Growth factors and their receptors in cancer metastasis

Growth Factors and their Receptors in Cancer Metastasis

Cancer Metastasis – Biology and Treatment

VOLUME 2

Series Editors Richard J. Ablin, Innapharma, Inc., Parkridge, U.S.A. Wen G. Jiang, University of Wales College of Medicine, Cardiff, U.K.

Advisory Editorial Board Harold F. Dvorak Phil Gold Ian R. Hart Hiroshi Kobayashi Robert E. Mansel Marc Mareel

Growth Factors and their Receptors in Cancer Metastasis Edited by

Wen G. Jiang University of Wales College of Medicine, Cardiff, The United Kingdom

Kunio Matsumoto University of Osaka Medical School, Osaka, Japan

and

Toshikazu Nakamura University of Osaka Medical School, Osaka, Japan

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: Print ISBN:

0-306-48399-8 0-7923-7141-0

©2004 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print ©2001 Kluwer Academic Publishers Dordrecht All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's eBookstore at:

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Table of Contents

Contributors The Role of Leukemia Inhibitory Factor in Cancer and Cancer Metastasis FARHAD RAVANDI AND ZEEV ESTROV Interleukin-2 and Its Receptors in Human Solid Tumours: Immunobiology and Clinical Significance THERESA L. WHITESIDE, TORSTEN E. REICHERT, AND QING PING DOU.

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Interleukin-8 and Angiogenesis TRACEY A. MARTIN.

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The Role of Interleukin-11 in the Formation of Bone Metastases. NAOYA FUJITA AND TAKASHI TSURUO.

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Therapeutic Potential of Adenovirus Mediated Interleukin-12 Gene Therapy for Prostate Cancer SHIN EBARA, YASUTOMO NASU, TAKEFUMI SATOH, SATORU SHIMURA, CHRIS H. BANGMA, GERALD W. HULL, MARK A. MCCURDY, JIANXIANG WANG, GUANG YANG, TERRY L. TIMME, AND TIMOTHY C. THOMPSON. Fibroblast Growth Factors and Their Receptors in Metastases of Prostate and Other Urological Cancers ZORAN CULIG, MARCUS V. CRONAUER, ALFRED HOBISCH, GEORG BARTSCH AND HELMUT KLOCKER.

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Insulin-Like Growth Factor Axis Elements in Breast Cancer Progression EMILIA MIRA, ROSA ANA LACALLE, CARLOS MARTÍNEZ-A. AND SANTOS MAÑES. The Role of Platelet Derived Growth Factor (PDGF) and Its Receptors in Cancer and Metastasis SARA WEISS FEIGELSON, CHERYL FITZER-ATTAS, LEA EISENBACH.

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TGF receptor Signaling in Cancer and Metastasis MARTIN OFT.

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VEGF-C/VEGFRS and Cancer Metastasis YUTAKA YONEMURA, YOSHIO ENDOU, TAKUMA SASAKI, KAZUO SUGIYAMA, TETUMOURI YAMASHIMA, TAINA PARTANEN, KARI ALITALO.

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HGF-c-met receptor Pathway in Tumor Invasion-Metastasis and Potential Cancer Treatment with NK4 KUNIO MATSUMOTO AND TOSHIKAZU NAKAMURA.

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Growth Factor Receptors and Cell Adhesion Complexes in Cytoskeletal Assembly/Anchorage GAYNOR DAVIES, MALCOLM D. MASON AND WEN G. JIANG

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Index

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Contributors

Alitalo, Kari. Molecular/Cancer Biology Laboratory, Haatman Institute, University of Helsinki, PL 21, 00014 Helsinki, Finland Bangma, Chris H. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA Bartsch, Georg. Department of Urology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria Cronauer, Marcus V. Department of Urology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria Culig, Zoran. Department of Urology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria Davies, Gaynor. Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN.UK. Dou, Qing Ping. H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA Ebara, Shin. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA Eisenbach, Lea. Weizmann Institute of Science, Department of Immunology, Rehovot, Israel Endou, Yoshio. Experimental Therapeutics Cancer Research Institute, Kanazawa University, Takara-Machi 13-1, Kanazawa 920, Japan Estrov, Zeev. Department of Bioimmunotherapy, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas, U.S.A

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Feigelson, Sara Weiss. Weizmann Institute of Science, Department of Immunology, Rehovot, Israel Fitzer-Attas, Cheryl. Weizmann Institute of Science, Department of Immunology, Rehovot, Israel Fujita, Naoya. Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Hobisch, Alfred. Department of Urology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria Hull, Gerald W. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA Jiang, Wen G. Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK. Klocker, Helmut. Department of Urology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria Lacalle, Rosa Ana. Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Universidad Autónoma de Madrid, E-28049 Madrid, Spain. Mañes, Santos. Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Universidad Autónoma de Madrid, E-28049 Madrid, Spain. Martínez, Carlos. Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Universidad Autónoma de Madrid, E-28049 Madrid, Spain. Mason, Malcolm D. Department of Medicine, Section of Clinical Oncology, Velindre Hospital, Cardiff, CF4 7XL. UK Martin, Tracey A. Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN. UK. Matsumoto, Kunio. Division of Biochemistry, Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan McCurdy, Mark A. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA

Contributors

Mira, Emilia. Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Universidad Autónoma de Madrid, E-28049 Madrid, Spam. Nakamura, Toshikazu. Division of Biochemistry, Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan Nasu, Yasutomo. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA Oft, Martin. UCSF Cancer Center - Box 0875, University of California, San Francisco, 2340 Sutter Street, Room S271, San Francisco, CA 941430128, USA Partanen, Taina. Molecular/Cancer Biology Laboratory, Haatman Institute, University of Helsinki, PL 21, 00014 Helsinki, Finland Ravandi, Farhad. Department of Bioimmunotherapy, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas, U.S. A Reichert, Torsten E. University of Mainz, Mainz, Germany Sasaki, Takuma. Experimental Therapeutics Cancer Research Institute, Kanazawa University, Takara-Machi 13-1, Kanazawa 920, Japan Satoh, Takefumi. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA Shimura, Satoru. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA Sugiyama, Kazuo. Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuoo-Ku, Tokyo 104, Japan Thompson, Timothy C. Scott Department of Urology, Cell Biology and Radiology, Baylor College of Medicine, Houston, Texas, USA Timme, Terry L. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA Tsuruo, Takashi. Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Wang, Jianxiang G. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA Wang Yang, Guang. Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA

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Whiteside, Theresa L. University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA Yamashima, Tetumori. Department of Neurosurgery, School of Medicine, Kanazawa University, Takara-Machi 13-1, Kanazawa 920, Japan Yonemura, Yutaka. Second Department of Surgery, School of Medicine, Kanazawa University, Takara-Machi 13-1, Kanazawa 920, Japan

Chapter 1 THE ROLE OF LEUKEMIA INHIBITORY FACTOR IN CANCER AND CANCER METASTASIS

Farhad Ravandi and Zeev Estrov Department of Bioimmunotherapy, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas, U.S.A

Key words:

