR-induced apoptosis, CD437 was also demonstrated to induce apoptosis via activation of the ER stress pathway (Watanabe et al. 2008). Thus, the synthetic retinoid related molecule CD437 appears to be a potent inducer of apoptosis and it will be of major interest whether combinations of CD437 and other anti-cancer agents will be powerful in eliminating cancer cells.

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13.6 Differential Effects of ATRA, 4HPR and CD437 on Gene Expression in Normal and Malignant Head and Neck Squamous Cells The synthetic retinoids 4HPR and CD437 appear to differentially affect cancer cells compared to their normal counterparts. For example, 4HPR appeared to selectively induce apoptosis in vitro in transformed cells while sparing their corresponding normal cells (Hail et al. 2006). We have previously shown that low pharmacologically achievable doses of 4HPR alone or in combination with the nonsteroidal antiinflammatory drug, Celecoxib, inhibited growth and colony formation, and induced apoptosis in immortalized, transformed and tumorigenic lung epithelial cells constituting a human in vitro lung carcinogenesis system (Schroeder et al. 2006). These effects were significantly lower in treated normal human bronchial epithelial (NHBE) cells or small airway epithelial cells (SAEC) (Schroeder et al. 2006). Our group also showed previously that CD437 induced rapid apoptosis in lung cancer cells while sparing their normal counterparts (Sun et al. 2002). In the same report, low concentrations of CD437 (less than 0.5 mM) induced substantial apoptosis in NSCLC cells but only caused minor cell growth arrest in NHBE and SAEC cells (Sun et al. 2002). In addition, CD437 acted differently on cutaneous SCC and normal human epidermal keratinocytes where it induced apoptosis in the malignant cells and G1 arrest in the normal cells (Hail and Lotan 2001b). Recently we found that 4HPR induces apoptosis, evidenced by PARP cleavage, in UMSCC-22B head and neck squamous cancer cells but not in normal human gingival keratinocytes (NHGK), despite generation of reactive oxygen species in both cell types (Kadara et al. 2008a).We performed global gene expression analysis of the transcriptome of NHGK and UMSCC-22B HNSCC cells treated with vehicle dimethyl sulfoxide (DMSO) or one of the three retinoids for 24 h at pharmacologically achievable doses previously shown to inhibit cell growth (Sun et al. 1999a; Kim and Lotan 2004). It was readily observed that 4HPR modulated the expression level of a larger number of genes compared to both CD437 and ATRA in both the UMSCC-22B HNSCC and NHGK (Table 13.1). In addition, only 37 and 20 genes were modulated similarly by the three retinoids in the UMSCC-22B HNSCC cells Table 13.1 The number of differentially expressed genes including those up-regulated and down-regulated in UMSCC-22B HNSCC cells and NHGK following treatment of cells with 1mM ATRA, 1 mM CD437 and 5 mM 4HPR for 24 h Total number Number of Number of Cells Retinoid of genes increased genes decreased genes UMSCC-22B CD437 91 63 28 4HPR 269 144 125 ATRA 93 67 26 NHGK CD437 57 24 33 4HPR 210 125 85 ATRA 64 28 36

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Fig. 13.1 Common and distinct genes differentially expressed following treatment of normal and malignant head and neck cells with ATRA, CD437 and 4HPR. Venn diagrams illustrating common and unique genes differentially expressed in UMSCC-22B HNSCC cells (a) and NHGK (b) treated with 1 mM ATRA, 1mM CD437 or 5mM 4HPR for 24 h. c, Venn diagram depicting genes whose expression was modulated by 4HPR in either UMSCC-22B cells or NHGK and genes differentially expressed in both cell types.

and the NHGK, respectively (Fig. 13.1a, b). Moreover, 4HPR appeared to be unique in its modulation of gene expression compared to both CD437 and the natural retinoid ATRA (Fig. 13.1a and b). Furthermore, the expression levels of only 63 genes were modulated by 4HPR in both the normal and malignant cells with the majority of the genes altered distinctly in each cell type (Fig. 13.1c). These analyses indicate that the apoptogenic effects of 4HPR and CD437 in squamous normal and malignant head and neck cells are mediated by distinct molecular mechanisms. This contention is supported by several reports that had previously hinted at a different mode of action between both synthetic retinoid derivatives (Ponzanelli et al. 2000; Appierto et al. 2001; Holmes et al. 2002). Functional pathways analysis using Ingenuity Pathway Analysis® (IPA) (http://www.ingenuity.com) also highlighted the modulation in function of cancerrelated gene sets and pathways in UMSCC-22B HSNCC cells treated with the three retinoids compared with DMSO-treated cells (Fig. 13.2a and b). It is noteworthy that 4HPR displayed the most significant modulation in gene sets with cancerrelated biological and molecular functions indicated in Fig. 13.2a. Interestingly, UMSCC-22B cells exposed to 4HPR also exhibited distinct changes in the function of cancer-related canonical pathways with significantly less modulation of the RAR activation pathway compared to the same cells treated with CD437 or ATRA and significantly higher alterations in the functions of the nuclear factor (erythroidderived 2)-like 2 (NRF2) oxidative stress response (OSR) and the G1/S checkpoint

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Fig. 13.2 Functional expression analyses of genes differentially expressed in normal and malignant head and neck cells exposed to ATRA, CD437 and 4HPR. Functional pathways analyses of genes differentially expressed by at least 2-fold in UMSCC-22B HNSCC cells and NHGK treated with the indicated retinoids for 24 h relative to the same cells treated with DMSO using global functional categories from IPA®. The value of –log(significance) represents the inverse log of the p-values of the modulation of the depicted gene sets and functional pathways in the UMSCC-22B cells (a and b, respectively) and NHGK (c and d, respectively). The number of genes displaying more than two-fold change in each category is indicated above each bar.

regulation pathways (Fig. 13.2b). Similar differential patterns were observed in NHGK-treated cells (Fig. 13.2c). NHGK cells treated with 4HPR exhibited significantly higher functional changes in NRF2-mediated OSR and protein ubiquitination pathways compared to the same cells treated with ATRA or CD437 (Fig. 13.2d). Interestingly, many molecules within the NRF2 pathway are implicated in the survival arm of the endoplasmic reticulum stress response, namely the unfolded protein response (UPR), and function in maintaining the proper folding of proteins following exposure of cells to stressful stimuli (Wu and Kaufman 2006; Lau et al. 2008). It appears that the up-regulation by 4HPR of a plethora of prosurvival and cell homeostasis maintaining proteins, such as heat shock proteins, occurs to alleviate the stressful effects of ROS generation by this synthetic retinoid and to prevent subsequent apoptosis induction.

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It is worthwhile to mention that while ATRA and CD437 decreased the e xpression of several markers of squamous cell differentiation, such as cornifin and translgutaminase I, 4HPR did not. Our recent observations following transcriptome profiling of retinoid-treated normal and malignant squamous head and neck cells highlight the unique modulation of gene expression by 4HPR compared with ATRA and CD437 (unpublished observations).

13.7 Concluding Remarks Although retinoids, in particular natural derivatives, have been shown to clearly inhibit squamous differentiation of both normal and malignant squamous cells mainly by retinoid receptor-dependent mechanisms, their synthetic derivatives or related molecules exert their anticancer effects mostly through apoptosis induction independently of retinoid receptor activation. Furthermore, previous findings by other groups and our current observations pinpoint differences in the molecular mechanisms of action among synthetic retinoids such as 4HPR and CD437. It is plausible to suggest that combinations of natural and synthetic retinoids or of different synthetic retinoid derivatives could confer increased anticancer efficacy compared to each retinoid molecule alone. Our recent findings on the differential gene expression mediated by 4HPR in normal and malignant cancer cells shed light on novel mechanisms that may govern the selective killing of head and neck squamous carcinoma cells by this synthetic retinoid. It will be of interest and importance to investigate the role of the 4HPR-induced stress response pathways in malignant cells resistant to 4HPR. Despite the resistance of normal human gingival keratinocytes to 4HPR-induced apoptosis, the continuous generation of ROS in these cells should be further investigated to examine any possible affects on cellular genome integrity. We and others have previously demonstrated the implication of redox-maintaining genes in resistance to 4HPR. It is reasonable to propose that targeting the NRF2 oxidant stress response may increase the apoptogenic activity of 4HPR in squamous cancer cells especially those resistant to this synthetic retinoid. In conclusion, synthetic retinoids such as 4HPR, due to their multifaceted mechanisms of apoptosis induction, have promising clinical applications, in single or in combination with other agents, for treating cancers such as SCCs.

References Abou-Issa H, Moeschberger M, el-Masry W et al. (1995) Relative efficacy of glucarate on the initiation and promotion phases of rat mammary carcinogenesis. Anticancer Res 15:805–810 Altucci L, Gronemeyer H (2001) The promise of retinoids to fight against cancer. Nat Rev Cancer 1:181–193 Appierto V, Cavadini E, Pergolizzi R et al. (2001) Decrease in drug accumulation and in tumour aggressiveness marker expression in a fenretinide-induced resistant ovarian tumour cell line. Br J Cancer 84:1528–1534

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Chapter 14

Regulation of Keratinocyte Differentiation by Vitamin D and Its Relationship to Squamous Cell Carcinoma Arnaud Teichert and Daniel D. Bikle

Abstract Vitamin D is a fat-soluble steroid hormone originally described as c ontributing to the maintenance of normal levels of calcium and phosphorus in the bloodstream. Strictly speaking, it is not a vitamin because human skin can manufacture it, but it is referred to as one for historical reasons. Vitamin D aids in the intestinal absorption of calcium, helping to form and maintain bone mineralization in concert with a number of other vitamins, minerals and hormones. Thus, vitamin D prevents rickets in children and osteomalacia in adults–skeletal diseases that result in defects that weaken bones. Recent investigations have shown that vitamin D also functions as regulator of cellular growth and differentiation in various tissues, including the skin. The mechanisms by which 1,25 dihydroxyvitamin D3 (1,25(OH)2D3 or calcitriol), the active vitamin D metabolite, alters keratinocyte differentiation are multiple and overlap with the mechanisms by which calcium regulates keratinocyte differentiation. The antiproliferative, prodifferentiating effects of 1,25(OH)2D3 raise the hope that it may prevent malignant transformation of keratinocytes just as it appears to do in many other tissues. In particular, vitamin D has been evaluated for its potential anticancer activity because of the presence of vitamin D receptor (VDR) in most normal and malignant cells including basal and squamous-cell carcinomas and melanomas, and the susceptibility of VDR null mice to develop skin tumors. Physiological and pharmacological actions of 1,25(OH)2D3 in various systems have indicated potential applications of VDR ligands in inflammation, cancer and autoimmune disorders. As such, a better understanding of the metabolism and mechanism of action of vitamin D in the skin has opened up new perspectives for therapeutic application of vitamin D analogs in a number of skin diseases including the prevention of malignancy.

A. Teichert (*) Endocrine Unit, University of California, San Francisco, CA, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_14, © Springer Science+Business Media, LLC 2011

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14.1 Structure of the Skin The skin is comprised of the dermis and epidermis, with hair follicles as a specialized structure of the epidermis extending into the dermis. Fibroblasts, including the specialized group of fibroblasts forming the dermal papilla at the base of the hair follicle, are the principal cells of the dermis. The keratinocyte is the main cell of the epidermis which also includes the Langerhan cell, a major contributor to the immune function of skin, and melanocytes, the product of which provides important protection from ultraviolet (UV) damage to the stem cells and proliferating keratinocytes of the stratum basale. For the purpose of this chapter, only the epidermis will be considered. But the vitamin D receptor (VDR) is known to play a critical role in the control of hair follicle cycling, and hair follicle stem cells can in some situations contribute to the epidermal keratinocyte population. The epidermis is divided into four layers marked by different stages of differentiation. The basal layer (stratum basale), just above the basal lamina, contains the stem cells and proliferating (transient amplifying) keratinocytes, providing the cells for the upper differentiating layers. They express different keratins but principally keratin K5 (58 kDa) and K14 (50 kDa) (Moll et al. 1982). Cells migrate from this basal layer, and undergo terminal differentiation as they pass through the suprabasal layers until the fully differentiated corneocyte is formed which is eventually sloughed off. The layer above the basal cells is the spinous layer (stratum spinosum). These keratinocytes produce the keratins K1 and K10, which are the keratins characteristic of the more differentiated layers of the epidermis (Eichner et al. 1986). Cornified envelope precursors such as involucrin (Warhol et al. 1985) and the enzyme transglutaminase, responsible for the e-(g-glutamyl)lysine cross-linking of these substrates into the insoluble cornified envelope (Tracher and Rice 1985), appear in the spinous layer. The next layer of keratinocytes, the granular layer (stratum granulosum, SG), is characterized by electron-dense keratohyalin granules, which are of two types (Steven et al. 1990). The larger contains profilaggrin, the precursor of filaggrin, a protein thought to facilitate the aggregation of keratin filaments (Dale et al. 1985). The smaller granule contains loricrin, a major component of the cornified envelope (Mehrel et al. 1990). The granular layer also contains lamellar bodies (LB), lipidfilled structures that fuse with the plasma membrane at the junction of the stratum granulosum and stratum corneum (SC), divesting their contents into the extracellular space where the lipid contributes to the permeability barrier of the skin (Elias et al. 1988). This barrier also provides a defense to invasion by infectious organisms via its expression of the innate immune system. As the cells pass from the granular layer to the cornified layer (SC), they undergo destruction of their organelles with further maturation of the cornified envelope into an insoluble, highly resistant structure surrounding the keratin-filaggrin complex and linked to the extracellular lipid milieu (Hohl 1990).

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VDR has been found expressed in keratinocytes of all epidermal layers except those of the SC (Milde et al. 1991).

14.2 Regulation of Epidermal Keratinocyte Growth and Differentiation by 1,25(OH)2D3 14.2.1 Metabolism of 1,25(OH)2D3 The hormonal or bioactive form of vitamin D is 1,25(OH)2D3. It is generated from sequential hydroxylations of vitamin D3, a secosteroid precursor that is obtained from the diet or produced in the skin from 7-dehydrocholesterol upon exposure to UV light. During exposure to sunlight, epidermal keratinocytes are the site of UVBinduced photochemical conversion of 7-dehydrocholesterol (provitamin D3) to previtamin D3. Provitamin D3 is thermodynamically unstable and converts to the more thermodynamically stable vitamin D3 (cholecalciferol) (Holick 2004). Vitamin D3 is hydroxylated by the vitamin D3-25 hydroxylase (25OHase) to form 25-hydroxyvitamin D3 and then by the 25 hydroxyvitamin D3-1 alpha hydroxylase (1OHase) to form the biologically active metabolite 1 alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol) (Bikle et al. 1986b; Lehmann et al. 2001). These sequential hydroxylations of vitamin D3 to form 1,25(OH)2D3 were originally thought to take place in the liver and kidney, respectively, but are now known to occur in a number of tissues, most importantly for this discussion in the epidermis. In particular, keratinocytes appear to be the only cell in the body capable of the entire pathway from 7-dehydrocholesterol to 1,25(OH)2D3. The vitamin D-25 hydroxylase in keratinocytes is the same mitochondrial enzyme (CYP27A1) that converts vitamin D to 25OHD in the liver (Lehmann et al. 1999; Masumoto et al. 1988). Its expression is increased by vitamin D and UVB irradiation (Lehmann et al. 1999). Similarly the 25OHD-1a-hydroxylase in the epidermis, which is responsible for 1,25(OH)2D3 production, is the same enzyme (CYP27B1) as that found in the kidney (Fu et al. 1997). Its expression and enzymatic activity are tightly regulated and coupled to the differentiation of these cells. The epidermis is likely to contribute to the total 1,25(OH)2D3 production as human keratinocytes rapidly and extensively convert 25OHD to 1,25(OH)2D3, although it is not clear how much of the 1,25(OH)2D3 produced by the epidermis actually enters the circulation. Peak levels of 1,25(OH)2D3 are reached in the cell within 1 h after adding 25OHD. By 1 h, 1,25(OH)2D3 is the main metabolite observed; however, other metabolites appear with continued incubation, many of which represent degradation products of 1,25(OH)2D3. When renal production of 1,25(OH)2D3 is normal the circulating levels of 1,25(OH)2D3

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are sufficient to limit the contribution from epidermal production. This appears to be due to the induction of 25OHD-24-hydroxylase (CYP24) in the keratinocyte by 1,25(OH)2D3, which catabolizes the endogenously produced 1,25(OH)2D3 and as such may protect the body from excessive 1,25(OH)2D3 production (Xie et al. 2002b).

14.2.2 Hormonal Regulation of 1,25(OH)2D3 Both the formation and catabolism of 1,25(OH)2D3 are under hormonal control. Parathyroid hormone (PTH) exerts a modest stimulation of 1,25(OH)2D3 production. Surprisingly, cAMP does not stimulate 1,25(OH)2D3 production in keratinocytes which also lack the classic PTH receptor. On the other hand the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) inhibits 1,25(OH)2D3 degradation (Bikle et al. 1986b). In combination, PTH and IBMX markedly increase the amount of 1,25(OH)2D3 that accumulates within the keratinocyte following the addition of 25OHD. In renal cells, PTH directs a more acute stimulation of 1,25(OH)2D3 production (Rasmussen et al. 1972), and cAMP appears to play a second messenger role (Rost et al. 1981). Thus, the regulation of 1,25(OH)2D3 production in keratinocytes by PTH and cAMP differs from their regulation of 1,25(OH)2D3 production in renal cells. The mechanism by which PTH stimulates 1,25(OH)2D3 production in keratinocytes is unclear. The hormone 1,25(OH)2D3 negatively regulates its own levels within the keratinocyte. This negative feedback loop is similar to that observed in the kidney, but it differs from that seen in the macrophage, which lacks this feedback loop. In the keratinocyte, this feedback inhibition is not mediated by an effect on 1,25(OH)2D3 production but is due solely to stimulation of 1,25(OH)2D3 catabolism through induction of the enzyme 25OHD 24-hydroxylase that converts 25OHD and 1,25(OH)2D3 to 24,25(OH)2D and 1,24,25(OH)3D, respectively (Fu et al. 1997). An important difference in the regulation of 25OHD metabolism by 1,25(OH)2D3 between keratinocytes and renal cells is that the concentration of 1,25(OH)2D3 required to induce the 24-hydroxylase in renal cells appears to be several orders of magnitude greater than that required for comparable effects in keratinocytes (Bikle et al. 1986b; Spanos et al. 1978; Trechsel et al. 1979). Thus, the detectable 1,25(OH)2D3 production by keratinocytes is exquisitely sensitive to exogenous 1,25(OH)2D3. This difference in sensitivity to feedback inhibition by 1,25(OH)2D3 between keratinocytes and renal cells may account for the observation that following acute nephrectomy extrarenal production of 1,25(OH)2D3 is very low (Reeve et al. 1983; Shultz et al. 1983); only with time after renal production has ceased does extrarenal production emerge. A similar observation was made in pig skins perfused with 25OHD; the amount of 1,25(OH)2D3 produced was initially low but increased after 4–8 h of perfusion (Bikle et al. 1994).

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14.2.3 Effects of Differentiation on 1,25(OH)2D3 Levels In the presence of adequate levels of calcium, keratinocytes progress from rapidly proliferating cells to cells capable of making cornified envelopes, one of the most distinctive features of epidermal terminal differentiation. As the cells differentiate in culture there is a sequential increase in involucrin, transglutaminase, and cornified envelope formation (Pillai et al. 1988a, b). 25OHD 1a-hydroxylase and 24-hydroxylase also change with differentiation (Pillai et al. 1988a). Preceding the rise in transglutaminase and involucrin is a rise in 25OHD 1a-hydroxylase activity; the 1a-hydroxylase activity, transglutaminase activity, and involucrin then fall as cornified envelopes appear. The appearance of cornified envelopes and the fall in 1a-hydroxylase activity coincide with a rise in 24-hydroxylase activity (Pillai et al. 1988a). The change in activity reflects a change in expression of the gene, although the means by which the expression of the 1a-hydroxylase is controlled during differentiation has not yet been determined. Growing the cells in 0.1 mM calcium, which retards differentiation (Pillai et al. 1988b), permits the cells to maintain higher 1a-hydroxylase activity than when they are grown in 1.2 mM calcium (Bikle et al. 1989), although acute changes in calcium have little effect on 1,25(OH)2D3 production (Bikle et al. 1986b). These changes in 1a-hydroxylase expression in vitro are consistent with the finding of higher levels of 1a-hydroxylase in the stratum basale than in the suprabasal levels of the epidermis in vivo (Zehnder et al. 2001).

14.2.4 Regulation of 1,25(OH)2D3 by Cytokines Both tumor necrosis factor-a (TNFa) and interferon-g (IFN-g) promote the differentiation of keratinocytes (Morhenn and Wood 1988; Pillai et al. 1989). Both cytokines regulate 1,25(OH)2D3 production by these cells in a manner consistent with their effects on differentiation (Bikle et al. 1989, 1991b). Unlike PTH and 1,25(OH)2D3, these cytokines must be incubated with the keratinocytes for at least 1 day (not hours) before their effects on 1,25(OH)2D3 production are observed. These cells are exquisitely sensitive to IFN-g, with maximal stimulation of 1,25(OH)2D3 production at concentrations less than 10 pM. Higher concentrations are inhibitory, but such concentrations also profoundly inhibit the proliferation of these cells and limit their ability to differentiate. Keratinocytes grown in 0.1 mM calcium are more sensitive to IFN-g than cells grown in 1.2 mM calcium (Bikle et al. 1989). Serum markedly reduces the potency of IFN-g in this system, for reasons that are unknown. TNFa stimulates 1,25(OH)2D3 production and transglutaminase activity in preconfluent cells (Bikle et al. 1991b), and it can reverse the inhibition seen with the higher concentrations of IFN-g. The effects of TNFa and IFN-g are not additive at the lower and stimulatory concentrations of IFN-g. When TNFa is added after the cells have reached confluence, a time after which 1,25(OH)2D3 production (and transglutaminase activity) has peaked, TNFa inhibits 1,25(OH)2D3 production (and transglutaminase activity) even as it stimulates cornified envelope

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formation. Although IFN-g is not made in keratinocytes, TNFa is produced by these cells, and its synthesis is stimulated by UV light (Trefzer et al. 1993) and barrier disruption (Wood et al. 1994). Thus, environmental perturbations could enhance 1,25(OH)2D3 production in the skin, and the increased levels of 1,25(OH)2D3 could play a role in the recovery from UV damage and/or barrier repair (Bikle et al. 2004).

14.2.5 1,25(OH)2D3 Response and Production in Transformed Keratinocytes Keratinocytes from squamous cell carcinomas (SCC) do not differentiate normally in response to calcium (Rheinwald and Beckett 1980) or 1,25(OH)2D3 (Bikle et al. 1991a) even though in these cells differentiation marker genes can be induced by serum (Rasmussen et al. 1972). Nevertheless, these cells produce 1,25(OH)2D3 [and 24,25(OH)2D], and in some cases the rates of production are comparable with those of normal keratinocytes (Bikle et al. 1991a). Furthermore, the SCC lines respond to exogenous 1,25(OH)2D3 with a reduction in 1,25(OH)2D3 production and an increase in 24,25(OH)2D production, although in some cases the sensitivity of the SCC line to 1,25(OH)2D3 is less than normal (Bikle et al. 1991a). The levels of the VDR mRNA and protein in SCC are comparable to those in normal keratinocytes (Rasmussen et al. 1972), suggesting that the reason why 1,25(OH)2D3 can regulate 25OHD metabolism but not differentiation in SCC lies in other transcription factors required for calcium and 1,25(OH)2D3 regulation of the differentiation pathway. We have shown that SCC overexpress the coactivator DRIP205 (vitamin D Receptor Interacting Protein 205), which anchors a large complex of proteins to the VDR and the RNA II polymerase complex at the transcription start site. The DRIP complex is functional primarily in proliferating keratinocytes, and is replaced by the steroid receptor coactivator (SRC) mediated complexes as keratinocytes differentiate. Thus one possibility for the limited differentiation of SCC is their failure to achieve the transition from the DRIP coactivation of VDR to SRC coactivation required for terminal differentiation. The role of these coactivators is discussed below.

14.3 Actions of 1,25(OH)2D3 14.3.1 Binding of 1,25(OH)2D3 to the Vitamin D Receptor Actions of 1,25(OH)2D3 and its synthetic analogs are mediated through the VDR, which belongs to the superfamily of nuclear hormone receptors. The VDR contains several functional domains, including a ligand-binding domain (LBD), that mediates ligand-dependent gene regulation (Carlberg and Polly 1998). A critical step in 1a,25(OH)2D3 action is the induction of an LBD conformational change to form activation function 2 (Moras and Gronemeyer 1998), which serves as a binding

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surface for coactivators (Feng et al. 1998). Unliganded nuclear receptor dimers associate with corepressors (Chen and Evans 1995; Kurokawa et al. 1995) and associated histone deacetylases (Heinzel et al. 1997; Nagy et al. 1997). These proteins function as adaptors to convey a repressive signal to the transcriptional apparatus by maintaining a closed chromatin structure (Belandia and Parker 2003). Ligand binding promotes the dimerization of the VDR with the retinoid X receptor alpha (RXRa) (Koli and Keski-Oja 1993), the release of corepressors and the binding of coactivators, enhancing the transcription of specific genes (Robyr et al. 2000). Some coactivators, such as the SRC family (Hong et al. 1996; Voegel et al. 1996; Zhu et al. 1996), recruit other coregulators with histone acetylase activity and remodel chromatin structure. Other coactivators, such as the DRIP factors (Rachez et al. 1999; Yuan et al. 1998), interact with the basal transcriptional machinery. Another coregulator, Hairless (Hr), is of great interest because null mutations in either VDR or Hr induces alopecia, in both the mouse and human (Cichon et al. 1998; Li et al. 1997; Yoshizawa et al. 1997) and Hr can repress VDR mediated transcription (Hsieh et al. 2003; Xie et al. 2006). The interaction of VDR with these coactivators and corepressors will be discussed further below. However, it is not clear that all actions of VDR require 1,25(OH)2D3. Hair follicle cycling, which is impaired in mice lacking the VDR, is normal in mice lacking the ability to produce 1,25(OH)2D3 (CYP27B1 knockouts). A recent study even suggests that some of the best known 1,25(OH)2D3 dependent actions of VDR such as CYP24 induction can occur in primary keratinocytes independent of 1,25(OH)2D3 through a 1,25(OH)2D3-independent VDR-RXR heterodimerization that is sufficient to drive transactivation of the 24-hydroxylase promoter (Ellison et al. 2007).

14.3.2 VDR Interactions with Coactivators We recently demonstrated that VDR, DRIP, and SRC are all required for promotion of both early and late keratinocyte differentiation (Hawker et al. 2007). Additionally, each keratinocyte differentiation marker assayed has a different specificity for the coactivators that regulate its expression. These results suggest the selective use of coactivators by VDR for selective regulation of gene expression in keratinocyte proliferation and differentiation. This leads to the concept in which the coactivator complex binding to the VDR in SCC and proliferating keratinocytes is the DRIP complex (Bikle et al. 2003; Oda et al. 2007). As keratinocytes differentiate, components of the DRIP complex are no longer produced, whereas SRC 3 (steroid receptor coactivator 3) increases in levels and binding to the VDR. This transition does not take place in SCC. The 24-hydroxylase gene appears to be activated by VDR bound to either DRIP or SRC3, whereas other vitamin D-regulated genes involved with differentiation (e.g., involucrin) prefer VDR bound to SRC3. Moreover, permeability barrier formation, a process correlated with keratinocyte differentiation, is controlled by VDR and SRC3 but not DRIP205 (Oda et al. 2008). Similarly the innate immune response induced by 1,25(OH)2D3 requires SRC3 but not DRIP205 (Schauber et al. 2007b).

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14.3.3 Action of 1,25(OH)2D3 on Cellular Proliferation 1,25(OH)2D3 blocks keratinocyte proliferation at the G0/G1 to S transition, p resumably following an up-regulation of the cell-cycle inhibitors p21 and p27 (Kira et al. 2003; Pinette et al. 2003; Sebag et al. 1992) and a reduction in the mRNA levels for c-myc (Matsumoto et al. 1990). Furthermore, 1,25(OH)2D3 also increases transforming growth factor beta1 and beta2 production by keratinocytes (Verlinden et al. 1998), that indirectly could mediate the antiproliferative effects of 1,25(OH)2D3. At subnanomolar concentrations, 1,25(OH)2D3 has been found to promote proliferation in some studies (Bollag et al. 1995; Gniadecki 1996; Itin et al. 1994), although antiproliferative actions are most frequently observed, especially when concentrations above 10−9 M are employed. The mechanisms underlying the proliferative actions are not known. 1,25(OH)2D3 also regulates expression of the skin calcium binding protein (RizkRabin and Pavlovitch 1988), causes a decrease in parathyroid hormone-related peptide (Kremer et al. 1991) and EGF receptor gene expression (Matsumoto et al. 1990), and increases in plasminogen activator inhibitor (Hashiro et al. 1991) gene expression. Where examined, these effects of 1,25(OH)2D3 can be reproduced by 25OHD (Matsumoto et al. 1991), presumably because of endogenous conversion of 25OHD to 1,25(OH)2D3, but are not observed with the biologically inactive b isomer of 1,25(OH)2D3, the natural isomer of la, 25(OH)2D3 (Smith et al. 1986).

14.3.4 Actions of 1,25(OH)2D3 on Epidermal Differentiation The observation that 1,25(OH)2D3 induces keratinocyte differentiation was first made by Hosomi et al. (Hosomi et al. 1983) and provided a rationale for the previous and unexpected finding of 1,25(OH)2D3 receptors in the skin (Stumpf et al. 1979). 1,25(OH)2D3 is likely to be an autocrine or paracrine factor for epidermal differentiation since it is produced by the keratinocyte, but under normal circumstances keratinocyte production of 1,25(OH)2D3 does not appear to contribute to circulating levels (Bikle et al. 1986a, b). VDR expression and the production of 1,25(OH)2D3 vary with differentiation (Horiuchi et al. 1985; Merke et al. 1985; Pillai et al. 1988a) in a manner that suggests feedback regulation; both are reduced in the later stages of differentiation. 1,25(OH)2D3 increases involucrin, transglutaminase activity, and cornified envelope formation at subnanomolar concentrations in preconfluent keratinocytes (Bikle et al. 1991a; Hosomi et al. 1983; McLane et al. 1990; Pillai and Bikle 1991; Smith et al. 1986). VDR controls the permeability barrier formation by regulating the expression of multiple enzymes involved in the glucosylated ceramide synthetic pathway and through its action on stratification and lamellar bilayer formation, LB formation, contents and secretion (Oda et al. 2008). Furthermore, 1,25(OH)2D3 is critical for the innate immune response of the skin, which is part of the barrier function of the skin, by regulating the expression of microbial pattern recognition receptors TLR2, CD14, and the antimicrobial peptide cathelicidin (Schauber et al. 2007a, b).

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1,25(OH)2D3 action on epidermal differentiation overlaps with different mechanisms regulating keratinocyte differentiation, the best studied of which is calcium. 14.3.4.1 Calcium-Regulated Differentiation of Epidermal Keratinocytes Calcium is the best studied prodifferentiating agent for keratinocytes. In vivo, a calcium gradient exists in the epidermis such that in the basal and spinous layers calcium is primarily intracellular and in low amounts, but in the upper granular layers calcium accumulates in large amounts in the cell and intercellular matrix (Menon et al. 1985). This gradient of calcium may provide the driving force for differentiation in the intact epidermis (Elias et al. 2002). Disruption of the permeability barrier by removing the SC or extracting its lipids leads to a loss of this calcium gradient (Mauro et al. 1998) resulting in increased LB secretion but reduced expression of loricrin, profilaggrin, and involucrin genes (Elias et al. 2002). In keratinocyte cell cultures, within minutes to hours of an increase in extracellular calcium concentration ([Ca]o), morphological changes are apparent, with rapid development of cell to cell contact (Hennings et al. 1980), desmosome formation (Hennings and Holbrook 1983), and a realignment of actin and keratin bundles near the cell membrane at the point of intercellular contacts (Zamansky et al. 1991). The cells begin to make involucrin (Pillai et al. 1990; Rubin et al. 1989; Su et al. 1994), loricrin (Hohl et al. 1991), transglutaminase (Pillai et al. 1990; Rubin et al. 1989; Su et al. 1994), keratins K1 and K10 (Yuspa et al. 1989), and filaggrin (Yuspa et al. 1989), and they start to form cornified envelopes (Pillai et al. 1990; Yuspa et al. 1989). Calcium response regions have been identified in the involucrin (Ng et al. 1996) and Kl (Huff et al. 1993) genes. The redistribution of integrin isoforms within days following the calcium switch (Guo et al. 1991; Marchisio et al. 1991; Ryynanen et al. 1991) may participate in the mechanism by which cells begin to stratify. The intracellular free calcium ion concentration ([Ca]i) increases as keratinocytes differentiate, correlating closely with their ability to form cornified envelopes (Pillai and Bikle 1991). Raising [Ca]o increases [Ca]i (Huff et al. 1993; Ng et al. 1996; Pillai and Bikle 1991; Su et al. 1994; Yoneda et al. 1990; Yuspa et al. 1989), through mechanisms involving at least the E-cadherin adherent junction and the calcium sensing receptor, as discussed further below. Calcium and 1,25(OH)2D3 interact in their ability to inhibit proliferation and stimulate involucrin and transglutaminase gene expression (Su et al. 1994). The higher the [Ca]o, the more sensitive is the keratinocyte to the antiproliferative effect of 1,25(OH)2D3 (and vice versa) (McLane et al. 1990). The interaction on gene expression is more complex. Both calcium (in the absence of 1,25(OH)2D3) and 1,25(OH)2D3 (at 0.03 mM [Ca]o) raise the mRNA levels for involucrin and transglutaminase in a dose-dependent fashion. The stimulation is synergistic at intermediate concentrations of calcium (0.1 mM) and 1,25(OH)2D3 (10−10 M), but inhibition is observed in combination at higher concentrations. The synergism is more apparent at earlier times after the calcium switch (4 h) than later (24–72 h), when increased turnover of the mRNA by the higher combined concentrations of calcium and 1,25(OH)2D3 becomes dominant. At least one explanation for the synergism in the

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induction of involucrin is that the calcium response element (CaRE) and vitamin D response element (VDRE) in the involucrin promoter are quite close spatially (Bikle et al. 2002). Mutations in the AP-1 site within the CaRE block both calcium and 1,25(OH)2D3 induction of the involucrin gene, but mutations of the VDRE block only its response to 1,25(OH)2D3. 14.3.4.2 Phosphoinositide Metabolism Phosphoinositide metabolism potentially provides additional second messengers for mediating keratinocyte differentiation (Jaken and Yuspa 1988; Lee and Yuspa 1991; Moscat et al. 1989; Tang et al. 1988). The main enzymes involved are phospholipase C (PLC) b and g1 which hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to the important second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG). Activation of PLC-g1 is required for calcium-induced keratinocyte differentiation (Xie and Bikle 1999). Both calcium and 1,25(OH)2D3 induce PLC-b and -g1 (Pillai et al. 1995; Xie and Bikle 1999), which will increase IP3 formation (Punnonen et al. 1993). Raising [Ca]o induces the co-localization of a-, b- and p120-catenin with E-cadherin at the intercellular adherent junctions (AJs) (Pokutta and Weis 2007), and the recruitment of phosphatidylinositol 3 kinase (PI3K) to this complex (Xie and Bikle 2007). The recruitment of PI3K is required for the calcium-induced PLC-g1 activation and, ultimately, keratinocyte differentiation (Xie and Bikle 2007), by stimulating the conversion of PIP2 to phosphatidylinositol trisphosphate (PIP3), which binds to and activates PLC-g1 (Xie et al. 2005). Raising [Ca]o also induces the recruitment of phosphatidylinositol-4-phosphate 5-kinase 1a (PIP5K1a), which is required for calcium-induced keratinocyte differentiation, by the E-cadherin-catenins complex (Xie et al. 2009). PIP5K1a converts phosphatidylinositol monophosphate (PIP) to PIP2, which is then used by PI3K to form PIP3 and thus activate PLC-g1 (Xie et al. 2009). Furthermore, PLC-g1 is required for the activation of store operated calcium influx (Tu et al. 2005), through its interaction with the transient receptor potential channel 1 (TRPC1), in association with the inositol 1,4,5-trisphosphate receptor (IP3R). This association is calcium dependant and decreases as [Ca]i increases. An acute increase in [Ca]i associated with an acute increase in phosphoinositide turnover (producing a rise in both IP3 and DAG) has been observed in several studies involving 1,25(OH)2D3 (Bittiner et al. 1991; MacLaughlin et al. 1990; Tang and Ziboh 1991; Tang et al. 1987; Yada et al. 1989). Not all investigators have been able to reproduce these acute effects of 1,25(OH)2D3 (Bikle et al. 1996), although a gradual rise in [Ca]i and cornified envelope formation is observed (Pillai and Bikle 1991). 14.3.4.3 Interaction with the Calcium Sensing Receptor The rise in [Ca]i following increased [Ca]o is believed to be through plasma membrane calcium channels and through store-operated channels. One of the key players

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in the keratinocyte response to calcium is a seven-transmembrane domain, GTP binding-protein-coupled calcium sensing receptor (CaSR) (Brown et al. 1993; Garrett et al. 1995). Activation of CaSR with calcium activates phospholipase C (PLC), which converts phosphatidylinositol 4,5-bisphosphate into DAG and IP3. IP3 binds to its receptor, IP3R, in the endoplasmic reticulum and Golgi apparatus membrane and triggers release of calcium from internal stores, resulting in an increase in [Ca]i (Berridge 1993). CaSR is a necessary component of the calcium-sensing apparatus in keratinocytes as the epidermis of mice lacking the CaSR contains markedly lower levels of terminal differentiation markers, and those keratinocytes fail to respond to calcium with a substantial rise in [Ca]i (Oda et al. 2000). In human keratinocyte culture, the glycosylated form of the CaSR forms a complex with phospholipase C g1, IP3R and the Golgi Ca2 + -ATPase and regulates keratinocyte differentiation in part by modulating [Ca]i stores (Tu et al. 2007). The stimulation of E-cadherin/PI3K signaling by calcium is mediated in part by the calcium-induced expression and activation of Src and Fyn families of tyrosine kinases (Calautti et al. 1995, 1998; Carpenter and Ji 1999; Xie et al. 2005). Following the calcium switch, CaSR stimulates the phosphorylation of Fyn and its association with the E-cadherin/PI3K complex (Tu et al. 2008). Moreover, CaSR is required for the calcium-induced AJ formation, membrane translocation and the complex formation of E-cadherin with catenins and PI3K (Tu et al. 2008). 1,25(OH)2D3 increases the CaSR mRNA levels and prevents their decrease with time (Ratnam et al. 1999). Furthermore, 1,25(OH)2D3 potentiates the ability of these cells to respond to [Ca]o with a rise in [Ca]i (Ratnam et al. 1999). Thus, the CaSR is an important mediator in the [Ca]i response of keratinocytes to [Ca]o, and provides a mechanism by which 1,25(OH)2D3 can regulate calcium-induced epidermal differentiation. 14.3.4.4 Action on the Permeability Barrier Formation One of the functions of the differentiated keratinocyte is to form a permeability barrier, made by a cornified envelope, in which proteins such as involucrin and loricrin are crosslinked into an insoluble matrix, and long chain lipids, glucosylceramides in particular, are produced and secreted into this matrix to control transepidermal water movements. This barrier is formed by the stratification of corneocyte layers enclosed by a cornified envelope formed by lipid secretions from LB produced by the SG (Holleran et al. 2006; Schurer and Elias 1991). In human epidermal keratinocyte cultures, VDR regulates the expression of multiple enzymes involved in the glucosylated ceramides synthetic pathway, including the epidermal specific GlcCer (acylGlcCer), through a mechanism involving SRC3 but not DRIP205 (Oda et al. 2008). VDR controls sphingol synthesis by regulating the expression of the desaturase enzymes, DES1 and DES2. Both VDR and SRC3 control the elongation of fatty acids by regulating the expression of the elongase ELOVL4, a key step in acylCer and acylGlcCer synthesis. They also control ceramide glucosylation by regulating the expression and enzyme activity

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of ceramide glucosyl transferase, which catalyzes the conversion of de novo synthesized Cer into GlcCer. Finally, VDR and SRC3 regulate the expression of the lipid transporter ABCA12, involved in lipid loading into the LB. VDR also controls stratification and lamellar bilayer formation, as well as LB numbers and contents (Oda et al. 2008). In addition, very little LB secretion occurs into the extracellular spaces in cells lacking VDR. Similar results are obtained in the absence of SRC3 suggesting a close interaction between VDR and SRC3 in the control of these processes. Again, these data suggest that DRIP205 and SRC3 play differential roles in VDR regulation during keratinocyte differentiation, with SRC3 playing the dominant role as keratinocytes differentiate. When examined in VDR null mouse, similar results have been observed, with a reduction in LB numbers, failure of those LBs to fuse with the membrane separating the SG and SC, resulting in abnormal junction formation and a defective barrier (Oda et al. 2008).

14.3.5 Action on Skin Innate Immunity The barrier function of the epidermis includes its resistance to infection by pathogenic organisms. Part of this defense relies on innate immune responses, which involve the activation of toll-like receptors (TLRs) in polymorphonuclear cells, monocytes and macrophages that leads to the expression of antimicrobial peptides, such as cathelicidin, and reactive oxygen species, to kill the pathogen. 1,25(OH)2D3 is critical for this system, in part by inducing the expression of microbial pattern recognition receptors TLR2, as well as their coreceptor CD14, and the antimicrobial peptide cathelicidin, through a mechanism that requires SRC3 but not DRIP205 (Schauber et al. 2007a, b). Skin wounding enhances 1,25(OH)2D3 synthesis through the induction of CYP27B1 expression in the skin (Schauber et al. 2007a). It also induces, through 1,25(OH)2D3, the expression of TLR2 and CD14, complementing an increase in cathelicidin expression (Schauber et al. 2007a). Vitamin D analogs can also stimulate differentiation of immature precursor immune cells, expressing CD34, into dendritic cells capable of antigen presentation (Lathers et al. 2004). This approach has been considered to induce, in SCC tumors, an immune response, otherwise inhibited by the production of immune suppressive factors and immune inhibitory cells (Young and Lathers 2005).

14.3.6 Mouse Models The recent availability of mice lacking either the VDR or the 1a-hydroxylase has expanded our understanding of the role of 1,25(OH)2D3 in epidermal differentiation. Although the most striking feature of the VDR-null mouse is the development of alopecia (also found in many patients with mutations in the VDR referred to as

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hereditary vitamin D resistance), these mice also exhibit a defect in epidermal differentiation as shown by reduced levels of involucrin and loricrin and loss of keratohyalin granules (Xie et al. 2002a). Moreover, as observed in cultured keratinocytes, VDR null mice display a reduction in LB numbers, failure of those LBs to fuse with the membrane separating the SG and SC, resulting in abnormal junction formation and defective barrier. A similar phenotype is observed in the 1a-hydroxylase null mouse. These mice also show a reduction in levels of the epidermal differentiation markers (Bikle et al. 2004). Furthermore, the 1a-hydroxylase-null animals have a retarded recovery of barrier function when the barrier is disrupted, which on ultrastructural examination is associated with a reduction in LB secretion and an impaired reestablishment of the calcium gradient in the epidermis (Bikle et al. 2004). These mice also fail to induce the expression of CD14 in response to wounding, a critical step in innate immune response, confirming the critical role of 1,25(OH)2D3 in the control of this response (Schauber et al. 2007a). However, the 1a-hydroxylase-null animals do not have a defect in hair follicle cycling. The difference in phenotypes between these genotypes is surprising and points to the possibility that the 1a-hydroxylase in the epidermis may be doing more than making 1,25(OH)2D3, just as the phenotype in the VDR-null animal suggests that the VDR may have functions independent of 1,25(OH)2D3.