Leukemia inhibitory factor (LIF), Cancer, Metastasis

Abstract:

Leukemia inhibitory factor (LIF) is a cytokine that exerts pleiotropic activities. LIF is a member of the interleukin-6 family of cytokines which share a similar receptor complex and signal through the gp 130 receptor subunit. Several neoplastic cells originating from various tissues express either LIF, its receptor, or both and respond to this cytokine. Data accumulated thus far provide a complex picture of LIF activities with LIF being stimulatory, inhibitory or having no effect, depending on the system in which it is studied. LIF appears to play an important role in stimulating the growth of certain tumours, and in affecting the surrounding tissue and the target organ of tumour metastases, particularly bone and skeletal tissue. Overproduction of LIF is likely to have significant constitutional effects. Studies using animal models have shown that LIF induces cachexia, metastatic-type bone calcifications, thrombocytosis, and an abnormal immune response. It is therefore possible that suppression of LIF activity might have a beneficial effect in some cancer patients.

induce proliferation, inhibit proliferation or cause apoptosis, depending on the system in which this cytokine is studied. Several studies have shown that LIF’s divergent physiological effects have been adopted by a variety of neoplastic cells and that LIF takes part in the pathophysiology of cancer. In many neoplasms LIF, produced by either normal tissue or tumour cells, provides the cancerous process with growth and survival advantage. LIF was initially characterized by its ability to induce differentiation of the

1. INTRODUCTION Leukemia inhibitory factor (LIF) is a pluripotent cytokine with pleiotropic activities. LIF is a member of a family of cytokines that includes the ciliary neurotrophic factor (CNTF), interleukin (IL)-6, IL-11, oncostatin-M (OSM), and cardiotropin-1 (1,2). These cytokines are grouped as a family because of their shared helical bundle structure (3-7), shared subunits of their receptor complexes, and in some cases, overlapping functions (8,9). As other members of this family, LIF can either 1

W.G. Jiang et al. (eds.), Growth Factors and their Receptors in Cancer Metastasis, 1–25. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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murine myeloid leukemia cell line M1 (10-15) and was cloned from a murine Tcell library (12,16). Independently, a human molecule in the supernatant of Tcell clones was identified and termed human interleukin for DA cells (HILDA) (17-20). Once cloned, this molecule was found to be homologous to its murine counterpart (21-23). Subsequently, additional characteristics of LIF were described, and it was given several other names, including differentiation factor (Dfactor) (24-25), differentiation-inducing factor (DIF) (26), differentiation inhibitory activity (DIA) (27), differentiation-retarding factor (DRF) (27,28), hepatocyte-stimulating factor III (HSF III) (29), melanoma-derived lipoprotein lipase inhibitor I (MLPLI) (34), cholinergic neural differentiation factor (31), and osteoclast-activating factor (OAF) (26,32) (Table 1). However, because LIF exerts a broad spectrum of activities and despite its diverse and sometimes opposing effects on different leukemia cell lines (21,26,27,29,32,3335), LIF has become the official name of this cytokine (1). The effects of LIF on various tissues provide several clues to its possible role in cancer. For example, LIF stimulates embryonic stem cell proliferation (36-40). It affects blastocyst implantation (36-41) and influences the development of peripheral nerves from their precursors in the embryonic neural crest (32,42), which implies that LIF can stimulate immature cells and probably tumour cells with immature cell characteristics. In addition, LIF was shown to induce a catabolic state

Chapter 1 and cachexia in nude mice and in primates (43-45). It stimulated the release of acutephase proteins from hepatocytes, (45-47) and affected bone metabolism by inducing both osteoblastic and osteoclastic activities (48-52). These effects are characteristic clinical features of patients with neoplastic diseases likely to be induced by various cytokines including LIF. In this chapter we describe the physiological characteristics and the pathophysiological role of LIF in cancer and cancer metastasis. 2. MOLECULAR AND CELLULAR CHARACTERISTICS 2.1 LIF Distribution in Cells LIF is expressed in cells of different tissues, including osteoblasts, keratinocytes, thymic epithelium, T cells, monocytes, skin fibroblasts, embryonic stem cells, bone marrow stroma cells, central nervous system cells, hepatocytes, and a number of tumour cell lines that have become a source for this cytokine (1,20,21,26,37,53-57) (Table 2). 2.2 Structure and Genetics Naturally occurring LIF appears as a monomeric glycoprotein with a molecular weight is between 40 and 70 kDa despite a polypeptidic core of 22 kDa (3,21,58). This is due to the presence of several putative sites of N-glycosylation in the primary structure of the molecule allowing extensive post-translational modifications (29).

Abbreviations: LIF, leukemia inhibitory factor; LIFR, LIF receptor; TNF, tumour necrosis factor; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; OSM, oncostatin M; IFN, intrerferon; IL, interleukin; IFN, interferon, CNTF, ciliary neurotrophic factor; CFU, colony forming unit; MM, multiple myeloma; TGF, transforming growth factor; G-CSF, granulocyte colony-stimulating factor; HSF, hepatocyte stimulating factor; SP, neuropeptide substance P; CNS, central nervous system

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LIF is encoded by genes localized at chromosome 11A1 in mice and chromosome 22ql2 in humans (59,60). Although the location of the human gene and the high incidence of a translocation involving t(11;22)(q24q12) in Ewing’s sarcoma stimulated considerable interest, further analysis using somatic cell hybrids and pulse-field gel electrophoresis has shown that the gene is located distal to the breakpoint and is not involved in this translocation (61). The sequences of cloned LIF genes from four mammalian species are highly conserved in the coding regions (62,63). Murine and human LIF have the complete nucleotide sequence of 8.7 and 7.6 kilobase pairs, respectively (12,14,16, 64,65). Both genes consist of three exons, two introns, and an unusually large 3’untranslated region that is 3.2 kilobase pairs (65). The LIF transcript is 4.2 kilobases in length and predicts a sequence with 179 residues for the mature protein and a 79% homology between the murine and human products (12,14,64). This is the primary and biologically active form of LIF. The promoter region of the LIF gene contains four highly conserved TATA elements, with two identified start sites of transcription (62). Three regions within the 5’ flanking region have been identified as important to the function of the LIF promoter (21,62). The structure of LIF has been determined (66). The main chain fold comprises four α-helices linked by two loops. There are two regions of the LIF molecule involved in receptor interaction and biological function. The first is located within the D helix and comprises residues 161-180, and the second is located between residues 150 and 160 at the C-terminus of the CD loop.