14.4 1,25(OH)2D3 and Cutaneous Cancer 1,25(OH)2D3 has been evaluated for its potential anticancer activity for approximately 25 years (Eisman et al. 1979). The list of malignant cells that express VDR includes basal cell (BCC) and SCCs (Kamradt et al. 2003; Ratnam et al. 1996) as well as melanomas (Colston et al. 1981). The accepted basis for the promise of 1,25(OH)2D3 in the prevention and treatment of malignancy includes its antiproliferative, prodifferentiating effects on most cell types. Epidemiologic evidence supporting the importance of adequate vitamin D nutrition (including sunlight exposure) for the prevention of a number of cancers including those of the colon, breast, and prostate (Bostick et al. 1993; Garland et al. 1985, 1990; Hanchette and Schwartz 1992; Kearney et al. 1996) is strong. However, several large epidemiologic surveys have not shown such a correlation with skin cancers (Hunter et al. 1992; van Dam et al. 2000; Weinstock et al. 1992), although this issue is being reexamined. One potential complication is that UVB radiation, one of the main causes of cutaneous cancer, has the dual effect of promoting vitamin D3 synthesis in the skin (which can be further converted to 1,25(OH)2D3) and increasing DNA damage leading to skin cancer. On the other hand recent studies in animals indicate that vitamin D plays a protective role in the skin with respect to carcinogenesis. VDR KO mice treated with the carcinogen 7,12 dimethylbenzanthracene (Indra et al. 2007; Zinser et al. 2002) or exposed to UVB radiation (Ellison et al. 2008) develop skin tumors at a much

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higher rate than their wildtype controls. Interestingly, VDR KO mice treated with DMBA developed mostly sebaceous, squamous and follicular papillomas and several BCC (Indra et al. 2007; Zinser et al. 2002), unlike RXRa null mice which developed both BCC and SCC (Indra et al. 2007). Conversely, VDR KO mice treated with UVB radiation developed SCC (Ellison et al. 2008). Those differences most likely illustrate the complex and interconnected regulatory mechanisms by VDR in the skin acting through multiple pathways. Each of these carcinogenic agents likely triggers the activation of a different sub-set of pathways. Additionally, a recent publication (Palmer et al. 2008) shows that expression levels of VDR correlates with the type of tumor arising in the skin. The development of trichofolliculomas by b-catenin overexpression can be blocked by an analog of 1,25(OH)2D3, and in the absence of VDR, b-catenin overexpression induces BCC formation rather than trichofolliculomas (Palmer et al. 2008). As mentioned previously some transformed cells over express the DRIP complex, whose levels in normal keratinocytes decrease with differentiation, a mechanism that does not take place in SCC (Kim et al. 1992; Oda et al. 2003). This over expression may block the ability of 1,25(OH)2D3 to promote their differentiation.

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Weinstock MA, Stampfer MJ, Lew RA, Willett WC, Sober AJ (1992) Case-control study of melanoma and dietary vitamin D: implications for advocacy of sun protection and sunscreen use. J Invest Dermatol 98:809–811 Wood LC, Elias PM, Sequeira-Martin SM, Grunfeld C, Feingold KR (1994) Occlusion lowers cytokine mRNA levels in essential fatty acid-deficient and normal mouse epidermis, but not after acute barrier disruption. J Invest Dermatol 103:834–838 Xie Z, Bikle DD (1999) Phospholipase C-gamma1 is required for calcium-induced keratinocyte differentiation. J Biol Chem 274:20421–20424 Xie Z, Bikle DD (2007) The recruitment of phosphatidylinositol 3-kinase to the E-cadherincatenin complex at the plasma membrane is required for calcium-induced phospholipase C-gamma1 activation and human keratinocyte differentiation. J Biol Chem 282:8695–8703 Xie Z, Komuves L, Yu QC, Elalieh H, Ng DC, Leary C, Chang S, Crumrine D, Yoshizawa T, Kato S, Bikle DD (2002a) Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth. J Invest Dermatol 118:11–16 Xie Z, Munson SJ, Huang N, Portale AA, Miller WL, Bikle DD (2002b) The mechanism of 1, 25-dihydroxyvitamin D(3) autoregulation in keratinocytes. J Biol Chem 277:36987–36990 Xie Z, Singleton PA, Bourguignon LY, Bikle DD (2005) Calcium-induced human keratinocyte differentiation requires src- and fyn-mediated phosphatidylinositol 3-kinase-dependent activation of phospholipase C-gamma1. Mol Biol Cell 16:3236–3246 Xie Z, Chang S, Oda Y, Bikle DD (2006) Hairless suppresses vitamin D receptor transactivation in human keratinocytes. Endocrinology 147:314–323 Xie Z, Chang SM, Pennypacker SD, Liao EY, Bikle DD (2009) Phosphatidylinositol-4-phosphate 5-kinase 1alpha mediates extracellular calcium-induced keratinocyte differentiation. Mol Biol Cell 20:1695–1704 Yada Y, Ozeki T, Meguro S, Mori S, Nozawa Y (1989) Signal transduction in the onset of terminal keratinocyte differentiation induced by 1 alpha, 25-dihydroxyvitamin D3: role of protein kinase C translocation. Biochem Biophys Res Commun 163:1517–1522 Yoneda K, Fujimoto T, Imamura S, Ogawa K (1990) Distribution of fodrin in the keratinocyte in vivo and in vitro. J Invest Dermatol 94:724–729 Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, Kawakami T, Arioka K, Sato H, Uchiyama Y, Masushige S, Fukamizu A, Matsumoto T, Kato S (1997) Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet 16:391–396 Young MR, Lathers DM (2005) Combination docetaxel plus vitamin D(3) as an immune therapy in animals bearing squamous cell carcinomas. Otolaryngol Head Neck Surg 133:611–618 Yuan C, Ito M, Fondell J, Fu Z, Roeder R (1998) The TRAP220 component of a thyroid hormone receptor- associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc Natl Acad Sci USA 95:7939–7944 Yuspa SH, Kilkenny AE, Steinert PM, Roop DR (1989) Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol 109:1207–1217 Zamansky GB, Nguyen U, Chou IN (1991) An immunofluorescence study of the calcium-induced coordinated reorganization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. J Invest Dermatol 97:985–994 Zehnder D, Bland R, Williams MC, McNinch RW, Howie AJ, Stewart PM, Hewison M (2001) Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase. J Clin Endocrinol Metab 86:888–894 Zhu Y, Qi C, Calandra C, Rao MS, Reddy JK (1996) Cloning and identification of mouse steroid receptor coactivator-1 (mSRC-1). as a coactivator of peroxisome proliferator-activated receptor gamma. Gene Expr 6:185–195 Zinser GM, Sundberg JP, Welsh J (2002) Vitamin D(3) receptor ablation sensitizes skin to chemically induced tumorigenesis. Carcinogenesis 23:2103–2109

Chapter 15

Epidermal Growth Factor Receptor-Targeted Therapies Sun M. Ahn, Seungwon Kim, and Jennifer R. Grandis

Abstract The epidermal growth factor receptor (EGFR) has been implicated in the progression and maintenance of various solid tumors. Efforts in understanding EGFR biology and related signaling cascades have lead to the development of anti-EGFR agents. The two main approaches of inhibition that have been studied most extensively are monoclonal antibodies and tyrosine kinase inhibitors. Despite clear evidence of antitumor activity in preclinical models, only a subset of cancer patients show clinical responses to EGFR inhibitors. Additionally, a majority of patients who demonstrate an initial response become refractory to continued therapy. Possible mechanisms of resistance to anti-EGFR therapy include EGFR overexpression, redundant parallel growth factor receptors, ErbB family heterodimerization, EGFR mutations, the presence of nuclear EGFR, and constitutive activation of downstream signaling mediators. Understanding the molecular mechanisms of resistance is critical in the development of combination therapies to target resistance pathways and improve clinical outcomes. Furthermore, identification of biomarkers that indicate sensitivity or resistance to anti-EGFR therapy will enable the selection of patients who are more likely to benefit from EGFR-targeted therapy. The epidermal growth factor receptor (EGFR) tyrosine kinase plays an important role in the progression of many human cancers, and molecular inhibitors that target EGFR have been extensively investigated for anti-cancer therapy. Anti-EGFR agents have shown promising activity in clinical trials and are now Food and Drug Administration (FDA)-approved for use in selected groups of cancer patients. However, most patients ultimately develop resistance to these agents. In this chapter, we will discuss the therapeutic potential of targeting EGFR, the effects of using EGFR inhibitors, and possible mechanisms of resistance to EGFR inhibitors.

J.R. Grandis (*) Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_15, © Springer Science+Business Media, LLC 2011

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15.1 Therapeutic Potentials of Targeting EGFR 15.1.1 Biology of EGFR Family The EGFR (or ErbB1 or HER1) is one of four members of the ErbB/HER family of transmembrane tyrosine kinase receptors found in normal epithelial tissues. EGFR plays a crucial role in tissue development and homeostasis, as evident from analyses of mice lacking EGFR (Miettinen et al. 1995). The other three members in the family include ErbB2/HER2/neu, ErbB3/HER3, and ErbB4/HER4. All ErbB family members share the same basic structure, consisting of an extracellular ligand-binding domain, a transmembrane lipophilic domain, and an intracellular domain with intrinsic tyrosine kinase activity. Ligands for EGFR include EGF, transforming growth factor alpha (TGF-a), and amphiregulin (Harris et al. 2003). These growth factors are produced as precursors tethered to the cell membrane that can be cleaved by proteases in a process called ectodomain shedding (Wakatsuki et al. 2004). The most important cell-surface proteases involved are matrix metalloproteinase (MMP) and a disintegrin and metalloprotease (ADAM) (Izumi et al. 1998). Upon the binding of a soluble ligand, the receptor undergoes conformational changes that allow homo- and heterodimerization between EGFR molecules and its family members. Dimerization induces autophosphorylation of tyrosine residues in the EGFR intracellular domain, permitting access of the kinase domain by substrates and signaling adaptor molecules. EGFR autophosphorylation activates intracellular signal transduction cascades that lead to increased mitogenic, invasive, migratory, antiapoptotic, and angiogenic phenotypes. A few of the major pathways involved in these downstream effects are the mitogen-activated protein kinsase (MAPK), the phosphatidylinositol 3-kinase (PI3K)-AKT, the signal transducer and activator of transcription (STAT), and the SRC tyrosine kinase pathways (Fig. 15.1) (Yarden and Sliwkowski 2001; Quesnelle et al. 2007; Egloff and Grandis 2008). Dimerized receptors are then internalized in clathrin-coated pits to be recycled back to the cell membrane or in a clathrin-independent lipid raft to be ubiquitylated and degraded (Sigismund et al. 2005, 2008).

15.1.2 The Role of EGFR in Tumorigenesis Although ErbB family receptors are essential for normal tissue development and physiology, aberrant ErbB receptor signaling has been implicated in the development and progression of a wide range of tumors. Thus, they are excellent candidates for selective anticancer therapeutic targets, especially in tumors of squamous cell origin, such as non small-cell lung cancers (NSCLC) and head and neck squamous cell carcinoma (HNSCC). Of the four ErbB receptor family members, EGFR was the first tyrosine-kinase receptor to be linked directly to human cancer

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(Gschwind et al. 2004). Receptor overexpression, gene amplification, activating mutations, and ligand overproduction are some of the abnormalities associated with oncogenic functioning of EGFR pathway. Specifically, receptor overexpression, which is thought to be the main mechanism behind the enhancement of EGFR signaling, has been shown in colon, lung, breast, cervical, prostate, and head and neck cancer. The level of overexpression varies between different types of tumors but is most consistently elevated in HNSCC, where over 90% of tumors show EGFR expression (Grandis and Tweardy 1993). The precise mechanism of overexpression is incompletely understood and may be due to gene amplification or changes in transcription or protein expression (Sunpaweravong et al. 2005). In addition to receptor overexpression, the increased production of EGFR ligands, including EGF and TGF-a, either by the tumor cells themselves or by the surrounding stromal cells, may lead to constitutively active EGFR signaling (Salomon et al. 1995). Therefore, ectodomain shedding of EGFR proligands is crucial in regulating EGFR activation. Proteases including ADAMs and MMPs have been associated with the overproduction of EGFR ligands in cancer cells following the activation of other signaling pathways involving G-protein-coupled receptors (GPCR) (Daub et al. 1996; Prenzel et al. 1999; Gschwind et al. 2003). For example, in prostate cancer, deregulated expression of GPCR and its ligand lysophosphatidic acid have been linked to chronic activation of EGFR and tumor development, primarily through EGFR ligand shedding and subsequent stimulation of cell proliferation (Daaka 2004). Mutations in the EGFR gene leading to constitutive deregulation have been found in selected cancer types. The most common variant of EGFR is the truncated mutant EGFRvIII resulting from the deletion of 2–7 exons in the extracellular ligand-binding domain. This variant is expressed in gliomas, as well as carcinomas of the breast, lung, ovary, and HNSCC (Pedersen et al. 2001). EGFRvIII activation is ligand independent, and the mutated receptor is not internalized and degraded (Ekstrand et al. 1994). Somatic mutations in the intracellular tyrosine-kinase domain of EGFR have been identified in NSCLC that may contribute to response to anti-EGFR therapy (Lynch et al. 2004). Dysregulation of the EGFR pathway leads to enhanced downstream signaling and is fundamental to cancer development and progression. The up-regulation of EGFR activity is associated with cell-cycle progression, increased cell proliferation, inhibition of apoptosis, stimulation of invasion and migration, induction of angiogenesis, and poor response to treatment. Furthermore, tumors with altered EGFR signaling are more aggressive and are associated with poor clinical outcome (Ratushny et al. 2009). For instance, in a large correlative study looking at EGFR overexpression in patients with advanced HNSCC, high levels of EGFR expression was found to be a strong independent prognostic indicator of reduced overall survival and higher rates of loco-regional relapse. Additionally, a subset of HNSCC patients who manifested increased EGFR gene copy number exhibited poor clinical outcome (Chung et al. 2006; Temam et al. 2007). Another study also found that overexpression of multiple ErbB receptors, including EGFR, in the same tumor correlated with metastatic disease (Bei et al. 2004). Given the apparent role of

PLC- γ

P

VEGFR

P

P

E.

C.

P

mTOR

AKT

PI3K

Grb 2

Sos

Apoptosis Resistance Cell Migration/Invasion

PIP3

PKC

P P

P P

IRS1

P

P

IGF-IR

Erk

MEK

Raf

Ras

STAT

Src

Cell Survival Proliferation

B.

PLC- γ

P

Gab1 P

A. EGFR

P

P

STAT

STAT

Src

P

Grb 2

P

P P P

P

P

Gab1 P

STAT

Sos

Sos

c-MET

Cell Cycle Progression Angiogenesis Apoptosis Resistance

D.

Grb 2

EGFR HER2 HER3 HER4

EGF-like ligand

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Fig. 15.1 EGFR downstream signaling cascades and crosstalks between EGFR and other parallel growth factor receptors. (a) Upon the binding of EGF, EGFR forms homo- and heterohydimers with its family members. Dimerization induces autophosphorylation of tyrosine residues, activating downstream signaling cascades. A few of the major pathways involved are the ras/raf/MEK/Erk, the phosphatidylinositol 3-kinase (PI3K)-Akt, the STAT, and the Src tyrosine kinase pathways, resulting in increased mitogenic, invasive, migratory, anti-apoptotic, and proangiogenic phenotypes. (b) By acting through a common adaptor molecule Grb2, parallel receptors IGF-IR and c-MET drive the proliferation and pro-survival signals during EGFR inhibition. (c) VEGFR activates the same proliferation and pro-survival signaling cascade through PKC. (d) EGFR, IL-6R, and c-MET share the common downstream effector molecules of the STAT family. (e) When EGFR is inhibited, PI3K-Akt is activated by parallel growth factor receptors VEGFR, IGF-IR, and c-MET. c-MET mesenchymal-epithelial transition factor, EGF epidermal growth factor, EGFR epidermal growth factor receptor, Erk extracellular signal-regulated kinase, Gab1 Grb2-associated binding protein 1, gp130 glycoprotein 130, Grb2 growth factor receptor-bound protein 2, HER human epidermal receptor, IGF-IR insulin-like growth factor I receptor, IL-6R interleukin-6 receptor, IRS1 insulin-receptor substrate-1, JAK Janus kinase, MEK mitogen-activated protein kinase kinase, mTOR mammalian target of rapamycin, PKC protein kinase C, PLC-g phospholipase C-g, STAT signal transducer and activator of transcription, VEGFR vascular endothelial growth factor receptor.

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EGFR in tumor tumorigenesis, therapies that include targeting the EGFR pathway represent a rational anti-cancer strategy in treating patients with tumors overexpressing EGFR.

15.2 Effects of Targeting EGFR for Cancer Therapy The concept of targeting EGFR as a cancer therapy was initially proposed by Sato et al. (1983) over 25 years ago when they observed that a monoclonal antibody against EGFR led to the inhibition of A431 epidermal carcinoma cell proliferation. Since then, many therapeutic strategies have been developed to inhibit the EGFR pathway. These include monoclonal antibodies (mAbs), tyrosine kinase inhibitors (TKIs), cytotoxin-conjugated ligands, and antisense strategies. The most extensively studies approached include mAbs and TKIs. The mAbs interfere with ligandreceptor binding interaction in the extracellular domain, thereby inhibiting downstream signal transduction. The TKIs compete with ATP in the intracellular tyrosine kinase domain of the receptor, leading to inhibition of autophosphorylation and subsequent downstream signaling. Both mAbs and TKIs are generally well tolerated by patients, with the most common side effects being skin reactions and diarrhea (Baselga et al. 2000, 2002; Herbst et al. 2002). Table 15.1 summarizes EGFR-targeted antibodies and kinase inhibitors, some of which are already approved for the treatment of cancer patients while others are under investigation in various clinical trials.

15.2.1 Current Clinical Inhibitors of EGFR 15.2.1.1 Anti-EGFR Antibodies Initial efforts to develop neutralizing anti-EGFR antibodies utilized a murine mAb called M225. Cetuximab (Erbitux or C225) is a chimeric monoclonal antibody that was engineered by scaffolding a human IgG1 Fc region to the murine M225 Fv (EGFR-binding) region. It is highly selective for EGFR, resulting in a potent inhibition of its ligand binding and the conformational shift required for dimerization (Li et al. 2005). Although cetuximab binding appears to result in autophosphorylation, no further downstream signaling cascades are activated (Mandic et al. 2006; Yoshida et al. 2008). Therefore, the treatment of tumor cells with cetuximab has been shown to block the downstream activation of many intracellular pathways that are essential for cancer development and progression. It has been demonstrated that cetuximab blocks the proliferation of tumor cells and the growth of tumor xenografts in nude mice (Overholser et al. 2000). To reduce the occurrence of antibody-associated reactions, new mAbs against EGFR that are either humanized

Humanized mAb

Humanized mAb

Humanized mAb

Reversible TKI

Reversible TKI

Dual TKI

Panitumumab (Vectibix)

Zalutumumab

Matuzumab

Gefitinib (Iressa)

Erlotinib (Tarceva)

Lapatinib (Tykerb)

EGFR, HER2

EGFR

EGFR

EGFR

EGFR

EGFR

Table 15.1 EGFR inhibitors in clinical use or development Names Class Targets Cetuximab (Erbitux) Chimeric mAb EGFR

GlaxoSmithKline

Genentech/OSI/Roche

AstraZeneca

Merck

Genmab

Amgen

Company ImClone/Merck/BristolMyers Squibb

(continued)

FDA approved for breast cancer. Phase III trials underway for HNSCC and gastric cancer and phase II trials for NSCLC, glioma, ovarian, and endometrial cancer

Status and comments FDA approved for CRC and HNSCC. Ongoing phase II/III trials for NSCLC, esophageal, and breast cancers FDA approved for CRC. Phase II/ III trials underway for HNSCC, NSCLC, and breast cancer Phase III trials underway for HNSCC and phase II trials for NSCLC and CRC Phase I/II trials underway for esophagogastric cancer and NSCLC FDA approved for NSCLC. Withheld from the US market in June 2005 after preliminary results from ISEL trial failed to show survival advantage FDA approved for NSCLC and pancreatic cancer. Phase II/III trials underway for HNSCC, HCC, ovarian cancer, and CRC

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Dual TKI

Dual TKI

Multi-TKI

Irreversible TKI

Tovok/BIBW 2992

Vandetanib (Zactima)

AEE788

Canertinib/CI-1033

EGFR, HER2, VEGFR EGFR, HER2

EGFR, VEGFR

EGFR, HER2

Targets

Pfizer

Novartis

AstraZeneca

Boehinger-Ingelheim

Company

Phase III trials underway for NSCLC and phase II trials for HNSCC, CRC, glioma, breast, and prostate cancer Phase III trials underway for NSCLC and phase II trials for GBM, HNSCC, CRC, HCC, thyroid, prostate, kidney, breast, and ovarian cancer Phase I/II trials underway for GBM

Status and comments

Phase I/II trials completed in NSCLC and breast cancer EKB-569 Irreversible TKI EGFR, HER2 Wyeth Phase II trials underway for NSCLC and CRC CRC colorectal cancer, EGFR epidermal growth factor receptor, FDA Food and Drug Administration, GBM glioblastoma multiforme, HCC hepatocellular carcinoma, HER2 human epidermal receptor 2, HNSCC head and neck squamous cell cancer, ISEL, Iressa Survival Evaluation in Lung Cancer, mAb monoclonal antibody, NSCLC non-small-cell lung cancer, TKI tyrosine kinase inhibitor, VEGFR vascular endothelial growth factor receptor

Class

Table 15.1 (continued) Names

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or fully human have been developed. Of the mAbs, cetuximab has received the most attention and was first approved by the FDA for treatment of patients with advanced colorectal cancer in 2003. Bonner et al. (2006) compared the efficacy of radiotherapy alone with radiotherapy plus cetuximab in patients with newly diagnosed HNSCC and found that the mean overall survival and progression free survival were improved by approximately 20 months and 5 months, respectively, by the addition of cetuximab to radiotherapy. The median duration of locoregional control was also improved by 9.5 months with the addition of cetuximab to radiotherapy. However, it is important to note that most of the improvement observed was limited to the major cohort of oropharyngeal subsite, suggesting that other site-related factors not included in this study may contribute to sensitivity or resistance to cetuximab. Nevertheless, this demonstration of significant improvement with the addition of cetuximab to radiotherapy led to the rapid FDA approval of cetuximab for use in head and neck cancer in 2006. Panitumumab, a fully human IgG2 monoclonal antibody against EGFR, was approved in 2006 for the treatment of metastatic colorectal cancer resistant to conventional chemotherapy regimen. Zalutumumab, another fully human IgG1 monoclonal antibody against EGFR is currently being evaluated in phase II/III studies as monotherapy and in combination with chemoradiation in patients with HNSCC and NSCLC. Mechanisms of Inhibition by Anti-EGFR Antibodies While direct blockade of EGFR-ligand interaction is considered the primary mechanism of EGFR inhibitory activity by cetuximab in vitro, other mechanisms are likely to play an important role in cetuximab’s anti-tumor activity in vivo. One such mechanism is the depletion of surface EGFR through the internalization of the antibody-bound receptor through altered endocytosis trafficking that is distinct from that of EGF-induced receptor internalization (Sunada et al. 1986). The binding of cetuximab antibody results in a significant reduction in surface EGFR to approximately 65% of control levels and also induces a partial down-regulation in total EGFR to about 60% of control (Jaramillo et al. 2006). In addition, whereas EGF weakly binds EGFR in an endosomal acidic pH condition (Ferguson et al. 2003), cetuximab remains associated with EGFR even in the acidic environment of an endosome (Jaramillo et al. 2006). Therefore, cetuximab is not likely to dissociate from EGFR in endosomes, and the antibody-receptor receptor is more likely be targeted for degradation rather than being recycled. In addition to blocking receptor activation, monoclonal antibodies can also mediate antitumor immunity. One such mechanism is antibody-dependent cellular cytotoxicity (ADCC). Through the direct interaction of the Fc domain on the immunoglobulin molecule with Fc receptors (FcgR) on monocytes and natural killer cells, the host immune effector cells cause tumor cell lysis in response to antibody-coated tumor cells (Lee et al. 2009). The importance of ADCC in the clinical efficacy of mAb-based therapies has been established in rituximab

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(Weng and Levy 2003), a mAb for treating lymphoproliferative disease, and trastuzumab (Varchetta et al. 2007), a mAb against HER2 in breast cancer. There are several lines of evidence that support the concept of mAbs-inducing ADCC in the treatment of EGFR-expressing tumors. In HNSCC, López-Albaitero and Ferris (2007) demonstrated that EGFR-specific mAbs, cetuximab and panitumumab, does not induce tumor cell lysis in vitro unless human lymphocytes are added to the media. The degree of in vitro ADCC activities was found to be correlated with EGFR expression levels in the HNSCC cell lines (Kimura et al. 2007; López-Albaitero et al. 2009). Furthermore, a correlation between clinical response to cetuximab treatment and polymorphisms in FcgR has been identified in both colorectal cancer (Zhang et al. 2007) and HNSCC (Taylor et al. 2008; López-Albaitero et al. 2009). Complement-dependent cytolysis (CDC) is another process by which EGFR mAbs can elicit an immune response against tumor cells. Evidence in CDC has been inconclusive to date, with earlier studies detecting no CDC activity (Naramura et al. 1993; Kimura et al. 2007) while a more recent study reported a drastic CDC activity with a combination therapy of two anti-EGFR mAbs cetuximab and matuzumab (Dechant et al. 2008). Thus, further work is necessary to address whether CDC plays an important role in the anti-tumor effects of mAbs specific for EGFR. In addition to direct activation of natural killer cells and complement lysis of tumor cells, anti-EGFR mAbs may also stimulate cytotoxic T lymphocytes to recognize and lyse tumor cells through the interaction with FcgRs on antigen- presenting cells. A recent report by Banerjee and Rothman (1998) describes the enhancement of this specific T-cell activation mediated by dendritic cells in glioma cancer cells treated with cetuximab. It is clear that mAbs directed against EGFR can activate a wide range of immune system responses, and the potential for clinical efficacy of mAbs may include other mechanisms in addition to inhibition of receptor-ligand interactions.

15.2.1.2 Tyrosine Kinase Inhibitors In 1994, Ward et al. identified a new structural class of anilinoquinazoline tyrosine kinase inhibitors as potential agents for treatment of cancer that overexpress EGFR. Two quinazolin derivatives, erlotinib and gefitinib, which are first-generation reversible, ATP-competitive inhibitors of the EGFR tyrosine kinase have been investigated in many tumor models. Similar to cetuximab, these TKIs have been shown to inhibit EGFR activation and reduce tumor growth in xenograft models. Unlike mAbs, TKIs have little effect on EGFR internalization and do not induce ADCC. However, TKIs have the ability to target constitutive ligand-independent receptor signaling, such as seen in the EGFRvIII mutation variant. Gefitinib was first approved by the FDA as a third-line monotherapy in patients with advanced NSCLC in 2003. Subsequently, erlotinib was approved by the FDA in 2004 as a monotherapy for the treatment of locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen. In contrast to cetuximab, there are

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no published results of phase III studies with EGFR TKIs in HNSCC. Other TKIs under clinical investigation include dual kinase inhibitors, such as lapatinib and AEE788, and several second-generation TKIs that are irreversible.

15.2.2 Therapeutic Response to EGFR Inhibition Of all the existing anti-EGFR mAbs, cetuximab has been most extensively studied. In HNSCC, cetuximab contributes a clinical benefit in both locoreginoally limited and metastatic tumors when combined with radiotherapy or chemotherapy. In patients with recurrent, refractory, or metastatic HNSCC, the general response rate to combined cetuximab and chemotherapy has ranged from 6% to 26% (Baselga et al. 2005; Burtness et al. 2005; Chan et al. 2005; Herbst et al. 2005; Bonner et al. 2006). In a phase III randomized, placebo-controlled trial by Burtness et al. (2005), recurrent or metastatic HNSCC patients given cetuximab in combination with cisplatin had a response rate of 26% as compared with a 10% response rate of patients given placebo with cisplatin. However, there was no progression-free and overall survival advantage in patients in the cetuximab plus cisplatin group. Bonner et al. (2006) reported that the treatment of locoregionally advanced HNSCC with a combination radiotherapy plus cetuximab improves locoregional control and reduces mortality. Cetuximab also has activity in previously treated patients refractory to platinum-based chemotherapy (Baselga et al. 2005; Herbst et al. 2005). Although one pilot phase II study of concurrent cetuximab, cisplatin, and radiotherapy was halted because of significant adverse events, the cetuximab-related safety profile in general has been acceptable (Pfister et al. 2006). Another phase III study found that the addition of cetuximab to radiotherapy in patients with HNSCC significantly improved overall survival without adversely affecting the quality of life (Curran et al. 2007). Among the TKIs undergoing clinical evaluation gefinitib and erlotinib have been studied most thoroughly. The overall clinical response to erlotinib in NSCLC in phase II trials was between 9% and 25%, depending on patient’s previous treatment history and tumor histology (Pérez-Soler et al. 2004; Pao and Miller 2005). Subsequently, in a large phase III randomized, placebo-controlled trial, erlotinib was found to prolong survival by 2 months (p < 0.001) in NSCLC patients after chemotherapy (Shepherd et al. 2005). In contrast, erlotinib achieved a modest response rate of 4.3% as a monotherapy in advanced recurrent or metastatic HNSCC (Soulieres et al. 2004). Recent phase II trials in HNSCC patients seem to suggest that erlotinib may have a better response rate when combined with other therapies, such as chemotherapy or VEGF-targeted therapy (Siu et al. 2007; Cohen et al. 2009). A double-blinded, randomized phase II trial reported that, gefitinib, at 250 mg, improved disease-related symptoms and induced radiographic tumor regression with a response rate of 18% just as well as 500 mg dose with less frequent adverse events in patients whose NSCLC progressed after chemotherapy (Kris et al. 2003). In HNSCC, gefitinib was found to be effective at 500 mg with a

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response rate of 10.6% while having no significant effect at the 250 mg dose (Cohen et al. 2003, 2005). Following the phase II trials in NSCLC, gefitinib was approved by FDA as third-line monotherapy in patients with advanced NSCLC in 2003. However, in light of a recent phase III trial in NSCLC, gefitinib has since been placed on alert status, and the future of this TKI remains uncertain. In this large phase III trial, there was no significant benefit conferred by adding gefitinib to the current first-line chemotherapy regimen in NSCLC patients (Giaccone et al. 2004; Herbst et al. 2004). There are several possible reasons for the differences in clinical outcome between mAbs and TKIs directed against EGFR. First, unlike mAbs, TKIs do not elicit immune effects. Also, TKIs exhibit broader target specificity while mAbs are very selective in its targeting. Finally, the differences in pharmacokinetics, pharmacodynamics, and pharmacogenetics give rise to the range of clinical responses in patients treated with mAbs or TKIs.

15.3 Mechanisms of Resistance to EGFR Targeting Despite clear evidence of objective clinical activity of anti-EGFR therapies in patients with tumors overexpressing EGFR, clinical benefits have been modest. This suggests that the majority of patients have tumors that are in part refractory to the treatment due to intrinsic or acquired resistance pathways. Therefore, understanding the molecular mechanisms of resistance is crucial in the development of combination therapies to overcome resistance and improve clinical outcomes. This knowledge will also be critical in predicting and selecting patients who are more likely to benefit from specific EGFR-targeted therapy. Some of the possible mechanisms of resistance and predictors of response discussed here are as follows (Fig. 15.2): (1) EGFR expression level, (2) redundant parallel growth factor receptors, (3) ErbB family heterodimerization, (4) EGFR mutations, (5) nuclear EGFR functions as transcription factor, and (6) constitutive activation of mediators downstream, such as K-ras.

15.3.1 Levels of EGFR Expression EGFR expression has been determined to correlate with outcome, but whether it can serve as a predictive biomarker for response to anti-EGFR therapies is yet to be determined. The levels of EGFR expression likely result from various molecular mechanisms including gene amplification and gene polymorphisms and transcriptional activation. In several studies investigating the clinical response to therapies directed against EGFR in patients with NSCLC and colorectal cancer, low EGFR gene copy number was associated with worse clinical response (Cappuzzo et al. 2005; Moroni et al. 2005; Sartore-Bianchi et al. 2007). In contrast, studies in

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Fig. 15.2 Possible mechanisms of resistance to clinical agents targeting EGFR. (a) EGFR e xpression level has been determined to correlate with outcome, but whether it can serve as a predictive biomarker for response to anti-EGFR therapies is yet to be known. (b) Tumor cells retain the ability to compensate for the loss of EGFR function by signaling through parallel growth factor receptors, such as VEGFR, c-MET, and IGF-IR. (c) EGFR can form heterodimers with the other members of the ErbB receptor family in order to overcome EGFR inhibition. (d) Type III EGFR deletion mutation (EGFRvIII) lacks amino acid residues 6–273 (exons 2–7) in the extracellular domain, and although it is unable to bind EGF-like ligands, it still undergoes dimerization and is constitutively phosphorylated. The kinase domain mutation is comprised of in-frame deletions and amino acid substitutions clustering around the ATP binding pocket of EGFR and serves as a predictor of increased sensitivity to anti-EGFR therapy. (e) Although studies have suggested prognostic significance of nuclear the EGFR level, the exact role of nuclear EGFR on the response of tumor cells to EGFR-targeted therapies has yet to be elucidated. (f) Constitutive activation of signaling mediators downstream of EGFR, such as K-ras, can lead to dysregulation of tumorigenic signaling pathways despite the use of clinical inhibitors to silence the upstream receptor. c-MET mesenchymal-epithelial transition factor, EGF epidermal growth factor, EGFR epidermal growth factor receptor, HER human epidermal receptor, IGF-IR insulin-like growth factor I receptor, VEGFR vascular endothelial growth factor receptor.

HNSCC and colorectal cancer have indicated that EGFR gene copy number does not correlate with clinical response to mAbs or TKIs against EGFR (Wirth et al. 2005; Lenz et al. 2006). Hence, the relationship between EGFR gene amplification and response to EGFR-targeted therapies needs further clarification. In addition to gene amplification, polymorphisms in the EGFR gene and promoter have been associated with increased levels of EGFR expression and altered response to EGFR inhibition. For example, in NSCLC, a polymorphism in the promoter region of EGFR was found to be associated with increased promoter activity, increased EGFR mRNA levels, and improved progression-free survival and overall survival when treated with erlotinib (Liu et al. 2005, 2007). Another

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type of polymorphism that has been studied extensively lies in intron 1 of EGFR. This region has a variable number of cytosine-adenine (CA) single sequence repeats, and the number of CA repeats affects the transcription of the EGFR gene. In a small study in HNSCC patients, higher number of CA repeats had lower expression levels of EGFR mRNA and protein, and was less sensitive to the inhibitory effects of erlotinib (Gebhardt et al. 1999; Amador et al. 2004). Epigenetic modifications of EGFR may play a role in EGFR expression and response to EGFR inhibition. Montero et al. (2006) demonstrated that CpG island hypermethylation in the EGFR gene was associated with transcriptional silencing of EGFR and that blocking methylation with dacitabine, a DNA methyltransferase inhibitor, restored EGFR expression and rendered tumor cells sensitive to gefitinib. Histone acetylation is another epigenetic modification that has been implicated in transcriptional modulation of EGFR and response to anti-EGFR therapy. A recent study in NSCLC demonstrates that the addition of romidepsin, a histone deacetylase inhibitor, increases the sensitivity to erlotinib synergistically (Zhang et al. 2009). EGFR expression levels have not consistently predicted clinical responses to EGFR inhibition. In HNSCC patients, one study found that patients with high EGFR staining were less responsive to cetuximab therapy (Burtness et al. 2005) while another study reports no association between EGFR level and response to the same cetuximab therapy (Chan et al. 2005). Decreased levels of EGFR protein expression were associated with poor clinical outcome with gefitinib therapy in NSCLC (Cappuzzo et al. 2005). Therefore, further evaluation is merited to determine the possible association of EGFR expression level and response to EGFR inhibition.

15.3.2 Redundant Parallel Growth Factor Receptors When examining the response to EGFR-targeted therapies, it is important to consider that most tumors of epithelial origin overexpress multiple receptors of the ErbB family and also co-overexpress one or more EGFR ligands. Therefore, it is likely that the activation of more than one ErbB family member is responsible for the aggressive phenotypes observed in tumors expressing EGFR and autocrine EGF-related ligands. A study by Motoyama et al. in 2002 demonstrated that a proliferative block induced by a decrease in EGFR activity through an EGFR-specific TKI can be overcome by the addition of EGF-related ligands. More importantly, this phenomenon was attenuated when the cells were treated with a dual-EGFR/HER2 TKI. This supports the notion that tumor cells retain the ability to compensate for the loss of one receptor function by dimerization among other members of the ErbB family to activate an overlapping set of downstream effector molecules. Therefore, the efficacy of targeting EGFR may be improved by designing strategies that simultaneously inhibit multiple members of the ErbB receptor family.

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Moreover, additional parallel growth factor receptors (GFRs) and cytokine receptors, such as vascular endothelial growth factor receptor (VEGFR), mesenchymal-epithelial transition factor (c-MET), insulin-like growth factor I receptor (IGF-IR), and interleukin-6 receptor (IL-6R) may contribute to the resistance of selective EGFR inhibition (refer to Fig. 15.1). Evidence indicates that GFRs and cytokine receptors may all contribute to varying degrees to coactivation the MAPK, PI3K-AKT and STAT3 pathways that promote the malignant phenotype of SCCs. For example, by acting through a common adaptor molecule Grb2, parallel receptors IGF-IR and c-MET may drive proliferation and pro-survival signals during EGFR inhibition (Montagut and Settleman 2009). Alternatively, VEGFR and c-MET activate the same and additional proliferation and pro-survival signaling cascades through protein kinase C (PKC) (Shu et al. 2002; Worden et al. 2005). Likewise, EGFR, IL-6 and c-MET share among their common downstream effector molecules the signal transducers and activator of transcription (STAT) family that, once phosphorylated, translocate to the nucleus and activate the transcription of genes involved in cell cycle progression, angiogenesis, and apoptotic resistance. Hence, even when the EGFR signaling pathway is inhibited by a mAb or a TKI, the c-MET and IL-6 signaling axis may still able to phosphorylate STAT, specifically STAT3 (Song et al. 2003; Lee et al. 2006). Another common signaling axis involving phosphatidylinositol 3-kinase-protein kinase B (PI3KAKT) is activated by parallel growth factor receptors VEGFR, IGF-IR, and c-MET when EGFR is inhibited (Chakravarti et al. 2002; Bianco et al. 2008). For instance, in NSCLC, hepatocyte growth factor induces gefitinib resistance by binding c-MET and restoring phosphorylation of AKT, and downregulation of c-MET reversed gefitinib resistance and phosphorylation of AKT (Yano et al. 2008). Thus, not only may it critical to target multiple ErbB receptors, it may also be crucial to simultaneously target other growth factor receptors contributing to the common effector signaling pathways.

15.3.3 Escaping Through Heterodimerization In addition to homodimerization upon EGF activation, EGFR can form heterodimers with the other members of the ErbB receptor family or parallel growth factor receptors in order to overcome EGFR inhibition. Furthermore, endocytosis of HER2, 3, and 4 occurs at a slower rate than EGFR, and it has been shown that the EGFR/HER2 heterodimer is endocytosed less frequently and is redirected to be recycled to the cell membrane rather than being degraded in endosomes (Baulida et al. 1996; Lenferink et al. 1998). Of the ErbB family members, ErbB2 (HER2) is the preferred partner of heterodimerization for the other activated ErbB receptors (Graus-Porta et al. 1997). Interestingly, the structure of HER2 is fixed in a conformation that resembles the ligand-activated state (Garrett et al. 2003). More specifically, HER2 is the only receptor in the ErbB family that has no natural EGF-related ligand that it binds and is in a permanently activated state ready for interaction with

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another ligand-bound receptor. There is a wealth of information demonstrating the importance of HER2 in the development and malignancy of human cancers, especially breast and ovarian cancers. In one study, 39% of HNSCC had high surface membrane levels of HER2 and this correlated with lymph node positivity and inversely with disease free survival (Cavalot et al. 2007). Many studies have investigated the preclinical and clinical significance of ErbB heterodimers in tumor cells overexpressing EGFR. One in vitro study demonstrated that a pan-ErbB TKI inhibited tumor cell proliferation by blocking EGFR/HER2 heterodimer signaling (Wong et al. 2006). Another study reported that increased levels of IGF-IR/EGFR heterodimer counteracts the anti-tumor effect of erlotinib (Morgillo et al. 2006). Wheeler et al. (2008) demonstrated that in NSCLC and HNSCC cells, prolonged exposure to cetuximab not only induced upregulation of EGFR through impaired endocytosis but also caused enhanced heterodimerization of EGFR with HER2, HER3, and cMET, resulting in acquired resistance to cetuximab therapy. Furthermore, expression levels of HER2 and HER3 in HNSCC have been associated with resistance to gefitinib while combining gefitinib with pertuzumab, an antibody targeting HER2 heterodimerization, provided additional growth-inhibitory effects over gefitinib alone (Erjala et al. 2006). Another group reported that HER2 expression levels may be a predictor of sensitivity to cytotoxic agents, including 5-fluorouracil and cisplatin, in HNSCC (Hasegawa et al. 2007). Hence, it is evident that dual targeting of EGFR and other transmembrane receptor tyrosine kinases, especially other members of the ErbB family, may have great potential for clinical benefit in treatment of HNSCC. Given that HER2 is the preferred dimerization partner for EGFR, two recent phase II trials were undertaken to evaluate the effects of targeting HER2 alone or in combination with EGFR inhibition in patients with HNSCC. Gillison et al. (2006) reported that trastuzumab, a mAb against HER2, did not improve the response rate to standard chemotherapy in patients with advanced HNSCC. Of note, the detectable staining of HER2 on tumor samples in this trial was particularly low at 7%. Furthermore, Abidoye et al. (2006) reported that lapatinib, a dual EGFR/HER2 TKI, did not have significant clinical activity in HNSCC patients when used as a single agent. It may be that targeting HER2 alone or using a dual TKI as a monotherapy in HNSCC patients may not result in significant tumor suppression. Additional studies investigating the effects of combination therapy using a dual inhibitor and a standard chemotherapy regimen should be undertaken to evaluate the value of targeting multiple receptor tyrosine kinases in patients with tumors overexpressing EGFR.