Chapter 1 2.3 Biological Forms of LIF Two forms of LIF were detected: the “diffusible” (D) LIF glycoprotein and an “immobilized” (M) form incorporated into the extracellular matrix (67). Both D- and M-LIF forms are produced by the expression of alternative transcripts that diverge throughout the first exon and use different promoters. The two LIF forms are encoded by mRNAs that are spliced differently at the exon 1/exon 2 boundary. The transcript D encodes the diffusible form and the transcript M encodes the matrix-associated form. Splicing a 5’ exon to exons 2 and 3 of the LIF transcription unit produces the latter. The two transcripts co-migrate on agarose gel and therefore can be distinguished by ribonuclease protection analysis but not by Northern blot analysis. The molecular organization of the gene for LIF can explain the different localization of its two forms. Exons 2 and 3 produce the core hydrophobic secretory sequences, whereas the extracellular localization is determined by the first exon. Therefore, changes in the amino terminal of the translocation product direct the formation of a mature, functional LIF with extracellular matrix localization (reviewed in 1 and 2). Although the reported molecular weight of LIF ranges from 38 to 67 kDa, this heterogeneity can be explained by variable glycosylation of the protein (35,64). Recombinant forms of LIF displaying varying patterns of glycosylation (yeast-derived and Escherichia coli-derived) are active (12,64). 2.4 LIF Receptors and Their Signaling The IL-6 cytokine family members share common signaling components i.e. the LIF receptor (LIFR) and the receptor

1. LIF and cancer metastasis subunit gp130 (68). The LIFR was first isolated and found to be structurally related to the gp130 component of the IL6 receptor and the granulocyte colonystimulating factor (G-CSF) (69). This receptor is now termed LIFR It binds with low affinity to gp130, whereas LIF binds with high affinity to the LIFRß/gp130 complex, initiating its signal trasduction (70). Other components of this receptor complex, used by other members of the IL-6 cytokine family, have been identified. For example, the receptor component CTNFR is utilized by CTNF(71,72). LIF binds to a variety of cells from different tissues (24,33,56,73,74). Following receptor binding, signaling pathways involving both protein tyrosine and serine/threonine kinases are activated. Both the Janus-kinase-signal transducer and activator of transcription (JAKSTAT) and mitogen-activated protein kinase (MAPK) pathways are activated (39,72,75) (Figure 1). Activated STAT molecules dimerize and translocate to the nucleus. Although there are at least six STAT proteins, STAT3 tends to be the protein that is activated by LIF (76). The LIFRß is essential for motor neuron development as demonstrated in studies with the LIFRß knockout mouse model (77) (see below). 3. BIOLOGICAL EFFECTS OF LIF 3.1 Effects on the Reproductive System and Embryogenesis

The mammalian embryo develops from a quasi-stem cell system controlled by regulatory factors, one of which is LIF (13,32,78,79). LIF is expressed in both embryonic and maternal tissue. LIF transcripts were also detected in mouse blastocysts, implying its role as a regulator of embryonic stem cells and a

5 mediator of the trophoblast development (36,74). In the LIF knockout mouse model, homozygous and heterozygous null mice for a functional LIF gene enabled investigating the role of LIF in the reproductive system (80). Male -/- LIF mice were fertile, but female mice, although able to produce viable blastocytes, failed to implant and were therefore sterile. However, the injection of LIF into homozygous -/- female restored blastocyte implantation (81). Male mice engrafted with a LIFproducing cell line showed complete absence of spermatogenesis, whereas female mice had reduction or complete absence of corporae luteae (47). Recent data confirm the crucial role of LIF during implantation and pregnancy in primates such as monkeys (82) and western spotted skunk (83). In addition, it has been demonstrated that LIF has a crucial role in the maintenance of pregnancy in humans (84,85). 3.2 Effects on Bone Metabolism

Several studies have clearly demonstrated the role of LIF in bone remodeling (86). Both osteoclast and osteoblast activities are either stimulated or suppressed by LIF depending on the developmental stage of the respective cells. When the local effect of LIF was studied in mice by injecting the cytokine over one hemicalvaria, two major effects were observed: 1) increased osteoclastic activity and bone resorption in the injected right hemicalvaria and 2) increased total mineralization, including the periosteal area in the non-injected left hemicalvaria (87). Additional studies using an array of laboratory assays showed that LIF inhibited osteogenic calcification (88), affected osteoclast migration (89), increased osteoclast differentiation (90),

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1. LIF and cancer metastasis inhibited bone module formation (91), and reduced bone calcification (92). Engraftment of mice with LIF-producing cells yielded results similar to those described above (87). The engrafted mice had increased calcifications in both skeletal and extra-skeletal tissues such as the myocard (47). In vitro stimulation of bone resorption by LIF was accompanied by the release of calcium from prelabelled mouse calvaria. This effect, caused by an increase in the number of osteoclasts, could be inhibited by indomethacin, indicating that it is mediated through the activation of prostaglandins. The prostaglandindependent bone-resorptive effect of LIF is similar to that of other cytokines such as IL-1, tumour necrosis factor (TNF), and transforrming growth factor (TGF)-ß (93,94). 3.3 Effects on Lipid Metabolism LIF, like TNF, IL-1, and interferon (IFN)can inhibit the enzyme lipoprotein lipase, which is a key enzyme in triglyceride metabolism (95,96). High levels of LIF induced a fatal catabolic state with cachexia in mice and monkeys (12,33,43-45,97). It is likely that this effect of LIF is mediated by its ability to suppress adipogenic processes through its enzyme inhibitory effect. Thus, the inhibition of lipoprotein lipase is likely to reduce the intake of fatty acids by adipocytes and lead to cachexia. 3.4 Role of LIF in Inflammation and Tissue Injury A number of studies demonstrated the role of LIF in inflammation. LIF was found to have both a pro- and an antiinflammatory role in a variety of inflammatory disorders (98). LIF mRNA increased in various mouse tissues during systemic inflammation triggered by the

7 injection of either endotoxin or lipopolysaccharide (LPS) (99). Interestingly, passive immunisation against LIF prior to LPS injection protected the mice from the lethal effect of high-dose LPS (100), indicating that LIF is one of the agents associated with the lethality of septic shock. Surprisingly, LIF injection prior to a challenge with high dose LPS protected against the lethal dose of LIF (101,102). This dual effect of LIF was found in different diseases in humans, such as rheumatoid arthritis (103,104). LIF is highly elevated in the synovial tissue and fluids of patients with rheumatoid arthritis. In addition, human articular chondrocytes and synovial tissue produce LIF that in turn may upregulate proinflammatory cytokines (105-109). Injection of LIF-binding proteins into a goat joint atenuates the inflammatory reaction caused by a prior injection with LIF (110). LIF has also been detected in the pleural effusion of patients with tuberculosis (111) and in the bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome (112). Local inflammatory processes have been shown to be mediated by LIF (113115). On the other hand, the response to injection of complete Freud’s adjuvant is significantly augmented in adult LIF knock-out mice (116). Some of the differences among these studies could be explained by dissimilar experimental designs, dose of LIF, and species and age of the studied animals. However, divergent effects of LIF on the thymus and on T and B lymphocytes (see below) may also contribute to dissimilar results in various experimental models. LIF also plays a role in tissue repair in cases such as stab wound injury and injury to the central and peripheral nervous systems (117-120). LIF mRNA was shown to be upregulated after muscle