15.3.4 Mutations of EGFR – EGFRvIII Mutations in the EGFR gene have been reported in various types of tumors, the most frequent being deletions in the extracellular domain. There are at least seven variant classes of extracellular domain mutations identified first in gliomas and

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later in other tumor models. They all contain deletion mutations of specific exons in the extracellular domain that lead to ligand-independent constitutive activation of EGFR and impaired receptor downregulation. The most common of these truncated receptor mutations is type III EGFR deletion mutation (EGFRvIII), which lacks amino acid residues 6–273 (exons 2–7) in the extracellular domain (Pedersen et al. 2001). This is thought to arise from error in transcript splicing, presumably arising during EGFR gene amplification process or alternative splicing (Sugawa et al. 1990). The functional consequence of this mutation is that it mimics the conformational change induced by ligands in wild-type EGFR (Moscatello et al. 1996). Therefore, although EGFRvIII is unable to bind EGF-like ligands, it still undergoes homo or hetero-dimerization and is constitutively phosphorylated (Fernandes et al. 2001). Furthermore, EGFRvIII also fails to be down-regulated upon autophosphorylation due to slight differences in conformational changes compared to wild-type EGFR. Although cetuximab is able to bind to EGFRvIII, the internalization of mAb-bound EGFRvIII is significantly decreased and delayed. Interestingly, EGFRvIII expression is unique to human tumors–brain, breast, lung, ovary, prostate, and now head and neck cancer–and has not been detected in normal tissue (Grandis and Sok 2004; Sok et al. 2006). The detection of this mutant variant in a wide range of human tumors suggests that there may be selection pressure for EGFRvIII-expressing cells during tumorigenesis. The expression of EGFRvIII seems to increase gradually with the progressive transformation from normal to malignant cells (Olapade-Olaopa et al. 2000), and the direct transfection of EGFRvIII into normal cell lines has been shown to transform the cells into malignant phenotypes (Batra et al. 1995; Tang et al. 2000; Damstrup et al. 2002). In gliomas, EGFRvIII expression correlates with increased tumor growth in vivo (Nishikawa et al. 1994) and poor clinical prognosis (Shinojima et al. 2003). EGFRvIII also enhances the invasive phenotype in glioblastoma cell lines by inducing extracellular matrix components, metalloproteases, and serine protease genes (Lal et al. 2002). Additionally, in vitro studies have demonstrated that EGFRvIII-expressing cells are more resistant to chemo- and radiotherapy, partly due to decreased apoptosis through the up-regulation of Bcl-XL (Nagane et al. 1998; Lammering et al. 2003; Sok et al. 2006) and increased growth through constitutive activation of PI3K-Akt signaling pathway (Moscatello et al. 1998; Li et al. 2004). In fact, the PI3K-Akt pathway, which is critical for cell survival, motility, and invasion, is persistently activated in tumor cells expressing EGFRvIII, and thus may play a role in predicting sensitivity to EGFR inhibition by TKIs. Tumor cells expressing EGFRvIII are chronically dependent on PI3K-Akt signaling, termed “pathway addiction (Weinstein and Joe 2008),” and disruption of this pathway addiction by TKIs leads to cell death. Lastly, the loss of tumor-suppressor protein PTEN (phosphatase and tensin homolog deleted in chromosome 10), an inhibitor of the PI3K-Akt signaling pathway, is associated with resistance to EGFR kinase inhibition (Bianco et al. 2003). Mellinghoff et al. (2005) reported that the expression of EGFRvIII sensitizes gliomas to EGFR TKIs, the loss of PTEN predicted resistance to TKIs, and that the best predictor of clinical response to EGFR TKI was the coexpression of EGFRvIII and PTEN.

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The presence of EGFRvIII as a predictive biomarker for EGFR targeted therapy has not been firmly established. While there are a few studies suggesting a decreased response to gefitinib therapy in patients with tumors that express EGFRvIII (Learn et al. 2004), a phase II trial of gefitinib in recurrent glioblastoma found no correlation between EGFRvIII expression and overall survival in patients treated with gefitinib (Rich et al. 2004). In contrast, Heimberger et al. (2005) reported that EGFRvIII was an independent negative prognostic indicator only in patients surviving more than 1 year. Despite the uncertainty behind the importance of EGFRvIII as a prognostic predictor, EGFRvIII is a promising candidate for anti-cancer therapy since it has not been detected in normal tissues. A variety of therapeutic approaches are currently being investigated to target EGFRvIII, including EGFRvIII-specific mAbs (Mishima et al. 2001; Modjtahedi et al. 2003; Perera et al. 2005), immunotherapy/vaccination (Heimberger et al. 2003; Wu et al. 2006; Schmittling et al. 2008), and small molecule inhibitors (Trembath et al. 2007).

15.3.5 Mutations of EGFR – Kinase Domain Mutations Mutations in the kinase domain (KD) of EGFR have been well established as a modulator of response to EGFR inhibition in a subset of patients with NSCLC. Unlike mutations in the extracellular domain, mutations in the KD have been shown to be a predictor of increased sensitivity to anti-EGFR therapy (Lynch et al. 2004). These mutations typically are comprised of in-frame deletions and amino acid substitutions clustering around the ATP binding pocket of EGFR. The mutations not only result in increased access to ATP and sustained response to EGF-like ligands but also stabilize interactions between the tyrosine kinase domain and the TKIs (Paez et al. 2004). Thus, patients with KD mutations show higher response rate to both erlotinib or gefitinib therapy and have a significantly better outcome (Riely et al. 2006). These mutations have been reported to be present in 10% to 30% of NSCLC (Pao and Miller 2005) but have not been detected to date in other cancers including HNSCC (Lee et al. 2005; Loeffler-Ragg et al. 2006). Most patients who demonstrate a significant clinical response and radiographic regression eventually experience clinical progression of disease (Fukuoka et al. 2003; Kris et al. 2003). Additionally, the resistance of tumors to a specific TKI cannot be overcome by increasing the dose of the TKI or switching to a different TKI (Costa et al. 2008). Recent studies have discovered a secondary mutation in exon 20 that impairs the binding of TKIs to the EGFR kinase domain (Gleich et al. 1998; Kobayashi et al. 2005; Pao et al. 2005). This mutation was not seen in the original diagnostic biopsy specimen prior to TKI treatment, indicating that the resistance through a secondary mutation is acquired or selected for after treatment with TKIs (Kobayashi et al. 2005; Pao et al. 2005). Several in vitro and in vivo studies have suggested that second-generation of irreversible TKIs that covalently crosslink to the receptor may be effective against tumors resistant to the first-generation reversible TKIs erlotinib and gefitinib (Kwak et al. 2005; Ji et al. 2006; Sequist et al. 2007).

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Phase I and II clinical trials are currently underway to investigate the effectiveness of the newer TKIs in patients with and without acquired resistance to first-generation TKIs (Riely 2008).

15.3.6 Nuclear EGFR The concept of transmembrane receptors’ role as a transcription factor in the nucleus has been described for many receptors, including receptors for insulin (Rosenzweig et al. 1993), fibroblast growth factor (Stachowiak et al. 2007), growth hormone (Waters et al. 1994), and erbB4/HER4 (Srinivasan et al. 2000). Therefore, it is not surprising that EGFR can also translocate to the nucleus to play a role in modulating gene expression. Although the presence of EGFR and its ligands has been observed in the nucleus of different tumor types for more than a decade (Lipponen and Eskelinen 1994), the functional role of nuclear EGFR had not been explored until a study by Lin et al. in 2001. In this study, they demonstrate that nuclear EGFR is preferentially present in highly proliferative tissues, including tumor tissues. In addition, a DNA-binding site was identified within the EGFR protein that, once stimulated by EGF, directly interacts with the promoter region of the cyclin D1 gene. Cyclin D1 is an important cell cycle regulator, and this may explain why nuclear EGFR is strongly correlated with highly-proliferating tissues. Subsequently, another study has successfully identified the presence of nuclear localization sequence in the juxtamembrane domain of EGFR (Hsu and Hung 2007). Recent reports indicate that EGFR internalization and nuclear translocation are Src kinase-dependent and occur following ligand binding and exposure to oxidative stress or radiation (Khan et al. 2006; Dittmann et al. 2008). Another proposed mechanism of intracellular receptor trafficking involves the translocation of EGFR to the endoplasmic reticulum and subsequent extraction of intact receptors into the cytoplasm and transport to the nucleus (Liao and Carpenter 2007). These data suggests the existence of new EGFR signaling pathway that escapes traditional transduction cascades. Thus, nuclear EGFR could represent a mode of resistance to anti-EGFR therapy since intracellular EGFR may bypass inhibition by extracellularly located mAbs. It is also unclear whether phosphorylation of EGFR is required for the transcription of target genes by nuclear EGFR. Several studies have investigated the physiologic and pathologic importance of nuclear translocation by identifying putative target genes. In addition to cyclin D1, inducible nitric oxide synthase has been suggested to be a transcriptional target of nuclear EGFR through the direct interaction with another transcription activator STAT3 (Lo et al. 2005a). Studies examining the prognostic significance of nuclear EGFR have reported that in patients with HNSCC, high nuclear EGFR expression was correlated with higher local recurrence and lower disease-free survival after radiotherapy (Psyrri et al. 2005). Similar inverse correlation between high nuclear EGFR and overall survival was observed in breast cancer (Lo et al. 2005b) and ovarian cancer patients (Xia et al. 2008). Moreover, nuclear EGFR correlated

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positively with increased levels of cycle D1 and Ki-67, both indicators of cell proliferation (Lo et al. 2005b; Xia et al. 2008). Although these studies seem to suggest an important role of nuclear EGFR in cancer, it is difficult to confirm the specific significance of nuclear EGFR in regulating cancer biology since high levels of nuclear EGFR tend to accompany high levels of overall EGFR. Additionally, little is known about the role of nuclear EGFR on the response to tumor cells to EGFR-targeted therapies. Therefore, additional investigation is warranted to better understand the functional significance of nuclear EGFR in human cancer and response to therapies targeting EGFR.

15.3.7 Constitutive Activation of Downstream Mediators: K-ras Mutation Constitutive activation of signaling mediators downstream of EGFR can lead to dysregulation of tumorigenic signaling pathways despite the use of clinical inhibitors to silence the upstream receptor. Of the downstream mediators, the clinical relevance of K-ras mutations has been extensively evaluated, especially in metastatic colorectal cancer. A member of the Ras family, K-ras is a GTPase oncoprotein that modulates signal transduction cascades, such as Raf-MEK-Erk pathway, required for cell proliferation and survival (Ellis and Clark 2000), Thus, activating mutations in K-ras result in EGFR-independent activation of these pathways that lead to malignant transformation of tumor cells. K-ras mutation is found in approximately 15–30% of patients with NSCLC and 40–45% of patients with colorectal cancer (CRC) (Dempke and Heinemann 2009). The direct impact of K-ras mutations on patient outcome is controversial, as the studies have been conflicting (Jimeno et al. 2009). However, data supporting the role of K-ras mutation status in predicting response to anti-EGFR therapy in patients with CRC are compelling. In a large phase III CRYSTAL trial, Cutsem el al. reported that CRC patients with wild-type K-ras demonstrated significant improvement in survival and overall response with the addition of cetuximab to a combination of chemotherapy compared to CRC patients with mutated K-ras (Van Cutsem et al. 2008). Another randomized phase II study confirmed that benefit from addition of cetuximab to standard chemotherapy treatment is higher in the CRC patient population with wild-type K-ras compared to those with mutated K-ras (Bokemeyer et al. 2008). Similar results were observed regarding K-ras mutation status when using panitumumab treatment in patients with CRC (Amado et al. 2008). In NSCLC, Jackman et al. (2008) reported that K-ras mutations were associated with resistance to EGFR TKI therapy. However, further trials need to be completed in patients with NSCLC to confirm the value of screening patients with K-ras mutations to select patients who will benefit from anti-EGFR therapies. Overexpession of K- and H-ras along with constitutive STAT3 and NF-kB nuclear activation has been reported in association with loss of TGFb receptor II and its inhibitory signaling in murine and human HNSCC (Lu 2006; Cohen et al. 2009). These data indicate that methods of

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detecting K-ras mutation, expression and activation, along with that of other pro-survival pathways, may be critical in selecting patients whom will benefit from therapies targeting EGFR.

15.4 Conclusion Despite clear evidence of the protumorigenic role for EGFR in many tumor models, agents targeting EGFR have shown inconsistent results to date in the clinical setting. Elucidation of the molecular mechanisms of resistance to anti-EGFR agents is required for the development of rationally designed combination therapies that can overcome clinical resistance. In addition, a more detailed understanding of the underlying mechanisms that confer resistance or sensitivity to anti-EGFR agents will allow for tailoring of individualized therapy for cancer patients based baseline factors in the patient’s tumor that can predict sensitivity. Acknowledgment This work was supported by the American Cancer Society, P50CA097190, R01CA77308, and R01CA098372, to JRG.

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Chapter 16

Targeting UVB Mediated Signal Transduction Pathways for the Chemoprevention of Squamous Cell Carcinoma G. Tim Bowden and David S. Alberts

Abstract The high incidence of skin cancer in the USA is directly related to solar radiation exposure. There is also a disturbing increase in the incidence of both nonmelanoma and melanoma skin cancers in the USA. There is evidence that the use of sunblocks or sunscreens is not totally effective in preventing skin cancer. Therefore, there is a need for new mechanism-based approaches to prevent solar radiation-induced skin cancers. Ultraviolet (UV) light can initiate skin tumor development through DNA damage and critical mutations, for instance in the p53 tumor suppressor gene. UV light can also promote the clonal expansion of these initiated cells through signal transduction pathways to give rise to benign tumors. Some of these signaling pathways mediate the downstream activation of the transcription factor complex, activator protein 1, which has been shown to play a functional role in UV induced skin carcinogenesis. Targeting of these signaling pathways using natural products and small molecule inhibitors is being explored for early chemoprevention of nonmelanoma skin cancer.

16.1 Ultraviolet Light and Skin Cancer Ultraviolet (UV) light from the sun is an important environmental carcinogen responsible for a high incidence of nonmelanoma skin cancers (NMSC) in exposed populations (Fry and Ley 1989). Over 1,000,000 new cases of skin cancer occur yearly in the US accounting for approximately 40% of all new cancers diagnosed. The incidence of skin cancer in the USA has been increasing over the last few years and is expected to continue to increase as the US population ages and larger amounts of UV reach the earth’s surface due to depletion of the ozone layer (Johnson et al. 1998). The majority of skin cancers are NMSCs and are either basal G.T. Bowden (*) Department of Cell Biology and Anatomy, Arizona Cancer Centre, University of Arizona, College of Medicine, University of Arizona, Tucson, AZ, USA and Arizona Cancer Center, University of Arizona, Tucson, AZ, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_16, © Springer Science+Business Media, LLC 2011

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cell carcinomas (BCC) or squamous cell carcinomas (SCC). Approximately 80% of skin cancers are BCCs, 16% SCCs and 4% are melanomas. NMSCs occur primarily on chronically sun-exposed regions of the body including the dorsal surfaces of the arms, the neck, and the face. There are a number of important risk factors for NMSCs which include occupation, life-style, geographic location, genetic factors, and skin pigmentation. The UV part of the light spectrum is subdivided into three parts. The parts include: (1) UVC 200–280 nm; (2) UVB 280–320 nm; (3) UVA = 320–400 nm. Ozone found in the earth’s stratosphere absorbs all of the UVC and much of the UVB (Matsui and DeLeo 1995). Depletion of the ozone layer could result in enhanced fluences of UVB reaching the earth’s surface and a significant increase in the incidence of NMSC and melanoma skin cancers. Much of what is known about the biology and molecular mechanisms of UV-induced skin carcinogenesis has come from studies conducted using mouse skin. The experimental induction of skin tumors in mice using UV as a complete carcinogen was reported as early as 1928 (Findlay 1928). Subsequently, researchers have taken advantage of the multistage nature of mouse skin carcinogenesis to ask which wavelengths of UV were effective in the different stages of carcinogenesis. The mouse skin model of carcinogenesis has been subdivided into three stages: (1) initiation, (2) promotion, and (3) progression (Boutwell 1974). UVB irradiation can initiate and promote the formation of mouse skin tumors while UVA acts primarily as a tumor-promoting agent (Willis et al. 1981; Strickland 1986). Both UVB and UVA can act as complete carcinogens producing SCCs when given repeatedly to the skin of mice. Therefore, both UVA and UVB are capable of accomplishing all three stages of skin carcinogenesis.

16.2 Genomic Mechanisms of UV Light-Induced Skin Cancer Mechanistically, UV light induces both genotoxic effects such as DNA damage and mutations as well as epigenetic events such as induction of gene expression. DNA damage is induced directly though UV light absorption by DNA resulting in the induction of cyclobutane pyrimidine dimers, (6–4) pyrimidine dimers, cytosine photohydrates, and purine photoproducts. UV of longer wavelengths can induce indirectly DNA single-strand breaks and DNA-protein crosslinks through reactive oxygen species. Wavelengths of UV in the UVA part of the spectrum have been shown to produce superoxide anions, singlet oxygen, and hydrogen peroxide (Danpure and Tyrrell 1976). Recently it has been shown that UVA light can also induce pyrimidine dimers in the DNA of mammalian cells. Several lines of evidence indicate that DNA is an important target for the mutagenic and carcinogenic effects of UV irradiation (Ananthaswamy and Pierceall 1990; Elmets 1992). The initiation and progression stages of skin carcinogenesis involve stable genetic changes involving mutation of critical genes such as protooncogenes and tumor suppressor genes. Furthermore, specific UV induced DNA lesions give rise through DNA replication to specific “signature” mutations (Brash et al. 1991). These mutations are C to T and CC to TT, which may result from misincorporation

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of deoxyadenosine opposite pyrimidine dimers due to the “A rule” (Tessman 1976). Activating point mutations in the ras family of oncogenes have been found in human skin SCCs and BCCs (Brash et al. 1991; Pierceall et al. 1991). Activating ras mutations have also been observed in UV-induced mouse skin tumors (Pierceall et al. 1992). Mutations in the p53 tumor suppressor gene are often observed in UV-induced skin tumors. Brash and coworkers (Ziegler et al. 1993) have reported that 58% of human skin cancers contain p53 mutations while others have reported 88% of human skin cancers with p53 mutations (Kanjilal et al. 1995). Of the ten mutational hot spots in the p53 gene (Tornaletti et al. 1993; Ziegler et al. 1993), three are at positions where UV-induced pyrimidine dimers localize. Epidermal cells with p53 mutations have been detected in sun damaged, uninvolved skin and in benign lesions called actinic or solar keratoses (AK) (Campbell et al. 1993; Fry et al. 1994; Ziegler et al. 1994). Many skin SCCs are derived from AKs. In parallel with human skin cancers, UV-induced mouse skin cancers also display “signature mutations” at pyrimidine-rich sequences of the p53 gene (Kress et al. 1992; Kanjilal et al. 1993). Therefore, inactivation of the p53 tumor suppressor gene by “signature mutations” appears as a very early event in UV-induced epidermal carcinogenesis. In contrast, activation of a protooncogene such as ras appears to be an early event in chemical skin carcinogenesis. The second stage in the development of SCC, tumor promotion, has been hypothesized to involve clonal expansion of the initiated cells with an activated oncogene or inactivated tumor suppressor gene to give rise to a benign tumor (Yuspa 1994). The clonal expansion appears to involve selective outgrowth of the “initiated” cells (Finch and Bowden 1996). This selective growth involves both hyperproliferation and a resistance of the “initiated” cells to induced terminal differentiation as in the case of chemically “initiated” epidermal cells and a resistance of UV “initiated” cells to induced apoptotic cell death. Therefore, repeated UV irradiations would provide a growth advantage and a positive selection for epidermal cells with p53 mutations.

16.3 Signal Transduction Pathways Activated by UV Light in Skin Cancer Development Although UVA has been thought mostly to be a tumor promoting agent, mediating its effects through epigenetic modifications and UVB has been thought to act as an initiating agent, it has been shown that UVB has significant epigenetic effects on membrane lipids and can increase the formation of eicosanoids, inhibit epidermal growth factor (EGF)-binding and activate EGF receptor in a ligand independent manner (De Leo et al. 1984; Punnonen et al. 1987; Matsui et al. 1989; Miller et al. 1994). Considerable evidence has been obtained in cultured cells (Sachsenmaier et al. 1994; Warmuth et al. 1994; Huang et al. 1996; Rosette and Karin 1996; Bender et al. 1997) and also in human skin (Fisher et al. 1998) that UV radiation activates cell surface growth factor and cytokine receptors; therefore, mimicking the natural ligands for these receptors. Fisher et al. (Fisher et al. 1998) demonstrated

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activation of EGF receptors, the guanosine-5¢-triphosphate-binding regulatory protein p21Ras, and mitogen-activated protein (MAP) kinases, ERK, JNK, and p38 in solar UV-irradiated human skin. Following UV irradiation of human skin there was increased phosphorylation of JNK and p38 leading to activation of transcription factors c-JUN and ATF-2, which bound to the c-JUN promoter and up-regulated c-JUN expression. An increase in metalloproteinases in skin resulted from elevated activator protein 1 (AP-1) activity due to c-JUN upregulation (Fisher et al. 1998). In addition, UVB irradiation of human skin in vivo activates the phosphatidylinositol-3-kinase (PI3K/AKT) survival pathway via EGF receptors (Wan et al. 2001). The involvement of EGFR in cultured human keratinocytes in response to UV was demonstrated using two EGFR tyrosine kinase inhibitors, PD15035 and AG1478 and the involvement of PI3K in AKT activation using LY294002 and wortmanin. UVB induced formation of reactive oxygen species initiated activation of the PI3K/AKT pathway, which was prolonged by feedback activation of p38 induced through release of cytokines in response to UVB irradiation (Zhang et al. 2001). Additionally there is evidence for UV irradiation induced crosstalk between the EGF receptor and cytokine receptors such as interleukin-1 receptor that causes activation of c-JUN kinase in human keratinocytes (Wan et al. 2001). The epigenetic effects of UV are also thought to be mediated by generation of reactive oxygen species with subsequent damage to cell membranes and modu lation of cell proliferation, differentiation, and apoptosis. Both UVA and UVB irradiation of cultured cells increased the levels of the signaling metabolite, diacyl glycerol (DAG) (Hanson et al. 1989; Punnonen and Yuspa 1992). These alterations in signaling molecules mediated by UV irradiation result in enhanced gene expression and this increase in the expression of certain genes involved in cell proliferation and apoptosis or “UV response” plays a role in UV-irradiation mediated tumor promotion (Herrlich 1989; Ronai et al. 1990; Holbrook and Fornace 1991; Herrlich et al. 1992; Angel 1995). There are at least two transcription factor complexes that have been implicated in mediating the “UV response,” AP-1 and NF-kB (Angel 1995; Liebenlist et al. 1995). A third transcription factor, cyclic AMP-binding protein (CREB) also mediates part of the “UV response” in that it is involved in inducing the expression of a protooncogene, c-FOS, (Gonzales and Bowden 2002c) that is part of the AP-1 complex. CREB also helps mediate the expression of cyclooxygenase II (COX-2), a critical eicosanoid biosynthetic enzyme (Chen et al. 2001), that has been implicated in UV-induced skin carcinogenesis.

16.3.1 Function and Mechanism of AP-1 Activation in Response to UV 16.3.1.1 Role of AP-1 in UV-Induced Skin Carcinogenesis AP-1 is a protein dimer that consists of JUN and FOS proteins (Shaulian and Karin 2002). There are three Jun proteins (c-JUN, JUN-B, and JUN-D) and four FOS proteins (c-FOS, FOS-B, FRA-1, and FRA-2). The AP-1 complexes could be either

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JUN-JUN homodimers or JUN-FOS heterodimers (Angel 1995). In human keratinocytes the AP-1 complex that binds to the AP-1 cis element in response to UVB irradiation consists of a heterodimer between c-FOS and JUN-D (Chen et al. 1998). UVB induction of AP-1 in keratinocytes may play a role in cell proliferation and therefore, in UVB mediated skin tumor promotion. In support of the role of AP-1 in tumor promotion and growth, it has been shown that AP-1 was only activated in tumor promotion sensitive JB-6 cells and not in tumor promotion-resistant cells (Bernstein and Colburn 1989). Blocking tumor promoter-(TPA-induced) AP-1 activity in these cells inhibited neoplastic transformation. In vivo studies have shown that blocking constitutively activated AP-1 transactivation in malignant mouse SCC cell lines inhibits their ability to form tumors upon subcutaneous injection into athymic nude mice (Domann et al. 1994). More directly, in a UV-induced mouse skin tumor assay, inhibition of UVB induced AP-1 activation with aspirin, sodium salicylates (Bair et al. 2002), or perillyl alcohol (Barthelman et al. 1998b) correlated with inhibition of skin tumor formation. When SKH-1 hairless mice expressing both a dominant negative c-JUN (TAM-67) mutant transgene under the control of the human keratin 14 promoter and an AP-1 luciferase reporter gene were exposed to a single UVB dose there was a significant decrease in UVB induced AP-1 activation compared to non-TAM-67 littermates (Young et al. 1999; Thompson et al. 2002; Cooper et al. 2003). In a chronic UVB skin carcinogenesis study, expression of TAM-67 delayed the appearance of skin tumors, reduced the number of tumors per mouse and reduced the size of the tumors. Thus blocking c-JUN with the TAM-67 transgene inhibited UVB induced AP-1 activation in the epidermis and UVB induced skin tumor development. These results suggest that the UVB signaling pathway leading to AP-1 activation is good molecular target for the development of new chemoprevention strategies to prevent sunlight induced skin cancers.

16.3.1.2 Signaling Pathways Leading to Increased AP-1 Activity There is evidence that the induction of AP-1 activity in keratinocytes by UV occurs primarily through increased levels of c-FOS. c-FOS is an immediate early-response gene that is constitutively expressed at low levels in many cell types and is induced by a wide variety of stimuli (Ruther et al. 1987; Ruther et al. 1989; Saez et al. 1995). Thus c-FOS was expressed at low basal levels in human keratinocytes, and c-FOS gene expression and protein increased following UVB irradiation correlating with AP-1 activation (Chen et al. 1998). The mitogen-activated protein (MAP) kinase family are important mediators leading to c-FOS transcription in response to UVB (Bode and Dong 2003). The MAP kinases are important regulators of various transcription factors including AP-1 which is a phosphorylation target of several MAPK signaling cascades (Coso et al. 1995). The MAP kinase family includes JNKs, ERKs, and p38, with multiple subtypes in each family. JNKs are encoded by three genes generating ten protein kinase polypeptides, ERK has two members, p44 and p42, and there are five p38 subtypes, p38a, p38b, p38b2, p38g, and stress-activated protein kinase 4 (SAPK4).

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These MAP kinases are part of parallel kinase cascades that may be activated independently or simultaneously, as well as interact with each other. In human keratinocytes UVB irradiation activates p38 and ERK MAP kinases (Chen and Bowden 1999) (Fig. 16.1). Treating these cells with the p38 MAP kinase inhibitor, SB202190, inhibited UVB induced p38 MAP kinase activation but not UVB induced ERK activation. Likewise, treating the cells with the MEK-1 inhibitor, PD98059, inhibited UVB-induced ERK activation but not UVB-induced p38 MAP kinase activation. Further studies demonstrated that blocking p38 almost completely abrogated UVB-induced c-FOS gene transcription and c-FOS protein synthesis, as well as UVB-induced AP-1 transactivation and DNA binding as determined by gel mobility shift assays (Chen and Bowden 2000). Of interest, SB202190 is known to inhibit two of the five isotypes of p38 MAP kinase, a and b1. This could mean that UVB mediates c-FOS transcriptional activation primarily through these two isotypes of p38 MAP kinase. Inhibiting ERK partially abrogated UVB-induced c-FOS transcriptional and protein levels. Suppression of both p38 and ERK not only completely blocked UVB-induced c-FOS expression but also decreased c-FOS basal gene expression. Thus, p38 may play a more important role than ERK in UVB-induced c-FOS expression in human keratinocytes. In addition, we have demonstrated that SB202190 strongly inhibited UVB-induced

Chen and Bowden Molecular Carcinogenesis, 1999

Gonzales and Bowden Molecular Carcinogenesis, 2002

UVB

EGCG

NDGA PI-3 Kinase

p38

LY294002 PI-3,4,5, –P3

SB202190

PI-4,5, –P2 PDK p Thr308 p Ser473 p Ser9

AKT GSK-3b

Ser133 CRE

p FAP1

p CREB

Ser129

CRE

COX-2

c-Fos

Tang et al., Cancer Research, 2001

Gonzales and Bowden Oncogene, 2002

Fig. 16.1 UVB Signal Transduction Pathways Leading to COX-2 and c-Fos Expression.

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AP-1 transactivation as well as UVB-induced AP-1 DNA binding as determined by gel mobility shift assays (Chen and Bowden 2000). Because c-FOS expression appears to play an important role in UVB-induced AP-1 activation and AP-1 activation is known to play a role in tumor promotion, both p38 and ERK could be potential targets for chemoprevention of skin cancer. 16.3.1.3 Regulation of c-FOS Transcription in Response to UVB It is not surprising that the signal pathways that lead to c-FOS induction vary according to the cell system and source of stimuli used in each study. A number of published studies have identified four cis elements that are critical for the induction of the c-FOS promoter (Robertson et al. 1995; Iordanov et al. 1997; Wang and Prywes 2000). These elements include: (1) the sis inducible element (SIE); (2) the serum response element (SRE); (3) the c-FOS AP-1 site (FAP1); and (4) the cyclic AMP response element (CRE). To investigate how c-FOS expression is regulated by UVB irradiation in human keratinocytes, the role of each of the four cis-elements within the c-FOS promoter was evaluated (Gonzales and Bowden 2002a, b, c). Using the human keratinocyte cell line, HaCaT, and transient transfection of a chimeric human c-FOS promoter driving luciferase with clustered point mutations we found that all four cis elements including the SIE, SRE, FAP1, and CRE sites were involved in UVB induction of c-FOS transcription. Our data indicated that all four cis elements are required for maximum promoter activity. The CRE and FAP1 elements were the two most active cis elements that mediate the UVB transactivation of c-FOS (Fig. 16.1). Homodimers of phosphorylated cAMP response element binding protein (CREB) were induced by UVB irradiation to bind to each of these elements. Therefore, CREB may function as an important regulatory protein in the UVB-induced expression of c-FOS.

16.4 Epigallocatechin-Gallate, an Inhibitor of the UVB-Induced p38 MAP Kinase Pathway Many epidemiological studies have suggested a potential causal relationship between green tea consumption and a reduced risk of some cancers (Mukhtar et al. 1992; Yang et al. 1997). Tissue culture and animal models have provided evidence for chemoprevention activity of green tea polyphenols (GTPs). The GTPs have been shown to inhibit tumor initiation, promotion, and progression in various model systems (Conney et al. 1992; Huang et al. 1992; Mukhtar et al. 1992; Wang et al. 1992; Gensler et al. 1996; Yang et al. 1997). The major polyphenolic compound with chemoprevention activity in green tea is (-)-epigallocatechin-(3)-gallate (EGCG) (Huang et al. 1992; Mukhtar et al. 1992; Wang et al. 1992; Yang et al. 1997). GTP and EGCG have antioxidant properties and this may explain their inhibition of UV- and TPA-induced skin damage, including edema, hyperplasia, H2O2 production, and inflammation (Huang et al. 1992; Katiyar et al. 1995a b). In JB6

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mouse epidermal cells, EGCG has been shown to inhibit epidermal growth factor and TPA-induced AP-1 activity (Dong et al. 1997). Through a sealing effect, EGCG blocks binding of tumor promoters to their receptors (Suganuma et al. 1996; Kitano et al. 1997). In BALB/3T3 cells EGCG is hypothesized to inhibit okadaic acid (OA)- induced tumor necrosis factor-a (TNFa) expression and release by inhibiting AP-1 DNA binding and interaction between OA and its cellular receptor (Suganuma et al. 1996). In nonmelanoma skin cancer models, EGCG applied topically to mouse skin inhibits UVB-induced complete photocarcinogenesis and immunosuppression caused by UVB (Gensler et al. 1996). Oral GTP has been shown to block both UVB-induced initiation and promotion (Wang et al. 1992). EGCG has also been shown to block cell transformation induced by TPA, EGF, and ionizing radiation (Dong et al. 1997). In cultured human keratinocytes UVB-induced AP-1 activity is inhibited by EGCG in a dose range of 5.45 nM to 54.5 mM (Barthelman et al. 1998a). EGCG was effective at inhibiting AP-1 activation when applied before, after or both before and after UVB irradiation. In vivo studies using transgenic mice expressing a luciferase reporter driven by two sets of TRE elements showed that repeated topical treatment with EGCG prior to and once after UVB irradiation inhibited the UVBinduction of AP-1 transactivation by 60% compared with vehicle treated mice. By inhibiting AP-1 activity in UVB-irradiated mouse skin, EGCG may be preventing nonmelanoma skin cancer at the level of tumor promotion. GTP can inhibit UVB-induced ornithine decarboxylase and cyclooxygenase, and restore UVB-inhibited catalase, GSH, and glutathione peroxidase in epidermal cells (Agarwal et al. 1993). Such effects are likely a result of its antioxidant abilities, lipid peroxidation, changes in endogenous antioxidants, membrane damage, and hyperproliferation. Interestingly, EGCG has been shown to interact directly with membranes in liposome studies, where it can inhibit PKC activity (Kitano et al. 1997). UVB signals are transduced through other cascades (Radler-Pohl et al. 1993; Huang et al. 1996; Assefa et al. 1997), including atypical PKCs, which are also EGCG sensitive. Through its antioxidant properties, EGCG could be acting at numerous points in the UVB-signaling cascade, including RAS/RAF and MAPK/ JNK/p38 activity which mediate UVB-induced AP-1 activity. Although in mouse epidermal JB6 cells, EGCG inhibits EGF-and TPA-induced c-JUN phosphorylation (Dong et al. 1997), it has been reported that higher doses cause an increase in JNK1 and ERK2 (Yu et al. 1997). EGCG has been found to induce the expression of JunD, c-FOS, and Fos-B mRNA only after okadaic acid stimulation of BALB/3T3 cells (Suganuma et al. 1996) but stimulate c-FOS and c-JUN expression in unstimulated HepG2 cells (Yu et al. 1997). Based on these diverse findings it must be concluded that EGCG activity is dependent on EGCG dose, as well as the state and nature of the cellular model. In the cultured human keratinocyte cell line HaCaT EGCG inhibited in a dose dependent manner UVB-induced increases in c-FOS steady state message transcriptional activation and protein accumulation (Chen et al. 1999). EGCG significantly inhibited UVB-induced p38 MAP kinase activation, but not JNK or ERK MAP kinase activation. It is possible that UVB-induced activation of p38 MAP

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kinase occur through generation of ROS because these have been shown to stimulate p38 MAP kinase activity and c-FOS gene expression in human fibroblast cells (Numazawa et al. 1997) and to partially mediate p38 MAP kinase activity in murine keratinocytes (Tao et al. 1996). The potent scavenger activity of EGCG against superoxide anion and hydroxyl radicals (Yang and Wang 1993) which have been implicated in UVB signal transduction pathways leading to AP-1 activation (Hsu et al. 2000) support this mechanism for activation of p38 MAP kinase activation by UVB.

16.5 Activation of COX-2 by UVB 16.5.1 COX-2 and Prostaglandin Synthesis in UVB Carcinogenesis Another target gene for UVB mediated signal transduction that is thought to play a role in UVB-induced skin carcinogenesis is COX-2 (Fischer et al. 1999; Pentland et al. 1999). COX-2 is one of the key enzymes involved in the synthesis of prostaglandin E2 (PGE2) from arachidonic acid (Smith et al. 1996). There are two isoforms of this enzyme: COX-1 and COX-2 (Smith et al. 1996; Vane et al. 1998). These two isoforms have a similar function but COX-1 is constitutively expressed in most tissues while COX-2 is inducible by tumor promoting agents in many tissues. COX-2 protein and prostaglandin production increases after UVB exposure in both human skin and cultured human keratinocytes (Buckman et al. 1998; An et al. 2002). In addition, immunohistochemistry and western analysis of human SCC biopsies exhibited strongly enhanced expression of COX-2 protein when compared with normal nonsun exposed control skin. Enhanced expression of COX-2 was also observed in human skin epidermal cancer cell lines and transfection with COX-2 antisense oligonucleotides into the cancer cells suppressed COX-2 protein expression and significantly inhibited cancer cell growth (Higashi et al. 2000). A pathological overexpression of COX-2 resulting in excessive prostaglandin production has been found in early stages of carcinogenesis and appears to be a consistent feature of neoplastic development in a wide variety of tissues (Marks and Furstenberger 2000). In vivo studies have shown a significant reduction of UV-induced skin tumors in hairless mice by selective COX-2 inhibition (Fischer et al. 1999; Pentland et al. 1999). Similarly, several studies have directly implicated prostaglandins in carcinogenesis in general (Bennett et al. 1982; Rigas et al. 1993; Sheng et al. 1998; Tsujii et al. 1998), and UV-induced prostaglandin synthesis in UV-induced skin carcinogenesis (Grewe et al. 1993; Fischer et al. 1999). Enhanced prostaglandin synthesis can promote angiogenesis (Tsujii et al. 1998) and increase cell proliferation (Sheng et al. 1998). Another regulated enzyme involved in biosynthesis of prostaglandins, including PGE2, is phospholipase A2 that facilitates the release of arachidonic acid from plasma membrane

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phospholipids (Gresham et al. 1996). UV has been reported to activate PLA2 and thus provide substrate for cyclooxygenases (Kang-Rotondo et al. 1993; Gresham et al. 1996).

16.5.2 Regulation of COX-2 Expression by MAPK We have investigated the role of p38 MAP kinases and ERK in mediating UVBinduced COX-2 gene expression in cultured human keratinocytes (Chen et al. 2001). In particular we have investigated the roles of p38 MAP kinases and ERK in UVB induced COX-2 gene expression (Fig. 16.1). We found that UVB irradiation of the cultured human keratinocyte cell line, HaCaT, significantly increased COX-2 gene expression at both the protein and mRNA levels. As we had observed previously, p38 and ERK kinases were significantly activated after UVB irradiation in HaCaT cells. In addition, treating the cells with the p38 MAP kinase inhibitor, SB202190 or the MEK inhibitor, PD98059 specifically inhibited UVB-induced p38 or ERK activation, respectively. We found that SB202190 strongly inhibited UVB-induced COX-2 gene expression at different time points and various UVB dose levels. Furthermore, SB202190 markedly inhibited UVB-induced COX-2 mRNA. Using the PD98059 inhibitor we found that ERK did not play a role in UVB-induced COX-2 gene expression. These results suggested, for the first time, that activation of p38 kinase is required for UVB-induced COX-2 gene expression in human keratinocytes. Since COX-2 expression plays an important role in UV carcinogenesis, p38 could be a potential molecular target for chemoprevention of skin cancer.

16.5.3 Regulation of COX-2 Transcription by UVB We have also investigated the cis-elements in the human COX-2 promoter that may be responsible for the UVB induction of COX-2 (Tang et al. 2001a) (Fig. 16.1). Analyses with the human COX-2 promoter region revealed that the cyclic AMP responsive element near the TATA box was essential for both basal and UVB induced COX-2 expression. This was further supported by studies using a dominant negative mutant of CREB, cyclic AMP response element binding protein, which strongly inhibited the activity of the COX-2 promoter. Electrophoretic mobility shift assays indicated that CREB and ATF-1 were the major proteins binding to the COX-2 CRE. CREB and ATF-1 were phosphorylated upon UVB treatment. SB202190, the p38 MAP kinase inhibitor, decreased phosphorylation of CREB/ ATF-1 and suppressed COX-2 promoter activity. Enhanced binding of phosphoCREB/ATF-1 to the COX-2 CRE was observed after UVB irradiation of the cells. Thus one signaling pathway for UVB induction of human COX-2 gene involves activation of p38, subsequent phosphorylation of CREB/ATF-1, and activation of the COX-2 CRE through enhanced binding of phosphorylated CREB/ATF-1.

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16.6 PI3K/AKT Pathways in the UV Response and UV-Induced Skin Cancer 16.6.1 PI3K/AKT Pathway Activation by UVB PI3K and its downstream signaling pathways are also activated by UVB (Kabuyama et al. 1998). Specific properties of class I PI3K suggest that it is likely to function as a mediator signaling molecule in UVB-induced signaling pathways that up-regulate c-FOS transcription and AP-1 activation. PI3K, a heterodimer that is composed of a catalytic subunit (p110) and a regulatory subunit (p85) functions as a key upstream regulatory enzyme for a number of signaling pathways that are deregulated in carcinogenesis (Carpenter et al. 1990; Roymans and Slegers 2001). PI3K activity can be induced by a number of stimuli including tumor promoting agents such as the phorbol ester, TPA, and UVB irradiation. UVB irradiation induces PI3K activity in cultured human keratinocytes (Gonzales and Bowden 2002a, b, c), and inhibition of PI3K activity, via expression of a mutant p85 subunit or treatment with the pharmacological inhibitor, wortmanin, resulted in decreased levels of c-FOS promoter activity and c-FOS protein. Two members of the PI3K signaling pathway, AKT and GSK-3b were found to affect c-FOS transcription. Expression of dominant negative AKT or wild type GSK-3b significantly inhibited UVB-induced c-FOS promoter activity. In addition, when GSK-3b activity was inhibited by lithium chloride, both c-FOS promoter activity and protein levels increased. These results demonstrated that both AKT activation and GSK-3b inactivation are required for maximal UVB induction of c-FOS expression. Inhibition of both UVB-induced p38 MAP kinase activation and PI3K activation using simultaneous treatment with SB202190 and wortmanin nearly abrogates UVB-induced c-FOS transcription and protein.

16.6.2 Regulation of COX-2 by PI3K /AKT Pathway UVB-mediated activation of the PI3K/AKT pathway impacts transcriptional regulation of the COX-2 gene (Tang et al. 2001b). In cultured human keratinocytes UVB caused AKT phosphorylation at both threonine 308 and serine 473 that was inhibited by LY294002, a PI3K inhibitor. LY294002 also decreased the UVB induction of COX-2 protein and COX-2 promoter activity. We found that UVB caused the phosphorylation of GSK-3b on serine 9 and presumably inactivation of GSK-3b. Inhibition of GSK-3b by lithium induced endogenous COX-2 protein expression and COX-2 promoter activity. Finally, overexpression of a dominantnegative AKT mutant or wild-type GSK-3b suppressed UVB-mediated induction of the COX-2 promoter. GSK-3b also phosphorylates CREB, but only when CREB is first phosphorylated at serine 133 and phosphorylation of CREB by GSK-3b decreases its binding to, for instance, the somatostatin gene CRE. We had shown

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that upon UVB irradiation, CREB/ATF-1 are phosphorylated at serine 133 through activation of p38 MAP kinase and their activation contributes to the UVB induction of COX-2 transcription (Fig. 16.1). From our studies we speculate that UVB also prevents inactivation of CREB through a pathway that involves activation of AKT and inactivation of GSK-3b. Maximal activation of CREB may require its phosphorylation at serine 133 through the p38 MAP kinase pathway and blocking of its inactivation by GSK-3b. Finally, because the PI3K pathway plays an important role in the regulation of cell growth, proliferation and survival and is activated by UVB suggests that members of this pathway are potential targets for chemoprevention of skin cancers.

16.6.3 AKT/mtor Pathway and Skin Cancer UVB irradiation of hairless mouse skin leads to activation of AKT in the epidermis (Bachelor et al. 2005). Activated AKT is known to lead to the activation of mTOR through the inhibition of the tumor suppressor gene products, TSC1/TSC2 (Kopelovich et al. 2007). mTOR is the target of rapamycin and a serine-threonine kinase with lipid kinase activity (Fig. 16.2) and rapamycin is a macrolide antibiotic and immnuosuppressant. mTOR is a component of two protein complexes, mTORC1 and mTORC2. mTORC1 consists of mTOR, mLST8 and raptor (regulatory-associated protein of mTOR) and mTORC2 consists of mTOR, mLST8, mSIN1, and rictor (rapamycin insensitive companion of mTOR). mTOR signaling is activated when the environmental and genetic landscape is optimal and decreases under stressful conditions including insufficient energy or growth factors as well as after DNA damage. Therefore, mTOR protein is essential for cell development and growth and is essential in cell cycle progression, cell migration and cell survival. In addition, mTOR protein regulates autophagy, a process in which organelles and proteins are degraded during nutrient deprivation (Lum et al. 2005). mTOR activity regulates a number of downstream targets (Fig. 16.2). These include ribosomal protein S6 kinases (S6K1 and S6K2). S6K1 is activated by mTOR and regulates ribosomal protein translation and ribosome biogenesis. There is also a role for S6K1 in feedback inhibition of insulin and insulin-like growth factor (IGF) induced PI3K activation (O’Reilly et al. 2006). S6K1, which can be stimulated by activated mTOR, phosphorylates insulin receptor substrate proteins, inhibiting their function, which in turn diminishes signaling through the PI3K/AKT pathway. Thus cells regulated by this mechanism can become resistant to mTOR inhibition as S6K1 production decreases and AKT rebounds. Therefore, there is rationale to use combinations of drugs to inhibit mTOR as well PI3K/AKT in both treatment and chemoprevention of cancer. Another important downstream target for mTOR is the translation initiating factor (eIF-4E) binding protein, 4EBP1 which is phosphorylated and inactivated by mTOR releasing eIF-4E to increase translation of cap-dependent mRNAs (Kopelovich et al. 2007).