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crash injury (121) consistent with LIF’s role as a stimulator of human muscle precursor-cell proliferation (122,123). In LIF knock-out mice, infiltration by neutrophils, macrophages, and mast cells is delayed in lesions of both the central and peripheral nervous systems (124), suggesting that LIF could be chemotactic for inflammatory cells. 3.5 Effects on Hepatic Function The hepatocyte-stimulating factor (HSF) III initially detected in cultured keratinocytes and squamous carcinoma cell lines was found to be identical to LIF (29,125). The liver secretes acute-phase proteins into the circulation upon various stimuli including those induced by several cytokines including LIF (126). Injection of LIF into rhesus monkeys strongly increased the levels of acute-phase proteins (127). This molecule is able to induce the production of a number of acute-phase proteins by the hepatocytes, an ability that it shares with other cytokines including IL-6 and TNF. Thus hepatocytes produce LIF which is capable of inducing the production of acute-phase proteins by the liver, suggesting an autocrine role for LIF. 3.6 Effects on the Nervous System Cholinergic neuronal differentiation factor, a protein acting on sympathetic neurons to induce acetylcholine synthesis and cholinergic function, is now known to be identical to LIF (32). LIF affects the development of peripheral neurons from their precursors in the embryonic neural crest (42). LIF also participates in the regulation of the neuropeptide substance P (SP) in sympathetic neurons, increasing SP in both neuronal cell cultures and cultures containing a mixture of neuronal and non-neuronal cells (126). LIF acts as a survival factor on mature sensory

Chapter 1 neurons (127). Neuronal differentiation of spinal-cord precursors is dependent on a functioning LIFRß (128). In the LIFRß knock-out mouse model, mice die shortly after birth, and they reveal a profound loss of astrocytes in the brain stem and spinal cord, and neurons with pycnotic nuclei and cytoplasmic vacuoles (77,129). These findings and the distribution of the LIFR mRNA in the brain and spinal cord suggests that LIF affects neuronal cells in the adult as well as during development (128). LIF can prevent the death of axotomised sensory and motor neurons (129,130). In the Wobbler mouse model, the animals develop lower-motor neuropathy. Injection of LIF has a sparing effect, improving the neuropathy (131), further demonstrating the complex effects of LIF in the nervous system. 3.7 Effects on the Hematopoietic System LIF has been characterized by its ability to induce differentiation and suppress the growth of M1 myeloid leukemia cells (12-15,48,132). However, in subsequent studies, LIF stimulated, inhibited or had no effect on leukemia cells, depending on the cell line or the system in which LIF’s activity was investigated (133-137). Similarly, LIF induces a divergent effect on normal hematopoietic progenitors. Exposure to LIF reduces the proliferative capability and survival of normal hematopoietic progenitors (138). Although LIF had no effect on CD34+ human bone marrow cells, it enhanced the stimulating effect of IL-3 (139). In another study, LIF stimulated the growth of colony-forming units granulocyte-erythroid-macrophagemegakaryocyte (CFU-GEMM) and CFUeosinophil (CFU-Eo), and burst-forming units-erithroid (BFU-E) colony-forming cells (140). Similar results were obtained

1. LIF and cancer metastasis with CD34+ cells stimulated with IL-3 and IL-6. LIF augmented the effect of megakaryocyte colony-forming cell stimulators and enhanced a chemoattractant effect on human and mouse eosinophils (19,20,73). Bone marrow stroma cells constitutively express LIF mRNA (53). Exposure of hematopoietic stroma to either , or increased the level of LIF mRNA (53,141). Stroma obtained from marrow cells of patients with chronic myelogenous leukemia who had high levels of expressed high levels of LIF (53,142). The first clue of the role LIF plays in normal hematopoiesis in vivo came from experiments carried out in mice (47,48). Mice tranfected with LIF-producing cells exhibited thymic atrophy and extramedullary hematopoiesis (47). Daily injection of LIF caused granulocytosis and an increase in megakaryocytes and platelets (46). Transgenic mice constitutively expressing diffusible LIF displayed B-cell hyperplasia, profound disorganization of the thymus, and loss of cortical CD4+ and CD8+ lymphocytes. Transplantation of transgenic bone marrow into wild-type mice recipients transferred the thymic and lymph node defects (143). Knock-out of the LIF gene significantly impaired the hematopoietic system (80). Both early and mature hematopoietic progenitors were dramatically reduced and a dose effect was seen because heterozygotes were less affected. However, mature hematopoietic elements in the marrow, spleen, and peripheral blood were normal, indicating that the defect was in the stem cell pool rather than in differentiation as found in other studies (144-149). Homozygous null mice for gp130 die mainly of cardiac defects due to the elimination of

9 cardiotrophin-1 signaling (3). In this mutant, the number of mononuclear cells in fetal liver was drastically reduced, as were the numbers of both early and mature CFUs. The thymuses were 50 percent smaller, consistent with other studies showing LIF’s role in hematopoiesis. 4. ROLE OF LIF IN CANCER Several tumour cell lines and neoplastic cells from various tissues produce LIF and express LIF receptors. However, the functional significance of either LIF or LIFR in human neoplasia is not fully understood. LIF can stimulate growth, induce differentiation, or trigger apoptotic cell death of various tumour cells (1,141,150,151) and data on the mechanisms controlling this diverse array of effects are scanty. Results of in vivo animal trials shed light on some of the possible roles of LIF in cancer and cancer metastasis. Cachexia (43,44), subcutaneous and abdominal fat loss, and elevated leukocyte and platelet counts often found in patients with metastatic cancer were induced by LIF in both mice and monkeys (46-48). In addition, at a high dose, LIF induced myelosclerosis whereas a low dose induced megakaryocytosis, reduced marrow cellularity and caused lymphopenia (48) suggesting a possible role for LIF in the pathogenesis of myeloproliferative disorders such as myelofibrosis and in marrow sclerosis. Furthermore, mice engrafted with FDS-P1 cells that produce high levels of LIF developed a fatal syndrome with cachexia, atrophy of liver and kidney, and excess bone formation with increased osteoblastic activity that resulted in metastatic-type calcifications (47) implying a role for LIF in bone tumours and neoplasms metastasizing to bone.

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Chapter 1

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Several in vitro studies were performed to delineate the effects of LIF on various tumours from different tissues. Though studies of cell lines often yielded conflicting results, experiments with fresh tissue confirmed LIF’s role in tumour growth, disease progression, and tumour metastasis (Table 3). 4.1 Hematological Malignancies LIF was originally characterized by virtue of its ability to induce differentiation in the murine myeloid leukemia cell line Ml, a property that it shares with IL-6 (13-15). However, LIF had no effect on the murine leukemia WEHI 3BD+ cell line that differentiates in response to IL-6 (150,192) whereas it stimulated the growth of the murine IL-3dependent DA-1 myeloid leukemia cell line (19,152). When injected into mice that had been implanted with T-22 cells, a subclone of the M1 cell line, it prolonged the animals’ survival by inducing differentiation (157). LIF is also produced by the THP-1 human monocytic leukemia cell line (58). LIF was found to be expressed in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cultured bone marrow stroma cells (155) and in human leukemia cell lines (140,154). Although LIF stimulated human normal marrow hematopoietic progenitor cell growth (139,140,193) and stroma-derived macrophage proliferation (194), it inhibited human leukemia cell growth (156,157). LIF also affects cells of the lymphoid lineage. T-cell clone (alloreactive) from lymphocytes rejecting kidney allografts and thymic epithelial cells (55) were found to produce LIF (reviewed in 1 & 2). Whereas normal human T lymphocytes did not bind radio-iodinated LIF (164), cells infected with human T-cell leukemia

Chapter 1 virus (HTLV)-I and –II expressed LIF (159) and proliferated in response to this cytokine (160). Similarly, various lymphoma cell lines were found to produce LIF (158), and LIF production was upregulated by IL-1 (66). Similar to IL-6, LIF plays a role in multiple myeloma (MM) cell proliferation. Human MM cell lines (161) and myeloma and plasmacytoma cells express LIF (162), LIFR, and the gp130 receptor subunit (163) and proliferate when exposed to LIF (163,164). Thus, similarly to IL-6 LIF may act as an autocrine growth factor for MM cells. The capability of LIF to induce both lytic and osteogenic effects in skeletal tissue, suggest that the osseous abnormalities typically found in MM are induced, among other factors, by LIF-producing myeloma cells. 4.2 Bone Tumours The effects of LIF on bone remodeling with LIF inducing both osteoclastic and osteoblastic activities suggest that LIFproducing tumour cells may significantly alter bone and skeletal tissue. Because the LIF gene was found to be mapped to chromosome 22q11-q12.2 (60), a question arose whether this site might be affected by chromosomal translocations that are related to tumours of neural-crest origin such as Ewing’s sarcoma and peripheral neuroepithelioma cytogenetically characterized by t(11;22)(q24;q12). It was found that the LIF gene is located far away from the Ewing’s sarcoma translocation (61,195). Nevertheless, bone tumours were found to produce high levels of LIF. Marusic et al. tested various rodent and human immortalized malignant bone tumour cell lines and found that LIF is constitutively expressed in several cell lines and is cytokine-inducible in others (165). LIF

1. LIF and cancer metastasis and LIFR were found in the cytoplasm of multinucleated giant tumour cells. Furthermore, LIF-stimulated giant tumour cells displayed osteoclast immunocytochemical features and resorbed large amounts of dentin (167,168). Additional indirect evidence for the role of LIF in bone tumours was provided by Gouin et al. who detected LIF in 34.7% of urine samples obtained from patients with a variety of bone tumours. They also found high LIF protein levels in supernatants of both neoplastic and benign bone tumour cells (166). Although LIF provides various bone tumours with a proliferation advantage and modulates their effects on bone tissue in either an autocrine or paracrine fashion, several studies showed that tumour cells that metastasize to bone may utilize similar mechanisms. 4.3 Breast Cancer Because LIF affects bone tissue and is produced by marrow stroma cells (86,155), several investigators asked whether LIF has a role in tumours such as breast cancer which metastasizes to this site (196). This was further emphasized by the study of Akatsu et al. who showed that the mouse mammary cell line MMT060562 produces LIF and supports osteoclast formation via a stroma celldependent pathway (197). Studies in breast cancer cell lines showed that some of these cells produce LIF, others express LIFR, and the cells may or may not respond to LIF. The diversity of cell lines and cell line clones that may have different features in different laboratories present a wide array of complex biological characteristics. For example, the estrogen-dependent breast cancer cell lines MCF-7 and T47-D do not produce LIF however their growth is stimulated by this cytokine (169-171).

13 MCF-7 cells bind LIF and, like several other breast cancer cell lines (172), express the gp130 subunit (169). In contrast, MDA-231 cells that express neither estrogen nor progesterone receptors produce LIF but their growth is not affected by this cytokine (170). Interestingly, progesterone treatment of MDA-231 cells co-transfected with both estrogen and progesterone induced the expression of LIF’s promoter (198). LIF also stimulated the estrogen-dependent T47D and the estrogen-independent SKBR3 and BT20 cell lines; inhibited, according to one study, MCF-7 cells (172), but had not effect on normal mammary epithelial cell growth (169,171). Interestingly, the SV40transformed mammary epithelium cell line HBL 100 was found to produce LIF (58). Breast cancer cells from 6 of 6 tumour samples expressed LIF transcripts (174) and widespread LIFR mRNA expression was found in primary breast tumours (172). Immunostaining of tumour samples obtained from 50 breast cancer patients detected LIF in 78% and LIFR in 80% of the samples. The presence of LIF correlated with a low S-phase fraction of the cell cycle and diploidy, whereas the presence of LIFR correlated with dipoidy, low S-phase fraction, and of estrogen receptor positivity. LIF and LIFR were also expressed in normal breast epithelium in 87% and 77% of the specimens, respectively (173). LIF stimulated colony formation of breast cancer cells obtained from five different patients in a dosedependent fashion (169) and the growth stimulation correlated with the presence of LIFR in these specimens (173). Taken together the data suggest a complex role of LIF and LIFR in breast cancer growth regulation. Because the bone marrow stroma produces LIF (155) and other cytokines such as stem cell

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factor that stimulate breast cell proliferation (169), cells that express LIFR and respond to these cytokines may have a growth advantage in the bone marrow microenvironment. 4.4 Kidney Cancer Renal carcinoma, like breast cancer, frequently metastasizes to bone. In addition, systemic symptoms, such as weight loss and fever, are common in kidney cancer and likely to result from overproduction of inflammatory cytokines. Moreover, the process of mouse nephrogenesis involves at least two distinct stages that can be blocked by LIF (199), and rat and human mesangial cells produce LIF and respond to this cytokine by transiently expressing the immediateearly genes c-fos, jun-B, and Egr-1 (200). These data suggest that LIF affects renal cell proliferation. Studies with cell lines have shown that both the primary kidney cancer line A-498 and the ACHN cell line established from pleural effusion of metastatic renal carcinoma produce LIF. Anti-LIF antibodies suppressed the cells’ growth and the inhibitory effect was reversed by exogenous LIF. These data suggest that the endogenously produced LIF stimulated kidney cancer cell line proliferation (170). 4.5 Prostate Cancer Prostate cancer cells selectively metastasize to the axial skeleton to produce osteolytic lesions. Laboratory data suggest that LIF plays a role in this disease. Paracrine-mediated growth factors may play a role in prostate cancer growth and development (201). In addition, IL-6, often expressed in parallel with LIF (202), was found to be expressed in prostate tissue (175) and might stimulate prostate cancer growth during

Chapter 1 disease progression (203). The hormoneindependent cancer cell lines TSU, PC-3 (204), and DU 145 (170) produce LIF and express gp130 (204). DU 145 cells did not proliferate in response to this cytokine (170,204) however, anti-LIF antibodies inhibited the cells’ growth (170). Thus, although only a few studies investigated the effect of LIF on prostate cancer cells and no data on binding of LIF to cellular LIFR are available, results from the above-described studies suggest that LIF plays a role in prostate cancer. 4.6 Malignant Melanoma In 1989, Mori et al. found that a factor produced by the melanoma cell line SEKI induced cachexia in tumour-bearing nude mice and inhibited lipoprotein lipase. This factor designated melanoma-derived lipoprotein lipase inhibitor was found to be identical to LIF (34,176). Subsequent studies found that LIF mRNA is expressed in various melanoma cell lines of which several produce the protein (58,177). Interestingly, oncostatin-M, another member of the IL-6 cytokine family, significantly increased LIF production by melanoma cells (205). LIF was detected in more than 60% of human melanoma samples and was found to enhance the expression of the intracellular adhesion molecule (ICAM)-1 in melanoma cells (177). Shedding of the soluble form of ICAM-1 from tumour cells impairs immune recognition and leads to tumour escape. Therefore, LIF may provide melanoma cells with a survival advantage. Furthermore, melanoma cells transfected with LIFR showed increased tumour growth suggesting that LIF may directly stimulate the growth of melanoma cells that express LIFR and provide them with a survival and growth advantage.