PRAS40 eIF4e Ribosome biogenesis

S6K1

Rapamycin & analogs

Cox-2 mRNA stability

AMPK

AMPK activators: metformin AICAR resveratrol

mTOR c1

Cap-dependent translation

Rheb GTP

PDK1

Akt

Rheb GDP

PI3K

4EBP1

LKB1

TSC1TSC2

EGFR

Fig. 16.2 Solar UV Signal Transduction Pathways Leading to mTOR Activation.

mTOR c2

PI3K inhibitors: LY 294002 solenopsin

IRS1

InsR

Solar UV

eIF4b

RSK2

ERK

MEK

Raf

Ras

EGFR

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Aberrant activation of the PI3K/AKT signaling pathway has been implicated in the development of nonmelanoma skin cancer (i.e., SCC development). Segrelles et al. 2002 have shown in a DMBA/TPA model of mouse skin carcinogenesis that AKT activity increases throughout the entire process and its early activation is detected prior to increased cyclin D1 (CycD1) expression. This early increase in AKT activity was due to raised PI3K activity. These authors further confirmed the involvement of AKT in the process by transfecting nonmalignant PB mouse keratinocytes with AKT and xenografting these cells into athymic nude mice. The expression of AKT accelerated tumorigenesis and contributed to increased malignancy. The same group of researchers (Segrelles et al. 2006) determined molecular determinants of AKT-induced keratinocyte transformation and found increased and nuclear localization of DNp63, b-catenin, and Lef1. They also found increased expression of c-MYC and CycD1, both targets of the beta-catenin/Tcf pathway. The authors point out that such increase is associated with increased phosphorylation and stabilization of c-MYC and CycD1 due to mTOR activation. Based on their observation in cell culture that rapamycin decreases the expression of CycD1 and c-myc, they performed subcutaneous injection of AKT-transfected keratinocytes in nude mice where a subcutaneous pump allowed continuous flow of rapamycin and saw the complete inhibition of tumor growth. These results imply that mTOR-mediated translation is essential for AKT-mediated keratinocyte transformation. In two transgenic mouse models of epidermal directed expression of wild type AKT or constitutively activated AKT, it was found that transgenic mice with the highest levels of AKT expression/activity developed spontaneous tumors and those mice with lower expression/activity of AKT showed heightened sensitivity to two-stage skin carcinogenesis, especially to the tumor promotion stage (Segrelles et al. 2007). While it is generally believed that AKT contributes to tumorigenesis by inhibiting apoptosis, Skeen et al. 2006 showed that AKT is required for normal cell proliferation and susceptibility to oncogenesis independently of its antiapoptotic activity. These researchers found that partial ablation of AKT-1 activity by deleting AKT-1 inhibits cell proliferation and oncogenesis in cultured cells while AKT-1 null mice are resistant to MMTV-v-H-ras induced tumors and to DMBA/TPA induced mouse skin tumors. Activated AKT and mTOR is also found in CD34+/K15+ keratinocyte stem cells during multistage mouse skin carcinogenesis (Affara et al. 2006). Within DMBA/TPA induced benign papillomas, pAKT, and pmTOR were expressed in the suprabasal cells of the tumor. These results suggest that pAKT and pmTOR may allow the respective cell populations to evade terminal differentiation and to persist for long periods of time in their specific niche. Finally, the authors suggest that strategies that target pAKT and pmTOR may deplete both cells within the CD34+/K15+ keratinocyte stem cell compartment, as well as impacting survival of non-proliferating suprabasal cells within premalignant papillomas. 16.6.3.1 Chemopreventive Agents Targeting mtor Genetic alterations including mutations or amplification of mTOR have not been found in human cancers. However, dysregulation in mTOR signaling pathways

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have been documented in premalignant and malignant human tissues (Vivanco and Sawyers 2002; Sawyers 2003; Faivre et al. 2006). This suggests that mTOR is a promising target for cancer prevention strategies. Actinic keratoses or solar keratoses display disregulated mTOR signaling pathways (Chen et al. 2009). Most of the data suggesting that disregulated mTOR signaling play a functional role in tumorigenesis comes from studies in which mTOR has been pharmacologically inhibited by rapamycin and its analogs RAD-001 (CCI-779) and AP23573 (Rao et al. 2004; Faivre et al. 2006; Sabatini 2006). In models of drug treatment of cancers, these selective mTOR inhibitors suppress the growth of a broad range of cancer types by slowing or arresting cells in the G1 phase of the cell cycle, promoting apoptosis or affecting angiogenesis pathways. There are a number of published studies which have shown that rapamycin and its analogues have chemoprevention activities in preclinical mouse SCC models. Most relevant to skin SCC is a published report that everolimus, RAD-001, inhibits in vivo growth of murine SCCs (SCC VII) (Khariwala et al. 2006). In this published study everolimus was given i.p. and SCC VII cells were injected either intradermally or intravenously. Everolimus significantly inhibited the growth of SCC VII cells growing as intradermal tumors or as pulmonary metastases. Supporting the idea that rapamycin or its analogues can prevent the development of skin cancer in patients are published studies showing that the use of a rapamycin analog called sirolimus may be beneficial in protecting renal transplant patients from skin cancer (Euvrard et al. 2004; Mathew et al. 2004).

16.6.4 AMP Kinase/mtor Pathway and Skin Cancer There are a number of upstream signaling pathways that converge on the tuberous sclerosis complex (TSC) which consists of the tumor suppressor protein, hamartin, encoded by the TSC1 gene and tuberin encoded by the TSC2 gene (Fig. 16.2). Rheb, Ras homolog enriched in brain, which can bind directly to and up-regulate mTOR, is inactivated by the GTPase activity of TSCs. Growth factors including insulin, IGF, EGF, and platelet-derived growth factors interact with mTORC2 by activating PI3K. PI3K activation is blocked by PTEN as well as by S6K1. The lipid product of PI3K localizes AKT to the plasma membrane where it is phosphorylated and activated by PDK1 and mTORC2. mTORC1 signaling is then activated by active AKT directly phosphorylating and inactivating TSC2. UVB-mediated activation of PI3K/AKT is through ligand independent activation of the EGF receptor (Fisher et al. 1998). Another important upstream signal that impacts on TSC complex activity is the energy sensing pathway involving AMP-activated protein kinase (AMPK) and its activator LKB1 (Fig. 16.2). Besides the net amount of ATP being an energy sensor, the high ratio of AMP to ATP is an even more sensitive indicator of energy deprivation. Small increases in the ratio of AMP to ATP leads to the activation of AMPK. In turn, activated AMPK phosphorylates and enhances the ability of TSC2 to inhibit mTORC1 signaling. LKB1, a tumor suppressor gene product, can directly phosphorylate and activate AMPK and under conditions of

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energy stress is required for mTORC1 suppression. Therefore, low energy leads through AMPK and LKB1 to a decrease in mTORC1 activity and decreases in translation of ribosomal proteins and cap-dependent translation of mRNAs. 16.6.4.1 Activators of AMP Kinase as Chemoprevention Agents Inhibition of mTOR can also occur indirectly by altering upstream signaling such as the pathway from LKB1 and AMP kinase (Woods et al. 2003; Corradetti et al. 2004; Shaw et al. 2004). Potential chemoprevention activity could theoretically occur via the energy-sensing component of mTOR signaling by activating AMPK which in turn enhances the suppressor activity of TSC1/TSC2 on mTORC1 (Fay et al. 2009). It has been shown that resveratrol increases the levels of AMPK in mice fed a high calorie diet, resulting in increased insulin sensitivity and reduced IGF-1 levels (Baur et al. 2006). Two antidiabetic drugs, rosiglitazone and Metformin, can activate AMPK and have chemoprevention activity in mouse models of cancer. Rosiglitazone, a peroxisome-proliferator-activated receptor-g agonist, increased PTEN expression, inhibited AKT phosphorylation, increased AMPK phosphorylation, and decreased S6K1 phosphorylation in non-small-cell lung carcinoma cells (Han and Roman 2006). It has been found that oral administration of rosigliazone and a related thiazolidinedione called troglitazone inhibited IGF-1-induced mouse skin tumorigenesis (He et al. 2006). Another AMP kinase activator, the antidiabetic drug Metformin, has chemopreventive activity in experimental animal models of cancer. Interestingly, systemic treatment with Metformin selectively impairs p53-deficient colon cancer cell growth (Buzzai et al. 2007), and this potentially could be important mechanism for effective chemoprevention of UV-induced skin cancers that display early inactivation of p53. This work was carried out with paired isogenic colon cancer cell lines HTC116 p53 +/+ and HCT116 p53 −/−. Treatment with Metformin selectively suppressed the tumor growth of HCT116 p53−/− xenografts. It was also observed that there was increased apoptosis in p53 −/− tumor sections. Treatment with AICAR, another potent AMPK activator, also showed a selective ability to inhibit p53 −/− tumor growth in vivo. In the presence of either of the two drugs HTC116 p53+/+ cells but not HCT116 p53−/− cells activated autophagy. It is concluded in this work that Metformin treatment forces a metabolic conversion that p53−/− cells cannot execute and thus the p53−/− cells undergo apoptosis. It is true that one cannot equate p53 null with mutated p53 which is found in UV-irradiated epidermis and skin tumors induced by UV. However, there is another rationale to use AMPK activators as chemoprevention agents to prevent UV-induced skin cancer. AMPK activators have also had a negative effect on mTOR signaling. It is of interest that there are two reports in the literature that link antidiabetic treatment using Metformin with a lower risk of cancer in patients (Evans et al. 2005; Bowker et al. 2006). It also has been shown that energy deprivation in the form of caloric restriction inhibits mouse skin carcinogenesis induced by both chemical carcinogens and UV light (Birt et al. 2004). Caloric or fat intake reduction

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(i.e., 20%) in patients for 2 years resulted in a significant decrease in the number of actinic keratoses (Black et al. 1994). The mechanism for these effects of caloric restriction on skin carcinogenesis could involve activation of AMP kinase and inhibition of mTOR downstream signaling leading to apoptosis and inhibition of tumor cell growth. Recently we have shown that UVB irradiation of human HaCaT cells in culture leads to stabilization of the COX-2 mRNA through inhibition of AMP kinase (Zhang and Bowden 2008). It has been shown that the RNA binding protein HuR is involved in stabilizing COX-2 mRNA by binding to the 3¢ UTR of the message and protecting the RNA from degradation. It has also been shown HuR localization in the cell is regulated by AMP kinase. Specifically, upon activation, AMP kinase phosphorylates importin alpha-1, which binds to HuR and shuttles it into the nucleus. An inhibitor of AMP kinase, Compound C, as well as UVB treatment of HaCaT cells leads to cytoplasmic localization of HuR and stabilization of the COX-2 mRNA. These published data support a novel mechanism for UVB-mediated COX-2 expression in human keratinocytes. These data also provide a rationale for using activators of AMP kinase to reverse the UVB-mediated stabilization of the COX-2 mRNA and enhanced expression of the COX-2 protein which plays a functional role in UVB-induced skin carcinogenesis.

16.7 NDGA, an Inhibitor of the UVB-Induced PI3K/AKT Pathway Nordihydroguaiaretic acid (NDGA) is a polyphenolic lignan from the Larrea tridentate bush that has been reported to show chemopreventive activity in chemical carcinogenesis studies. NDGA prevents mammary, bowel, and skin tumor formation (Nakadate et al. 1985; Birkenfeld et al. 1987; McCormick and Spicer 1987). In these studies, NDGA was reported to exert its anticancer effects by inhibiting ornithine decarboxylase activity or DNA synthesis. There are several lines of evidence to suggest that NDGA may inhibit UVB-induced signaling pathways that contribute to the development of skin carcinogenesis. In HaCaT cells NDGA inhibited in a dose dependent manner UVB-induced AP-1 activation and c-FOS promoter activation (Gonzales and Bowden 2002a, b, c). In addition, NDGAinhibited UVB-induced accumulation of c-FOS protein. NDGA also inhibited the UVB-induced AP-1 DNA binding to a TRE containing oligonucleotide. As a lignan, NDGA possesses a wide variety of biological properties, including the ability to inhibit enzyme activity. Previous published work from our laboratory indicated that UVB induction of c-FOS expression involved UVB-induced PI3K signaling(Gonzales and Bowden 2002a, b, c). In PI3K test tube assays, addition of NDGA to enhanced PI3K activity due to cell exposure to UVB-inhibited kinase activity in a dose-dependent manner indicating that NDGA inhibited PI3k activity. In HaCaT cells NGDA also inhibited UVB induced phosphorylation of AKT at serine 473 and threonine 308, AKT activity and c-FOS expression (Gonzales and

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Bowden 2002a, b, c). These results suggested that negative regulation by NDGA of UVB-induced PI3K signaling extended to downstream components of the PI3K pathway. It has been reported that NDGA inhibits lipoxygenase signaling and lipoxygenase-mediated signal transduction pathways that are important for induction of c-FOS, c-JUN and AP-1 transactivation (Glasgow et al. 1992; Rao et al. 1996). However, there was no inhibition of UVB induced c-FOS expression or AP-1 activation in HaCaT cells treated with the with lipoxygenase inhibitor, phenidone. Thus, our data suggest that lipoxygenase activity does not play a role in UVB-induced c-FOS and AP-1 transactivation and that the effects of NDGA on UVB-induced c-FOS expression and AP-1 transactivation is largely through inhibition of the PI3K signaling pathway. Topical NDGA application has been shown to inhibit DMBA initiated, TPA promoted mouse skin tumor formation (Nakadate et al. 1982), and Park et. al. (Park et al. 1998) reported that NGDA inhibited formation of a FOS-JUN DNA complex. A positive clinical trial has also been conducted with a topical NDGA treatment, masoprocol or Actinex, for actinic or solar keratoses (Olsen et al. 1991; Odom 1998). Because NDGA also has antioxidant properties that can inhibit redox signaling (Kemal et al. 1987), it is possible that the action of reactive oxygen species in UVB-induced signaling pathways may be prevented in the presence of NDGA. Collectively these studies suggested that the diverse biochemical properties of NDGA make it a strong candidate for chemoprevention of UVB-induced skin carcinogenesis.

16.8 Translation of UVB Signal Transduction Findings into the Clinic We have demonstrated in mouse skin and in cultured human keratinocytes that UVB irradiation activates the p38 MAPK pathway and the PI3K pathway that lead downstream to activation of the transcription factor complex, AP-1, and enhanced expression of COX-2. We have determined whether activation of these pathways also occurs in human skin exposed to 4X minimal erythemic dose of UVB (Einspahr et al. 2008). Skin biopsies were taken prior to, half hour, 1 and 24 h after irradiation. We found in agreement with the mouse studies the earliest UVB-induced changes in the epidermis were in phospho-CREB and in phospho-MAPKAPK-2. At 1 h phospho-cJUN and phospho-p38 MAPK were increased. Increases in c-FOS and COX-2 were not seen until 24 h postirradiation. These findings in human epidermis are in agreement with our findings in mouse epidermis after UVB irradiation. Validation of mouse models in human skin will aid in the development of effective skin cancer chemoprevention strategies. Because the incidence of nonmelanoma skin cancers in the USA have been rising in the past years, and primary prevention strategies such as the use of sun screens or blocks have not been totally effective, much attention has been paid to developing new chemoprevention strategies to prevent nonmelanoma skin cancers (Baade et al. 1996). As discussed above, the initiation process in UVB-induced skin carcinogenesis

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appears to involve UV-induced DNA damage which if unrepaired, can be replicated to generate UVB-induced signature mutations including those in the tumor suppressor gene, p53. This process of initiation is irreversible and occurs at any time during a person’s life (Tornaletti et al. 1993). Therefore, strategies to prevent initiation by drug intervention are very difficult to envision and to design. In contrast, UVBmediated skin tumor promotion which involves clonal expansion of the initiated cells through UVB-mediated signal transduction pathways to give rise to benign lesions is a reversible process (Brash et al. 1991). Therefore, chemoprevention of the reversible process of skin tumor promotion is a much more attractive strategy using a chemoprevention drug approach. Because UVB mediates skin tumor promotion through signaling pathways that downstream lead to clonal expansion of initiated cells, a thorough understanding of these pathways would help in designing new strategies for chemoprevention of skin cancer. Early intervention in SCC development can result in a considerable delay in the onset of malignant tumor development by preventing the appearance of benign skin lesions, i.e., actinic keratoses. The development of new chemoprevention agents for general population use involves the execution of six different clinical trials including phases 0, I, IIa, IIb, III and IV (Lippman et al. 1990; Einspahr et al. 2002). Phase 0 trials have been designed to allow brief exposure to an experimental agent for the purpose of initial pharmacokinetic analysis or to determine if a specific molecular target in the tissue of interest was “hit”. Phase I is a short-term (1 month) hypothesis testing and safety trial. Phase IIa is a short-term (e.g., 1–3 months) dose finding randomized, placebo controlled trial. Phase IIb is an intermediate biomarker randomized, double-blinded, placebo-controlled trial. Phase III is a controlled intervention trial and phase IV is a defined population trial. At the University of Arizona a decision tree is utilized to funnel new potential chemoprevention agents into clinical trial (Einspahr et al. 2003). Agent leads from basic science and epidemiology are put through UVB mouse skin carcinogenesis studies and preclinical toxicology testing in mice and if successful they are placed in a phase I trial. The University of Arizona Cancer Center has been over the years involved in testing in clinical trials certain chemoprevention agents. These agents include oral vitamin A, injectable Melanotan, difluoro-methyl-ornithine (DFMO), topical myristyl nicotinate, topical EGCG and topical perillyl alcohol (Table 16.1). The one agent that has been taken through a phase III trial is oral vitamin A or retinol. This was a large randomized placebo-controlled trial performed in subjects at high risk of developing non-melanoma skin cancers (Moon et al. 1997). This trial was 5 years in duration with 2,297 subjects with moderate to severe AK who were taking 25,000 IU/day. This intervention resulted in a significant reduction in SCCs but no effect on BCCs. Of interest, the retinoids are known to transrepress the AP-1 transcription factor complex by competing for the coactivator, CBP/p300 (Li et al. 1996). Of further interest was the preclinical toxicology testing and phase I trials with topical EGCG. A 10% (w/w) EGCG formulation in hydrophilic ointment USP was prepared and used to determine pharmacokinetic parameters following topical application to full thickness mouse or human skin in vitro. Intradermal uptake of 9% and 19% of the applied dose was found in human and mouse skin respectively,

MCR-1 receptor

ODC

p38 MAPK & AP-1

Farnesyl Transferase & AP-1 PPAR-g

Melanotan (sub Q)

a-DFMO (topical)

EGCG (topical)

Perillyl Alcohol (topical) Myristyl nicotinate

+ (Barthelman et al. 1998b) + (Jacobson et al. 2007)

+ (Gensler et al. 1996)

+ (Gensler et al. 1996)

+

+(Stratton et al. 2008) Phase 1 completed

+

+ (Levine et al. 1999; Dorr et al. 2004) + (Alberts, Dorr et al. 2000) Local Toxicity**

+ (Dvorakova et al. 1999) +

+

+

Table 16.1 Chemoprevention agents previously and currently under study at the Arizona Cancer Center Non-toxic in preclinical Chemo-preventive Active in UVB mouse models Safe in phase 1/2a trials agent Molecular target model Vit A (oral) AP-1 transcription + (Gensler et al. 1990) + + (Goodman, Alberts et al. 1983)

Beginning phase 2a

Currently in phase 2

Active in phase 2b trials + in Phase 3 (SCC) (Moon et al. 1997; Alberts et al. 2004) Phase 3 (Australia, Europe, US pending) Currently in Phase 2b combo. w/ diclofenac Phase 1 completed

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while transdermal penetration was observed only in mouse skin (Dvorakova et al. 1999). The 10% EGCG formulation was evaluated in a phase I clinical study. The topical EGCG was applied to buttock skin and an SPF of 3.6 was recorded. When applied to the posterior forearm skin, no systemic toxicities were seen in the 19 patients who completed the study. However, 42% of the participants reported moderately severe skin reactions and histological evaluation corroborated the clinical findings. Therefore, another formulation is required for human studies due to toxicities seen and evidence of partial oxidation of the formulation. Another potential chemoprevention agent that we found in preclinical studies to inhibit both UVB-induced AP-1 activation and UVB-induced mouse skin carcinogenesis is perillyl alcohol (Barthelman et al. 1998b). This is an alcohol derivative of d-limonene and d-limonene is derived from orange and lemon peel. Perillyl alcohol has as one of its activities the ability to inhibit ras-farnosylation. This activity could explain the inhibitory effects of this agent on UVB-induced AP-1 activation and UVB-induced mouse skin carcinogenesis. A stable cream formulation of perillyl alcohol has been made and dermal toxicity tests in SKH-1 hairless mice showed no significant dermal or systemic toxicity after 6 months. The agent was stable at 4°C and 80°C for up to 6 months and high concentrations of the agent were absorbed into mouse skin. These are excellent indicators that should facilitate the testing of perillyl alcohol in phase II studies. A 1 month, first-in-human, phase 1 trial of topically applied perillyl alcohol in cream in human subjects was conducted at the Arizona Cancer Center (Stratton et al. 2008). Endpoints included safety and evaluation of any histopathological changes in skin after 1 month use of perillyl alcohol in cream. We randomized 25 subjects with normal, healthy skin with little or no sun damage and no history of skin cancer in a double-blind fashion to receive topical perillyl alcohol (0.76% wt/wt) on one forearm with placebo cream applied to the other forearm twice daily for 30 days. Subjects were monitored for toxicity, and a 4 mm punch biopsy in the treated area was performed at the end of study for histopathological evaluation. The topical cream was well tolerated and no serious cutaneous toxicities, systemic toxicities, or histopathological abnormalities were observed. Based on this successful phase 1 study, a phase 2 clinical trial for efficacy using biomarkers is underway at the Arizona Cancer Center. Finally, NDGA has already been clinically tested for its ability to treat actinic keratoses (Olsen et al. 1991; Odom 1998). It has been reported that topical NDGA, Actinex, or masoprocol resulted in a significant decrease in the number of actinic keratoses. However, to date, NDGA has not been tested for its chemoprevention activity in for SCCs clinical trials.

16.9 Toward Patient Tailored and Pathway-Driven Therapy for Prevention of SCC Recent studies show that for a number of solid tumors the enormous and seemingly incongruous number of mutational differences between patients tumors manifest themselves in distinct and interlinked protein pathways that are far less

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complicated (Wulfkuhle et al. 2008). This finding provides a firm basis for the discussion of human cancer as a pathway-driven disease, and for the treatment of human cancer by pathway disruption and modulation. This new concept can be taken one step further through the use of novel and unique genomic and proteomic techniques to identify druggable pathway changes to prevent the development of SCC. The guiding aim of this translational research should be the elucidation of these pathway changes as skin epithelium transitions from early UV-induced damage to frank SCC with a critical evaluation of the causal significance of these pathway changes through the utilization of targeted therapeutic clinical trials. Thus, collaborative research should take advantage of human tissue study sets from prospective clinical trials, along with animal tissue to study fully the mechanistic details of SCC tumorigenesis and translation of these findings to the bedside in a unique one-of-a-kind approach to the new era of molecular, targeted medicine.

References Affara NI, Trempus CS et al (2006) Activation of Akt and mTOR in CD34+/K15+ keratinocyte stem cells and skin tumors during multi-stage mouse skin carcinogenesis. Anticancer Res 26(4B):2805–2820 Agarwal R, Katiyar SK et al (1993) Protection against ultraviolet B radiation-induced effects in the skin of SKH-1 hairless mice by a polyphenolic fraction isolated from green tea. Photochem Photobiol 58(5):695–700 Alberts DS, Dorr RT et al (2000) Chemoprevention of human actinic keratoses by topical 2-(difluoromethyl)-dl-ornithine. Cancer Epidemiol Biomarkers Prev 9(12):1281–1286 Alberts D, Ranger-Moore J et al (2004) Safety and efficacy of dose-intensive oral vitamin A in subjects with sun-damaged skin. Clin Cancer Res 10(6):1875–1880 An KP, Athar M et al (2002) Cyclooxygenase-2 expression in murine and human nonmelanoma skin cancers: implications for therapeutic approaches. Photochem Photobiol 76(1):73–80 Ananthaswamy HN, Pierceall WE (1990) Molecular mechanisms of ultraviolet radiation carcinogenesis. Photochem Photobiol 52(6):1119–1136 Angel P (1995) The role and regulation of the Jun proteins in response to phorbol esters and UV light. In: Baeuerle PA (ed) Inducible gene expression. Birkhouser, Boston Assefa Z, Garmyn M et al (1997) Differential stimulation of ERK and JNK activities by ultraviolet B irradiation and epidermal growth factor in human keratinocytes. J Invest Dermatol 108(6):886–891 Baade PD, Balanda KP et al (1996) Changes in skin protection behaviors, attitudes, and sunburn: in a population with the highest incidence of skin cancer in the world. Cancer Detect Prev 20(6):566–575 Bachelor MA, Cooper SJ et al (2005) Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model. Mol Cancer Res 3(2):90–99 Bair WB 3rd, Hart N et al (2002) Inhibitory effects of sodium salicylate and acetylsalicylic acid on UVB-induced mouse skin carcinogenesis. Cancer Epidemiol Biomarkers Prev 11(12):1645–1652 Barthelman M, Bair WB 3rd et al (1998a) (-)-Epigallocatechin-3-gallate inhibition of ultraviolet B-induced AP-1 activity. Carcinogenesis 19(12):2201–2204 Barthelman M, Chen W et al (1998b) Inhibitory effects of perillyl alcohol on UVB-induced murine skin cancer and AP-1 transactivation. Cancer Res 58(4):711–716

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Chapter 17

Molecular-Targeted Chemotherapy for Head and Neck Squamous Cell Carcinoma Harrison W. Lin and James W. Rocco

Abstract The head and neck region hosts the intersection of several essential organ systems which provide the vital functions of respiration, swallowing, communication and airway protection. Therapeutic modalities designed to treat squamous cell carcinoma (SCC) of the head and neck, including surgery, chemotherapy, and radiation therapy can therefore result in devastating physical and psychological consequences. Recent advances in our understanding of cancer biology have revealed many potential therapeutic targets and have started to provide clinicians with a selective means to treat patients with head and neck SCC, limiting treatment morbidity while maintaining patient survival. Despite this initial promise, only a few of these molecular-targeted chemotherapies are currently in established clinical use, with the majority under ongoing preclinical and clinical trial evaluation. Future molecular-targeted chemotherapy will compliment existing therapeutic strategies before ultimately reducing the morbidity of current treatment options while maintaining or improving survival. We review the current status of the preclinical and clinical work on these putative cancer-associated molecules and the proposed strategies to exploit them for targeted tumor cell death.

17.1 Introduction The role of medical therapy for cancer has evolved considerably in the past half century since the introduction of chemotherapy during the post-World War II era. When leukocytopenia was noted in a group of people accidentally exposed to the chemical warfare agent mustard gas, it was correctly inferred that a drug that significantly decreased rapidly-proliferating white blood cell populations could have a similar effect on patients with leukemia and lymphoma (Joensuu 2008). While the anti-mitotic and proapoptotic principles of these and other chemotherapy agents still hold today, considerable advances in cancer research have created an extensive body of knowledge on J.W. Rocco (*) Department of Surgery, Massachusetts General Hospital, Jackson 904G, 55 Fruit Street, Boston, MA 02114, USA e-mail: [email protected]

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the fundamental rules that govern the transformation of normal cells to cancer cells and accordingly, have revolutionized modern chemotherapy. In their landmark paper, The Hallmarks of Cancer, Hanahan and Weinberg outline a set of six functional capacities cancer cells acquire during their development and through various mechanistic strategies (Hanahan and Weinberg 2000). Each of these cellular facilities, including selfsufficiency of growth signals, evasion of apoptosis, sustained angiogenesis, limitless replicative potential, tissue invasion and metastasis, and insensitivity to antigrowth signals, are achieved through aberrant molecular signaling pathways. The identification of the key intracellular and extracellular molecules essential to these signaling pathways and correspondingly, to cancer cell survival, has introduced a new realm of targets with the potential to both improve and individualize head and neck cancer therapy (Fig. 17.1). Although most systemic chemotherapeutic drugs in clinical use today are directed at all rapidly proliferating cells by impairing mitosis and thereby affect both normal and cancer cells, many of the recently developed anticancer agents employ a more elegant and targeted approach to achieve cancer cell-specific death. Each of these strategies utilizes a technology that directly or indirectly destines the affected cancer cell for cell death. For instance, recent advances in treating recurrent (R) and metastatic (M) head and neck squamous cell carcinoma (HNSCC), among other cancers, have been achieved with the use of monoclonal antibodies raised against molecules expressed in cancer cells. These antibodies bind to specific membrane proteins and consequently occupy survival-promoting ligand binding sites and exploit the immune system to facilitate targeted cell destruction. Similarly, the use of vaccines, antibody-toxin conjugates, Gene addition therapy p53-expressing adenovirus Extracellular receptor inhibition Cetuximab Gene disruption therapy RNA interference Intracellular receptor inhibition

Cell signaling pathway inhibition Bortezomib

P Epigenetic modification therapy microRNA modification Vascular growth inhibition Bevacizumab

Fusion protein antibody Proxinium Oncogenic protein inhibtion Small molecule Bcl-2 inhibition

Fig. 17.1 Potential methods of molecular-targeted chemotherapeutic interventions. Cartoon of a cancer cell and its supportive collection of molecules and vascular networks known to promote cancer cell survival and proliferation. An example of a chemotherapeutic drug or intervention currently in clinical use or development is provided in italics.

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molecular radiotherapy and antibodies designed against molecules critical for vascular support of tumors are alternative means of achieving cancer cell death. In addition, recent work has also focused on small molecule inhibitors of cytoplasmic and nuclear proteins essential for cancer cell function and survival. These and other molecules are potential targets for other methods of intervention at the nucleic acid level, including the use antisense oligonucleotides, gene therapy and RNA interference (RNAi). Although a substantial amount of preclinical and clinical research awaits most if not all of the novel medical agents with a potential application for HNSCC, the promise of molecular-targeted therapies is patently contrasted against the significant morbidity and limited success of the chemotherapeutic drugs in standard use today. The heedlessly cytotoxic nature of current HNSCC agents has not only exhibited side effects that at times question their designation as an “organ-preserving” therapy, its role as a definitive therapy has been generally confined to patients with advanced and unresectable cancers. In contrast, molecular-targeted therapies hold the theoretical potential to serve as definitive chemotherapy for all stages of HNSCC and as “maintenance” therapy to impede cancer progression, delay cancer-related death and minimize side effects that may significantly impact quality of life. Furthermore, additional research on the molecular biology of HNSCC tumorgenesis may provide further insight into the molecular profiles specific to a patient and thereby identify people with an increased likelihood of developing cancer and provide individualized guidance on drug selection. We begin this overview of molecular-targeted chemotherapy by providing a brief review of SCC of the head and neck. We then discuss the process of transitioning a candidate drug or molecule from the laboratory to the clinical realm and follow this discussion with a review of the molecular-targeted agents that have successfully bridged this gap and are currently in prevalent use or in late stage clinical trials. Next, up and coming approaches to cancer cell-specific chemotherapy that are in preclinical or early clinical testing are introduced to provide an overview of novel or borrowed technologies that may hold considerable promise in HNSCC treatment. Finally, we discuss new concepts in HNSCC therapy that are emerging as efforts aimed at providing individualized surgical and chemoradiation therapies to cancer patients gain momentum and advance the frontiers of medical oncology.

17.2 Head and Neck Squamous Cell Carcinoma HNSCC is an aberrant proliferation of the squamous epithelium lining the mucosal surfaces of the head and neck. Primary tumors can develop in any mucosa-lined region, including the tympanic cavity, paranasal sinuses, nasal cavity, naso- and oropharynx, oral cavity, and larynx. Left untreated, these tumors typically metastasize through lymphatic channels to the lymph nodes of the neck, which are situated in the deep cervical fascia coating important anatomic structures. When cervical nodal spread is present, the patient is considered to have locoregional disease, which in many instances can be successfully treated with combined surgical and chemoradiation therapies. However, when tumor is found inferior to the neck, most frequently in the

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lungs or liver, the patient is regarded as having distant metastatic disease and is therefore not a traditional candidate for surgery. Patients such as these must rely on established or experimental chemotherapies to address their tumor burden. Additionally, patients who have been previously treated with surgery and chemoradiation therapies but subsequently develop significant recurrent disease will characteristically respond poorly to traditional chemoradiation therapy. Consequently, these patients are oftentimes offered experimental treatment modalities. SCC can be found in a variety of organs in the body, including skin, esophagus, lung, breast, and vagina. Unlike most of these anatomic locations, however, the head and neck is readily visible and is essential for all social and interpersonal interactions. A disfiguring tumor or surgical defect can have a significant impact on the psychological state of HNSCC patients. Concerted efforts to maintain a “normal” appearance for the patient add an additional level of complexity to surgicalplanning already made difficult by both the intricate neuromuscular and vascular anatomy and the many difficult-to-access regions of the head and neck, such as the skull base or oropharynx. Furthermore, many of the structures of the head and neck provide essential functions of respiration, deglutination, sensation, and communication. As the crossroads of multiple organ systems, including neurologic, cardiovascular, respiratory, alimentary, and endocrine systems, the head and neck exhibits very little redundancy and consequently, oncologically sound surgery that maintains high levels of both form and function is difficult to achieve. Accordingly, the development of novel interventions designed to control tumor growth and thereby minimize the extent of or eliminate the need for surgery is the next challenge for clinicians and scientists investigating HNSCC therapy. Efforts to create treatment modalities to directly address tumor cells will undoubtedly be driven by advances in the understanding of HNSCC tumor biology, which will not only provide innovative strategies to kill or slow the proliferation of cancer cells, but may also provide clinicians with reliable biomarkers circulating in the blood or measured directly from tumor biopsies that may serve to guide therapy or indicate the extent of disease, prognosis and response to treatment. Therapeutic approaches that specifically target HNSCC cells may also benefit from emerging knowledge of the role of the human papilloma virus (HPV) in the development of certain types of HNSCC. Proteins found to be expressed by or upregulated by HPV infection may serve as targets of chemotherapy agents or may guide clinicians in their treatment selection. Given the intricacy of head and neck anatomy, moleculartargeted therapies presently in development will be anticipated to reduce tumor load and thereby obviate the need for extensive and disfiguring surgery. These novel chemotherapies are currently being tested in patients with R/M disease, but perhaps may have a role in the primary treatment of early stage disease, as well.

17.3 Benchtop to Bedside The development of novel therapeutic agents or modalities must begin with a thorough understanding of the aberrant biology of tumor cells. Specifically, knowledge of proteins either overexpressed in or vital to cancer cells provides investigators

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with a list of potential targets at which to direct a molecularly designed chemotherapy agent. Once produced, research into its efficacy as an anticancer drug often begins with preclinical, benchtop studies utilizing the numerous immortalized HNSCC cell lines that are available to investigators (Lin et al. 2007). The clinical data of each cell line, including patient demographic information, tumor site of origin, tumor stage, cellular growth rate, and genetic or mutational information are frequently provided for each of the over 300 lines currently reported in the literature. Several of these cell lines have been demonstrated to be effective for introduction of cultured cells as xenografts into athymic, immunodeficient nude mice. This technique serves as an animal model that accurately simulates the complex interactions between cancer and its host and can be used to test the efficacy of novel anticancer regimens in reducing tumor size and extent of metastasis (Lin et al. 2007). Once an agent is identified in preclinical studies as having sufficient anticancer properties, investigators can begin the first stage of testing in human subjects, in a phase I clinical trial. Typically, a group of 20–80 volunteers with advanced HNSCC will be selected to receive the novel drug in an effort to assess its safety, tolerability, pharmacokinetics, and if appropriate, its efficacy. Patients will often receive escalating doses of the drug to determine the dose range for therapeutic use and any dose-limiting toxicities. Following confirmation of initial drug safety, phase II trials can then be performed on larger groups (20–300) to directly assess the ability of the drug to establish a clinical response, which is often subclassified into disease stabilization, partial response, or complete response. Other measures including median time to progression and overall survival duration can also be determined. In addition, phase II trials often continue phase I safety and dosing studies in the larger group of patients. With continued demonstration of drug efficacy and safety, a multicentered randomized, controlled trial on hundreds to thousands of patients is then conducted in a phase III clinical study designed to definitively assess drug efficacy in direct comparison with current “gold standard” therapies. Once a drug has been proved satisfactory in one or more phase III trials, Food and Drug Administration (FDA) approval for commercial distribution and use and advancement to a postmarketing surveillance (phase IV) trial can be obtained.

17.4 Current Therapeutic Molecular Targeting Approaches in Head and Neck Cancer 17.4.1 Epidermal Growth Factor Receptor We begin our overview on the molecular-targeted therapies in development and in use with the only current molecular-targeted chemotherapy for HNSCC with a clear and documented role for clinical use in the USA. Cetuximab is a monoclonal antibody raised against the extracellular domain of the epidermal growth factor receptor (EGFR), a type I tyrosine kinase receptor that regulates critical cellular functions in carcinomas. Also referred to as ErbB1 and HER1, EGFR is expressed in over 90% of cases of HNSCC and high levels of expression have been associated with

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poor prognosis (Ang et al. 2002). Phase III trials demonstrating added survival benefit to HNSCC patients with advanced disease with the addition of cetuximab to radiation (Bonner et al. 2006), or R/M disease with cisplatin (Burtness et al. 2005), and a platinum-based agent with 5-fluorouracil (5-FU) (Vermorken et al. 2008), all provided evidence contributing to the first approval of new agent for HNSCC by the FDA in several decades. In addition, a 46% disease control rate reported by a phase II trial employing cetuximab as a single agent for patients with R/M disease who previously failed platinum-based therapy furthermore established a role for cetuximab in platinum-refractory patients (Vermorken et al. 2007). Ongoing phase II and III trials will likely reveal additional groups of HNSCC patients who may benefit from cetuximab and/or the many other EGFR-antibody therapies currently in development (Table 17.1). Specifically designed small molecules that are able to freely pass through the cellular plasma membrane and into the cytoplasm can also accomplish disruption of protein functions essential for cancer cell survival and growth. Several small molecule inhibitors of the intracellular tyrosine kinase domain of EGFR are currently in advanced clinical testing, including erlotinib, which has been studied in phase II trials as a single agent (Soulieres et al. 2004) and in combination with cisplatin (Siu et al. 2007). Both studies demonstrated excellent patient tolerance and encouraging anticancer activity. Gefitinib, another EGFR tyrosine kinase inhibitor, has also demonstrated some degree of promise in published phase I-III trials (Cohen et al. 2003, 2005; Stewart et al. 2009), including efficacy as a single agent equivalent to methotrexate and with better quality of life measures and favorable toxicity profiles (Stewart et al. 2009). More recently, another member of this class of agents, lapatinib, provided a remarkable 81% clinical response in a phase I study of patients with locally advanced HNSCC (Harrington et al. 2009) and is currently in phase II trials (Abidoye et al., 2006). Many experts agree that EGFR represents a molecule with tremendous promise to play a major role in the future of HNSCC therapy and that continued translational work into agents targeting EGFR is needed (Karamouzis et al. 2007a; Sattler et al. 2008; Egloff and Grandis 2008).

17.4.2 Vascular Endothelial Growth Factor In accordance with the rapid and unrestrained cellular growth and proliferation seen in tumors, the angiovascular network needed to maintain tumor viability must also be augmented. This is often achieved with tumor-stimulated release of proangiogenic factors such as vascular endothelial growth factor (VEGF). Accordingly, interventions to limit the VEGF levels have the potential to slow the growth of rapidly proliferating cancer cells, but because this approach does not specifically target tumor cells, anti-VEGF therapies are frequently used in combination with other agents with different mechanisms of action. For example, a phase I/II study investigating the combination of bevacizumab, an antibody designed against VEGF, and erlotinib

Table 17.1 Summary of the most advanced clinical trials of several molecular-targeted therapies in R/M HNSCC Monotherapy Phase Target MOA Patient population Comments 46% disease control rate Cetuximab II EGFR Antibody R/M pts who failed platinum therapy Gefitinib III EGFR Antibody R/M pts Equivalent to methotrexate monotherapy Erlotinib II EGFR Kinase inhibitor R/M pts Prolonged disease stabilization comparable to other palliative therapies Intratumoral antisense I EGFR DNA Advanced refractory 29% response rate DNA disease Sorafenib II VEGF Kinase inhibitor R/M Modest monotherapy activity, should be used in combination Semaxanib III VEGF Kinase inhibitor R/M Responses rare, no further testing should be done Proxinum I EpCAM Fusion antibody R/M 71% response rate Double agent therapy Cetuximab Versus XRT alone, significantly + XRT III EGFR Antibody Locoregionally improved LRC, PFS, OS advanced HNSCC pts + Cisplatin III EGFR Antibody R/M pts Significantly improved objective response rate; no survival benefit Erlotinib + Cisplatin II EGFR Kinase inhibitor R/M pts Versus other combination therapies, better side-effect profile with equivalent response/survival rates (continued)

Siu et al. (2007)

Burtness et al. (2005)

Bonner et al. (2006)

Macdonald et al. (2009)

Fury et al. (2007)

Elser et al. (2007)

Lai et al., (2009)

Soulieres et al. (2004)

Stewart et al. (2009)

References Vermorken et al. (2008)

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VEGF

I/II

III

I

I

Bevacizumab + Erlotinib

Triple agent therapy Cetuximab + 5-FU & Platinum

Lapatinib + XRT & Cisplatin

Bevacizumab + 5-FU & XRT

VEGF

EGFR

EGFR

Target

Table 17.1 (continued) Monotherapy Phase

Anti-angiogenesis

Kinase inhibitor

Antibody

Anti-angiogenesis

MOA

R/M pts

Locoregionally advanced HNSCC pts

R/M pts

R/M pts

Patient population

OS comparable with other regimens; 21% fistula/ necrosis rate

81% response rate

Significantly improved OS, PFS, RR

Complete responses associated with target expression levels

Comments

Seiwert et al. (2008)

Harrington et al. (2009)

Vermorken et al. (2008)

Cohen et al., (2009)

References

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was reported to provide several patients with sustained clinical benefit, including complete responses. Furthermore, the pretreatment expression profiles of putative targets within tumor tissue were highly associated with response status (Cohen et al. 2009). However, in a reflection of its systemic antiangiogenic properties, a phase I trial combining bevacizumab, 5-FU and radiation therapy for a cohort of advanced HNSCC patients that included treatment-naïve patients reported a 21% rate of tissue necrosis and fistula formation (Seiwert et al. 2008). Additional work on both the benefits and complications of these and other molecular-targeted approaches against angiogenesis are under way, including a phase II trial of the VEGFR tyrosine kinase inhibitor, AZD2171 (Recentin) in patients with unresectable HNSCC (http://clinicaltrials.gov/ct2/show/NCT00458978). These and other trials should provide further insight into the role of these agents in HNSCC therapy.

17.4.3 Other Molecular Targets Recent preclinical benchtop research and early clinical work have shed considerable light on the potential efficacy of molecular chemotherapy agents that are either newly developed or proved in other types of cancers. The biological rationale for the use of drugs in these early stages of development is exceptionally sound but definitive clinical data supporting their efficacy in HNSCC patients are still lacking. Human epidermal growth factor receptor 2 (HER-2/neu), or ErbB2, is a protein overexpressed in 15–20% of breast cancers, and in HNSCC, HER-2/neu is the preferred dimerization partner of EGFR. Heterodimers of HER-2/neu and EGFR are believed to potentiate receptor signals and contribute to HNSCC resistance to EGFR inhibitors. Trastuzumab (Herceptin®) is a humanized monoclonal antibody targeting HER-2/neu that has already made a significant impact in the treatment of breast cancer, and in HNSCC cell lines, has shown to augment gefitinib inhibition of cell proliferation (Kondo et al. 2008). A phase II clinical trial is currently underway (Gillison et al. 2006). Similarly, insulin-like growth factor type I receptor (IGF-IR) is a receptor tyrosine kinase that has been shown to heterodimerize with EGFR in HNSCC. IMC-A12, a human monoclonal antibody raised against IGF-IR, together with cetuximab demonstrated a synergistic reduction in HNSCC cell line proliferation and migration (Barnes et al. 2007) and is also currently in a phase II trial. Although phase I/II trials of other novel agents with promising anticancer activity in the laboratory have thus far similarly failed to demonstrate efficacy as a single agents, including inhibitors to farnesyl transferase such as lonafarnib (Yang et al. 2005) or to intracytoplasmic proteins involved in the PI3K/AKT/mTOR pathway such as perifosine (Argiris et al. 2006) and temsirolimus (Raymond et al. 2004), continued research on these agents in combination with other moleculartargeted drugs has been suggested (Le Tourneau et al. 2009). Preclinical studies on HNSCC tumor biopsies and cell lines and early clinical studies on non-HNSCC patients have revealed a number of molecules involved in signaling pathways vital to cancer cell growth and survival that may represent

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potential targets for novel chemotherapy agents against HNSCC. For example, inhibitors of Src kinase and focal adhesion kinase have demonstrated apoptotic activity in HNSCC cells and are currently in phase I/II trials in advanced solid tumors (Egloff and Grandis 2008). Bortezomib, an inhibitor to the 26S subunit of a proteasome involved in the NF-kB pathway (Allen et al. 2007), has been shown to have single-agent activity in patients with non-small cell lung carcinoma and in HNSCC cell lines (Sunwoo et al. 2001). An early Phase I trial of bortezomib demonstrated enhanced apoptosis in post-treatment HNSCC tumor biopsies (Allen et al. 2008). Other molecular targets whose inhibition may have roles in future anticancer therapies, including protein kinase C, aurora kinases, cyclin-dependent kinases, histone deacetylases, polo-like kinases and heat shock proteins, are eloquently reviewed by Le Tourneau and Siu (2008)

17.4.4 Unconventional Methods of Treatment Delivery Direct intratumoral injections of novel molecular-targeted chemotherapies have demonstrated substantial potential to achieve both robust clinical responses and minimal adverse effects despite their current inability to treat systemic (noninjected) disease. Proxinium (VB4-845), a recombinant fusion protein combining an antibody against epithelial cell adhesion molecule (EpCAM) and the exotoxin A protein from the bacterium Pseudomonas aeruginosa, reduced or stabilized tumors in over 71% of patients with cancers expressing EpCAM (Macdonald et al. 2009). Additionally, intratumoral injection of plasmid DNA expressing antisense oligonucleotides against the EGFR translational start site achieved a 29% clinical response rate with no dose-limiting toxicities in a recent phase I trial (Lai et al. 2009). Of note, patients demonstrating disease control were found to have significantly higher levels of EGFR expression compared to patients with progressive disease, suggesting that a priori knowledge of EGFR expression levels may influence the indications for this or other modes of molecular-targeted therapy. Additional work in this and other areas of nucleic acid chemotherapy may provide further insight into the role of gene therapy in the treatment of HNSCC.

17.5 Up and Coming Therapies and Targets 17.5.1 Genetic Manipulation As previously discussed, the delivery of therapeutic nucleic acids (TNAs) directly into tumors represents a novel treatment approach that may replace or compliment the various chemoradiotherapeutic regimens currently administered to HNSCC patients. By reducing the expression of proteins vital to either cancer cell survival or chemoradiotherapy resistance, the application of TNAs to cancer cells may

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c ontribute to cell death and cell sensitization and thereby increase the efficacy of a standard chemoradiotherapy regimen or reduce the dosing required to achieve an expected level of response. Although clinical use of TNAs in HNSCC is currently premature, benchtop research has opened a number of promising avenues. For example, decreased cellular invasion potential, viability, proliferation activity or resistance to other chemotherapies has been demonstrated in HNSCC cell lines with the application of RNAi against several key genes, including EGFR (Nozawa et al. 2006), EpCAM (Yanamoto et al. 2007), MET receptor (Seiwert et al. 2009), Cyclin D1 (Oridate et al. 2005), NF-kB p65 (Duan et al. 2007), VEGF, telomerase reverse transcriptase, Bcl-xl (Wang et al. 2008) and p63 (Rocco et al. 2006). Additionally, the manipulation of gene expression within cancer cells using DNA gene therapy techniques has shown promise in both preclinical and early clinical studies. While there are a number of approaches to achieving tumorspecific cell death or growth inhibition through gene therapy, including gene excision, “suicide” gene transfer, pro-apoptotic gene and anti-angiogenic gene therapy, among others, “corrective” gene therapy, or gene addition therapy, has to date been the most extensively utilized technique for HNSCC research and therapy. Notably, in 2003 a phase III trial conducted in China on patients with advanced HNSCC comparing radiotherapy alone versus radiotherapy with intratumoral injections of a recombinant adenovirus expressing p53, a key regulator of the cell cycle and the most commonly mutated gene in HNSCC, showed an impressive 93% response rate in the latter group, with 64% of patients exhibiting a complete response (Peng 2005). Of note, 85% of the 135 patients enrolled, however, had nasopharyngeal carcinoma primaries. This study, along with others (Zhang et al. 2003, 2005), led China to become the first nation to approve the adenoviral-p53 therapy, trademarked Gendicine® (Shenzhen SiBiono GeneTech, Shenzhen, China), for commercial production (Karamouzis et al. 2007b). A similarly-designed adenovirus expressing p53 (INGN-201) has likewise shown considerable promise in early clinical studies in the USA (Clayman et al. 1998, 1999) and is currently in phase III testing. Additional genetic approaches to HNSCC treatment, including immunomodulatory cancer gene therapy and chemoprotective/chemoresistance gene therapy, among others, are articulately reviewed by Karamouzis and colleagues (Karamouzis et al. 2007b).

17.5.2 Human Papilloma Virus Recent studies demonstrating the presence of HPV as an etiologic agent in roughly half of HNSCCs arising from the tonsil or base of tongue have provided investigators with an additional potential target for novel chemotherapeutic agents. Of particular note is the creation of a DNA vaccine linking HPV E7, an oncogenic protein, to a Mycobacterium tuberculosis heat shock protein (HSP) in an effort to amplify the cytotoxic T-cell mediated immune response to the HPV protein. In animal models, mice challenged with lung cancer cells expressing E7 demonstrated

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dramatically decreased pulmonary metastatic nodules when subsequently treated with the E7/HSP vaccine (Hsu et al. 2001). A clinical trial using this vaccine as adjuvant therapy in patients with HPV-associated HNSCC is currently underway (Psyrri et al. 2009). Additionally and taking from research on SCC of the cervix, a cancer that has similarly been intimately linked to HPV, studies on the impact of HPV vaccines such as Gardasil (Merck & Co., Inc., Collegeville, PA) and Cervarix (GlaxoSmithKline, Research Triangle Park, NC) on the incidence and natural history of oral and oropharyngeal HPV infection will also be forthcoming (Vidal and Gillison 2008). Laboratory studies on cervical cancer cell lines with moleculartargeted therapies against HPV, however, have already demonstrated considerable success. The use of a variety of TNAs including catalytic ribozymes, antisense nucleotides, and RNAi directed against key HPV genes have been shown to successfully exhibit anticancer activity in cultured cervical cancer cells (Alvarez-Salas and DiPaolo 2007). Future in vitro and in vivo work into these and other therapeutic approaches will provide further insight into the role of anti-HPV therapies in the treatment of both cervical and HPV-associated HNSCC.

17.5.3 Emerging Gene Technologies Significant advances in gene expression profiling technologies have provided scientists with an opportunity to not only examine the diagnostic and therapeutic potential of molecules known to be dysregulated in HNSCC but also to identify novel genes and gene expression modifiers which may be targets of future molecular-based therapies. Large scale transcriptomic methods to identify molecules with potential relevance in carcinogenesis such as complimentary DNA microarray (Colombo et al. 2009; Ogawa et al. 2009) and Serial Analysis of Gene Expression (Silveira et al. 2008) have revealed a host of candidate genes which may serve as early diagnostic markers, molecular targets for therapy, or both. The recent discovery of non-coding RNAs (ncRNAs), functional transcripts predominantly composed of small double-stranded RNAs critical for gene expression regulation that do not code for proteins, may provide clinicians with the ability to predict disease status and follow the development, progression or regression of disease. Of all the ncRNAs, microRNAs (miRNAs), 18–24 nucleotide molecules with epigenetic influence on gene expression and a carcinogenic role in many cancers, including HNSCC, are the most widely studied and characterized. To date, more than 600 human miRNAs having been identified. These ncRNAs tend to be highly conserved, typically act as negative regulators of gene regulation and have potential roles as both biomarkers in cancer detection and as direct therapeutic targets (Negrini et al. 2009). The usefulness of miRNAs as a potential biomarker for cancer detection stems from the fact that their expression patterns tend to be tissue specific and reflect developmental lineage and differentiation states of tumors. Notably, miRNAs have been successfully used to classify poorly differentiated tumors with much higher accuracy than conventional mRNA expression profiling. Expression profiles of

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various HNSCC-specific miRNAs have been shown to be highly sensitive and specific in distinguishing cancer from normal epithelium (Chang et al. 2008; Avissar et al. 2009; Childs et al. 2009). The therapeutic potential of targeting miRNAs is a consequence of multiple studies demonstrating differential expression patterns in a wide variety of human tumors and their capacity to function as both oncogenes and tumor suppressor genes. Several studies have shown specific miRNA expression can repress known oncogenes such as ras and Bcl-2, while in other contexts miRNAs can act as an oncogene, presumably by down-regulating tumor suppressor genes (Lee and Dutta 2006). Therapeutic approaches to modify miRNA levels using miRNA mimetics and antisense oligonucleotides are currently being developed (Visone and Croce 2009). Despite their tremendous potential, additional studies are needed to understand their precise role in diagnosis and treatment (Chua et al. 2009; Costa 2009). In addition, the discovery of the polycomb group of genes believed to play a vital role in epigenetic transcriptional regulation through histone modification and chromatin remodeling in both cancer and stem cells have inspired considerable interest in these genes as both markers of cancer and targets for therapy. Of note, the enhancer of zeste homolog 2 (EZH2) gene has been the subject of substantial investigation for its involvement in a wide array of human cancers, and most recently, EZH2 was found to be highly expressed in both HNSCC cell lines and tumor specimens. Moreover, higher levels of EZH2 expression significantly correlated with increased tumor size, rate of locoregional metastases, clinical stage and shorter overall survival duration, indicating that this polycomb gene may serve as a biomarker of disease severity and a predictor of prognosis (Kidani et al. 2009). Studies targeting this and other epigenetic modifiers in cultured cancer cells are currently underway (Fiskus et al. 2009).

17.5.4 Small Molecular Inhibitors Small molecule inhibitors such as the aforementioned drugs engineered to disrupt the function of various receptor tyrosine kinases represent a class of agents with tremendous promise to target proteins known to lead to carcinogenesis, and collaborations with basic scientists will continue to generate these novel compounds crafted by teams from an array of research fields. For example, combined work by structural biologists, medicinal chemists, computer programmers and clinicians have for the past decade produced a host of small, organic molecules designed to inhibit Bcl-2, a potent antiapoptotic protein shown to be highly expressed in a number of cancers, including HNSCC, and associated with poor outcomes (Michaud et al. 2009). Several of these agents are based on the structure of gossypol, naturally occurring product of cotton seeds and roots shown in HNSCC cell culture (Oliver et al. 2004) and animal xenograft models (Wolter et al. 2006) to induce apoptosis and inhibit growth. Other synthesized Bcl-2 inhibitors, including TW-37 (Ashimori et al. 2009) and ABT-737 (Li et al. 2009) have been shown to

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have anti-HNSCC activity in vitro, while other investigational drugs targeting the Bcl-2 family, including oblimersen sodium, AT-101, ABT-263 and GX15-070 have already begun testing in early clinical trials on non-HNSCC cancers (Kang and Reynolds 2009; Azmi and Mohammad 2009). The results of these phase I/II trials, combined with further work on determining the efficacy of these novel agents to inhibit HNSCC growth, will likely generate additional clinical trials examining the role of small molecule Bcl-2 inhibitors in HNSCC therapy.

17.5.5 Cancer Stem Cells The recent discovery of small subpopulations of highly tumorigenic “cancer stem cells” (CSC) within solid tumors, including HNSCC, has introduced a new line of research into HNSCC tumor and cell line biology. It is well known that all stem cells require a stable and specific microenvironment to maintain their undifferentiated state, and it is believed that the matrix that composes the majority of tumor bulk, including fibroblasts, inflammatory and endothelial cells, provides CSCs with their needed supportive environment via intercellular contacts or secreted factors. Additionally, studies on HNSCC tumor specimens and cell lines have identified a series of cellular proteins specific to or upregulated in CSCs, including CD44+, BMI1 (Prince et al. 2007) and TERT (Okamoto et al. 2009), among others. Taken together, molecular-targeted therapies directed against elements vital to the maintenance of the CSC microenvironment and/or against CSC markers and upregulated gene products may prove to contribute to the efficacy of current standard chemoradiotherapy protocols.

17.6 Future Trends As our understanding of the molecular biology of cancer become further refined, the approaches to address both the diagnosis and management of cancer patients have similarly become more advanced and sophisticated. It is hoped that one day simple salivary (Lallemant et al. 2009) or blood (Colnot et al. 2004) tests will be able to not only provide an accurate diagnosis and prognosis of a patient with or suspected of having HNSCC, but also guide individualized preventive, medical or surgical therapy. Reflective of these efforts is the plan by the Massachusetts General Hospital to genetically screen the tumors of all new cancer patients for the limited number of genetic abnormalities for which chemotherapy drugs targeting these mutations either already exist or are currently in advanced development (Kaiser 2009). Clinicians will be following the efficacy and economics of routine genetic fingerprinting of tumors, currently estimated to be roughly $2,000 per patient, with great interest. Additionally, efforts to improve patient compliance of prescribed chemotherapy regimens could be directed at advancing technologies on effective enteral

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chemotherapy drug delivery. Due to their pan-cytotoxic nature, many of the chemotherapies currently in routine use for HNSCC patients, including carboplatin, cisplatin and fluorouracil, among others, require frequent intravenous administrations and/or central venous line placement and thereby obligates patients to numerous hospital and clinic visits. Conversely, many of the molecular-targeted therapies in development, most notably the small molecule inhibitors, are readily absorbed by the gastrointestinal system and may therefore permit select HNSCC patients to self-administer their medications in pill form. Furthermore, advances in the development of biomaterials optimized for local and sustained delivery of drugs will also benefit future generations of patients by reducing the frequency of intratumoral injections and the need for systemic treatment. Further research into the efficacy of these targeted drug delivery systems in animal models will be needed to provide additional insight into the role of localized, molecular-targeted chemotherapy in the context of surgical and radiation therapies.

References Abidoye OO, Cohen EE, Wong SJ et al. (2006) A phase II study of lapatinib (GW572016) in recuurent/metastatic (R/M). squamous cell carcinoma of the head and neck (SCCHN). J Clin Oncol 24:5568 [abstract] Allen CT, Ricker JL, Chen Z, Van Waes C (2007) Role of activated nuclear factor-kappaB in the pathogenesis and therapy of squamous cell carcinoma of the head and neck. Head Neck 29:959–971 Allen C, Saigal K, Nottingham L et al. (2008) Bortezomib-induced apoptosis with limited clinical response is accompanied by inhibition of canonical but not alternative nuclear factor-k B subunits in head and neck cancer. Clin Cancer Res 14:4175–4185 Alvarez-Salas LM, DiPaolo JA (2007) Molecular approaches to cervical cancer therapy. Curr Drug Discov Technol 4:208–219 Ang KK, Berkey BA, Tu X et al. (2002) Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res 62:7350–7356 Argiris A, Cohen E, Karrison T et al. (2006) A phase II trial of perifosine, an oral alkylphospholipid, in recurrent or metastatic head and neck cancer. Cancer Biol Ther 5:766–770 Ashimori N, Zeitlin BD, Zhang Z et al. (2009) TW-37, a small-molecule inhibitor of Bcl-2, mediates S-phase cell cycle arrest and suppresses head and neck tumor angiogenesis. Mol Cancer Ther 8:893–903 Avissar M, Christensen BC, Kelsey KT et al. (2009) MicroRNA expression ratio is predictive of head and neck squamous cell carcinoma. Clin Cancer Res 15:2850–2855 Azmi AS, Mohammad RM (2009) Non-peptidic small molecule inhibitors against Bcl-2 for cancer therapy. J Cell Physiol 218:13–21 Barnes CJ, Ohshiro K, Rayala SK et al. (2007) Insulin-like growth factor receptor as a therapeutic target in head and neck cancer. Clin Cancer Res 13:4291–4299 Bonner JA, Harari PM, Giralt J et al. (2006) Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 354:567–578 Burtness B, Goldwasser MA, Flood W et al. (2005) Phase III randomized trial of cisplatin plus placebo compared with cisplatin plus cetuximab in metastatic/recurrent head and neck cancer: an Eastern Cooperative Oncology Group study. J Clin Oncol 23:8646–8654 Chang SS, Jiang WW, Smith I et al. (2008) MicroRNA alterations in head and neck squamous cell carcinoma. Int J Cancer 123:2791–2797

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Chapter 18

Effects and Therapeutic Potential of Targeting Dysregulated Signaling Axes in Squamous Cell Carcinoma: Another Kinase of Transcription and Mammalian Target of Rapamycin Cheryl Clark, Oleksandr Ekshyyan, and Cherie-Ann O. Nathan Abstract Head and neck squamous cell carcinoma (HNSCC) needs new approaches to treatment, as 500,000 new cases are seen worldwide annually, and recurrences and second primaries result in significant morbidity and poor survival. HNSCC is characterized by a persistent activation of the human v-akt murine thymoma viral oncogene homolog 1 (AKT)/mammalian target of rapamycin (mTOR) pathway that initiates a cascade of cellular events intrinsic to the carcinogenic process including cell survival, proliferation, cell cycle progression, cell growth, transcription and translation, angiogenesis, invasion, and metastasis. The AKT/mTOR pathway integrates a variety of signaling pathways involved in cell growth and division, and inhibitors of this pathway effectively starve the targeted cells.

18.1 Background Organ preservation chemoradiotherapy is commonly used in the management of advanced stage head and neck squamous cell carcinoma (HNSCC). Significant treatment-related toxicities and high rates of locoregional recurrences following therapy in advanced stage HNSCC have encouraged investigation of alternative and second-line treatment options. Several Food and Drug Administration-approved agents for molecular cancer therapy target proteins dysregulated through the phosphoinositide 3-kinase (PI3K) pathway. These include the epidermal growth factor receptor (EGFR) and human epidermal growth factor (HER2/neu) which activate PI3K and AKT1 (Lin et al. 1999; Wen et al. 2000).

C.-A.O. Nathan (*) Department of Otolaryngology-Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, USA and Feist-Weiller Cancer Center, Shreveport, LA, USA e-mail: [email protected]

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18.1.1 AKT/mTOR as a Signaling Axis The mammalian target of rapamycin (mTOR) signaling pathway appears dysregulated in 99% of HNSCC (Molinolo et al. 2007) and in a smaller percentage of squamous cell carcinoma of the lung (LSCC) (Mahalingam et al. 2009) and esophageal squamous cell carcinomas (ESCC) (Hay and Sonenberg 2004; Bianco et al. 2008). The AKT/mTOR pathway activity is regulated by nutrients and growth factors, but can also be activated by mutations that occur in carcinomas. mTOR can be activated through the EGF, insulin growth factor (IGF), and vascular endothelial growth factor (VEGF) receptors (Mahalingam et al. 2009), loss-of-function mutations in the phosphatase and tensin homolog (PTEN) tumor suppressor gene (a negative regulator of the PI3K/AKT pathway) (Wee et al. 2009), AKT overexpression, PI3K activating mutations, and amplification of the RAS-RAF-MEK pathway (Mahalingam et al. 2009). mTOR in turn dysregulates downstream targets of the translation machinery (Hay and Sonenberg 2004). The mTOR protein complex integrates a variety of intracellular signaling pathways by connecting the VEGF, hypoxia inducing factor (HIF1A) and HER family receptors (Bianco et al. 2008). mTOR inhibitors suppress oncogenesis by down-regulating translation of specific mRNAs required for cell cycle progression, cell proliferation, and angiogenesis (DeBenedetti et al. 1994; Shantz and Pegg 1994; Rosenwald et al. 1995; Yu et al. 2001; Huang and Houghton 2002; Lane et al. 2009). These pathways are often dysregulated in cancer, making mTOR inhibition an attractive antitumor target.

18.1.2 Rationale for Targeting the AKT/mTOR Pathway Signaling pathways upstream and downstream of mTOR appear dysregulated in all HNSCC (Molinolo et al. 2007). EGFR inhibitors such as cetuximab are FDAapproved as a single agent in metastatic HNSCC and in combination with radiotherapy in locoregionally advanced HNSCC (Astsaturov et al. 2006). It is believed that these inhibitors control apoptosis indirectly through effects on the PI3K/AKT pathway, which is activated by the EGFR (Wang et al. 2000). Activation of the PI3K/AKT/mTOR pathway may be involved in tumor cell chemoresistance (Wendel et al. 2004). Chemotherapy and radiotherapy can further upregulate pro-survival signaling in cancer cells (Kandel and Hay 1999) counteracting the cytotoxic effects of the therapy. The incidence of increased phosphorylation of AKT (p-AKT) doubles in patients with esophageal SCC after chemotherapy (from 25% to 51%; p = 0.0018); furthermore, innate and chemotherapy-induced p-AKT over expression is associated with poor prognosis in these patients (Yoshioka et al. 2008). The AKT/mTOR signaling pathway is also activated in the mucosa surrounding tumors. Overexpression of EIF4E, a downstream translation factor, when detected in tumor-free surgical margins, is an independent predictor of recurrence (Nathan et al. 2002), and is functionally activated by the AKT/mTOR signaling pathway (Nathan et al. 2004). In earlier studies on surgical margins, as the degree of dysplasia increased

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there was an increase in overexpression of EIF4E in the surgical margins of HNSCC patients (Nathan et al. 1999). Hence, inhibition of the AKT/mTOR pathway in premalignant oral cavity lesions could provide a rational approach to block and/or decrease carcinogenesis. Inhibition of the AKT/mTOR pathway inhibits two important downstream targets p70S6 kinase (RPS6KB1) and the EIF4E-binding protein (EIF4EBP1), both of which regulate protein translation (Fig. 18.1). The serine/threonine kinase RPS6KB1 is required for cell growth and cell cycle progression (Pullen and Thomas 1997; Dufner and Thomas 1999), and the translation repressor EIF4EBP1 inhibits cap-dependent translation initiation (Flynn and Proud 1996). When EIF4EBP1 is inactivated, the unphosphorylated EIF4EBP1 binds to EIF4E, a translation initiation factor of mRNAs with highly structured 5’ untranslated regions (5’UTRs) such as FGF2, VEGF, cyclin D1 (CCND1), and MMP9 (Rhoads 1993; DeBenedetti et al. 1994; DeBenedetti and Harris 1999; Nathan et al. 2004). The decrease in free EIF4E levels leads to a decrease in cap-dependent Growth Factors IRS-1

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Fig. 18.1 The PI3K-AKT-mTOR pathway. Components include another kinase of transcription (AKT) / mammalian target of rapamycin (mTOR) signaling pathway and inhibition. IRS1 insulin receptor substrate 1, PI3K phosphoinositide 3-kinase, PTEN phosphatase and tensin homolog, PDK1 3-phosphoinositide-dependent kinase-1, PDK2 3-phosphoinositide-dependent kinase-2, AKT another kinase of transcription, TSC2 tuberous sclerosis complex 2, TSC1 tuberous sclerosis complex 1, RHEB Ras homolog enriched in brain, mTOR mammalian target of rapamycin, mTORC1 mammalian target of rapamycin complex 1, mTORC2 mammalian target of rapamycin complex 2, RPTOR regulatory associated protein of mTOR, RICTOR RPTOR independent companion of mTOR, MLST8 mTOR associated protein, LST8 homolog (S. cerevisiae), MAPKAP1 mitogen-activated-protein-kinaseassociated protein 1, RPS6KB1 ribosomal protein S6 kinase, 70 kDa, polypeptide 1, EIF4EBP1 eukaryotic translation initiation factor 4E binding protein 1, EIF4E eukaryotic translation initiation factor 4E, P phosphorylated.

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translation of these specific mRNAs, thus leading to inhibition of cell proliferation and invasion, respectively.

18.2 AKT/mTOR Function 18.2.1 History mTOR inhibitors have been used as therapeutic agents since their initial isolation from the soil bacterium Streptomyces hygroscopicus (Sehgal et al. 1975) on Easter Island (Rapa Nui) in 1972 by researchers at Ayerst Pharmaceuticals in Montreal. Initially developed as an antifungal agent, unveiling of its immunosuppressive effects halted its development. Animal studies in the 1980s clarified the adverse profile and led to its role in transplant rejection therapy and the potential utility as a cancer therapeutic was initially recognized by the National Cancer Institute and soon realized by a number of pharmaceutical companies.

18.2.2 Function and Regulation of the AKT/mTOR Pathway 18.2.2.1 AKT AKT, also known as protein kinase B, is found in three structurally similar isoforms (AKT1, AKT2, and AKT3) that are widely expressed and activated by the threonine kinases 3-phosphoinositide-dependent kinase-1 and -2 (PDK1 and PDK2). PI3K-dependent AKT activation occurs downstream of G-protein-coupled receptors (Engelman et al. 2006). PI3K activates the lipid signaling second messenger system by phosphorylating phosphatidylinositol bisphosphonate (PIP2), to generate phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Dephosphorylation of PIP3 by PTEN negatively regulates the PI3K/AKT signaling pathway (Cantley and Neel 1999). Once activated, PIP3 binds to the plekstrin homology domain of AKT and PDK1, and is recruited to the plasma membrane in the rate-limiting step of AKT activation (Kandel and Hay 1999; Brazil and Hemmings 2001; Scheid and Woodgett 2001). PDK1 phosphorylates the activation loop of AKT at Thr308, which is essential for AKT activation (Alessi et al. 1996; Alessi et al. 1997). Further activation of AKT is required and achieved through Ser473 phosphorylation in the regulatory hydrophobic motif (Alessi et al. 1996). AKT family members are inactive in serum-starved primary and immortalized fibroblasts and activated by PDGF, insulin, and IGF1. AKT regulates and is regulated by the tuberous sclerosis complex (TSC1 and TSC2), also known as hamartin and tuberin. The TSC1 and TSC2 complex are tumor suppressors that stimulate specific GTPases in a cytosolic complex, possibly acting as a chaperone.

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18.2.2.2 mTOR A downstream target of AKT is the mTOR, also known as FK506 binding protein 12-rapamycin associated protein 1. TOR is a member of the phosphatidylinositol kinase-related kinase family and is an evolutionarily conserved serine/threonine kinase cloned in mammalian cells after discovery of the budding yeast TOR1 and TOR2 during a rapamycin-resistance screen (Kunz et al. 1993; Helliwell et al. 1994). mTOR receives input from multiple signaling pathways, although the main pathway is PI3K/AKT and can be either EGFR-dependent or EGFR-independent. Tissue array analysis confirmed PI3K/AKT/mTOR is activated in HNSCC but can be independent from EGFR activation (Molinolo et al. 2007), and can be regulated in an AKT-dependent and independent manner. mTOR is the catalytic subunit of two distinct complexes (Wullschleger et al. 2006): mTORC1 and mTORC2 (Fig. 18.1). In the mTORC1 complex, mTOR associates with raptor (regulatory associated protein of mTOR) and MLST8 (mTOR associated protein, LST8 homolog). AKT stimulates mTORC1 to phosphorylate targets RPS6KB1, RPS6KB1, EIF4EBP1 and EIF4EBP2 (Hay and Sonenberg 2004). The mTORC1 complex is specifically inhibited by rapamycin, and its activity is suppressed by the TSC1 and TSC2 heterodimer complex, low nutrient levels, growth factor deprivation, reductive stress, caffeine, rapamycin, farnesylthiosalicylic acid, and curcumin. Activity of the mTORC1 complex is stimulated by insulin, growth factors, serum, phosphatidic acid, amino acids, and oxidative stress. In the mTORC2 complex, mTOR associates with rictor (rapamycin-insensitive companion of mTOR), MAPKAP1 (mitogen-activated protein kinase associated protein 1; also known as mSin1) and MLST8 (GbL) to phosphorylate the AGC kinase family members AKT(Ser473) (Sarbassov et al. 2005) and PRKCA (PKCa) (Sarbassov et al. 2004; Guertin et al. 2006). mTORC2 regulates the cytoskeleton through F-actin, paxillin, RHOA, RAC1, CDC42, and PRKCA, and is regulated by insulin, growth factors, serum and nutrient levels.

18.3 AKT/mTOR and Cancer 18.3.1 Aberrant Signaling Activation of the AKT/mTOR pathway contributes to carcinogenesis by inducing cell survival, proliferation, and angiogenesis in a variety of cell types (Tsurutani et al. 2005; Dasqupta et al. 2009). The mammalian cell cycle is tightly regulated and divided into the DNA synthetic and mitotic phases that are immediately preceded by gap phases. Aberrant regulation of these cell division phases can either block cell division or induce hyperproliferation. Deregulation of Cyclin D1, a ratelimiting factor in progression through the gap phase, occurs frequently in cancer

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through its overexpression. Activated mTOR signaling occurs in a variety of other tumor types including Cowden’s syndrome, Peutz-Jeghers syndrome, and tuberous sclerosis complex indicating that mTOR signaling is activated in many cancer types and in conditions of proliferative dysregulation (Meric-Bernstam and GonzalezAngulo 2009).

18.3.2 AKT/mTOR Pathway Overexpression in Squamous Cell Carcinomas Many cancer-promoting events activate the AKT/mTOR pathway. It has been suggested that AKT activation is an early event during human HNSCC development (Moral et al. 2009). Head and neck cancer is characterized by persistent activation of the AKT/mTOR pathway that leads to phosphorylation of RPS6KB1 and EIF4EBP1 (Amornphimoltham et al. 2004). Constitutive activation of the mTOR pathway was found in all 20 cases of cervical squamous cell carcinoma (CSCC) (Feng et al. 2009). Nuclear translocation of p-mTOR was observed in all CSCC lesions and 90% of the cases displayed strong cytoplasmic p-mTOR staining, while normal cervical epithelium showed intermediate staining (Feng et al. 2009). In SCCs of other organ systems, the AKT/mTOR pathway is overexpressed in a smaller percentage of tumors. AKT and mTOR overexpression in LSCC was found in 49% and 40% of the specimens correspondingly (Dobashi et al. 2009), whereas only 25% of ESCC have positive p-AKT staining while 52% showed mTOR overexpression (Hay and Sonenberg 2004). In other studies, mTOR overexpression was found in 25% of ESCC (Bianco et al. 2008). A large international study by the NIDCR using tissue microarrays in HNSCC noted that unlike EGFR and p53, p-AKT was activated in 99% of tumors (Molinolo et al. 2007). This study confirmed that the PI3K/ AKT/mTOR pathway can be activated independently of the EGFR in HNSCC, suggesting that inhibitors of both signaling axes may be more therapeutically beneficial.

18.3.3 Development of AKT/mTOR Pathway Inhibitors The AKT/mTOR pathway integrates a variety of signaling pathways involved in cell growth and division, and inhibitors of this pathway effectively starve the targeted cells. Rapalogs are specific mTOR inhibitors, although the variety of mTOR complexes with a range of rapalog sensitivities suggests exploitable therapeutic opportunities in HNSCC. A major target of mTOR inhibition is angiogenesis. mTOR inhibitors including rapamycin and its derivatives have demonstrated their antiangiogenic properties in a variety of solid tumors (DelBufalo et al. 2006; Phung et al. 2006;

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Figlin et al. 2008; Marone et al. 2009) without cumulative toxicity after repeated administration. AKT and mTOR inhibitor monotherapy studies have expanded into combination therapies to inhibit as many signaling pathways as possible to overcome resistance due to the many parallel signaling pathways (Fig. 18.2). The most developed inhibitor of AKT is perifosine (NSC 639966), an oral alkylphospholipid. Perifosine targets the pleckstrin homology domain of AKT, thereby preventing its translocation to the plasma membrane (Kondapaka et al. 2003). In vitro, perifosine exerts antiproliferative properties by blocking cell cycle progression of HNSCC cell lines at G1/S and G2/M through an induction of CDKN1A (Patel et al. 2002). In the mouse HNSCC xenograft tumor model, perifosine induced a dose-dependent tumor growth delay and enhanced radiation-induced cytotoxicity (Vink et al. 2006). Despite promising results of the preclinical studies, perifosine monotherapy did not demonstrate any antitumor activity in a phase II clinical trial conducted on 19 patients with recurrent or metastatic HNSCC (Argiris et al. 2006). The most common therapy-related adverse events were gastrointestinal symptoms (constipation, nausea, vomiting) and fatigue. No objective responses were observed. The authors concluded that the use of perifosine as a single agent is not justified in HNSCC (Argiris et al. 2006). However, since AKT activation is implicated in the resistance to EGFR inhibitors (Cooper and Cohen 2009) and can

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Fig. 18.2 Targeted therapy in squamous cell carcinomas. EGFR epidermal growth factor receptor, HER2 human epidermal growth factor receptor 2, ERBB v-erb-b2 erythroblastic leukemia viral oncogene homologs, PI3K phosphoinositide 3-kinase, PTEN phosphatase and tensin homolog, AKT another kinase of transcription, mTORC1 mammalian target of rapamycin complex 1, VEGFR vascular endothelial growth factor receptor (also known as fms-related tyrosine kinase 1 (FLT1)), PDGFR platelet-derived growth factor receptor, RAF RAF kinases: v-raf-1 murine leukemia viral oncogene homolog 1 (RAF1) and v-raf murine sarcoma viral oncogene homolog B1 (BRAF), MAP2K mitogenactivated protein kinase kinase, ERK extracellular-signal-regulated kinase (MAPK), SRC v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian), STAT signal transducers and activator of transcription, PTK2 protein tyrosine kinase 2 (also known as focal adhesion kinase [FAK]).

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be induced during mTOR inhibitor therapy (Sun et al. 2005; Wan et al. 2007), the combination of perifosine with other molecular-targeted agents would be of potential interest. Triciribine phosphate (also known as API-2 and VQD-002), a tricyclic nucleoside and a specific inhibitor of AKT, has growth-inhibitory effects and induces apoptosis in cancer cells harboring constitutively activated AKT (Yang et al. 2004). High doses of triciribine phosphate exhibited serious side effects, including hepatotoxicity, hypertriglyceridemia, thrombocytopenia, and hyperglycemia, in phase I and II clinical trials conducted on advanced tumors of mixed entities and advanced CSCC in the 1980s and 1990s (Feun et al. 1984; Feun et al. 1993). Few patients had an objective response to the therapy and the authors concluded that triciribine phosphate has limited activity in metastatic or recurrent CSCC. Importantly, it was not known to be a specific AKT inhibitor when the first triciribine clinical trials were being conducted. By selectively enrolling patients whose tumors have upregulated p-AKT expression, the clinical response rate to triciribine might be enhanced. Four mTOR inhibitors are available for clinical trials: the prototype rapamycin (sirolimus) and three derivatives: Torisel® (also known as CCI-779 and temsirolimus), Afinitor® (also known as RAD001 and everolimus), and ridaforolimus (also known as AP23573, MK-8669, and formerly Deforolimus). Rapamycin derivatives are the result of relatively minor modifications to the structure of the prototype drug, involving substitution at the C40 hydroxyl position of rapamycin outside FKBP12- and mTOR-binding domains. These modifications decrease the immunosuppressive properties and increase the compounds solubility in aqueous solution (Ballou and Lin 2008; Chan et al. 2009). Rapamycin (sirolimus) was FDAapproved in 1999 to prevent renal transplant rejection (Faivre et al. 2006) and is a clinically approved immunosuppressive agent with promising antitumor activities in patients. Its prolonged and relatively safe use in renal transplant patients is well established (Morath et al. 2007; O’Donnell et al. 2008). mTOR inhibitors are relatively well tolerated with toxicities that include reversible dermatological side effects, such as herpes simplex lesions, acne-like rash, maculopapular rash, and nail disorders. Dose-limiting toxicities consist of mucositis/stomatitis, asthenia, and thrombocytopenia, although the most common adverse effects are myelosuppression and hyperlipidemia.

18.3.4 Targeting mTOR in Preclinical Tumor Systems The antitumor effects of everolimus on intradermal murine SCC tumor growth and pulmonary metastases have been evaluated (Khariwala et al. 2006). Tumor growth was inhibited by approximately 50% with everolimus treatment compared to the control group, and no significant difference in tumor size was noted between the two dose groups (0.5 and 1 mg/kg everolimus twice a day). Pulmonary metastasis induced by tail vein injection of SCC was inhibited by everolimus in a dose- dependent manner, with only the higher dose (1 mg/kg twice daily) reaching a significant difference from the untreated control (Khariwala et al. 2006).

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Rapamycin and temsirolimus cause significant mTOR signaling pathway inhibition in HNSCC in vitro and in vivo models (Amornphimoltham et al. 2005; Nathan et al. 2007a). Phosphorylation of the mTOR pathway targets RPS6 and EIF4EBP1 was dose-dependently inhibited by rapamycin and temsirolimus. Importantly, RPS6 appears to be a more sensitive marker of mTOR inhibition, since lower concentrations of rapamycin were needed to induce its decrease and maximal inhibition of the marker was achieved at low nanomolar concentrations (Amornphimoltham et al. 2005). Temsirolimus also decreased the expression of the angiogenic factors FGF2 and VEGF in FaDu HNSCC cells. The growth of FaDu cells was inhibited by temsirolimus in a dose-dependent manner at concentrations up to 100 ng/mL, with no further growth inhibition at higher doses (Nathan et al. 2007a). Concentrationdependent antiproliferative effects of rapamycin in other HNSCC cell lines have also been reported (Brown et al. 2006; Aissat et al. 2008). In the nude mouse established xenograft tumor model, rapamycin and temsirolimus showed marked growth inhibitory properties. These mTOR inhibitors caused significant xenograft growth inhibition established from five different HNSCC cell lines (Amornphimoltham et al. 2005; Nathan et al. 2007a). In the study by Nathan et al. (2007b), no significant differences were noted among four different temsirolimus groups tested (ranging from 5 to 20 mg/kg), suggesting that the optimal dose may be less than or equal to 5 mg/kg daily. Rapalogs are known cytostatic agents that induce a G1 arrest and apoptosis in HNSCC cell lines (Aissat et al. 2008). Accordingly, rapamycin inhibited DNA synthesis in vivo as evidenced by a decrease in the number of 5-bromo-2-deoxyuridine immunoreactive nuclei in xenograft tumors during the treatment course. The number of apoptotic nuclei in tumors treated with rapamycin peaked on days 2 and 3 by TUNEL assay, and decreased to control levels thereafter, demonstrating that mTOR inhibition can also induce apoptosis in HNSCC. Furthermore, rapamycin treatment displayed antiangiogenic properties (Amornphimoltham et al. 2005). Overexpression of the rapamycin-resistant form of mTOR in HNSCC cells rendered HNSCC xenografts completely resistant to rapamycin treatment indicating that tumor cells might be the primary target of rapamycin in vivo and suggests that the antiangiogenic effects of the rapalogs may be a consequence of mTOR inhibition in the tumor cells (Amornphimoltham et al. 2008). Preclinical studies in HNSCC have shown the mTOR inhibitor temsirolimus is effective as a single agent in a minimal residual disease model. There were significant differences in the tumor-free rate between the control (4%) and the treatment group (50%) of mice, and the median tumor-free time was 7 versus 18 days, respectively (p < 0.0001). In those animals that formed tumors, temsirolimus caused a significant decrease in the tumor volume. Temsirolimus significantly inhibited EIF4EBP1 and RPS6 phosphorylation in tumor tissues as well as in peripheral blood mononuclear cells (PBMCs), suggesting that PBMCs can serve as a surrogate marker of response in an adjuvant setting (Nathan et al. 2007b). Similar to the effects of mTOR inhibitors in HNSCC, rapamycin treatment of esophageal SCC reduced expression of p-RPS6KB1 and p-EIF4EBP1, inhibited proliferation in a dose- and time-dependent manner, and induced a G1 arrest and apoptosis (Hou et al. 2007). Rapamycin effectively inhibited mTOR signaling and the growth of nonsmall cell lung cancer (NSCLC) cells, including SCCL (Sun et al. 2005).