1. LIF and cancer metastasis 4.7 Hepatoma Only a few groups studied the effects of LIF in hepatoma. It was found that LIF is expressed in the HuH-7 and Hep-G2 hepatoma cell lines (179). LIF upregulated the expression of acute-phase proteins in the rat H-35 hepatoma cells (180) and activation of LIFR initiated signaling through the JAK pathway in Hep-G2 cells (181). 4.8 Gastrointestinal Malignancies The mRNA of LIF, LIFRß, and gp130 was detected in six stomach cancer, two colon cancer, one esophageal cancer, one gall bladder cancer, and seven pancreatic cancer cell lines (179). LIF induced apoptosis in the AZ-521 gastric and the GBK-1 gall bladder cancer cell lines and was detected in the MIA PACA pancreatic carcinoma cells (58). LIF did not affect the growth of either stomach or cancer cell lines; however, it stimulated the proliferation of two of seven pancreatic cancer cell lines (171). LIF is produced by the colon carcinoma cell lines SW948 and HRT18 (58). It has been shown to enhance human colon carcinoma HT24 cell proliferation suggesting that LIF facilitates the transition from ulcerative colitis to colon cancer (182). Because the results of cell line studies are inconsistent and since patient tumour tissue has not been studied yet, the biological significance of the cell line studies remains to be determined. 4.9 Central Nervous System Tumours Considering the variety of effects induced by LIF in the central nervous system (CNS), its involvement in CNS tumour growth is not surprising. LIF, LIFR, and the gp130 receptor subunit were detected in medulloblastoma tumour cells. Twelve of 12 tumour samples expressed LIF, and more than 90% of the

15 samples expressed LIFR and gp130. (185). In addition, LIF antisense inhibited medulloblastom cell proliferation (186). Taken together these data suggest that LIF acts as an autocrine growth factor in medulloblastoma. LIF was also studied in other CNS tumours. It either inhibited (183) or had no effect (184) on glioma cell lines. Meningioma cells expressed LIF transcripts; however, LIF did not affect the cells’ growth in vitro (206). 4.10 Other Neoplasms Several groups have reported LIF’s expression, production, and function in a variety of tumour cell lines. These studies implicate LIF’s role in the proliferation of neoplastic cells from several malignancies. Little is known about the role of LIF in tumours of the lung and the oral cavity. LIF is localized in the human airway mainly in fibroblasts, and IL-1ß can upregulate the expression of LIF’s mRNA and the release of LIF protein (207). LIF stimulated the growth of the metastatic human lung giant cell carcinoma PG cell line (187) and was found to be produced by the lung adenocarcinoma NCI-H23 cells (58) and the oral cavity carcinoma cell line OCC-1C (188). Because of LIF’s crucial role in the reproductive system, its effects on neoplasms originating from this system are of special interest. To our surprise, we were able to find only a limited number of studies addressing this issue. Bamberger et al. reported that LIF’s transcription is upregulated upon exposure of the SKUT1B uterine tumour cell line to a progesterone agonist (189). A soluble form of LIFR was detected in the supernatant of the choriocarcinoma cell line NJG, which also expressed LIF cDNA (190). Interestingly, human germ

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tumour cell lines express two forms of LIFR. Overexpression of LIFR of either form generated different levels of LIF protein activity, suggesting an autocrine role for LIF during germ cell tumourigenesis (191). LIF is also expressed in several other human tumour cell lines. Those include the bladder carcinoma line 5637, the epidermal carcinoma cell line HLFa (58) the squamous carcinoma line COLO-16 (29), and the SV40-transformed keratinocyte cell line SVK14 (58). The significance of these findings is yet to be determined.

5. CONCLUSION Similar to its diverse physiological effects, LIF exerts a broad spectrum of activities in various neoplastic cells, their surrounding tissues, and the cancer patient’s body as a whole. Although the accumulating data are incomplete and far from being conclusive, they indicate that LIF plays a major role in the pathophysiology of neoplasia. Tumours of bone, breast, kidney, the CNS, and other tissues, benefit from the presence of

Chapter 1 this cytokine. LIF, produced endogenously or by the tumours’ surrounding tissue, stimulates the cancer cells in an autocrine or paracrine fashion. In addition, LIF-producing tumour metastases, especially those metastasizing to bone, cause local distortion by inducing either blastic or lytic lessions. Moreover, overproduction of LIF is likely to be responsible for constitutional reactions such as an abnormal immune response; inflammatory and anti-inflammatory reactions, production of acute-phase proteins; abnormal responses of the hematopoietic system, including thrombocytosis; and neutrophilia and cachexia. Several groups worldwide have investigated the role LIF plays in normal physiology and in pathophysiology of cancer. These studies have revealed a wide array of complex effects that are not fully understood. Nevertheless, at least in a limited number of tumours, LIF appears to accelerate the cancerous process. Whether inhibition of LIF would be beneficial as an anticancer therapy remains to be seen.