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18.3.5 Clinical Evaluations The exploratory biomarker trial of temsirolimus in patients with advanced stage HNSCC showed that three 25 mg doses of temsirolimus significantly decreased p-RPS6 and p-EIF4EBP1, downstream targets of mTOR, in the post-treatment tumors compared to the pre-treatment tumors (Nathan et al. 2007b). The significant decrease in p-RPS6 was also noted in PBMCs, while the decrease of p-EIF4EBP1 in tumors was not mirrored in PBMCs. Hence down-regulation of p-RPS6 in PBMCs could be a surrogate marker of response to therapy in future clinical trials using mTOR inhibitors as adjuvant therapy for HNSCC patients (Nathan et al. 2007b). mTOR inhibitors have been evaluated in several clinical trials of mixed solid tumor entities that include SCC. A phase I study of temsirolimus administered weekly at doses ranging from 7.5 to 220 mg/m2 revealed a tolerable safety profile in a cohort of 24 patients. The most frequent drug-related adverse events were cutaneous toxicity (acne-like, maculopapular rashes) and mucositis (75% each). Four patients had dose-limiting grade 3 toxicities, consisting of thrombocytopenia in two patients at doses of 34 and 45 mg/m2, one patient with grade 3 manic-depressive syndrome with grade 3 stomatitis and grade 3 transaminase elevation, and another patient with grade 3 asthenia and stomatitis at the maximum dose of 220 mg/m2. All toxicities were manageable and reversible with treatment discontinuation, and partial responses were observed in two patients (Raymond et al. 2004). In a phase I dose escalation study of temsirolimus administered daily for 5 days every 2 weeks, a maximum tolerated dose of 15 mg/m2/day for patients with extensive prior treatment, and of 19 mg/m2/day for minimally pretreated patients were established. This intermittent temsirolimus dosing regimen was well tolerated without evidence of clinically relevant immunosuppression. The most frequently observed temsirolimus-related adverse events were asthenia (56%), mucositis (54%), nausea (41%), cutaneous toxicity (41%), hypertriglyceridemia (37%), and thrombocytopenia (33%) with thrombocytopenia being the most common cause for dose reductions and treatment delays. Dose-limiting toxicities (all grade 3: hypocalcemia, hyperglycemia, stomatitis, elevated transaminases, and vomiting with diarrhea and asthenia) were seen in a total of four out of 63 patients. Evidence of antitumor activity was noted in six patients, including one patient with nasopharyngeal carcinoma who received 4.5 mg/m2/day temsirolimus and achieved stable disease for more than 24 weeks (Hidalgo et al. 2006). Importantly, the toxicity spectrum of temsirolimus in daily and weekly regimens was similar, although the incidence of mucositis and skin reactions was higher in the weekly regimen (Raymond et al. 2004; Hidalgo et al. 2006). Preliminary results of a phase II study of temsirolimus administered at the intravenous dose of 25 mg/week to patients with advanced NSCLC were encouraging with 8% of the patients showing a partial response and 30% with stable disease (Molina et al. 2007). A partial response was observed in 5.3% and stable disease in 45% of the patients with advanced NSCLC who had previously received platinumbased chemotherapy in a phase II trial of everolimus administered at an oral dose of 10 mg/day until progression. Patients whose tumors had previously failed both

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chemotherapy and treatment with EGFR tyrosine kinase inhibitors had a somewhat lower response and stable disease rate. The most frequent adverse events included: stomatitis/mucositis, cough, dyspnea, rash, fatigue, anorexia, nausea, anemia, and diarrhea (Papadimitrakopoulou et al. 2007). Rapamycin showed promising results as an effective radiosensitizer in a case report of a post-liver transplant patient who switched to rapamycin for immuno suppression and subsequently underwent radiation therapy for SCC of the larynx. The patient had an unusually early response to a relatively low dose of radiation (1,400 cGy) with complete resolution of the tumor following treatment and no evidence of recurrence at the 1 year follow-up visit. However, the patient also developed mucositis and odynophagia earlier than expected and required a break from radiation that nevertheless did not preclude treatment completion (Shinohara et al. 2009). 18.3.5.1 Combination Therapy for SCC mTOR inhibitors as a single agent often demonstrate only modest therapeutic activity. Rapalogs can induce some apoptosis in HNSCC although their effects are believed to be primarily cytostatic rather than cytotoxic. The combination of mTOR inhibitors with cytotoxic treatments, such as radiation therapy or chemotherapy, may potentiate the antitumoral effects of mTOR inhibition. Combinations of rapamycin with the chemotherapeutic drugs paclitaxel and carboplatin have shown synergistic and additive effects in HNSCC cell lines (Aissat et al. 2008). Chemotherapeutic agent exposure prior to rapamycin enhanced the cytotoxic effects of the combined treatment in some cell lines (Aissat et al. 2008). Farnesyltransferase inhibitors, such as lonafarnib (SCH66336), showed promising anti-tumor activity against HNSCC in preclinical studies (Argiris et al. 2006), although the activity in patients with chemorefractory advanced HNSCC was limited (Hanrahan et al. 2009). It was identified that insulin-like growth factor 1 receptor (IGF1R) activation interferes with the anti-tumor activity of SCH66336 in HNSCC by inducing protein synthesis of surviving through the IGF1R/AKT/mTOR pathway. Among other approaches, rapamycin-induced mTOR inhibition was effective in overcoming resistance to SCH66336 and induced apoptosis (Oh et al. 2008). mTOR inhibition using temsirolimus or rapamycin had no radiosensitizing effects in several HNSCC cell lines in vitro (Ekshyyan et al. 2009; Shinohara et al. 2009). Conversely, in HNSCC established xenograft tumors, the combination of temsirolimus and radiation significantly augmented the in vivo growth inhibitory effects of radiotherapy or drug treatment alone (Ekshyyan et al. 2009). Radiotherapy activates the AKT/mTOR pathway in HNSCC xenograft tumors, and this is significantly attenuated by temsirolimus, causing increased apoptosis and inhibition of angiogenesis which may explain the mechanism of its selective radioenhancing effects in vivo but not in vitro. The combination of temsirolimus with radiotherapy also displayed superior anti-tumor activity compared to conventional chemoradiotherapy with cisplatin. Temsirolimus combined with radiotherapy was as

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effective as conventional chemoradio therapy in improving survival of mice bearing cisplatin-sensitive HNSCC xenografts, but also demonstrated potent antitumor activity against cisplatin-resistant HNSCC xenografts, suggesting mTOR inhibition may be especially beneficial against tumors with inner or acquired resistance to cisplatin (Ekshyyan et al. 2009). It is likely that a combination of molecular-targeted therapeutics may be required to fully evaluate the potential of mTOR inhibitors and that the addition of AKT or PI3K inhibitors to the mTOR inhibitors may be needed due to the activation of AKT during mTOR inhibition (Wan et al. 2007). The AKT/mTOR pathway activates p70S6K (RPS6KB1). A negative feedback loop that suppresses PI3K/ AKT signaling when RPS6KB1 is phosphorylated has been described. mTORactivated RPS6KB1 attenuates PI3K signaling by suppressing insulin receptor substrate-1 (IRS1) function (Carracedo and Pandolfi 2008). Rapamycin and its analogs readily inhibit the mTOR-raptor complex (mTORC1) potentially disrupting the aforementioned negative feedback loop via a decrease of RPS6KB1 phosphorylation. This phenomenon was observed in cell systems and patient biopsies (O’Reilly et al. 2006), and in glioblastoma clinical trials (Cloughesy et al. 2008). Many studies now show that the PI3K/AKT/mTOR pathway is not a linear pathway but rather intersecting pathways with considerable cross-talk and feedback regulation (Fig. 18.1). For example, PDK1/2 can regulate translation independently of AKT by directly phosphorylating and activating RPS6KB1, a downstream effector of mTOR (Wang et al. 2001). mTOR controls phosphorylation of AKT at its hydrophobic motif when bound to Rictor (mTORC2), potentially providing a level of positive feedback on the pathway. Importantly, phosphorylation of AKT at Ser473 by mTORC2 is essential for AKT activity (Sarbassov et al. 2005). Although temsirolimus disrupted the negative feedback loop and upregulated AKT phosphorylation in preclinical HNSCC studies, the increase in p-AKT by the combination of temsirolimus with radiotherapy was not significantly different from the increase in p-AKT noted with radiotherapy or cisplatin-based chemoradiotherapy. These results challenge the importance of rapalog-induced p-AKT activation in treatment regimens employing radiotherapy (Ekshyyan et al. 2009). In the temsirolimus clinical trial of patients with advanced stage HNSCC, a decrease in p-AKT in the tumors was noted after three 25 mg weekly doses of temsirolimus, that was close to significance (p = 0.06), and a significant inhibition of AKT(Ser473) phosphorylation in PBMCs (p = 0.04). This suggests that temsirolimus might have an inhibitory effect on mTORC2 in HNSCC. The absence of p-AKT upregulation in head and neck cancer patients could be due to short-term versus long-term effects of temsirolimus; intermittent dosing and/or HNSCC having constitutively higher baseline levels of AKT compared with other organ sites making it difficult to discern a further increase in the phosphorylation of AKT. AKT activation via the feedback loop may occur as an early event, i.e., within 24–48 h of treatment initiation, but this activation does not persist. In certain types of cancer, prolonged treatment with rapalogs may impair mTORC2 assembly and therefore reduce AKT phosphorylation (Sarbassov et al. 2006). Studies using leukemic cells have demonstrated that temsirolimus and everolimus inhibit mTORC2 formation (Zeng et al. 2007).

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Although addition of upstream inhibitors (PI3K, AKT) might be beneficial for cancer treatment as some studies suggest (Abraham and Gibbons 2007), it may not be essential especially in the adjuvant setting. In non-small-cell lung cancer cell lines, including SCCL, combining rapamycin with erlotinib (Tarceva®) down-modulated rapamycin-stimulated AKT activity and exerted additive or synergistic effects on growth inhibition depending on the cell line (Buck et al. 2006). Temsirolimus (20 mg/kg/day) showed superior growth inhibition to erlotinib (50 mg/kg/day) against two HNSCC cell lines established as xenograft tumors in nude mice. The combination treatment did not enhance the anti-tumor activity of temsirolimus in an erlotinib-resistant cell line, while tumor regression was observed in the erlotinib-sensitive xenograft model. A significant upregulation of p-AKT was observed in the erlotinib-resistant cell line after temsirolimus treatment that was abrogated by erlotinib. However, the combined treatment was only as effective as the best single agent (e.g., temsirolimus). In both xenograft models, the combination treatment significantly decreased HIF1A mRNA expression. This effect was greater and had significant antiangiogenic activity in the erlotinib-sensitive xenograft model where tumor regression was observed (Jimeno et al. 2007). In CSCC cell lines, rapamycin and erlotinib complemented each other in suppression of mTOR signaling as evidenced by almost complete inhibition of RPS6 phosphorylation after combination treatment in all three cell lines tested (Birle and Hedley 2006). In a murine xenograft study, there were beneficial effects of combining rapamycin and erlotinib as all CSCC xenografts were sensitive to rapamycin treatment. The combination treatment significantly enhanced the growth-inhibitory effects of single-drug therapies in erlotinib-sensitive SiHa xenografts and restored erlotinib sensitivity to erlotinib-resistant Me180 xenografts. There was no additional benefit of erlotinib addition in the CaSki xenograft model that was highly responsive to rapamycin. A significant antiangiogenic effect was achieved using a combination treatment but not single molecular-targeted agents (Birle and Hedley 2006). The results of these studies indicate that the combination of molecular-targeted therapeutics is a promising strategy. However, these studies highlight the problems of intertumoral heterogeneity in their sensitivity to combined molecular-targeted therapy and identification of predictive markers for sensitivity. The ongoing clinical trials evaluating the AKT and mTOR inhibitors in the treatment of patients with SCC and mixed solid tumor entities are listed in Table 18.1. A majority of the trials are evaluating mTOR inhibitors in combination with chemotherapeutic and other molecular-targeted agents. 18.3.5.2 mTOR Inhibitors in Other Cancer Types mTOR inhibitors are in various stages of development for a number of clinical trials. Most notably, mTOR inhibitors have demonstrated anti-tumor activity and an acceptable safety profile, where most of the toxicity data has been reported in patients with organ transplantation (Sankhala et al. 2009). The mTOR pathway integrates a variety of signaling networks, suggesting its utility as adjuvant

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Table 18.1 Ongoing clinical trials evaluating another kinase of transcription (AKT) and ammalian target of rapamycin (mTOR) inhibitors in squamous cell carcinomas (SCC) and mixed m solid tumor entities Phase Inhibitor Combination agents Cancer type I/II Perifosine None Non-small cell lung cancer, other solid tumors II Temsirolimus None Non-small cell lung cancer I Temsirolimus Topotecan Cervical carcinoma, other gynecologic malignancies I Temsirolimus Radiation therapy Non-small cell lung cancer I Everolimus None Non-small cell lung cancer I Everolimus Cisplatin HNSCC, other solid tumors I Everolimus Cisplatin and Radiation HNSCC therapy I Everolimus Vatalanib HNSCC, other solid tumors I Everolimus Pemetrexed Non-small cell lung cancer I/II Everolimus Erlotinib Non-small cell lung cancer Non-small cell lung cancer I/II Everolimus Carboplatin, Paclitaxel and Bevacizumab I/II Everolimus Docetaxel Non-small cell lung cancer I/II Everolimus Gefitinib Non-small cell lung cancer

anticancer therapy, and is now a clinically relevant treatment option in a variety of cancers. mTOR inhibitors have shown activity in neuroendocrine cancer (Wang et al. 2002; Phan and Yao 2008), glioblastoma multiforme (Maher et al. 2001; Cloughesy et al. 2008), endometrial cancer (Gadducci et al. 2008), sarcoma (Okuno 2006), gastric cancer (Cejka et al. 2008; Hashimoto et al. 2008), melanoma (Meier et al. 2007; Marone et al. 2009), breast cancer (DeGraffenried et al. 2004; Beeram et al. 2007), lung cancer (LaMonica et al. 2009), and renal cell carcinoma (Hudes et al. 2007; Motzer et al. 2008; O’Donnell et al. 2008; Amato et al. 2009). A benefit of mTOR inhibitors is the ability to overcome resistance to receptor-targeted inhibitors, which has been demonstrated in a variety of cancers. Studies combining mTOR inhibitors with EGFR inhibitors demonstrated a significant decrease in activation of MAPK and mTOR signaling pathways, although growth inhibition was induced rather than cell death activation (LaMonica et al. 2009). Breast cancer cells with activated AKT are resistant to hormonal therapy but sensitivity is restored with the addition of mTOR inhibitors (DeGraffenried et al. 2004; Beeram et al. 2007). Renal cell carcinoma studies indicate that mTOR inhibitors have a direct anti-tumor effect complementary to the anti-tumor effects of VEGFR inhibitors, which may lead to combining these agents for therapeutic benefit. Activation of the PI3K/AKT pathway further activates mTOR which induces the EGFR-resistant phenotype (Chakravati et al. 2002; Bianco et al. 2003; Janmaat et al. 2003; Rojo et al. 2006; Vivanco and Sawyers 2002). Because the EGFR tyrosine kinase inhibitor response rate when used as a single agent is only 4–11% (Soulieres et al. 2004; Cohen et al. 2005), and a majority of patients develop

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resistance to EGFR inhibitors (Rexer et al. 2009), it was hypothesized that combining EGFR and mTOR inhibitors would result in improved antitumor response. In support of this hypothesis, Bianco and associates demonstrated that EGFR resistance can be re-sensitized with mTOR inhibitors to respond to EGFR inhibitors (Bianco et al. 2008). The treatment-related skin toxicities from the combined EGFR inhibitors with radiotherapy (Lacouture 2006; Budach et al. 2007; Lord et al. 2008) is comparable to the transient mild to moderate toxicities observed in mTOR inhibitor clinical trials (Sankhala et al. 2009). The PI3K/AKT/mTOR pathway is overexpressed or mutated in approximately 30% of all human cancers (Cully et al. 2006) and overexpressed in 99% of HNSCC (Molinolo et al. 2007) suggesting inhibitors of this pathway are preferable in HNSCC. Although greater than 90% of HNSCC (Molinolo et al. 2007; Gold et al. 2009) have mutations in or overexpression of EGFR, it does not reliably predict therapeutic response to EGFR inhibitors (Gusterson and Hunter 2009). Together this suggests that combining EGFR and mTOR inhibitors may provide a more efficient antitumor therapy, especially in those cancers likely to develop EGFR resistance. Importantly, only 10–15% of HNSCC patients respond to EGFR inhibitors (Thariat et al. 2007). An improved response to EGFR inhibitors can be achieved in conjunction with chemotherapy or radiation (Baselga and Arteaga 2005). However most tumors eventually become refractory to EGFR inhibitor treatment and progress (Rexer et al. 2009), possibly due to mutations in EGFR or downstream effectors. EGFR variant III extracellular domain deletions may be responsible for inactivity of EGFR antibodies in approximately 40% of HNSCC (Sok et al. 2006). PI3K is altered in 12–15% of head and neck cancers by either mutation or amplification, and although PI3K inhibitors target this dysregulated pathway, further mutations can occur downstream rendering the affected cancer cells refractory to PI3K inhibitors (Laurent-Puig et al. 2009). Therefore, targeting the AKT/mTOR pathway that is dysregulated in HNSCC and downstream of EGFR and PI3K may prove to be a more effective therapy for HNSCC. As up to 50% of patients with advanced stage disease develop a recurrence of HNSCC, targeting any of the steps involved in the carcinogenic process merit investigation.

18.3.6 mTOR Inhibitors as Chemopreventive Agents The majority of HNSCC patients are diagnosed with advanced stage disease (Forastiere et al. 2001; Mao et al. 2004). Oral dysplasia carries a risk of progression to cancer from 6% to 36% (Reibel 2003) and increases considerably for high-grade dysplasia (Mehanna et al. 2009). Although surgical excision can decrease the risk of progression to cancer, the risk is not eliminated (Mehanna et al. 2009). Transformation to oral cancer occurs over many years suggesting a broad window of opportunity for intervention, therefore understanding the underlying mechanisms may guide development of rational chemotherapeutic agents. Identifying those at highest risk of transformation to HNSCC could reduce morbidity and mortality.

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Nakayama reported p-AKT was significantly activated in 36 untreated oral SCC biopsies and 15 oral epithelial dysplasia (Nakayama et al. 2001). Further evidence that activation of the AKT/mTOR pathway represents an early event in the carcinogenic process, AKT and mTOR are already detectable in dysplastic lesions of a chemical-induced murine model (Czerninski et al. 2009). Head and neck malignancies are a serious problem due to alcohol consumption and a prevalence of oral tobacco use which is increasing in adult minority and teen populations. Carcinogens induce AKT activation and lung carcinogenesis (Lippman and Heymach 2007), and AKT/mTOR inhibitors appear to have a potential in prevention of tumor progression, and is a promising approach already showing potential for controlling breast, colon and prostate cancer (Kopelovich et al. 2007). Gutkind et al. have demonstrated that chronic rapamycin administration promoted regression of advanced carcinogen-induced SCCs and inhibits premalignant lesion progression in oral carcinogeninduced (Czerninski et al. 2009) and genetically defined carcinogenesis (Raimondi et al. 2009). Using intermittent dosing schedules, stable disease and partial responses have been achieved in a variety of tumor type clinical trials (Rao et al. 2004; Faivre et al. 2006) with only mild to moderate toxicities observed. These studies demonstrate the role that the mTOR pathway plays in HNSCC progression and suggest a novel approach in application of mTOR inhibitors for HNSCC chemoprevention.

18.4 Conclusions A major challenge of clinical trials in HNSCC is the heterogeneity of the patient population due to recurrent and/or metastatic disease. Several issues are critical to a patients response to novel molecular-targeted agents: prior chemotherapy including platinum and anti-EGFR therapy, induction or concurrent therapy, nonmetastatic versus metastatic unresectable-locoregional recurrence, and molecular heterogeneity (Yun et al. 2007). Targeted molecular therapy is currently studied in the recurrent and metastatic patient, with prior chemotherapy, radiotherapy or both. Therefore, these patients have likely developed resistance and are less likely to respond to additional therapies. AKT/mTOR-targeted therapy is already undergoing clinical evaluation for HNSCC. Carefully selecting patients that are likely to respond to these inhibitors requires predictive biomarkers of response. Delivering combinations of targeted inhibitors may induce a more toxic chemoresistant anti-tumor effect than single agent therapy. Initial rapamycin studies elicit two major issues: immunosuppression and feedback activation of AKT leading to tumor cell survival. In certain types of cancer, prolonged inhibition of mTOR by rapamycin may impair mTORC2 assembly and AKT activation in vivo and it was suggested that rapamycin analogs can be celltype-dependent inhibitors of mTORC2 function (Sarbassov et al. 2006). PI3K and mTOR have been reported to play important roles in the immune system. Although the FDA has approved rapamycin as an immunosuppressant to prevent kidney transplant rejection, a black-box warning has been issued. Stability,

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exposure, and dosage frequency affect the efficacy of mTOR inhibitors. Using an intermittent dosing schedule, the immunosuppressive effects of mTOR inhibitors can be minimized (Kopelovich et al. 2007). Given that renal transplant patients were also taking cyclosporine and corticosteroids suggests that rapamycin administered as a single agent may be safe (Dennis 2009). Phase I trials with the mTOR inhibitor temsirolimus did not reach the maximum tolerated dose signifying a high therapeutic index with no immunosuppressive effects seen (Rowinsky 2004). Clinical trials have also shown that mTOR inhibitors are well tolerated and may induce prolonged stable disease and even tumor regression in a subset of patients (Dancey 2005; Vignot et al. 2005). HNSCC is one of the few cancers where mTOR is activated in almost all tumors. The molecularly driven approach may be the best way to design trials with targeted therapy compared with the empiric approach (Dancey 2002). Such an approach resembles HER2-targeted therapy in breast cancer. Hence patients with mTOR-activated residual cells may truly benefit from adjuvant therapy with mTOR inhibitors.

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Chapter 19

Head and Neck Cancer and the PI3K/Akt/ mTOR Signaling Network: Novel Molecular Targeted Therapies Panomwat Amornphimoltham, Vyomesh Patel, Alfredo Molinolo, and J. Silvio Gutkind

Abstract Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer among men worldwide. Despite significant advances in conventional therapies for other prevalent cancer types, the survival rate of HNSCC patients has barely improved over the past 3 decades, emphasizing the urgent need for the development of more effective treatment strategies. A better understanding of the mechanisms of tumorigenesis has led to novel molecular targeted options for cancer treatment. In this regard, we have observed that the persistent activation of the v-akt murine thymoma viral oncogene homolog 1Akt pathway is a frequent event in HNSCC. Akt promotes cell proliferation by coordinating mitogenic signaling with energy and nutrient-sensing pathways that control protein synthesis through the mammalian target of rapamycin (mTOR). This kinase, in turn, phosphorylates and activates key regulatory circuitries involved in mRNA translation, cell metabolism, and cell cycle control. Indeed, the activation of mTOR was found to be a widespread event in HNSCC, as judged by the detection of phospho-S6 (pS6), one of the most downstream targets of mTOR, in more than 80% of human HNSCCs. The promising development of mTOR inhibitors, including rapamycin (sirolimus) and its derivatives known as rapalogs, such as CCI-779 (temsilorimus), RAD001 (everolimus), and AP23573 (deferolimus), as antitumor agents prompted us to investigate the effects of rapamycin in HNSCC. Indeed, rapamycin treatment rapidly reduced the enhanced level of pS6 in vitro and in xenograft models. Furthermore, rapamycin displays a potent antitumor effect in vivo, as it induces apoptosis in HNSCC xenografts, and promotes the regression of chemically induced SCC lesions in skin and oral cancer models and in newly developed oral-specific, genetically defined mouse cancer models. In this chapter, we will describe a series of research efforts aimed at addressing dysregulated molecular mechanisms in HNSCC, focusing on the PI3K-Akt-mTOR signaling axis. We will also discuss recent and ongoing studies on the molecular targets of mTOR in HNSCC, and J.S. Gutkind (*) Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, 30 Convent Drive, Building 30, Room 212, Bethesda, MD 20892-4340, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_19, © Springer Science+Business Media, LLC 2011

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emerging experimental information supporting the effectiveness of mTOR inhibitors for the prevention and treatment of HNSCC.

19.1 Introduction Each year in the United States, more than 11,000 deaths are attributed to cancers of the oral cavity, salivary glands, larynx, and pharynx (Jemal et al. 2009). As more than 90% of these neoplastic lesions are of squamous cell origin, they are usually referred to, collectively, as head and neck squamous cell carcinomas (HNSCC). HNSCC is considered the sixth most common cancer among men in the world (Parkin et al. 2005). In spite of the recent advances in our understanding, prevention and treatment of other types of cancers, the 5-year survival rate after diagnosis for HNSCC is still very low, approximately 50%, which is considerably lower than that for other cancers, such as those of colorectal, cervix, and breast origin (Jemal et al. 2009). The reasons for the poor prognosis of HNSCC patients include failure to respond to available therapies, late presentation of the lesions, and the lack of suitable markers for early detection. While many of the common risk factors involved in HNSCC pathogenesis, including alcohol and tobacco usage, HPV infection and areca nut chewing are well recognized (Curado and Hashibe 2009; Psyrri et al. 2009) the mechanisms leading to the malignant conversion of oral epithelium into squamous carcinomas are not fully understood, thus preventing the development of molecular-targeted treatment options and chemopreventive strategies. However, recent discoveries have dramatically increased our understanding of the most basic mechanisms controlling normal cell proliferation and have also greatly enhanced our ability to investigate the nature of the biological processes that lead to cancer. We now know that the majority of cancer cells derive from the clonal expansion of a single or few stem cells that have acquired an aberrant program of cell growth. Whereas normal cells proliferate only when needed, as a result of a delicate balance between growth promoting and growth inhibiting factors and under the influence of biochemical cues provided by neighboring cells, cancer cells override these controlling mechanisms and follow their own internal program for timing their reproduction. These cells usually proliferate in an unrestricted manner, and over time they can acquire the ability to migrate from their original site, invade nearby tissues and metastasize at distant anatomical sites. The progressive changes in cellular behavior, from slightly deregulated proliferation to full malignancy, are a result of the accumulation of mutations in a limited set of genes. Among them, two classes of genes, oncogenes and tumor suppressor genes, play major roles in triggering and promoting cancerous growth (Chi et al. 1999; Hoffman and Liebermann 1998; Pearson and Van der Luijt 1998). Whereas activated oncogenes promote cell proliferation, tumor suppressor genes inhibit it and contribute to the carcinogenic process when inactivated by mutations and other genetic or epigenetic alterations. An emerging concept is that several

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activating and inactivating events must occur in oncogenes and tumor suppressor genes for the initiation and progression of many types of cancer (Urbain 1999). These changes occur in a multistep process (Partridge et al. 1997). Thus, if molecular markers representing early and late events could be isolated, it would be then possible to identify persons at high risk of HNSCC, namely, those whose lesions are progressing through the premalignant state. Furthermore, the availability of biochemical markers heralding malignancy would be key for monitoring cancer recurrence, as well as for the evaluation of the efficacy of novel chemopreventing agents. Clearly, the full elucidation of the genetic and epigenetic changes leading to the development of HNSCC will lead to improved molecular assays with important implications for the early diagnosis, therapy and prognosis of HNSCC patients.

19.2 Genetic Alterations in HNSCC While the genetic alterations at each step as HNSCC progress are still not fully elucidated, accumulated evidence led to the current HNSCC genetic progression model, thus providing a framework for the understanding of the molecular pathogenesis of this cancer type (Argiris et al. 2008; Califano et al. 1996; Mao et al. 2004). In this model, the sequential loss of chromosomal material is thought to result in changes leading to dysplasia (9p21, 3p21, 17p13), carcinoma in situ (11q13, 13q21, 14q31), and invasive tumors (4q26–28, 6p, 8p, 8q). These initial observations have been recently expanded by the use of a variety of highly sophisticated techniques, such as comparative genomic hybridization, fluorescence in situ hybridization, and the use of polymorphic microsatellite markers. The latter has helped identify a number of areas of loss of heterozygosity, including 3p, 4q, 5q21–22, 8p21–23, 9p21–22, 11q13, 11q23, 13q, 14q, 17p, 18q and 22q, thus suggesting the contribution of several known tumor suppressor genes in HNSCC, such as p16 (9p21), APC (5q21–22) and p53 (17p13), as well as the existence of many novel putative tumor suppressor genes affected in HNSCC (Forastiere et al. 2001; Hunter et al. 2005). An emerging concept is that activating and inactivating events must occur sequentially in numerous oncogenes and tumor suppressor genes for the initiation and progression of human cancer. Multiple chapters in this book provide a comprehensive review of most known molecular mechanisms involved in HNSCC cancerous growth and metastasis. In this chapter, however, we will center on recent findings supporting the key role of the activation of the PI3K-Akt-mTOR biochemical route as part of the process leading to the acquisition of the malignant phenotype in human HNSCC. We will first define the molecular components of this signaling route, and then focus on the analysis of the functional activity of the PI3K-Akt-mTOR pathway in HNSCC and the biological and biochemical consequences of its perturbation, which has provided the basis for the ongoing clinical exploration of mTOR inhibitors for HNSCC treatment.

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19.3 Phosphatidylinositol 3-Kinase (PI3Ks) The PI3K pathway has become one of the most frequent targeted pathways in all sporadic human cancer, with estimates suggesting that mutation in one or another PI3K component accounts for up to 30% of all human cancer (Bader et al. 2005; Cully et al. 2006) (Table 19.1). These genomic aberrations include mutations, amplifications, and rearrangements resulting in the dysregulation of cells proliferation control and survival, which contributes to a competitive growth advantage, metastasis competence and therapy resistance (Hennessy et al. 2005). PI3Ks are grouped into three classes (I–III) according to their substrate preference and sequence homology (Cantley 2002) (Fig. 19.1). Different classes of PI3K have distinct roles in cellular signal transduction, as do the different isoforms that can exist within each class. Class I PI3Ks. Class I PI3Ks are divided into two subfamilies, depending on the receptors to which they couple. Class IA PI3Ks are activated by growth factor receptors tyrosine kinases (RTKs), whereas class IB PI3Ks are activated by G protein-coupled receptors (GPCRs). Class IA PI3Ks are heterodimers of a p85 regulatory subunit and a p110 catalytic subunit. In mammals, there are numerous isoforms of each subunit (Fig. 19.1). Interestingly, the p110 isoform of class IA PI3K is regulated not only by the p85 regulatory subunit but also by binding to Gbg subunits of heterotrimeric G proteins. Therefore, the class IA p110 isoform might integrate signals from GPCRs as well as RTKs. p85 binds and integrates signals from various cellular proteins, including transmembrane tyrosine kinase-linked receptors and intracellular proteins such as protein kinase C, SHP1, Rac, Rho, Table 19.1 Aberrant P13K/AKT signaling in human cancer Molecule Alteration in tumors Cancer type Glioblastoma, melanoma, PTEN Somatic and germline mutation, hepatocarcinoma, breast, prostate, deletion, LOH promotor HNSCC, ovarian, skin, kidney, methylation liver, stomach, thyroid, many cancer-prone syndrome p85 Mutation Ovarian, colon, lymphoma, glioma, gliobalstoma PIK3CA Amplification, mutation Ovarian, cervix, lung, breast, HNSCC, brain, colon cervical, gastric, esophageal, endometrial, urinary tract, thyroid, liver AKT Amplification, mutation overexpression Gastric, ovarian, pancreas, breast, colorectal breast, colon, endometrial, glioblastoma, ovarian HNSCC TSC1/2 Mutation Tuberous sclerosis,HNSCC elF4E Overexpression Squamous cell, adenocarcinoma, HNSCC PTEN phosphatase and tensin homologue deleted on chromosome 10, p85 regulatory subunit of phosphotidylinositol 3-kinase (P13K); PIK3CA catalytic subunit of P13K, TSC1/2 tuberous sclerosis

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Fig. 19.1 Multistep process and genetic alterations characterizing head and neck squamous cell carcinoma (HNSCC). Long-term exposure to risk factors such as tobacco and alcohol causes multiple genetic and epigenetic alterations leading to genetic instability that may contribute to the progression from normal epithelium to epithelial dysplasia, carcinoma in situ, carcinoma and metastasis. The loss of 9p and inactivation of p16 is acquired at the early stages. Further progression to dysplasia is associated with p53 mutations or inactivation and cyclin D amplification. The loss of heterozygosity and the accumulation of multiple genetic and epigenetic defects in oncogenes and tumor suppressor genes leads to the development of an invasive phenotype and the metastatic spread of HNSCC.

hormonal receptors, mutated Ras and Src, providing an integration point for activation of p110 and downstream molecules. The SH2 domain of p85 has two major divergent functional activities: activation of small G-proteins and relief of transinhibition of p110 (Cantley 2002; Fruman et al. 1998; Hennessy et al. 2005). Upon activation, PI3Ks phosphorylate PIP2 (Fig. 19.2) to produce PIP3, a second messenger that binds a subset of pleckstrin-homology (PH), FYVE, Phox (PX), C1, C2 and other lipid-binding domains in downstream targets to recruit them to the activation at the membrane (Dhand et al. 1994; Fruman et al. 1998). The PH domain is one of the most studied protein domains involved in this interaction. Genetic screens in model organisms have identified Akt as the primary downstream mediator of the effects of PI3K; however, the presence of a large number of proteins with FYVE, PH, and other lipid-binding domains that interact with PIP3 suggest the existence of additional crucial targets. PIP3 is subsequently metabolized by SHIP-1 and -2 to generate PIP2, which regulates a separate subset of PH domains and thus downstream signaling molecules (Dhand et al. 1994; Fruman et al. 1998). PTEN dephosphorylates the 3’OH group phosphorylated by PI3K, acting as the tumor suppressor to the PI3K oncogene (Cully et al. 2006). There are three known isoforms of class IA p110 (p110a/p110b/p110d), which contain an aminoterminal p85/p55interacting region, a domain that binds to Ras, a “PIK domain” homologous to other phosphoinositide kinases, and a carboxy-terminal catalytic domain. There are seven known p85/p55 subunits generated by alternative splicing of three genes (p85a/ p85b/p55g); all can bind p110a/b/d1. Biochemical and gene knockout studies suggest that the different isoforms of p110 and p85 preferentially mediate specific signaling processes, with, however, a degree of redundancy. Only p110a and p85a have been found to be mutated and p85a to be translocated in tumors (see below). Class IB PI3Ks do not have p85 family regulatory subunits and therefore are not regulated by RTKs. They seem to be exclusively activated by GPCRs through interacting

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Fig. 19.2 Three classes of the phosphatidylinositol 3-kinase (PI3Ks) family. The domain organization and defining criteria of the three phosphatidylinositol 3-kinase (PI3K) classes. The catalytic subunit of class I enzymes (p110a, b, d, and g) contains an adaptor-binding domain, a Ras-binding domain (RBD), a C2 (protein-kinase-C homology-2) domain, a helical domain and a catalytic domain. Class IA enzymes are constitutively associated with an adaptor subunit to form a heterodimeric complex. There are three genes that encode at least five different adaptor subunits, all of which can bind to phosphorylated tyrosine in YxxM motifs through their SH2 (Rous-sarcoma-oncoprotein homology-2) domains. The only member of class IB is p110gamma, which associates with the p101 regulatory subunit or the recently identified p84 adaptor subunit. Class I enzymes are regulated by receptor tyrosine kinases and Ras, and have lipid substrates that include phosphatidylinositol-bisphosphates (PIP2), phosphatidylinositol phosphate (PIP) and phosphatidylinositol (PI). Class II enzymes are comprised of a PI3K-C2alpha, beta and gamma domains. They lack an adaptorbinding site but possess carboxy-terminal phox (PX) and C2 domains that could mediate binding to phosphoinositides that have been phosphorylated at position D3 and other membrane lipids. Class II enzyme substrates include PIP2, PIP and PI. Although their adaptor subunits have not been identified, class II enzymes are believed to be regulated by receptor tyrosine kinases and G-protein coupled receptors. Class III enzymes comprise analogs of the yeast Vps34p PI3K subunit and a p150 adaptor subunit, and accept only PI as a substrate. They are believed to be constitutively active, and all isoforms of class I PI3K also possess an intrinsic protein kinase activity.

directly with the G subunit of trimeric G proteins. Similar to class IA PI3Ks, class IB PI3Ks are heterodimers of regulatory and catalytic subunits. The p101 regulatory subunit facilitates the activation of the p101–p110 heterodimer by G proteins (Andrews et al. 2007). Class II PI3Ks. Class II PI3Ks consist of only a single p110-like catalytic subunit. In vitro, these enzymes preferentially phosphorylate phosphatidylinositol and, to a lesser extent, phosphatidylinositol-4-phosphate (PI-4-P). However, phosphatidylinositol-4, 5-bisphosphate (PI-4,5-P2) is a poor substrate for these enzymes. Class II PI3Ks bind clathrin and localize to coated pits, indicating a function in regulating membrane trafficking and receptor internalization. In addition, class II PI3Ks can be activated by RTKs, cytokine receptors, and integrins. Their specific functions in response to these activators, however, are not well understood (Falasca and Maffucci 2007). Class III PI3Ks. The yeast class III PI3K, Vps34, was originally identified in budding yeast as the gene product required for trafficking vesicles from the Golgi

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apparatus to the vacuole. Recently, Vps34 was found to regulate mammalian target of rapamycin (mTOR) activity in response to aminoacid availability, and so this enzyme, plays an important role in the ability of cells to respond to changes in nutrient conditions. In addition, class III PI3Ks are also required for the induction of autophagy during nutrient deprivation in both yeast and mammalian cells (Backer 2008; Yan and Backer 2007; and references therein).

19.4 Protein Kinase B (Akt) The PKB/Akt subfamily of the mammalian “cAMPdependent, cGMP-dependent, protein kinase C” (AGC) family of kinases consists of three members, PKBa/Akt1, PKBb/Akt2 and PKBg/Akt3 derived from distinct genes, whereas yeast, flies and worms have a single PKB/Akt (Hennessy et al. 2005). Family members share a high degree of structural and sequence conservation through evolution and comprise an N-terminal PH domain and a kinase domain (Osaki et al. 2004). All three Akt/PKB isoforms possess conserved threonine and serine residues (T308/S473 in Akt1, T309/S474 in Akt and T305/S472 in Akt3) that together with the PH domain are critical for Akt/PKB activation. The C-terminal regions between these three isoforms are more diverse (homology 73–84%) as compared with the kinase domain (homology 90–95%), suggesting that C-terminal regions may represent functional difference between Akt1, Akt2 and Akt3. PKB/Akt family members are regulated by signaling through PI3Ks, which are activated in response to various stimuli, including growth factors and G protein coupled receptor engagement (Dorsam and Gutkind 2007; Osaki et al. 2004). The direct products of PI3K activity, the lipid second messengers PtdIns(3,4)P2 and PtdIns(3,4,5) P3, are constituents of the inner leaflet of the plasma membrane and serve as docking sites for proteins that contain PH domains, including PKB/Akt proteins and phosphoinositide-dependent kinase 1 (PDK1) (Cohen et al. 1997), which phosphorylates PKB/Akt proteins within their catalytic domains in the so-called T-loop (Thr308 in PKBa/Akt1) and activates them. The second activation-specific PKB/Akt phosphorylation site lies within a hydrophobic motif proximal to the C-terminus (Ser473 in PKBa/Akt1) and is targeted by a distinct protein kinase(s), most likely the mammalian target of rapamycin (mTOR)–Rictor complex (Sarbassov et al. 2005).

19.5 Cellular Functions of Akt Pathway and its Dysregulation in Human Cancer The identification of PKB/Akt substrates has been the focus in recent years of studies to understand the mechanisms by which this kinase impacts on insulin signaling, cell growth, and apoptosis. The minimal consensus site for PKB/Akt phosphorylation as defined by comparison of its known substrate phosphorylation

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sites – consists of RXRXXS/T (using the one-letter aminoacid code, where X represents any aminoacid) and is a reasonable predictor of PKB/Akt targets (Chan et al. 1999). Two main functions of PKB/Akt have been linked to tumorigenesis and tumor progression as following; 1. Cell survival and anti-apoptosis. Akt signaling inactivates several proapoptotic factors (Fig. 19.2). These include BAD, procaspase-9 and Forkhead transcription factor family. Akt also activates transcription factors that upregulate antiapoptotic genes, including cyclic-AMP response element-binding protein, and activates IkB kinase to phosphorylate IkB (inhibitor of NF-kB), leading to its proteasomal degradation and NF-kB nuclear localization. Akt can inactivate p53 through Mdm2, contributing to centrosome hyperamplification and chromosome instability in cancer. Akt phosphorylation of many effectors regulates their localization, and thereby activity, by generating binding sites for 14-3-3 proteins, which are important in regulating cellular location and degradation of various molecules (Datta et al. 1999) 2. Cell growth, metabolism, translation and proliferation. The Akt pathway has diverse downstream effectors, resulting in multiple functional outcomes. The functional responses probably represent the effects of the spectrum, level and duration of activation of particular components of the PI3K pathway. However, different molecules in the pathway have been implicated as playing a dominant role in particular outcomes (Fig. 19.2). Akt-mediated activation of mTOR is important in stimulating cell proliferation. mTOR regulates translation in response to nutrients/ growth factors by phosphorylating components of the protein synthesis machinery, including the ribosomal protein S6 kinases (p70S6K) and 4E-binding protein (4EBP), the latter resulting in release of the translation initiation factor eIF4E, which is known to have transforming and anti-apoptotic activities in vitro (Fig. 19.3). In addition, eIF4E has been used as a prognostic biomarker to identify malignant activity in the surgical margins of head and neck cancers (Nathan et al. 2002; Sorrells et al. 1999). The tuberous sclerosis complex (TSC)-1: TSC2 complex opposes these effects by inhibiting p70S6K and activating 4E-BP1 to sequester eIF4E. However, Akt also phosphorylates and inhibits TSC2, and this pathway is controlled by complicated interactions and feedback loops. The TSC/Rheb/mTOR/ S6K cascade also regulates insulin receptor substrate 1/2 and PDGFR, which potentially comprises an additional important negative feedback loop (Vander Haar et al. 2007). Indeed, mTOR inhibition by rapamycin can activate upstream proteins including Akt in certain cells, which is probably a result of loss of feedback inhibition (Jacinto et al. 2006; Sarbassov et al. 2006). Activated Akt, in part via eIF4E, can then attenuate growth inhibition associated with rapamycin. Targeting the PI3K pathway at multiple sites might therefore be required to interrupt feedback loops, thus achieving optimal outcomes. Indeed, combining rapamycin with LY294002, a PI3K inhibitor, or with inhibitors of cell-surface tyrosine kinases enhances growth inhibitory activity in vitro, warranting the evaluation of combinatorial therapy in animal models and, eventually, patients. In addition, the PI3K/Akt pathway interacts with molecular mechanisms controlling cellular energy control and glucose metabolism. LKB1

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Fig. 19.3 Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K/Akt and related pathways are important in transducing the effects of external mitogenic stimuli of membrane tyrosine kinases. Activation of membrane kinases including epidermal growth factor receptor (EGFR). by growth factors initiates receptor dimerization, autophosphorylation and subsequent events to activate these intracellular pathways. Genetic aberrations of the molecules in this pathway are involved in cancer and cancer prone conditions. Akt is activated downstream of PI3K and has multiple targets. (BAD BCL2-antagonist of cell death, EGFR Epidermal growth factor receptor, FKHR Forkhead transcription factors, GSK3 Glycogen synthase kinase 3, IKK Inhibitor of nuclear factor kappa-B kinase, MDM2 Human homolog of double minute 2, mTOR mammalian target of rapamycin, NF-kB nuclear factor-kappaB, PDK1 3-phosphoinositide-dependent protein kinase 1, PIP2 phosphatidylinositol-3,4-diphosphate, PIP3 phosphatidylinositol-3,4,5-triphosphate, PTEN phosphatase and tensin homolog deleted on chromosome 10, TSC1/2 tuberous sclerosis complex 1/2).

(STK11)-mediated activation of AMP-activated protein kinase, an evolutionarily conserved sensor of the cellular ATP/ADP ratio, leads to inhibition of mTOR through TSC2 in response to energy depletion, thereby allowing energy conservation (Inoki and Guan 2006; Martin and Hall 2005). Germline mutations in genes encoding PTEN, TSC2 and LKB1 all result in similar clinical syndromes that are characterized by the presence of hamartomas at different sites and an increased risk of specific malignancies, providing evidence that deregulation of the PI3K/Akt pathway by inactivation of these crucial genes causes loss of cellular growth regulation (Fig. 19.3) (Inoki et al. 2005; Pan et al. 2004). Akt phosphorylates both cyclin-dependent kinase (CDK) inhibitors p21CIP1/WAF1 and p27KIP1. This results in their exclusion from the nucleus and subsequent cytoplasmic sequestration/degradation (Hennessy et al. 2005). This increases cellular proliferation due to

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decreased inhibition of cyclins and also likely due to novel cytosolic functions of the CDK inhibitors. Akt, directly or indirectly, phosphorylates and inhibits glycogen synthase kinase-3 (GSK3), phosphodiesterase-3B, protein phosphatase 2A and possibly Raf1, creating a complex intracellular network. GSK3 inhibition prevents phosphorylation of b-catenin, thereby impeding its degradation and resulting in its translocation to the nucleus where it stimulates transcription of target genes including c-JUN (Behrens 2000). In addition, cyclin D1 phosphorylation by GSK3 results in its destabilization (Alt et al. 2000). PI3K signaling also controls angiogenesis, growth, proliferation, senescence and other processes through mechanisms including the transcriptional activation of vascular endothelial growth factor (VEGF) and inducing hypoxia inducible factor-1a (HIF1a) expression (Bardos and Ashcroft 2004; Giatromanolaki and Harris 2001). The von Hippel Lindau tumorsuppressor protein, through its oxygen-dependent polyubiquitylation of HIF1a leading to HIF1a degradation, has a central role in the mammalian oxygen-sensing pathway by opposing the effects of the PI3K pathway.

19.6 Aberrant Function of the PI3Ks, PTEN, Akt, and mTOR Signaling Network is a Frequent Event in HNSCC Human cancers harbor frequently occurring oncogenic mutations (e.g., in the small GTPase Ras, PI3K, and receptor and nonreceptor tyrosine kinases) that result in the constitutive activation of Akt, or exhibit genetic and epigenetic alterations inhibiting the activity or expression of tumor-suppressor proteins (e.g., PTEN, TSC1, TSC2, and LKB1) that act by inhibiting the activity of Akt and its downstream targets. This underscores the critical role of the dysregulation of the Akt-pathway in cancer (Brazil et al. 2004; Inoki et al. 2005). We have recently shown that Akt is persistently activated in the vast majority of HNSCC cases, as phosphorylated, active forms of Akt can be readily detected in both experimental and human HNSCCs and in HNSCC-derived cell lines (Amornphimoltham et al. 2004). Furthermore, the blockade of PDK1, which acts upstream of Akt, can inhibit tumoral cell growth (Amornphimoltham et al. 2004; Patel et al. 2002). Activation of Akt is an early event in HNSCC progression, which can be observed in as many as 50% of tongue preneoplastic lesions (Massarelli et al. 2005), and activation of Akt represents an independent prognostic marker of poor clinical outcome in both tongue and oropharyngeal HNSCCs (Massarelli et al. 2005; Yu et al. 2007). Multiple genetic and epigenetic events may converge to promote the activation of the PI3K-Akt pathway in HNSCC (Fig. 19.4). These include EGFR overexpression and alterations in its coding sequence and the expression of oncogenic ras mutants (see above). Copy number gain and amplification at 3q26, where the PI3Ka gene is located, represents a frequent (~40%) and early genomic aberration in HNSCC (Woenckhaus et al. 2002), which may contribute with epigenetic events to

Fig. 19.4 Akt/mammalian target of rapamycin (mTOR) signaling control protein translation. Mammalian target of rapamycin (mTOR) is regulated by multiple inputs, such as growth factors, amino acids and intracellular energy levels, to control translation through the phosphorylation of two main downstream effectors; p70-S6 Kinase 1 (S6K1) which regulate the ribosomal biogenesis and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) which regulate the capdependent mRNA translation. The well-known cap-dependent mRNA targets include cyclinD1, hypoxia inducible factor 1 alpha (HIF1a), myc and ornithine decarboxylase (ODC). The external growth stimuli pathway is initiated by ligands binding to the receptor tyrosine kinase such as epidermal growth factor receptor (EGFR), and activates the downstream PI3K (p85 regulatory and p110 catalytic subunits), then recruiting 3-phosphoinositide-dependent protein kinase 1 (PDK1) and Akt to the plasma membrane. Then Akt is phosphorylated at residue T308 by PDK1 and inhibits the tuberous sclerosis complex (TSC) thereby activating the mTOR pathway by reducing its GAP activity toward the small GTPase Rheb. mTOR phosphorylates Akt in S473. Cells sense the low energy level in response to changes in the intracellular ATP/AMP ratio, which leads to the activation of the 5’AMP-activated protein kinase (AMPK) phosphorylation and activates TSC2, thereby inhibiting mTOR activation. In addition, LKB1, the gene inactivated in Peutz-Jeghers syndrome, is the upstream activating AMPK. The mTOR specific inhibitor, rapamycin, by forming complex with small protein called FK506-binding protein 12 (FKBP12) can bind to mTOR and effectively inhibit its function thereby inhibiting cell proliferation and promoting cell death.

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PI3Ka overexpression and Akt activation (Fenic et al. 2007; Pedrero et al. 2005). Interestingly, activating mutations in the PI3Ka gene, referred to as the PI3KCA oncogene, can be observed in a small fraction (< 10%) of HNSCC tumors (Kozaki et al. 2006; Murugan et al. 2008). In addition, PIP3 is rapidly metabolized by PTEN, and genetic alterations in PTEN, located at 10q23.3, occur in 5–10% of HNSCC lesions but, remarkably, loss of PTEN expression can be observed in approximately 30% of HNSCCs, and this may constitute independent indicator of poor clinical outcome (Lee et al. 2001; Squarize et al. 2002). Overall, Akt can be activated in HNSCC due to the overactivity of EGFR, ras mutations, PI3Ka gene amplification, overexpression, or activating mutations, together with defective PTEN activity due to genetic alterations or decreased expression. These multiple convergent pathways resulting in enhanced Akt function may explain why activation of this pathway represents one of the most frequent events in HNSCC (Molinolo et al. 2007).

19.7 mTOR, a Molecular Target in Squamous Cell Carcinomas of the Head and Neck Despite accumulating evidence supporting an important role for the Akt signaling network in the development of HNSCC, the nature of the biologically relevant pathway(s) through which Akt acts in this tumor type are still poorly understood. Of interest, recent findings suggest that the ability of Akt to coordinate mitogenic signaling with nutrient-sensing pathways controlling protein synthesis may represent an essential mechanism whereby Akt ultimately regulates cell growth (Sekulic et al. 2000; Shamji et al. 2003). We have recently provided evidence that the AktmTOR pathway plays a central role in HNSCC. Aberrant accumulation of the phosphorylated active form of S6 (p-S6), the most downstream target of the AktmTOR-p70S6K pathway, is a frequent event in clinical specimens from HNSCC patients and in HNSCC-derived cell lines (Amornphimoltham et al. 2005; Molinolo et al. 2007). We also found that the level of activated S6 was rapidly reduced when HNSCC cell lines were treated with rapamycin, which specifically inhibits mTOR (Amornphimoltham et al. 2005). Furthermore, p-S6 was dramatically reduced using clinically relevant doses of rapamycin in xenograft models of HNSCC. Concomitantly, we observed that rapamycin exerts a potent anti-tumor effect in vivo, as it inhibits cell proliferation and induces apoptotic cell death of HNSCC cells – ultimately promoting tumor regression (Amornphimoltham et al. 2005). Blockade of mTOR with rapamycin exerts a potent antitumoral effect and even prevents minimal residual disease in multiple human HNSCC xenograft models (Amornphimoltham et al. 2005; Nathan et al. 2007). These findings identify the Akt-mTOR pathway as a potential therapeutic target for HNSCC, thus raising the possibility of exploring the clinical activity of rapamycin and its analogs in HNSCC patients (Fig. 19.5).

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Fig. 19.5 (a) Tumor regression observed in HNSCC xenografts treated with rapamycin. HN12 cells were used to establish xenografts in athymic nu/nu mice, and tumor bearing animals were treated intraperitoneally for 5 consecutive days (i.p./qd x 5). with either rapamycin (10 mg/kg; n = 20). or equivalent volume of 5.2% Tween-80 and 5.2% PEG buffer (control; n = 20). Lesions dissected from HN12 xenograft 20 days after treatment with rapamycin or vehicle control are shown. (b) Tumor size from HNSCC xenografts in both rapamycin and vehicle treated groups from different cell lines (HN12, CAL27, UMSCC11B and HEp-2) were assessed daily, as indicated, and tumor weight was calculated. The results are expressed as mean tumor weight (mg) ± SE. Student’s t test was used to determine the difference between the rapamycin treated and control group (p < 0.0001) at day 20. Data are from a representative experiment that was repeated three times with similar results (Data are from Amornphimoltham et al. 2005).

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19.8 Effectiveness of mTOR Inhibitors in Halting Tumor Progression in Genetically-Defined and Chemically Induced Animal Models of SCC The effectiveness in human xenograft tumors is not always predictive of a clinical anticancer activity (Frese and Tuveson 2007; Lu et al. 2006). In this regard, while tumors originated in genetically defined and chemically induced animal models are often more difficult to treat than xenotransplanted human tumors in immunocompromised mice, they reflect better the more complex and challenging situation of the clinicalsetting (Becher and Holland 2006; Frese and Tuveson 2007; Lu et al. 2006). In initial studies, we showed that the epithelial-specific Pten conditional deletion (K14Cre PtenF/F mice) leads to multiple hyperproliferative lesions in the skin and oral cavity, concomitant with elevated activity of Akt and mTOR (Squarize et al. 2008). In this model, we observed that rapamycin treatment can rapidly revert the mucocutaneous papillomatous lesions in the face and limbs, acral keratosis, and deformities of nipples, among others, concomitant with a marked decreased of the elevated levels of pS6, which served as a suitable biomarker of drug efficacy in the target tissues. Furthermore, the early treatment with low dose of rapamycin prevented the development of lesion in mice in which Pten was excised, thus dramatically increasing their life expectancy (Squarize et al. 2008). In parallel, we took advantage of the ability of conditionally delete and activate genes in a temporally defined fashion to develop an oral-specific cancer model. For this we used mice expressing a tamoxifen-inducible Cre recombinase under the control of the cytokeratin 14 (K14) promoter (K14-CreERtam) and the ras oncogene from its own promoter after Cre excision of a stop signal (LSL-K-rasG12D mice) (Raimondi et al. 2009). These mice developed large papillomas exclusively in the oral cavity and hyperplasia in the tongue within 1 month of tamoxifen treatment. Furthermore, if these mice are crossed with mice harboring a floxed allele of p53 (p53flox/flox), all animals developed SCCs in the tongue within few days after the concomitant ras activation and p53 deletion by the administration of tamoxifen (Raimondi et al. 2009). The use of this mouse model, K14-CreERtam/LSL-K-rasG12D/+/p53flox/flox, in which animals develop oral cancer lesions in the absence of exogenously added tumor promoters, revealed that the ability to uncouple epithelial cell proliferation with differentiation and the activation of the Akt-mTOR signaling pathway resulting in pS6 accumulation represent early events in HNSCC progression. This model system also enabled us to explore whether interfering with the activity of mTOR could halt oral cancer development. In these studies, we found that decreasing mTOR activity by the administration of rapamycin after the genetic recombination leading to p53 deletion and ras activation was sufficient to prevent tumor progression (Raimondi et al. 2009). This two-hit oral cancer animal model system supported the effectiveness of mTOR inhibitors for HNSCC treatment, and it may also facilitate the development of complementary targeted approaches for HNSCC therapy. In parallel studies, we took advantage of the well-established two-step chemical carcinogenesis model, in which squamous carcinogenesis (SCC) is initiated by the topical application of a tobacco-related chemical carcinogen (7,12-dimethylbenz(a)

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anthracene) to the skin followed by the prolonged treatment with phorbol esters [12-O-tetradecanoylphorbol-13-acetate] (Califano et al. 1996) to explore the effectiveness of rapamycin for the treatment of skin SCC lesions. By targeting mTOR, rapamycin decreased the tumor burden of mice harboring early and advanced tumor lesions, and even recurrent skin SCCs (Figs. 19.6 and 19.7).

Fig. 19.6 The lentiviral expression of the rapamycin-insensitive mammalian target of rapamycin (mTOR) mutant (mTOR-RR) reverts the biochemical effects of rapamycin in head and neck squamous cell carcinoma (HNSCC) cells, and reveals that HNSCC cells are the primary targets of rapamycin in HNSCC xenografts. (a) Cartoon shows the domain structure of mTOR. The rapamycininsensitive mTOR mutant was obtained by mutating the amino acid at position Ser 2035 located in the FRB domain for Ile, which disrupts the binding of rapamycin-FKBP12 complex to wild type (wt). mTOR. (b) The mTOR-RR was expressed in HNSCC cell lines by lentiviral gene delivery, and cells were left untreated (−) or exposed to rapamycin (RP) (+) for 30 min. The expression of mTOR and the levels of total and phosphorylated S6 were analyzed Western blotting. (c) Cartoon depicting the experimental design. HNSCC cell lines expressing the mTOR-RR and GFP construct as a control were injected into the flanks of nude mice. After tumor development, rapamycin treatment was initiated for 5 consecutive days. The tissues were collected at every day of treatment for further analysis. (d) Top panel; Double IF staining of pS6 (in green) and CD31 (in red) were performed after 2 days of rapamycin treatment. Note the reduction of pS6 in the tissues from the control group expressing GFP (a), but not after expressing the mTOR-RR (b). The architecture of cancer and endothelial cells within the tumor mass was documented by the fluorophore labeling by E-cadherin (in green) and CD31 (in red), respectively, as shown in panels c and d. In parallel, the apoptotic response to rapamycin treatment detected by the IF staining of cleaved caspase-3 (in red) was found to overlap with the E-cadherin staining (in green) of tumor cells (e) in the control group but not in xenografts of mTOR-RR expressing cells (f) Data from Amornphimoltham et al. 2008b.

Fig. 19.6 (continued)

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Fig. 19.7 Mammalian target of rapamycin (mTOR) – dependent anti-tumor effect of rapamycin in the skin carcinogenesis model. Tumor number and size change as a function of rapamycin administration. Rapamycin (RP; 10 mg/kg) or vehicle (V) treatment started at week 9 during the tumor promotion. The second phase of the rapamycin treatment in the rapamycin-treated group was reinitiated at week 24. Bar, number of tumors divided by size difference as color indicated. Arrows, week of injection. X, week of the study termination. The representative of IF study in the tissues from corresponding vehicle and rapamycin treated animals. The phosphoS6 (pS6; in red) immunodetected the mTOR status and E-cadherin (in green) was used to label the tumor cells. Cell nuclei were stained with DAPI (in blue) (Data are from Amornphimoltham et al. 2008a).

The effectiveness of rapamycin in diminishing the tumor burden in this chemically induced squamous carcinogenesis model provided the opportunity to monitor and quantify the molecular signaling changes provoked in response to rapamycin in a temporal manner. We focused on molecules related to the Akt/mTOR pathway (pS6, pAkt Ser473, eIF4E), and biomarkers reflecting the status of cell metabolism, cell proliferation and cell death (p53, PCNA, CCND1, Glut1, and TUNEL assay), and tumor angiogenesis (VEGF, CD31, and CD34). For example, a decrease in the level of pS6 in rapamycin treated tumors clearly contrasted the high level of pS6 in the control group, reflecting the hyperactivity of the Akt/mTOR signaling pathway in SCC (Amornphimoltham et al. 2005; Yang et al. 2006). Concomitant with a reduction in pS6, we noted a decrease in cell proliferating cells that stained positive for PCNA and cyclin D1 (CCND1), and a reduction in cells expressing VEGF and Glut-1, reflecting the impact of mTOR in promoting angiogenesis and elevating glucose uptake (Amornphimoltham et al. 2008b). Unexpectedly, we also observed

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a reduction in the number of cells showing nuclear p53 in response to rapamycin treatment (Amornphimoltham et al. 2008a). While these emerging findings suggest that mTOR inhibitors may represent suitable candidates for the treatment of HNSCC, it is nonetheless imperative to further validate the effectiveness of candidate drugs using animal models that reflect the complexity of the human disease, before pursuing a clinical evaluation in HNSCC patients. This prompted us to search for defined animal models that closely resemble human HNSCC. In this regard, we have recently shown that 4NQO, a synthetic water-soluble carcinogen, can induce the sequential stages of oral cancer such as hyperplasia, dysplasia, in situ carcinoma and SCC (Czerninski et al. 2009) when administered to mice in the drinking water. High doses of 4NQO causes high incidence of esophageal cancers in mice (Kanojia and Vaidya 2006). Instead 4NQO at the lower dose of 50 mg/mL in drinking water, causes minimal esophageal tumorigenesis (microcancer or papilloma) in C57BL/6 mice, but leads to the progressive appearance of cancer lesions in the tongue and oral mucosa that were preceded by clearly identifiable premalignant events in 100% of these mice (Czerninski et al. 2009). An emerging body of data suggest that the progressive changes occurring in oral tumoral lesions of the 4NQO mouse model reflect changes that occur in human HNSCC, including altered expression of differentiation markers such as keratins, decreased expression or acquisition of potential inactivating mutations in tumor suppressor genes such as p16 and p53, increased expression of the epidermal growth factor receptor and cyclooxygenase-2, enhanced angiogenesis, increased levels of vascular endothelial growth factor, and increased activity of the PI3K–Akt–mTOR pathway (Czerninski et al. 2009; Hasina et al. 2009; Kanojia and Vaidya 2006; Tang et al. 2004). The remarkable efficacy of rapamycin in decreasing the size and number of HNSCC lesions in this animal model supported the emerging view that mTOR inhibitors may represent suitable chemopreventive and therapeutic agents for oral cancer prevention and treatment (Vitale-Cross et al. 2009).

19.9 Rapamycin Antitumor Mechanism in Head and Neck Cancer Models Overall, the emerging evidence supporting that the activation of the Akt-mTOR signaling network contributes to HNSCC progression has provided the rationale for exploring the therapeutic potential of inhibiting this pathway for HNSCC treatment. Rapamycin derivatives also diminish microscopic residual disease and enhance the effectiveness of EGFR inhibitors in experimental SCC models (Jimeno et al. 2007; Nathan et al. 2007). Unexpectedly, we observed that rapamycin does not exert growth suppressive or proapoptotic activities in HNSCC cells in vitro (Amornphimoltham et al. 2005), thus raising the possibility that its antitumoral effects may result from its actions on the stromal cells within the tumor microenvironment rather than by acting on HNSCC cells directly. We have recently utilized a retroinhibition approach to assess whether the cancer cell-autonomous actions of rapamycin contributes to its

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antitumoral effects. This consisted in the expression of a rapamycin-resistant form of mTOR (mTOR-RR) in HNSCC cells, while retaining wild-type mTOR alleles in host-derived endothelial and stromal cells, which hence remained rapamycin-sensitive. Lentiviral-mediated expression of mTOR-RR in HNSCC cells prevented the decrease in pS6 levels caused by rapamycin through mTOR in HNSCC cells but not in stromal cells, and rendered HNSCC xenografts completely resistant to the antitumoral activity of rapamycin. This reverse-pharmacology strategy enabled us to monitor the direct consequences of inhibiting mTOR in cancer cells within the complex tumor microenvironment. These studies revealed that mTOR controls the accumulation of HIF-1a and the consequent expression of VEGF and a glucose transporter, Glut-1, in HNSCC cells (Amornphimoltham et al. 2008b). These findings indicate that HNSCC cells are the likely primary target of rapamycin in vivo, and provide evidence that its antiangiogenic effects may represent a downstream consequence of mTOR inhibition in cancer cells (Amornphimoltham et al. 2008b). These observations may have important implications for the treatment of HNSCC and other solid tumors, and may explain the nature of the antiproliferative and proapoptotic effects of rapamycin, as the expression of HIF-1a is required for developing tumors to stimulate angiogenesis and other microenvironmental responses that allow continued expansion of the tumor tissue mass. This process involves the enhanced release of proangiogenic mediators, such as VEGF, whose expression is tightly regulated by HIF-1a (Semenza 2003), as well as the increased expression of glucose transporters and a myriad of cytosolic and mitochondrial proteins involved in the appropriate balance between aerobic and anaerobic glucose metabolism (Gatenby and Gillies 2004; Semenza 2003). In this scenario, it is tempting to speculate that by interfering with the mTOR-dependent regulation of HIF-1a expression, rapamycin may deprive HNSCC cells from an essential cell metabolic regulatory system, thus resulting in the progressive cancer cell death. This exciting possibility, as well as the precise mechanism by which rapamycin interferes with the expression of HIF-1a will certainly warrant further investigation.

19.10 Ongoing and Future Studies Addressing the Clinical Efficacy of mTOR Inhibitors In yeast and mammals, TOR has been shown to function as the catalytic subunit of two distinct molecular complexes in cells. Each composed of TOR, a regulatory subunit called LST8 and substrate-defining subunits, in TOR complex 1 (mTORC1) are raptor (regulatory associated protein of mTOR) and rictor (rapamycin-insensitive companion of mTOR) in TOR complex 2 (mTORC2). While rapamycin-sensitive mTORC1 represents the likely mediator of the antitumoral activity of rapamycin and its analogs, the mTOR-rictor complex may also play a role. mTORC2 has a PDK2 activity directly phosphorylating Akt at Ser473 site at c-terminal hydrophobic motif that is necessary for the full activation of Akt, which is analogous to the hydrophobic motif site in S6K that regulated by its sibling, mTORC1 (Guertin and Sabatini

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2007). Adding to the complexity is the recent finding that long term treatment of rapamycin can also inhibit mTORC2 activity and AktSer473 phosphorylation in a subset of tumor cells including HNSCC, while in certain tumors blockade of mTORC1 may lead to an increase in AktT308 phosphorylation by the disruption of a negative feedback loop (Sarbassov et al. 2006). The later was not observed in HNSCC, which may reflect the therapeutic response to rapamycin in this tumor type (Amornphimoltham et al. 2008b). It has been speculated that in cancer cells which rapamycin inhibit both mTORC1 and mTORC2, this results in overall Akt inhibition, and that tumors that respond to rapamycin as a monotherapy have drug-sensitive mTORC2 activity and depend on PI3K/Akt signaling (Sabatini 2006). As expected, these findings have also propelled the search for clinically useful mTOR inhibitors that may block both mTORC1 and mTORC2 complexes. Overall, the promising results obtained in a variety of models systems of HNSCC, and the antineoplastic activity of rapamycin and its analogs, CCI-779 and RAD001, in breast cancer, glioblastoma and mantle cell lymphoma patients (Chan et al. 2005; Galanis et al. 2005; Yee et al. 2006), together with the recent approval by the Food and Drug Administration of the use of CCI-779 (temsirolimus) and RAD001 (everolimus) in advanced renal carcinoma (Amato et al. 2009; Hudes et al. 2007; Motzer et al. 2008), have provided a strong rationale for the early evaluation of mTOR inhibitors as a molecular targeted approach to treat HNSCC. In this regard, several single institution and multicenter clinical studies are already underway or in the planning phase aimed at examining the biochemical consequences and clinical response of HNSCC patients to a variety of rapalogs as single agents and in combination with conventional and novel targeted agents. Certainly, we expect that the ability to investigate the clinical efficacy of mTOR inhibitors by multidisciplinary teams may soon help identify the HNSCC patients that may benefit the most from this promising group of therapeutic candidates.

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Chapter 20

High Throughput Molecular Profiling Approaches for the Identifications of Genomic Alterations and Therapeutic Targets in Oral Cancer Xiaofeng Zhou, Shen Hu, and David T. Wong Abstract Tumors, including oral squamous cell carcinoma (OSCC), develop through the combined processes of genomic instability, alteration, and selection, resulting in clonal expansion of cells that have accumulated the most advantageous set of genetic aberrations. With the human genome deciphered, high-throughput molecular profiling technologies are currently linking genome-wide transcriptome, proteome and mutation profiles with biological and disease phenotypes, which present an unprecedented opportunity for advancing the diagnosis and treatment of cancer. In this chapter, we aim to summarize modern genomics and proteomics technologies that can significantly facilitate our understanding of the molecular events underlying the development of OSCC. Understanding the molecular and genetic alterations in the pathogenesis of OSCC will help elucidate the mechanisms involved in tumor formation as well as identify potential targets for improved diagnosis and treatment of OSCC.

20.1 Introduction Oral cancer, predominantly oral squamous cell carcinoma (OSCC), is the sixth most common human cancer affecting over 300,000 people worldwide annually (Greenlee et al. 2000; Parkin et al. 2005). The American Cancer Society estimates that 35,310 new cases of oral cancer were diagnosed in 2008, and 7,590 people died from this disease (Oral cancer 2008). The main risk factors for oral cancer include tobacco use, alcohol consumption, and human papilloma virus infection. Despite the treatment advances in surgery, chemotherapy and radiotherapy, the survival rates for patients with oral cancer have not been significantly improved in the past few decades. The high mortality rate of oral cancer can be attributed to factors

X. Zhou (*) Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois at Chicago, Chicago, IL, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_20, © Springer Science+Business Media, LLC 2011

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including limited understanding of the disease mechanism, lack of clinical tools for early cancer detection, and nonresponsiveness to therapeutic treatments. OSCC evolves through a series of histopathological stages, including hyperplasia, dysplasia of varying degrees, carcinoma in situ, and eventually invasive SCC (Brinkman and Wong 2006). At the molecular level, the development of OSCC is initiated by chemical/biological insults to the normal oral epithelium by carcinogens such as tobacco and alcohol or human papilloma virus infection. This results in increasing genetic instability and a number of genetic alterations such as loss of heterozygosity, gene inactivation by methylation, and gene amplification. Many regulatory proteins such as p16, p53, Rb, cyclin D1, epidermal growth factor receptor, and transforming growth factor-alpha are also aberrantly altered (Deshpande and Wong 2008). These important signaling pathways promote cell proliferation, cell survival, and/or transformation capabilities, leading to the formation of preneoplastic lesions, subsequent loss of cellular organization, and eventually invasive penetration through the basement membrane. Nevertheless, our understanding of the related molecular mechanism is far from complete due to the cellular and molecular heterogeneity of OSCC development. Meanwhile, the fact that there are many additional genes potentially involved in oral carcinogenesis emphasizes the importance of studying gene alterations in a global scale by genomics and proteomics. In this book chapter, we aim to summarize modern genomics and proteomics technologies that can significantly facilitate our understanding of the molecular events underlying the development of OSCC. Understanding the molecular and genetic alterations in the pathogenesis of OSCC will help elucidate the mechanisms involved in tumor formation as well as identify potential targets for improved treatment of OSCC.

20.2 Genomic Analysis at Chromosomal Level Genetic aberrations at chromosomal levels can be analyzed using several highthroughput genomic techniques, including chromosome banding (also known as karyotyping), loss of heterozygosity (LOH), comparative genomic hybridization (CGH), digital karyotyping (Wang et al. 2002), fluorescence in situ hybridization, restriction landmark genome scanning (Imoto et al. 1994), and representational difference (Lisitsyn and Wigler 1993). These analyses enable the identification of a broad range of chromosomal abnormalities in cancer.

20.2.1 Comparative Genomic Hybridization (CGH) − Systematic Copy Number Analysis CGH technique was developed to detect gene copy-number changes (amplifications and deletions) between normal and neoplastic tissue or cells (Kallioniemi et al. 1992).

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In a typical CGH experiment, the DNA from test (disease) and reference (normal) samples are differentially labeled with different fluorescence dyes, and then cohybridized to the normal metaphase chromosomes to generate fluorescence ratios along the length of chromosomes. This ratio provides a cytogenetic representation of the relative DNA copy-number variation. CGH was the first effective tool to examine the entire genome for variations in DNA copy-number changes (Pinkel and Albertson 2005a,b). However, this earlier version metaphase chromosome-based CGH has a limited mapping resolution (~20 Mb). Array-based CGH is the second generation CGH in which fluorescence ratios on arrayed DNA elements provide a locus-bylocus gene copy-number measure (Pinkel et al. 1998; Ishkanian et al. 2004). While this approach increases mapping resolution, most array-CGHs utilize large genomic clones (e.g., bacterial artificial chromosomes) which limit spatial sensitivity. Furthermore, using large genomic clones may also lead to reduced specificity as a result of their inclusion of repeats (e.g., Alu) and segments of extensive sequence similarity (e.g., pseudogenes) (Mantripragada et al. 2004). Recently, several novel CGH platforms have become available with the completion of the human genome sequence. These include cDNA array-based CGH (Pollack et al. 1999; Zhou et al. 2004a), oligonucleotide array-based CGH (Brennan et al. 2004; Lucito et al. 2003), tiling array-based CGH (Ishkanian et al. 2004), and copy number analysis using highdensity SNP microarrays (Bignell et al. 2004; Zhao et al. 2004, 2005; Zhou et al. 2004c). The tiling array and the SNP array-based approaches have drawn more attention due to their remarkable mapping resolution. Tiling arrays have the potential to detect small chromosomal gains and losses (resolution ~40 kb, almost at single gene level) that might be overlooked by marker-based arrays (Ishkanian et al. 2004; Davies et al. 2005). We envision that in the near future, it will be possible to survey copy number changes at bp resolution using tiling arrays that contain billions of overlapping probes covering the entire genome. The SNP array-based CGH provides the unique advantage of combining CGH and LOH analysis in one single experiment, which will be discuss in the following section (Zhao et al. 2004; Zhou et al. 2004c).

20.2.2 Loss of Heterozygosity (LOH) − Systematic Allelic Imbalance Analysis Chromosomal aberrations such as allelic losses, which are caused by mitotic recombination, gene conversion, or nondisjunction, cannot be detected by CGH. These allelic imbalances can be detected based on LOH at polymorphic loci. This approach is based on the Knudson two-hit hypothesis (Knudson 1971, 1996) for tumor-suppressor genes. Examples include the discovery of the first tumor suppressor gene, RB1 (Friend et al. 1986), in which a recessive mutation was discovered in one allele, and the loss of the other wild-type allele was detected by LOH. Traditionally, polymorphic markers (e.g., restriction fragment length polymorphisms [RFLPs] and microsatellite markers) have been employed for LOH analysis (Vogelstein et al. 1989). However, these approaches are labor intensive and require

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large amount of DNA, allowing only a modest number of markers to be screened. The recent advances in human genome projects lead to the identification of millions of SNP loci (http://www.ncbi.nlm.nih.gov/SNP/), which makes them ideal markers for various genetic analyzes, including LOH. SNP markers have significant advantages over RFLPs and microsatellite markers in terms of abundance, spacing, and stability across the genome. Several high-density SNP arrays have recently been developed to support large-scale high throughput SNP genotyping (Wang et al. 1998). The LOH patterns generated by SNP array analysis have a high degree of concordance with previous microsatellite analyses of the same cancer samples (Lindblad-Toh et al. 2000), and have been utilized in a number of studies for the molecular classification of various types of cancers (Zhou et al. 2004b,c; Janne et al. 2004; Wang et al. 2004; Hoque et al. 2003; Lieberfarb et al. 2003). One unique advantage of this SNP array-based approach is that the intensity of sample hybridization to the array probes can also be used to infer copy number changes (similar to CGH) (Bignell et al. 2004; Zhao et al. 2004; Zhou et al. 2004c). This unique feature has been explored by algorithms implemented in several independent bioinformatics/statistical software packages, including dChipSNP (Zhao et al. 2004), Copy Number Analysis Tool (Huang et al. 2004), and FASeg (Yu et al. 2007). Based on these novel data analysis tools, we are able to perform concurrent copy number analysis and LOH analysis with a single experiment (Zhou et al. 2004c).

20.2.3 Cytogenetic-Based Approaches − The Chromosome Staining Techniques Cytogenetics is a set of relatively old techniques which are based on the chromosome-banding method introduced in 1969 (Caspersson et al. 1969a,b). One major drawback of these approaches is the requirement of in vitro culture and metaphase preparation of the cells. Nevertheless, cytogenetic approaches will always have their place in the genomic studies because they provide direct visualization of chromosomal abnormalities. Furthermore, these cytogenetic techniques complement the high-throughput techniques (e.g., CGH and LOH) by providing information on chromosomal structural rearrangements that are not readily resolved by DNA copy number analyses. For example, translocations are common genomic abnormalities in cancer (Futreal et al. 2004), but they cannot be detected by CGH or LOH. An experienced cytogeneticist, however, can easily detect many forms of chromosomal translocations using classical cytogenetic techniques, such as chromosome banding technique (also known as karyotyping). A typical karyotyping analysis involves blocking the cells in mitosis, staining the chromosomes with Giemsa dye (which stains AT rich regions of chromosomes and produces dark bands), and visualizing under a light microscope. Karyotype analyses are performed as part of standard clinical tests for prenatal and postnatal screening, as well as for the diagnosis of specific types of cancers (e.g., hematological malignancies). However, many cancer cells have complex karyotypes, and are difficult

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to interpret base on standard karyotyping images. Recently, a number of new cytogenetic labeling methods have been developed, including spectral karyotyping, multicolor fluorescence in situ hybridization, cross-species color banding, and multicolor chromosome banding. These techniques permit the simultaneous visualization of all chromosomes in different colors, and thus considerably improve the detection of translocations or deletions. With the introduction of these techniques (Schrock et al. 1996; Liyanage et al. 1996; Speicher et al. 1996), the comprehensive analysis of complex chromosomal rearrangements present in tumor karyotypes has been greatly improved.

20.3 Transcriptome Profiling Techniques The genome-wide transcriptome profiling became a reality when several epochmaking genomic techniques were introduced, including DNA microarray and Serial Analysis of Gene Expression (SAGE). Both DNA microarray and SAGE are powerful tools targeted at global gene expression. While the microarray technology requires prior knowledge of the sequence of the genes to be analyzed, SAGE technology can analyze gene expression in organisms with uncharacterized genomes. The obvious advantage of the microarray is the ability to measure gene expression in cell and tissue samples, and commercial platforms (e.g., GeneChip from Affymetrix, Inc.) are available for flexible research design. With the Human Genome Project completed at the beginning of the new millennium, the microarray takes center stage in investigating genome-wide gene expression in all aspects of human cancer. A DNA microarray is typically a small solid support (e.g., a glass microscope slide) on which known sequences of tens of thousands of genes are immobilized. Commonly used immobilization methods include “ink-jet” printing, pin-spotting, and direct synthesis. The parallel presence of so many genes (often covering the whole genome of an organism) on a single microarray has allowed genomic studies to be performed in a high-throughput fashion. For example, expression changes of all genes on a whole genome can be monitored simultaneously. Doing this “one gene at a time” would be unthinkable.

20.3.1 Transcriptome Profiling-Based Analysis on OSCC Metastasis – A Promising Example Microarray expression profiling has proven to be a powerful approach for characterizing the genome-wide expression changes associated with progression of OSCC, such as metastasis. To gain a better understanding of the underlying molecular biological processes that dictate the observed expressional changes, it may be more fruitful to focus on a higher level of biological information (e.g., alterations

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of group of genes, certain pathways, or biological processes in lymph node metastasis of OSCC), rather than focusing on specific genes. The following sections highlight the recent advances in the understanding of OSCC metastasis based on this genome-wide system biological approach. 20.3.1.1 Matrix Metalloproteases and Tissue Inhibitors of Metalloproteases Hyperactivation of matrix metalloproteases (MMPs) is a hallmark of invasive cancers for which it constitutes a mechanistic prerequisite for the degradation of the basement membrane and extracellular matrix (ECM) thus allowing tumor cells to leave the primary tumor site and enter blood or lymphatic vessels for dissemination (Bachmeier et al. 2005). The role of MMPs in metastasis of OSCCs is well established (de Vicente et al. 2005a,b; 2007; Kato et al. 2005; Kim et al. 2006; Lyons and Jones 2007; Patel et al. 2005; Roy et al. 2007; Ziober et al. 2006) and highlighted by a number of transcriptome profiling studies (Chiang et al. 2008; Lin et al. 2004; Ito et al. 2003; Kashiwazaki et al. 2008; Kondoh et al. 2008; Nagata et al. 2003; Roepman et al. 2005; Zhou et al. 2006). The biological activities of MMPs are regulated by four endogenous protease inhibitors of MMP: TIMP1, TIMP2, TIMP3, and TIMP4 (Jiang, et al. 2002). Since specific MMPs can promote cancer progression, it is reasonable to hypothesis that high levels of endogenous TIMPs would prevent cancer progression, and consequently, tumors with high TIMPs levels would have a better prognosis than those with low TIMPs levels. In OSCCs, several studies have shown that elevated expression of TIMPs, especially TIMP1 and TIMP2, in metastatic carcinoma have a good prognosis (Baker et al. 2006; Ikebe et al. 1999; Kurahara et al. 1999). However, contradictory findings have also been reported (de Vicente et al. 2005a; Nakamura et al. 2005; Katayama et al. 1984). These different findings may be because TIMPs are multifunctional proteins and that their effects on tumor progression are contextand concentration-dependent. Therefore, further studies are warranted to fully explore the roles of TIMPs in OSCC metastasis. The expressions of MMPs and their endogenous inhibitors are regulated by a variety of cytokines, growth factors, and transcription factors that participate in tissue remodeling. TIMP2 is an essential factor for efficient activation of proMMP2. TIMP2 accomplishes this activation by acting as a bridge between MMP2 and membrane type 1-MMP (MT1-MMP) on the cell membrane. This trimolecular complex allows a second MT1-MMP molecule to cleave the prodomain of MMP2 (Lander et al. 2001). In two independent studies, MT1-MMP was found to be up-regulated by laminin 5 (Yamamoto et al. 1986) and E1AF (an ets-oncogene family transcription factor) (Izumiyama et al. 2005) and therefore, activated the expression of MMP2 in tumor cells. Using a xenograft model, Miyazaki et al. (2008) demonstrated that both MMP2 and MMP9 levels were increased under hypoxic condition. MMP2 was predominantly expressed in the hypoxic region of tumor tissue, while MMP9 was mainly detected in neighboring

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stromal tissues containing blood vessels. Interestingly, the actions of MMPs and their inhibitors also depend on their concentrations. Baker et al. (2006) reported that tissue concentrations of a subset of these factors correlated with tumor progression, suggesting that it is the balance between MMPs and their corresponding TIMPs that control tissue degradation at each stage of tumor invasion and metastasis. These findings support the hypothesis that specific TIMPs, under specific conditions and at concentrations founded in vivo, may play a role in promoting rather than inhibiting cancer progression. Additional studies are currently underway to investigate the role of the MMP/TIMP system in tumor invasion and metastasis. 20.3.1.2 Urinary Plasminogen Activator and its Receptor Recently, the critical roles of urinary plasminogen activator (PLAU) (also known as urokinase-type plasminogen activator, uPA) in ECM remodeling, tumor invasion and metastasis have become evident. PLAU is a serine protease that binds to a surface-anchored receptor (PLAUR) (also known as uPAR), which localizes its proteolytic activity to the pericellular milieu. Furthermore, PLAU and PLAUR interact with a number of transmembrane proteins to regulate multiple signal transduction pathways and influence a wide variety of cellular behaviors, including cell adhesion, migration, chemotaxis and tissue remodeling (Blasi 1996, 1997; Shi and Stack 2007). The PLAUR expression levels in tumor tissues and their prognostic values have been studied in a number of cancer types, including breast (Han et al. 2005), lung (Volm et al. 1999), prostate (Shariat et al. 2007), ovarian (Begum et al. 2004) and colorectal cancers (Seetoo et al. 2003). Results from these studies have shown that elevated PLAUR expression correlates with poor prognosis, thereby making it a potential biomarker for molecular classification of cancers. Enhanced expression of PLAU and PLAUR has also been found in OSCC, and correlates with tumor differentiation grade, lymph node metastasis and prognosis (Shi and Stack 2007; Bacchiocchi et al. 2008; Baker et al. 2007; Hundsdorfer et al. 2005; Li et al. 2006). Functional study has shown that silencing the endogenous PLAUR expression in highly malignant OSCC cells resulted in a dramatic reduction of tumor cell proliferation, adhesion, migration and invasion in vitro (Weng et al. 2008). Recently, genome-wide profiling studies have identified the PLAU as a strong biomarker for predicting poor disease outcome of OSCC using a “gene signature” approach (Ziober et al. 2006; Nagata et al. 2003). Furthermore, Ghosh et al. demonstrated that PLAU expression and PLAUR relocalization are regulated by a3b1 integrin-activated Src/MEK/ERK signaling pathway in oral keratinocytes (Ghosh et al. 2000). Conversely, blocking PLAUR-a3b1 integrin interaction results in significant inhibition of PLAU expression, suggesting the functional relevance of PLAUR-a3b1 integrin association in protease regulatory pathways (Ghosh et al. 2006). Collectively, these works implicate an important role of the PLAU-PLAUR system in invasion and metastasis of OSCC.

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20.3.1.3 SDF-1/CXCR4 Signaling Axis It has been demonstrated that G-protein-coupled seven-span transmembrane receptor CXCR4 is expressed in numerous types of embryonic cells and the a-chemokine stromal-derived factor 1 (SDF-1) has chemoattractant effects on these cells (Knaut et al. 2003; McGrath et al. 1999; Rehimi et al. 2008). Animal models in which SDF-1/CXCR4 signaling have been interrupted exhibit a number of phenotypes that can be explained by inhibition on SDF-1-mediated chemoattraction of stem/progenitor cells (Ma et al. 1998; Mizuno et al. 1994; Tachibana et al. 1998; Zou et al. 1998). Furthermore, the expression patterns for both SDF-1 and CXCR4 are highly consistent with the possibility that they have shifted developmental patterns in the formation of many different tissues. These observations suggest a crucial role for the SDF-1/CXCR4 signaling axis in regulating the migration of different types of stem/progenitor cells. It is believed that cancer stem cells, much like normal stem/progenitor cells, can give rise to tumor cells in primary tumors and can also metastasize to seed tumors in a second site. In this case, one may postulate that the SDF-1/CXCR4 signaling axis may influence the biology of tumors and direct the metastasis of CXCR4-expressing tumor cells by chemoattracting them to organs that express high levels of SDF-1 (e.g., lung, liver, bones, and lymph nodes). Supporting this notion, it has been recently reported that several CXCR4-expressing cancers, including breast, prostate, ovarian cancer and neuroblastoma (Geminder et al. 2001; Gerber et al. 2003; Muller et al. 2001; Kikuchi et al. 2003), metastasize to specific organs in a SDF-1-dependent manner. The role for the SDF-1/CXCR4 signaling axis involved in lymphatic metastasis of OSCC was also investigated in the past several years (Almofti et al. 2004; Delilbasi et al. 2004; Ishikawa et al. 2006; Oliveira-Neto et al. 2008; Onoue et al. 2006; Uchida et al. 2003, 2004, 2007). SDF-1a expression was detected mainly in the stromal cells, but also occasionally in the tumor cells metastasized to the regional lymph nodes (Uchida et al. 2003). CXCR4 expression in metastatic cancer tissues was significantly higher than that in nonmetastatic cancer tissues, and its expression was strongly associated with invasion, recurrence, and lymph node metastasis. Additional studies have shown that SDF-1a rapidly activates extracellular signalregulated kinase (ERK) 1/2, Akt/protein kinase B (PKB) and Src family kinases in CXCR4-expressing cancer cells (Onoue et al. 2006; Uchida et al. 2003). More importantly, recombinant SDF-1a stimulates in vitro invasiveness and scattering in CXCR4-expressing OSCC cells, and induces metastasis of these cells to the cervical lymph node in an orthotopic nude mice model (Uchida et al. 2004). Taken together, these results indicate that SDF-1/CXCR4 signaling mediates the lymph node metastasis in OSCC via ERK1/2 and/or Akt/PKB pathway. 20.3.1.4 Epithelial-Mesenchymal Transition (EMT) EMT, in which epithelial cells lose their polarity and become motile mesenchymal cells, occurs during the development process and is also a key step in the tumor

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progression towards metastasis, including metastasis of OSCC (Onoue et al. 2006; Kudo et al. 2006; Takayama et al. 2009; Takkunen et al. 2008). Accumulating evidence supports that EMT can contribute to metastasis by changing the adhesive properties of tumor cells and promoting their motility, thereby increasing their invasiveness. More strikingly, a variety of EMT markers, including downregulation of Cadherin 1 (CDH1) (also known as E-cadherin) and cytokeratin (Onoue et al. 2006), increased expression of Cadherin 2 (CDH2) (also known as N-cadherin) (Pyo et al. 2007), MMPs (Kashiwazaki et al. 2008; Roepman et al. 2005; Zhou et al. 2006; Higashikawa et al. 2008) and transcription factors such as Snail 1 (Snail) (Sun et al. 2008), SIP1/ZEB2 (Maeda et al. 2005), NF-kb (Hu et al. 2007), and occludins (Bello et al. 2008), have also been found in lymph-node metastatic OSCC cells. Moreover, cadherin switching, which has been known to play a central role in the EMT, has also been implicated in OSCC metastasis (Pyo et al. 2007). The presence of the EMT markers in tumor tissues indicates an important role for EMT in promoting invasion and metastasis of OSCCs. The continuously evolving microarray technology has the potential to revolutionize the clinical practice. Physicians in the future may be empowered with a handheld device to monitor health status in real time during a routine physical examination to detect any health problems at an early stage and even to suggest the best treatment options based on the characteristics of an individual’s genome. With the powerful microarray technology for molecular profiling, we are witnessing the beginning of the personalized medicine era.

20.4 Small RNA and MicroRNA Profiling Technologies MicroRNAs are newly recognized, non-coding, regulatory RNA molecules, about 22 nucleotides in length. It is estimated that the human genome have approximately 800–1,000 microRNAs (Bentwich et al. 2005). While not involved directly in protein coding, microRNAs are believed to control the expression of more than one third of the protein-coding genes in the human genome (Lewis et al. 2003, 2005; Xie et al. 2005) Each microRNA can target and regulate the mRNA transcripts of hundreds of genes. One microRNA can have multiple target sites in the mRNA transcript of a gene, while one mRNA can be targeted by multiple microRNAs. Therefore, microRNAs act as a newly recognized level of regulation of gene expression. They are pivotal regulators of diverse cellular processes including proliferation, differentiation, apoptosis, survival, motility, and morphogenesis. High-throughput microRNA profiling is a technical challenge. The short length the microRNA render many conventional tools ineffective – very small RNA molecules are difficult to reliably amplify or label without bias. There are three common approaches for microRNA profiling: hybridization based methods, PCR-based detection, and cloning methods. Here, we will provide an overview of the technologies, and will also highlight the recent progresses in microRNA profiling.