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menstrual cycle and early pregnancy. Biol Reprod 2000;63(2);508-12 83. Passavant C, Zhao X, Das SK, Dey SK, Mead RA. Changes in uterine expression of leukemia inhibitory factor receptor gene during pregnancy and its Up-regulation by prolactin in the western spotted skunk. Biol Reprod 2000;63(1):301-07 84. Piccinni MP, Maggi E, Romagnani S. Role of hormone-controlled T-cell cytokines in the maintenance of pregnancy . Biochem Soc Trans 2000;28(2):212-15 85. Hsieh YY, Tsai HD, Chang CC, Hsu LW, Chang SC, Lo HY. Prolonged culture of human cryopreserved embryos with recombinant human leukemia inhibitory factor. J Assist Reprod Genet 2000;17(3):l31-34 86. Martin TJ, Allan EH, Evely RS, Reid IR. Leukaemia inhibitory factor and bone cell function. Ciba Foundation Symposium 1992;167:141-50 87. Cornish J, Gallon K, King A, Edgar S, Reid IR. The effect of leukemia inhibitory factor on bone in vivo. Endocrinology. 1993;132(3):1359-66 88. Malaval L, Gupta AK, Aubin JE. Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures. Endocrinology. 1995;136(4): 1411-18 89. Chandrasekhar S, Harvey AK. Modulation of PDGF mediated osteoblast chemmotaxis by leukemia inhibitory factor (LIF). J Cell Physiol 1996;169(3):481-90 90. Sabokbar A, Fujikawa Y, Brett J, Murray DW, Athanasou NA. Increased osteoclastic differentiation by PMMA particle-associated macrophages. Inhibitory effect by interleukin 4 and leukemia inhibitory factor. Acta Orthopaedica Scandinavica 1996;67(6): 593-98 91. Malaval L, Gupta AK, Liu F, Delmas PD, Aubin JE. LIF, but IL-6, regulates osteoprogenitor differentiation in rat calvaria cell cultures: modulation by dexamethasone. J Bone Miner Res 1998;13(2):175-84 92. Bohic S, Rohanizadeh R, Touchais S, Godard A, Daculsi G, Heymann D. Leukemia inhibitory factor and oncostatin M influence the mineral phases formed in a murine heterotopic calcification model: a Fourier transform-infrared microspectroscopic study. J Bone Miner Res 1998;13(10):1619-32 93. Thomson BM, Saklatvala J, Chambers TJ. Osteoblasts mediate interleukin 1 stimulation of bone resorption by rat osteoclasts. J Exp Med 1986;164(1):104-12 94. Thomson BM, Mundy GR, Chambers TJ. Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption. J Immunol 1987;138(3):775-79

Chapter 1 95. Beutler B, Cerami A. Cachectin: more than a tumor necrosis factor. N Engl J Med 1987;316(7):379-85 96. Beutler BA, Cerami A. Recombinant interleukin 1 suppresses lipoprotein lipase activity in 3T3L1 cells. J Immunol 1985;135(6):3969-71 97. Metcalf D. Disease states induced by hemopoietic growth factor excess: their implications in medicine. Int J Cell Cloning 1990;8 Suppl 1:374-87; discussion 387-90 98. Gadient RA, Patterson PH. Leukemia inhibitory factor, Interleukin 6, and other cytokines using the GP130 transducing receptor: roles in inflammation and injury. Stem Cells 1999;17(3):127-37 99. Brown MA, Metcalf D, Gough NM. Leukaemia inhibitory factor and interleukin 6 are expressed at very low levels in the normal adult mouse and are induced by inflammation. Cytokine 1994:6:300-09 100. Block MI, Berg M, McNamara MJ. Passive immunization of mice against D factor blocks lethality and cytokine release during endotoxemia. J Exp Med 1993;178:1085-90 101. Alexander HR, Wong GG, Doherty GM. Differentiation factor/leukemia inhibitory factor protection against lethal endotoxemia inmice: synergistic effect with interleukin 1 and tumor necrosis factor. J Exp Med 1992; 175:1139-42 102. Waring PM, Waring LJ, Billington T. Leukemia inhibitory factor protects against experimental lethal Escherichia coli septic shock in mice. Proc Natl Acad Sci USA 1995;92:133741 103. Carroll G, Bell M, Wang H. Antagonism of the IL-6 cytokine subfamily—a potential strategy for more effective therapy in rheumatoid arthritis. Inflamm Res 1998;47:1-7 104. Hui W, Bell M, Carroll G. Oncostatin M (OSM) stimulates resorption and inhibits synthesis of proteoglycan in porcine articular cartilage explants. Cytokine 1996;8:495-00 105. Lotz M, Moats T, Villiger PM. Leukeamia inhibitory factor is expressed in cartilage and synovium and can contribute to the pathogenesis of arthritis. J Clin Invest 1992;90:888-96 106. Okamoto H, Yamamura M, Morita Y. The synovial expression and serum levels of interleukin-6, interleukin-11, leukemia inhibitory factor, and oncostatin M in rheumatoid arthritis. Arthritis Rheum 1997;40:1096-05 107. Waring PM, Carroll GJ, Kandiah DA. Increased levels of leukemia inhibitory factory in synovial fluid from patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum 1993;36:911-15 108. Henrotin YE, De GD, Labasse AH. Effects of exogenous IL-1 beta, TNF alpha, IL-6, IL-8

1. LIF and cancer metastasis and LIF on cytokine production by human articular chondrocytes. Osteoarth Cart 1996;4:163-73 109. Chabaud M, Fossiez F, Taupin JL. Enhancing effect of IL-17 on IL-1-induced IL-6 and leukemia inhibitory factor production by rheumatoid arthritis synoviocytes and its regulation by Th2 cytokines. J Immunol 1998;161:409-14 Bell M, Carroll GJ, Chapman H. Leukemia 110. inhibitory factor (LIF) binding protein attenuates the phlogistic and abolishes the chondral effects of LIF in goat joints. J Rheumatol 1997;24:2394-02 111. Heymann D, Her E, Nguyen JM. Leukaemia inhibitory factor (LIF) production in pleural effusions: comparison with production of IL-4, IL-8, IL-10 and macrophage-colony stimulating factor (M-CSF). Cytokine 1996;410-16 112. Jorens PG, De JR, Bossaert LL. High levels of leukaemia inhibitory factor in ARDS. Cytokine 1996;8:873-76 Szepietowski JC, McKenzie RC, Keohane 113. SG. Leukaemia inhibitory factor: induction in the early phase of allergic contact dermatitis. Contact Dermatitis 1997; 36:21-25 114. McKenzie RC, Paglia D, Kondo S. A novel endogenous mediator or cutaneous inflammation: leukemia inhibitory factor. Acta Derm Venerol 1996;76:111-14 115. Thompson SW, Dray A, Urban L. Leukemia inhibitory factor induces mechanical allodynia but not thermal hyperalgesia in the juvenile rat. Neuroscience 1996;71:1091-94 116. Banner LR, Patterson pH, Allchorne A. Leukemia inhibitory factor is an antiinflammatory and analgesic cytokine. J Neurosci 1998;18:5456-62 117. Banner LR, Patterson PH. Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganlia. Proc Natl Acad Sci USA 1994;91:7109-13 118. Banner LR, Moayeri NN, Patterson PH. Leukemia inhibitory factory is expressed in astrocytes following corticla brain injury. Exp Neurol 1997;147:1-9 119. Curtis R, Scherer SS, Somogyi R. Retrograde axonal trnsport of LIF is increased by peripheral nerve injury: correlation with increased LIF expression in distal nerve. Neuron 1994;12:191-04 120. Kurek JB, Ausin L, Cheema SS. Upregulation of leukaemia inhibitory factor and interleukin-6 in transected sciatic nerve and musce following denervation. Neuromuscul Disord 1996;6:105-14

21 121.