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20.4.1 Hybridization-Based MicroRNA Profiling − Microarray The common hybridization based microRNA detection methods include Northern blotting, in situ hybridization, bead-based flow-cytometry, and more recently, microarray. The majority of the published studies reporting microRNA profiling analysis were performed using different microarray technologies. The differences in these microarray platforms are mainly in their probe design, probe immobilization chemistry, sample labeling, and signal detection methods (see (Yin et al. 2008) for comprehensive review on array-based microRNA profiling). Similar to the early mRNA microarrays, most of the early stage microRNA arrays were custom made. With the recent introduction of several commercially available microRNA array platforms, the study design and data analysis became more streamlined. While the currently available commercial microRNA arrays make profiling studies on microRNA much easier for biomedical investigators, new developments in the biotech field have emerged as potential opportunities to further improve the microRNA microarrays. The locked nucleic acid (LNA) has recently emerged as a popular tool in various biological and biomedical studies due to its high affinity and specificity to the complementary RNA. LNA is a conformational analogue of the RNA molecule that contains at least one LNA monomer. The unprecedented thermal stability between LNA molecules and their target RNAs enables visualization of microRNA by in situ hybridization. In addition, the LNA molecules are highly metabolic stable, which makes them ideal tools for novel therapeutic approaches by targeting cancer-associated microRNAs. The LNA-based probes have also been used in the design of microarrays for microRNA profiling (Castoldi et al. 2006, 2007, 2008), which appears to improve the mismatch discrimination. This array has recently been used in studying several malignancies, including chronic myeloid leukemia and breast cancer (Venturini et al. 2007; Sempere et al. 2007). A recent review by Stenvang el al. provided a comprehensive review on recent advances in LNA-based microRNA detection in cancer (Stenvang et al. 2008). Other attempts to improve the microarray-based microRNA profiling include the RNA-primed array-based Klenow enzyme (RAKE) assay (Nelson et al. 2004) and the modified versions of RAKE assay (Berezikov et al. 2006). The RAKE assay is based on the ability of an RNA molecule to function as a primer for Klenow polymerase-dependent extension when fully base-paired with a single-stranded DNA molecule. Combining with the microarray technology, RAKE assay appears to provide better specificity than other conventional microarray platforms. It has been reported that with this RAKE assay, microRNAs isolated from formalin-fixed paraffin-embedded tissue can be used to generate optimal quality microRNA profiles (Nelson et al. 2004, 2006), which leads new opportunities for analyses of small RNAs from archival clinical tissue samples.

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20.4.2 Quantitative Real-Time PCR (qRT-PCR)-Based MicroRNA Profiling While the microRNA microarrays described above provide excellent throughput and high coverage, these methods do not amplify the microRNA and thus often compromise the sensitivity. The qRT-PCR technology provides unparalleled sensitivity and specificity. However, it is technically challenging to amplify and quantify mature microRNA because the mature microRNA is only around 22 nucleotides in length, roughly the size of a typical PCR primer. Therefore, earlier versions of qRT-PCR assays are usually designed to quantify microRNA precursors. While the relative level of most mature microRNAs may be projected based on the level of corresponding precursors, additional tests will be needed to ensure that the levels of the mature microRNAs are reflected by the level of their precursors. Recently, the second generation of qRT-PCR assays has been developed to directly quantify the mature microRNA. These assays typically incorporate a target specific stem-loop, reverse transcription primer. This innovative design addresses a fundamental challenge in microRNA quantification: the short length of mature microRNAs (~22 nucleotides). The stem-loop structure provides specificity for the mature microRNA target and forms a RT primer/mature microRNA-chimera that extends the 3¢ end of the microRNA. The resulting longer RT product presents a template amenable to standard real-time PCR-based quantification using TaqMan Assays. These qRT-PCR assays are now commercially available (e.g., TaqMan MicroRNA Assay from Applied Biosystems). To improve the throughput, these qRT-PCR assays have been packaged into convenient, pre-configured micro fluidic cards that contain up to 384 unique TaqMan assays and they are compatible with most of the common qPCR instruments.

20.4.3 Cloning and Deep Sequencing-Based MicroRNA Profiling The microRNA profiling methods described above rely on primers or probes designed to detect known microRNAs. They can only detect known microRNA species that previously identified by sequencing or homology search. Moreover, the huge range of microRNA level from tens of thousands to just few molecules per cell complicates the detection of microRNAs expressed at low copy numbers. Therefore, many undetected microRNA may exist even in well-explored species. The cloning and deep sequencing based microRNA profiling approach allows both the quantification of expression levels and identification of new microRNAs at high speed and sensitivity and low cost.

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This approach is developed by combining aspects of microRNA cloning and SAGE technology, which lead to its original name − miRAGE (Cummins et al. 2006). Similar to traditional cloning approaches, miRAGE starts with the isolation of 18- to 26-base RNA molecules to which specialized linkers are ligated, and reverse-transcribed into cDNA. However, subsequent steps, including amplification of the complex mixture of cDNAs using PCR, tag purification, concatenation, cloning, and sequencing, have been performed by using SAGE methodology optimized for small RNA species. SAGE was originally designed to characterize gene expression profiles. It has a potential to be a high-throughput gene expression profiling tool. Over the years, much improvement has been made to increase sequencing efficiency and reduce input RNA amount requirement (Datson 2008; Matsumura et al. 2008; So et al. 2004; de Hoon and Hayashizaki 2008; Torres et al. 2008; Hene et al. 2007). Although it is not as popular as microarrays and qRT-PCR due to technological and economical challenges, this technology has the unique advantage of combining discovery and quantification. The introduced “next-generation” sequencing technologies, such as massively parallel signature sequencing and more recently the Roche/454 and Illumina’s GAII systems, offer inexpensive increases in throughput. With the added depth of sequencing now possible, we have an opportunity to identify low abundance microRNAs or those exhibiting modest expression differences between samples, which may not be detected by hybridization-based or qRT-PCRbased methods. The continuation of advances in the sequencing technologies, coupled with the unique features of microRNA (e.g., short length, difficult to amplify and label without introducing bias), tends to suggest that deep sequencing may be the optimal approach for high-throughput profiling of microRNA (and other small RNA).

20.5 Mass Spectrometry-Based Proteomics Proteomics is a novel molecular technology that may significantly accelerate oral cancer research. In fact, cellular functions are mainly performed by proteins and the majority of anticancer drugs are targeting at proteins. A promising application of oral cancer proteomics is to reveal key target proteins and signaling pathways underlying the development of oral cancer. The study may also identify novel therapeutic targets and discover protein biomarkers for cancer diagnosis and prognosis. Modern proteomics is primarily driven by mass spectrometry (MS), an exquisite analytical technology which measures the mass-to-charge ratio of ionized molecules. In early MS-based proteomics studies, most of the applications were focused on identification of proteins of interest. This can be done using either peptide mass fingerprinting (PMF) or tandem MS (MS/MS). In PMF, an isolated, unknown protein is cleaved using a proteolytic enzyme and the resulting peptides are usually measured by matrix-assisted laser desorption/ionization with time-of-flight MS (MALDI-TOF MS) (Pappin et al. 1993). The premise of PMS is that every unique

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protein will have a unique set of peptides and hence unique peptide masses. Identification is accomplished by matching the observed peptide masses to the theoretical masses derived from a sequence database. This technique is well suited for identification of proteins in two-dimensional gel spots where the protein purity is high. However, PMF protein identification can run into difficulties with a mixture of proteins, which typically requires the use of tandem MS to achieve confident identification. Tandem MS, also known as MS/MS, involves multiple stages of MS analysis, with some form of fragmentation occurring in between the stages (Aebersold and Mann 2003). It can be done using physically separated mass analyzers with a collision cell between these elements for molecule fragmentation. For example, one mass analyzer can isolate a peptide ion from many entering a mass spectrometer. The peptide is then broken into smaller fragments in the collision cell by collisioninduced dissociation (CID) and a second mass analyzer can measure the fragments produced from the peptide precursor. Tandem MS can also be done using ion trap or Fourier transform ion cyclotron resonance mass spectrometers, where precursor or fragment ions are trapped in a single mass analyzer with multiple MS steps taking place over time. Similar to the PMF approach, the obtained masses (including precursor and fragment ions) from tandem MS are then in silico compared to either a proteome or genome database to find the best matched protein. This is achieved by using a computer program for database searching (e.g., Mascot or Sequest), which calculates the absolute masses of theoretical peptides and fragments from each protein in the database and then compares the corresponding masses of the unknown protein to those of each protein in the database. Currently, large-scale identification of proteins in a specific proteome (e.g., plasma or saliva proteomes) mainly relies on tandem MS and database-searching algorithms for peptide and protein identification (Hu et al. 2005, 2006; Denny et al. 2008). As proteomics tools evolve, quantitative analysis/profiling of proteins in defined biological or disease samples (quantitative proteomics) becomes a central application of proteomics. A commonly used quantitative proteomics approach is based on the use of stable isotope labeling of proteins/peptides, followed by tandem MS to compare the relative abundance of the proteins in different samples. Stable isotope labeling with amino acids in cell culture is a straightforward approach for in vivo incorporation of isotope tags into cellular proteins for MS-based quantitative proteomics. The method relies on metabolic incorporation of amino acids with substituted stable isotopes (e.g., 13C, 15 N), and is particularly useful when studying cell line models (Ong et al. 2002). As for clinical samples such as tissue or body fluids from disease patients, quantitative proteomic analysis can be performed using tandem MS coupled with stable isotope labeling techniques such as isotope-coded affinity tagging (ICAT) (Gygi et al. 1999), isotope tagging for relative and absolute quantitation (iTRAQ) (Ross et al. 2004), isotope coded protein labeling (Schmidt et al. 2005) or proteolytic 18O labeling (Miyagi and Rao 2007). These isotope tags either label proteins or proteolytic peptides from different samples for comparative analysis. If the intact proteins get tagged (e.g., ICAT), the labeled samples are subsequently combined, digested with trypsin, and then analyzed with LC-MS/MS.

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Because isotope-labeled peptide pairs are chemically identical, they coelute during LC separation. The relative quantitation can be determined by the ratio of ion intensities from coeluting isotope-labeled peptides in the MS survey scan, which defines the ratio between parent proteins in the starting samples. Meanwhile, MS/MS analysis of the peptides allows the identification of the protein based on sequence database searching. If the proteolytic peptides are labeled with tandem mass tags, then both quantitation and identification rely on MS/MS spectra. For instance, iTRAQ utilizes isobaric tags that can be cleaved during CID to yield an isotope series (reporter ions) representing the quantity of a peptide from different samples. Because the peptide remains attached to the isobaric tags until CID is conducted, the resulted MS/MS spectrum allows for simultaneous identification (based on fragment ions) and quantitation (based on reporter ions) of the peptide. By using MS-based proteomics to investigate global protein alterations in patients with OSCC or OSCC-derived cell lines, a number of tumor-associated proteins have been identified (Hu and Wong 2007). Although the function of these proteins in oral carcinogenesis remains unclear, some of them indeed show a regulatory role in the development of OSCC and may have clinical or therapeutic implications for OSCC (Weng et al. 2008; Ralhan et al. 2009; Hu et al. 2008; Patel et al. 2008; Wang et al. 2008). The extensive protein alterations observed from these studies also indicate that multiple cellular and etiological pathways are involved in the process of oncogenesis, and suggest that multiple protein molecules should be simultaneously targeted as an effective strategy to counter the disease (Chen et al. 2004). Proteomics may play a significant role in anti-cancer drug discovery because this technology can be used to discover and validate therapeutic targets, to assess drug efficacy and toxicity, and to identify disease subgroups for targeted therapy. It is also promising for identifying protein targets of anti-cancer drug action, and therefore providing further insight for new drug development (Lee et al. 2006; Sung et al. 2006).

20.6 Summary Merely 20 years ago, the prevalent mode of biomedical research was centered on the “one gene at a time” model: cloning and characterizing a single gene or a few closely related genes. This had been the gold standard until the mid-1990s, when the “genomics era” began with the establishment of several epoch-making genomic techniques (e.g., DNA microarray). Together with the completion of the human genome project at the beginning of the new millennium, the resulting exponential boom of new knowledge brought us into the “post-genomics” era. Emerging genomics and proteomics technologies are rapidly reshaping cancer research, allowing a transition from the traditional genetic studies to a new paradigm based on systems biology. Compared with traditional studies, the systems biology approach allows us investigate the complex biological networks as a whole. Based on integration of data from multidimensional (genomic/transcriptomic/proteomic) analyses, the systems

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biology approach will also increase the reliability of discovering causative genes for complex diseases (Hu et al. 2009). Considering that multifactorial etiology and heterogeneity of oncogenic pathways of OSCC, a systems biology approach by integrating genomic and proteomic data may be necessary in order to have a more profound understanding the molecular mechanism underlying oral carcinogenesis. We are now witnessing an exciting new era in cancer research. By applying and translating this newfound knowledge, we are developing more efficacious treatments for OSCC that will be of great relevance to other cancers as well.

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Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J et al (1998) Large-scale identification, mapping, and genotyping of singlenucleotide polymorphisms in the human genome. Science 280(5366):1077–1082 Wang TL, Maierhofer C, Speicher MR, Lengauer C, Vogelstein B, Kinzler KW, Velculescu VE (2002) Digital karyotyping. Proc Natl Acad Sci USA 99(25):16156–16161 Wang ZC, Lin M, Wei LJ, Li C, Miron A, Lodeiro G, Harris L, Ramaswamy S, Tanenbaum DM, Meyerson M et al (2004) Loss of heterozygosity and its correlation with expression profiles in subclasses of invasive breast cancers. Cancer Res 64(1):64–71 Wang Z, Jiang L, Huang C, Li Z, Chen L, Gou L, Chen P, Tong A, Tang M, Gao F et al (2008) Comparative proteomics approach to screening of potential diagnostic and therapeutic targets for oral squamous cell carcinoma. Mol Cell Proteomics 7(9):1639–1650 Weng LP, Wu CC, Hsu BL, Chi LM, Liang Y, Tseng CP, Hsieh LL, Yu JS (2008) Secretome-based identification of Mac-2 binding protein as a potential oral cancer marker involved in cell growth and motility. J Proteome Res 7(9):3765–3775 Xie X, Lu J, Kulbokas EJ, Golub TR, Mootha V, Lindblad-Toh K, Lander ES, Kellis M (2005) Systematic discovery of regulatory motifs in human promoters and 3’ UTRs by comparison of several mammals. Nature 434(7031):338–345 Yamamoto T, Kamata N, Kawano H, Shimizu S, Kuroki T, Toyoshima K, Rikimaru K, Nomura N, Ishizaki R, Pastan I et al (1986) High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines. Cancer Res 46(1):414–416 Yin JQ, Zhao RC, Morris KV (2008) Profiling microRNA expression with microarrays. Trends Biotechnol 26(2):70–76 Yu T, Ye H, Sun W, Li KC, Chen Z, Jacobs S, Bailey DK, Wong DT, Zhou X (2007) A forwardbackward fragment assembling algorithm for the identification of genomic amplification and deletion breakpoints using high-density single nucleotide polymorphism (SNP) array. BMC Bioinform 8(1):145 Zhao X, Li C, Paez JG, Chin K, Janne PA, Chen TH, Girard L, Minna J, Christiani D, Leo C et al (2004) An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays. Cancer Res 64(9):3060–3071 Zhao X, Weir BA, LaFramboise T, Lin M, Beroukhim R, Garraway L, Beheshti J, Lee JC, Naoki K, Richards WG et al (2005) Homozygous deletions and chromosome amplifications in human lung carcinomas revealed by single nucleotide polymorphism array analysis. Cancer Res 65(13):5561–5570 Zhou X, Jordan RCK, Mok S, Birrer MJ, Wong DT (2004a) DNA copy number abnormality of oral squamous cell carcinoma detected by cDNA array-based CGH. Cancer Genet Cytogenet 151(1):90–92 Zhou X, Li C, Mok SC, Chen Z, Wong DTW (2004b) Whole genome loss of heterozygosity profiling on oral squamous cell carcinoma by high-density single nucleotide polymorphic allele (SNP) array. Cancer Genet Cytogenet 151(1):82–84 Zhou X, Mok SC, Chen Z, Li Y, Wong DT (2004c) Concurrent analysis of loss of heterozygosity (LOH) and copy number abnormality (CNA) for oral premalignancy progression using the Affymetrix 10 K SNP mapping array. Hum Genet 115(4):327–330 Zhou X, Temam S, Oh M, Pungpravat N, Huang BL, Mao L, Wong DT (2006) Global expressionbased classification of lymph node metastasis and extracapsular spread of oral tongue squamous cell carcinoma. Neoplasia 8(11):925–932 Ziober AF, Patel KR, Alawi F, Gimotty P, Weber RS, Feldman MM, Chalian AA, Weinstein GS, Hunt J, Ziober BL (2006) Identification of a gene signature for rapid screening of oral squamous cell carcinoma. Clin Cancer Res 12(20 Pt 1):5960–5971 Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR (1998) Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393(6685):595–599

Index

A Activator protein (AP-1) transcription factor cellular processes, 186 DNA binding domain, 186 functional integration of, 188 Jun and Fos proteins, 186 MAP kinase pathway, 187 oncogenic Ras, 186 skin development and homeostasis cytokine-regulated mesenchymalepidermal interaction, 190 keratinocyte differentiation/function, 189 tumor-associated target genes, 190 squamous cell carcinogenesis DNA enzyme strategy, 195 Fos protein functions, 192–193 gefitinib and erlotinib, 196 gene clustering, 195 genome-wide approaches, 194 Jun function, 191–192 pharmacophore modeling, 196 Podoplanin (Pdpn), 195 RAS-MAPK pathway, 196 skin cancer development, 193–194 therapeutic targets, 194 TPA-responsive elements, 185 ADCC. See Antibody-dependent cellular cytotoxicity Adipocyte differentiation-related protein (ADRP), 230 AKT. See Another kinase of transcription AKT/mTOR pathway aberrant signaling, 387–388 function and regulation, 386–387 inhibition, 385 inhibitor development, 388–390 signaling axis, 384

squamous cell carcinomas combination therapy, 393–395 overexpression, 388 tumor cell chemoresistance, 384 All-trans retinoic acid (ATRA), 262 Another kinase of transcription (AKT) activation, 416 EGFR inhibitors, 389 expression, 348 functional outcomes, 414 function and regulation, 386 members, 413 perifosine inhibitor, 389 transcription factors and TSC2 inhibition, 414 Antibody-dependent cellular cytotoxicity (ADCC), 313 Anti-EGFR antibodies antibody-dependent cellular cytotoxicity, 313 cetuximab, 310, 313 complement-dependent cytolysis, 314 inhibitors, 311–312 ligand interaction, 313 panitumumab and zalutumumab, 312 Anti-inflammatory effects PPARa, 227 PPARb/d, 230 PPARg, 234 AP-1 activation c-FOS transcription, 341 signaling pathways, 339–341 UV-induced skin carcinogenesis, 338–339 AP-1 transcription factor. See Activator protein (AP-1) transcription factor ATRA. See All-trans retinoic acid

A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3, © Springer Science+Business Media, LLC 2011

453

454 C cAMP-response element binding (CREB) protein, 135 Cancer stem cells (CSC), 378 Cancer therapy, EGFR anti-EGFR antibodies antibody-dependent cellular cytotoxicity, 313 cetuximab, 310, 313 complement-dependent cytolysis, 314 inhibitors, 311–312 ligand interaction, 313 panitumumab, 312 tyrosine kinase inhibitors, 314–315 zalutumumab, 312 therapeutic response, 315–316 CDC. See Complement-dependent cytolysis CD437 molecules anti-cancer effects, 269 cytochrome c releases, 271 mechanisms, 270 mitochondrial membrane permeability and oxygen consumption, 271 p53 tumor suppressor protein and RAR¡ receptors, 270 squamous cell differentiation, 264, 275 Cell adhesion molecules E-cadherin mediated signaling b-catenin, 12 JAK and Src signaling pathway, 11 multicellular spheroid model, 13 PI3K/AKT signaling, 14 SCC cells, 11 tumor nests, 10 tyrosine phosphorylation, 13 invasive tumor phenotype, 2 laminin-332 expression, SCC adhesion receptors, 4 a3b1 integrin, 7–8 a6b4 integrin, 8–10 cancer therapy, 4 cell migration/adhesion, 6 ECM development, 5 immunohistochemical staining, 3 keratinocytes, 4 matrilysin (MMP-7) and hepsin, 6 oral mucosal lesions, 4 plasmin and astacins, 5 proteolytic processing, 4 tumorigenesis, 5 malignant lesions, 2 Chemopreventive agents mTOR inhibitors, 397–398 mTOR targeting, 348–349

Index Comparative genomic hybridization (CGH), 432–433 Complement-dependent cytolysis (CDC), 314 Costello syndrome, 151 CREB protein. See cAMP-response element binding protein CSC. See Cancer stem cells Cyclooxygenase-2 (COX-2) arachidonic acid, 132 clinical studies, 142–143 EP receptors in skin carcinogenesis E-cadherin, 140 human keratinocytes, 139 immunohistochemical staining, 140 human squamous cell carcinomas PGE2 tumor levels, 133 premalignant lesions, 134 uterine cervix, 133 keratinocyte and SCC proliferation, PGE2 EP2/EP3 proliferation signals, 142 indomethacin inhibition of, 141 iNOS/guanylate cyclase inhibitors, 142 pathway-specific inhibitors, 141 MAPK expression, 344 mouse skin carcinogenesis models follicular keratinocytes, 135 genetic mutations, 134 papillomas, 135 UV irradiation treatments, 134 pathophysiological functions, 132 PGE2 EP receptors adenylate cyclase, 139 G protein-coupled receptors, 138 protein kinase A (PKA), 139 prostacyclin and thromboxane synthases, 132 prostaglandin synthesis, 343–344 regulation of arylhydrocarbon receptor (AhR), 135 curcumin and cocoa polyphenols, 136 HaCaT human keratinocyte cell line, 135 nuclear factor-IL6 (NF-IL6), 136 UVB irradiation, 135 SCC development celecoxib, 137 chemically-induced carcinogenesis, 138 DMBA/TPA-induced mouse skin tumor development, 136 endogenous tumor promoter, 138 indomethacin, 136 UV-induced skin tumorigenesis model, 137

Index D Diacylglycerol (DAG), 152 1,25 Dihydroxyvitamin D3 (1,25(OH)2D3) binding, 288–289 cellular proliferation, 290 cutaneous cancer, 295–296 cytokines regulation, 287–288 differentiation effects, 287 epidermal differentiation calcium-regulated differentiation, 291–292 calcium sensing receptor, 292–293 permeability barrier formation, 293–294 phosphoinositide metabolism, 292 hormonal regulation, 286 metabolism, 285–286 mouse models, 294–295 skin innate immunity, 294 squamous cell carcinomas, 288 VDR intraction, 289 DNp63 isoforms skin development adhesion, 250 basement membrane formation, 250 epidermal differentiation, 249–250 expression, 248 squamous cell carcinomas apoptosis, 253–254 tumor progression, 254 E EMT. See Epithelial-mesenchymal transition Enhancer of zeste homolog 2 (EZH2), 377 Epidermal growth factor receptor (EGFR) apoptosis and cell survival breast cancer cell line, 120 physiologic processes, 119 PI3K/AKT signaling, 120 biological significance of cellular response, 117 cutaneous keratinocyte, 116 epidermal-like pattern, 117 hair follicle cycling, 116 biology, 306, 308–309 cancer therapy clinical inhibitors, 310, 313–315 therapeutic response, 315–316 cell migration and invasion extracellular matrix degradation, 121 neoplastic cells, 120 proteolytic enzymes, 121 reepithelialization, 120 SCC progression, 121

455 cell proliferation benign and malignant cutaneous neoplasms, 118 cellular localization of, 119 epithelial monolayer, 117 goblet cell density, 118 inhibition/genetic deletion, 117 Nf2 tumor suppressor gene, 119 psoriasis vulgaris, 118 RET/PTC, 119 saccules development, 117 UV-induced actinic keratosis, 118 wound healing process, 117 differentiation in cell fate decision, 121 NOTCH signaling, 122 simple and stratified epithelium, 121 terminal differentiation program, 122 head and neck squamous cell carcinoma, 369–372 ligands and family members carcinogenesis, 115 EGF-motifs, 114 epithelial cancers, 115 ERBB receptors, 114 genetic alterations, 115 neuregulins, 114 plethora of actions, 116 signal-transduction pathway activation, 114 transactivation, 115 transphosphorylation, 114 mechanisms EGFRvIII mutation, 320–322 expression levels, 316–318 heterodimerization, 319–320 kinase domain mutations, 322–323 K-ras mutation, 324–325 nucleus functional role, 323–324 parallel growth factor receptors, 318–319 tumorigenesis dysregulation pathways, 307 ectodomain shedding, 307 ErbB receptor, 306 mutations, 307 receptor overexpression, 307 Epidermal keratinocytes calcium-regulated differentiation, 291–292 calcium sensing receptor, 292–293 permeability barrier formation, 293–294 phosphoinositide metabolism, 292 Epigallocatechin-gallate cultured human keratinocytes, 342 epidermal growth factor, 342

456 Epigallocatechin-gallate (cont.) green tea polyphenols, 341 nonmelanoma skin cancer, 342 p38 MAP kinase activation, 343 TPA-induced AP-1 activity, 342 Epithelial-mesenchymal transition (EMT), 438–439 Esophageal squamous cell carcinoma (ESCC), 100 Extracellular matrix (ECM), 2, 22 Extracellular signaling-regulated kinase 1/2 (ERK1/2), 136 EZH2. See Enhancer of zester homolog 2 F Fibroblast growth factor (FGF), 98 Focal adhesion kinase (FAK), 28 G G-protein-coupled receptors (GPCR) class IB PI3Ks activation, 410 prostate cancer, 307 Green tea polyphenols (GTPs) antioxidant properties, 341 inhibition, 342 H HDACs. See Histone deacetylases Head and neck squamous cell carcinoma (HNSCC), 1. See also AKT/mTOR pathway; Mammalian target of rapamycin (mTOR) aberrant function, 416–418 clinical trials, 398 genetic alterations, 409 genetic and epigenetic changes, 409 genetic manipulation, 374–375 molecular-targeted chemotherapy bortezomib inhibitor, 374 cancer stem cells, 378 epidermal growth factor receptor, 369–372 functions, 368 future benefits, 378–379 gene technologies, 376–377 genetic manipulation, 374–375 human papilloma virus, 375–376 preclinical studies, 369 small molecular inhibitors, 377–378 therapeutic approaches, 368 treatment modalities, 368

Index unconventional method, 374 vascular endothelial growth factor, 370, 373 mTOR anti-tumor effect, 421 clinical efficacy, 425–426 drug effectiveness, 424 lentiviral expression, 423 molecular target, 418–419 rapamycin treatment, 421 oncogenes and tumor suppressor genes, 408, 409 organ preservation chemoradiotherapy, 383 phosphatidylinositol 3-kinase (PI3Ks), 410–413 preclinical studies, 391 rapamycin antitumor mechanism, 424–425 risk factors, 408 Hepatocyte growth factor/c-MET signaling abnormality of, 99–101 c-MET, 92–94 genetic defects and oncogenic activation autocrine mechanism, 94 cell scattering, 95 chromosome 7q, 94 genetic alterations, 94, 95 human breast carcinomas, 95 RAS, 94 stromal cells, 95 transcriptional activation, 94 truncation mutation, 95 paracrine and autocrine regulatory networks, 97–98 scatter factor, 92 therapeutic targeting humanized antibodies, 101–102 small molecule inhibitors, 102–104 transcriptional regulation and cross-talk E-cadherins, 97 juxtamembrane domain, 95 phosphorylation, 97 RAS-related protein 1, 96 thrombospondin-1, 97 tyrosine1003 (pY1003), 95 vascular endothelial cell migration and angiogenesis, 97 Heterodimerization, 319–320 Histone deacetylases (HDACs), 262 HNSCC. See Head and neck squamous cell carcinoma HPV. See Human papilloma virus Human head and neck squamous cell carcinomas (HNSCC), 55 Human papilloma virus (HPV)

Index anticancer activity, 376 p53 tumor, 270 therapeutic approaches, 368 I Inducible nitric oxide synthase (iNOS), 142 Integrins anticancer therapies bidirectional signal transduction, 22 cancer progression, 23 cytoskeletal proteins, 22 intracellular pathways, 23 cell adhesion, 22 functional switch a3b1, 39 a6b4, 38–39 gain-of-function mutations, 39–40 growth factors integrin-dependent activation of, 33–34 integrin-dependent enhancement of, 34–35 signaling complexes, 35–36 laminin-332-binding integrins a3b1, 32–33 a6b4, 31–32 preclinical studies angiogenesis inhibitors, 40 cilengitide, 41 therapeutic targets, 42 tumor cells, 40 SCC expression, 27 signaling, 27–29 signal transduction pathways, 22 stratified epithelia basement membrane and tissue homeostasis, 24 tumor progenitor/stem cells, 25 avb6, 29–30 av integrin switch, 37–38 wound healing, 25–26 Interferon-g (IFN-g), 287 Interleukin-1 (IL-1), 202 J Janus kinase (JAK), 114 K Keratinocyte differentiation regulation characteristics, 284 1,25 dihydroxyvitamin D3 (1,25(OH)2D3)

457 binding, 288–289 cellular proliferation, 290 cutaneous cancer, 295–296 cytokines regulation, 287–288 differentiation effects, 287 epidermal differentiation, 290–294 hormonal regulation, 286 metabolism, 285–286 mouse models, 294–295 skin innate immunity, 294 squamous cell carcinomas, 288 VDR intraction, 289 Kinase domain (KD) molecule inhibitors, 370 mutation, 322 K-ras mutation, 324–325 L Laminin-332 a3b1 integrin beta-catenin and Smad signaling pathways, 8 cell spreading and motility, 7 epithelial-mesenchymal transition, 8 GTPases, 7 migratory/invasive cells, 8 RhoA/ROCK pathway, 7 a6b4 integrin cytoplasmic interactions, 9 hemidesmosomes, 8 mouse tumor-initiating cells, 9 oral malignancy, 10 plectin, 9 wound repair process, 8 SCC expression, 3–4 processing, 4–6 Lipopolysaccharide (LPS), 202 LNA. See Locked nucleic acid Locked nucleic acid (LNA), 440 Loss of heterozygosity (LOH), 433–434 M mAbs. See Monoclonal antibodies Mammalian target of rapamycin (mTOR) angiogenesis inhibition, 388 cancer types, 395–397 chemopreventive agents, 397–398 function and regulation, 387 inhibitors anti-tumor effect, 421 clinical efficacy, 425–426

458 Mammalian target of rapamycin (mTOR) (cont.) drug effectiveness, 424 lentiviral expression, 423 molecular target, 418–419 rapamycin treatment, 421 preclinical tumor systems, 390–391 signaling pathways aberrant activation, 348 chemopreventive agents, 348–349 keratinocyte transformation, 348 protein components, 346 TOR protein, 345 UVB activation, 345 Matrix metalloproteinases (MMPs), 26, 57 Medullary thyroid cancer (MTC), 103 MicroRNA profiling cloning and deep sequencing-based microRNA, 441–442 hybridization based microRNA, 440 quantitative real-time PCR, 441 Mitochondrial membrane permeability (MMP), 271 Mitogen-activated protein kinases (MAPKs), 151 COX-2 expression, 344 growth factor receptors, 319 mechanisms and cellular pathways, 271 MMPs. See Matrix metalloproteinases Molecular-targeted chemotherapy antimitotic and proapoptotic principle, 365 head and neck squamous cell carcinoma bortezomib inhibitor, 374 cancer stem cells, 378 epidermal growth factor receptor, 369–372 functions, 368 future benefits, 378–379 gene technologies, 376–377 genetic manipulation, 374–375 human papilloma virus, 375–376 preclinical studies, 369 small molecular inhibitors, 377–378 therapeutic approaches, 368 treatment modalities, 368 unconventional method, 374 vascular endothelial growth factor, 370, 373 methods, 366 systemic chemotherapeutic drugs, 366 Monoclonal antibodies (mAbs) anti-EGFR, 310 cytotoxic T lymphocytes, 314 mTOR. See Mammalian target of rapamycin

Index N N-(4-hydroxyphenyl)retinamide (4HPR) apoptogenic effects, 273 dependent and independent mechanisms, 267 gene expression, 272, 273 ROS effects, 269 side effects, 267 Nonmelanoma skin cancer animal models, 150 DNA repair/blistering disorders, 149 EGFR-Ras-MAPK Pathway chemokine/cytokine expression, 153 Costello syndrome, 150 DMBA/TPA tumor model, 151 genetic deletion, 153 human keratinocytes, 151 missense mutation, 152 ras-induced skin carcinogenesis, 154 Ras proteins cycle, 150 Tiam1 deficiency, 152 protein kinase C (PKC) basal keratinocytes, 156 intracellular receptor, 154 mitochondrial death pathway, 156 oncogenic Ras, 155 phorbol ester, 154 proinflammatory signaling pathways, 155 TNFa deficiency, 156 tumor microenvironment, 155 SCC and NF-kB connection chemokine receptor expression, 158 CXCR2 ligands, 157 DMBA/TPA chemical carcinogenesis model, 159 Oncogenic ras, 158 transcriptional targets, 157 ultraviolet radiation, 149 Nonmelanoma skin cancers (NMSCs) epigallocatechin-gallate, 342 risk factors, 335 Nonsteroidal anti-inflammatory drugs (NSAIDs), 136, 207 Nordihydroguaiaretic acid (NDGA), 351–352 Nuclear factor-kappa B (NF-kB) atypical activation pathways, 205–206 classical pathway of hepatocyte apoptosis, 204 lymphoid organogenesis, 202 lysine residues, 205 post-translational modifications, 204 proteolytic processing, 203 RelA phosphorylation, 204 tumorigenesis, 203

Index immunoglobulin k light-chain gene, 201 inflammation and cancer, 206–207 kappa gene expression, 202 MAPK pathways, SCC chronic skin inflammations, 208 cyclin dependent kinases (CDKs), 209 cyclosporine/UVB irradiation, 208 epidermal differentiation, 210 human epithelial cancers, 208 keratinocyte transformation, 207 molecular-targeted therapies, 210 oncogenic Ras, 207 p53 pathway cancer-promoting activity, 213 cytokine stimulation, 214 STAT pathway clinical trials, 211 HNSCC, pathogenesis of, 210 IL-6 expression, 211 interrelated signaling pathways, 210 TGFb pathway epithelial homeostasis, 211 IKKa expression, 212 SCC cell lines, 213 tumor microenvironment, 212 therapeutic strategies, 215–216 O Oral squamous cell carcinoma (OSCC) genomic analysis comparative genomic hybridization, 432–433 cytogenetic-based approaches, 434–435 loss of heterozygosity, 433–434 histopathological stages, 432 mass spectrometry-based proteomics, 442–444 risk factors, 431 small RNA and microRNA profiling technologies cloning and deep sequencing-based microRNA, 441–442 hybridization based microRNA, 440 quantitative real-time PCR, 441 transcriptome profiling techniques epithelial-mesenchymal transition, 438–439 matrix metalloproteases, 435–436 SDF-1/CXCR4 signaling axis, 438 tissue inhibitors metalloproteases, 435–436 urinary plasminogen activator and receptor, 437

459 OSCC. See Oral squamous cell carcinoma P Papillary thyroid cancer (PTC), 119 Parathyroid hormone (PTH), 286 Peptide mass fingerprinting (PMF), 442 Peroxisome proliferator-activated receptors (PPARs) PPARa anti-inflammatory effects, 227 AP-1 signaling, 226 expression, 225 fatty acid catabolism, 227 keratinocytes terminal differentiation, 226 skin tumorigenesis, 227 UV inhibition, 226 PPARb/d adipocyte differentiation-related protein, 230 anti-inflammatory activities, 230 cell growth regulation, 229 chemopreventive effects, 229 expression and effects, 228 inhibition of cell proliferation, 230 keratinocytes terminal differentiation, 230 ligand activation, 228 skin tumorigenesis, 229 PPARg anti-inflammatory activities, 234 cell growth inhibition, 233 ligand activation, 232 skin tumorigenesis, 233 transcriptional regulation cellular and physiological function, 224 expression, 225 features, 223 intracellular ligand and activation, 224 squamous epithelium, 225 Peroxisome proliferator response elements (PPRE), 224 p63 gene C-terminal sequences, 244–245 DNA binding domain, 243–244 embryonic development appendage development, 246–247 DNp63 isoforms, 248–250 epidermal development, 246 TAp63 isoforms, 247–248 expression pattern, 245–246 members of, 242 oligomerization domain, 244

460 p63 gene (cont.) squamous cell carcinomas apoptosis, 253–254 mutation and overexpression, 251 p53 interaction, 252–253 stabilization, 252 tumor progression, 251–252, 254 transactivation domain, 242–243 transcription factor, 242 Phosphatidylinositol 3-Kinase (PI3Ks) class I, 410 class IB, 411 class II, 412 class III, 412–413 human cancer, 410 Phospholipase C (PLC), 114 Phosphotidylinositol-3-kinase (PI3K), 114 PI3K/AKT pathways AMP kinase, 349–351 COX-2 regulation, 345–346 mTOR pathway aberrant activation, 348 chemopreventive agents, 348–349 keratinocyte transformation, 348 protein components, 346 TOR protein, 345 UVB activation, 345 nordihydroguaiaretic acid, 351–352 PMF. See Peptide mass fingerprinting PPARs. See Peroxisome proliferator-activated receptors Prostaglandins (PGs), 131 Protein kinase B anti-apoptosis and cell survival, 414 epidermal growth factor receptor, 415 metabolism and cell growth, 414 molecular mechanisms, 414 PI3Ks regulations, 413 translation and proliferation, 414 Protein kinase C (PKC) DNA synthesis, 165 E-cadherin, 167 epidermal homeostasis, 166 epidermal hyperplasia, 168 extracellular calcium, 166 mouse skin chemical carcinogenesis Hras activation, 169 Ras activation, 170 SCC etiology, 169 TPA, 168 tyrosine phosphorylation, 170 phospholipase C-coupled signaling events, 167 PKCa, 170–172

Index PKCd, 170–171 PKCe, 172–173 PKCh, 173 proinflammatory cytokines, 166 UV carcinogenesis, 173–174 R Retinoic acid receptors (RARs), 262 Retinoids ATRA treatment, 264 celecoxib, 272 preclinical and clinical studies chemopreventive effects, 266 limitations, 266, 267 RARb expression, 265 receptors ATRA and histone deacetylases, 262 vitamin A, 261 retinoic acid treatment, 264 squamous metaplasia, 263 synthetic retinoid CD437 molecules, 269–271 4HPR (fenretinide), 267–269 Retinoid X receptors (RXRs), 262 RNA-primed array-based Klenow enzyme (RAKE), 440 S Serial analysis of gene expression (SAGE), 435 Signal transducer and activators of transcription (STAT), 114 Skin cancer genomic mechanisms, 336–337 nonmelanoma skin cancers, 335 PPARa anti-inflammatory effects, 227 AP-1 signaling, 226 expression, 225 fatty acid catabolism, 227 human keratinocytes, 226 keratinocytes terminal differentiation, 226 mechanism, 226 skin tumorigenesis, 227 PPARb/d, 229 PPARg, 232 skin stages, 336 ultraviolet light AKT/mTOR pathways, 346–349 AMP Kinase/mTOR pathway, 349–351 AP-1 role, 338–339

Index COX-2 regulation, 345–346 genomic mechanisms, 336–337 PI3K/AKT pathways, 345 Skin tumorigenesis PPARa, 227 PPARa ligands, 225 PPARb/d, 229 PPARg, 233 Steroid receptor coactivator (SRC), 288 Synthetic retinoids CD437 molecules anti-cancer effects, 269 cytochrome c releases, 271 mechanisms, 270 mitochondrial membrane permeability and oxygen consumption, 271 RAR¡ receptors, 270 4HPR (fenretinide) dependent and independent mechanisms, 267 ROS effects, 269 side effects, 267 T TAp63 protein isoforms epidermal development, 247 TAp63a expression, 248 p53 intraction, 252–253 stabilization, 252 tumor progression, 251–252 12-O-Tetradeconylphorbol-13-acetate (TPA), 165 Therapeutic nucleic acids (TNAs), 374 Thromboxane A2 (TXA2), 138 Toll-like receptors (TLRs), 294 Transcriptome profiling epithelial-mesenchymal transition, 438–439 matrix metalloproteases, 435–436 SDF-1/CXCR4 signaling axis, 438 tissue inhibitors metalloproteases, 435–436 urinary plasminogen activator and receptor, 437 Transforming growth factor-(TGF)-b angiogenesis, 58 common mediator Smad, 80–81 epidermal wound repair autocrine/intrinsic activation, 57 E-cadherin expression, 58 epithelial cells, 56 Notch signaling pathway, 57

461 tissue injury, 56 homeostatic functions of ATM kinase, 56 cell cycle control, 55 genomic stability, 56 keratinocytes vs. DNA damage, 55 tissue homeostasis, 54, 55 tumor development, 55 human keratinocytes, 59–60 Human SCCs, 62 immune suppression, 58–59 inhibitor Smads, 81–82 receptor-associated Smads, 80 squamous carcinogenesis, mouse models of fibroblastoid phenotype, 61 TPA-induced benign skin tumor formation, 60 TbR-I human SCC cell lines and tumors, 63–73 somatic mutations, 74 tamoxifen, 62 TbR-II clinical outcome, 79 genomic alterations, 77 growth inhibitory signal, 76 immunohistochemical staining, 78 intragenic mutation, 79 keratinocytes, growth rate of, 75 lymph node metastases, 78 metastatic dissemination, 75 missense mutations, 76 pre-or post-translational level, 78 single nucleotide insertions/deletions, 77 stratified squamous epithelia, 76 Tuberous sclerosis complex (TSC), 349 Tumorigenesis EGFR dysregulation pathways, 307 ectodomain shedding, 307 ErbB receptor, 306 mutations, 307 receptor overexpression, 307 PPARa ligands, 225 skin modulation PPARa, 227 PPARb/d, 229 PPARg, 233 Tumor necrosis factor-a (TNFa), 287 Tyrosine kinase inhibitors (TKI) clinical evaluation, 315 inhibit EGFR activation, 314 tumor cell proliferation, 320

462 U Urinary plasminogen activator (PLAU), 437 UVB mediated signal transduction pathways AP-1 activation c-FOS transcription, 340 mitogen-activated protein, 339 p38 activation, 340 UV-induced skin carcinogenesis, 338–339 chemoprevention drug approach, 353, 354 COX-2 MAPK expression, 344 prostaglandin synthesis, 343–344 regulation, 344 EGFR tyrosine kinase inhibitors, 338 epigallocatechin-gallate cultured human keratinocytes, 342 epidermal growth factor, 342 green tea polyphenols, 341 nonmelanoma skin cancer, 342 p38 MAP kinase activation, 343 TPA-induced AP-1 activity, 342

Index PI3K/AKT pathway activation, 345–352 SCC prevention, 355–356 skin cancer genomic mechanisms, 336–337 nonmelanoma skin cancers, 335 skin stages, 336 transcription factor, 338 V Vascular endothelial growth factor receptor (VEGFR), 319 Vitamin D-25 hydroxylase, 285 Vitamin D receptor (VDR). See also Keratinocyte differentiation regulation carcinogenesis, 295 epidermal regulation, 285–295 functions, 283 hair follicle cycling and stem cells, 284 skin structure, 284–285 UVB radiation, 295