Kurek JB, Austin L, Cheema SS, Bartlett PF, Murphy M. Upregulation of leukaemia inhibitory factor and interleukin-6 in transected sciatic nerve and denervated muscle. Neuromusc Disor 1996;6:105-14 122. Austin L, Burgess AW. Stimulation of myoblast proliferation in culture by leukaemia inhibitory factor. J Neurol Sci 1991;101:193-97 123. Austin L, Bower J, Kurek J, Vakakis N. Effects of leukaemia inhibitory factor and other cytokines on murine and human myoblast proliferation. J Neurol Sci 1992;112:185-91 124. Patterson PH, Kou S-Y, Sugiura S. LIF coordinates neuronal and inflammatory response to nerve injury. Soc Neurosci Abstr 1997;23:393-33 125. Baumann H, Jahreis GP, Sauder DN, Koj A. Human keratinocytes and monocytes release factors which regulate the synthesis of major acute phase plasma proteins in hepatic cells from man, rat, and mouse. J Biol Chem 1984;259(11):7331-42 126. Kordula T, Rokita H, Koj A. Effects of interleukin-6 and leukemia inhibitory factor on the acute phase response and DNA synthesis in cultured rat hepatocytes. Lymphokine Cytokine Res 1991:10:23-26 127. Mayer P, Geissler K, Ward M. Recombinant human leukemia inhibitory factor induces acute phase proteins and raises the blood platelet counts in nonhuman primates. Blood 1993;81:3226-33 128. Richards LJ, Kilpatrick TJ, Dutton R, Tan SS, Gearing DP, Bartlett PF, Murphy M. Leukaemia inhibitory factor (LIF) and related factors promote the differentiation of neuronal and astrycytic precursors within the developing murine spinal cord. Eur J Neurosci 1996;8:29199 129. Li M, Sendtner M, Smith A. Essential function of LIF receptor in motor neurones. Nature 1995;378:724-27 130. Cheema SS, Richards LR, Murphy M, Bartlett PF. Leukemia inhibitory factor rescues motorneurones from axotomy-induced cell death. Neuroreport 1994;5:989-92 131. Ikeda K, Iwasaki Y, Shiojima T, Kinoshita M. Neuroprotective effect of cholinergic differentiation leukaemia inhibitory factor on Wobbler murine motor neurone disease. Muscle Nerve 1995:18:1344-47 132. Metcalf D. The induction and inhibition of differentiation in normal and leukaemic cells. Philos Trans R Soc Lond B Biol Sci 1990;327(1239):99-109 133. Shabo Y, Lotem J, Rubinstein M, Revel M, Clark SC, Wolf SF, et al. The myeloid blood cell

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differentiation-inducing protein MGI-2A is interleukin-6. Blood 1988;72(6):2070-73 134. Metcalf D. Suppression of myeloid leukemic cells by normal hemopoietic regulators. Recent Prog Cytokine Res 1989;10:11-19 135. Maekawa T, Metcalf D. Clonal suppression of HL60 and U937 cells by recombinant human leukemia inhibitory factor in combination with GM-CSF or G-CSF. Leukemia 1989;3(4):270-76 136. Maekawa T, Metcalf D, Gearing DP. Enhanced suppression of human myeloid leukemic cell lines by combinations of IL-6, LIF, GM-CSF and G-CSF. Int J Cancer 1990;45(2):353-58 137. Wang C, Lishner M, Minden MD, McCulloch EA. The effects of leukemia inhibitory factor (LIF) on the blast stem cells of acute myeloblastic leukemia. Leukemia 1990;4(8):548-52 138. Metcalf D, Hilton DJ, Nicola NA. Clonal analysis of the actions of the murine leukemia inhibitory factor on leukemic and normal murine hemopoietic cells. Leukemia 1988;2(4):216-21 139. Leary AG, Wong GG, Clark SC, Smith AG, Ogawa M. Leukemia inhibitory factor differentiation-inhibiting activity/human interleukin for DA cells augments proliferation of human hematopoietic stem cells. Blood 1990;75(10):1960-64 140. Verfaillie C, McGlave P. Leukemia inhibitory factor/human interleukin for DA cells: a growth factor that stimulates the in vitro development of multipotential human hematopoietic progenitors. Blood 1991;77(2):263-70 141. Estrov Z, Talpaz M, Wetzler M, Kurzrock R. The modulatory hematopoietic activities of leukemia inhibitory factor. Leuk Lymphoma 1992;8(1-2):1-7 142. Wetzler M, Kurzrock R, Lowe DG, Kantarjian H, Gutterman JU, Talpaz M. Alteration in bone marrow adherent layer growth factor expression: a novel mechanism of chronic myelogenous leukemia progression. Blood 1991;78(9):2400-06 143. Shen MM, Skoda RC, Cardiff RD, CamposTorres J, Leder P, Orntiz DM. Expression of LIF in transgenic mice results in altered thymic epitehlium and apparent interconversion of thymic and lymph node morphologies. EMBO J 1994;1375-85 144. Schaafsma MR, Falkenburg JH, Duinkerken N, Moreau JF, Soulillou JP, Willemze R, Fibbe WE. Human interleukin for DA cells (HILDA) does not affect the proliferation and differentiation of hematopoietic progenitor cells in human long-term bone marrow cultures. Exp Hematol 1992;20:6-10

Chapter 1 145.

Firkin FC, Birner R, Farag S. Differential action of diffusible molecules in long-term bone marrow culture on proliferation of leukaemic and normal haemopoietic cells. Br J Haematol 1993;84:8-15 146. Debili N, Masse JM, Katz A, Guichard J, Breton-Gorius J, Vainchenker W. Effects of the recombinant hematopoietic growth factors intermegakayocytic differentiation of CD34+ cells. Blood 1993;82:84-95 147. Gabutti V, Timeus F, Ramenghi U, Crescenzio N, Marranca D, Miniero R, Cornaglia G, Bagnara GP. Expansion of cord blood progenitors and use for hemopoietic reconstitution. Stem Cells 1993;11 Suppl: 105-12 148. Szilvassy SJ, Cory S. Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells. Blood 1994;84:74-83 149. Szilvassy SJ, Weller K.P, Kin W, Sharma AK, Ho AS, Tsukamoto A, Hoffman R, Leiby KR, Gearing DP. Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells. Blood 1996;87:4618-28 150. Metcalf D. Leukemia inhibitory factor – a puzzling poly-functional regulator. Growth Factors 1992; 7:169-73 151. Kamohara H, Sakamoto K, Ishiko T, Masuda Y, Abe T, Ogawa M. Leukemia inhibitory factor induces apoptosis and proliferation of human carcinoma cells through different oncogene pathways. Int J Cancer 1997;72(4):687-95 152. Moreau JF, Bonneville M, Peyart MA, Jacques Y, Soulillou JP. Capactiy of alloreactive human T clones to produce factor(s) inducing proliferation of the IL3-dependent DA-1 murine cell line. I. Evidence that this production is under IL2 control. Ann Inst Pasteur Immunol 1986; 137C(1):25-37 153. Abe T, Murakami M, Sato T, Kajiki M, Ohno M, Kodaira R. Macrophage differentiation inducing factor from human monocytic cells is equivalent to murine leukemia inhibitory factor. J Biol Chem 1989;264:8941 154. Xie P, Chan FS, Ip NY, Leung MF. Induction of gp130 and LIF by differentiation inducers in human myeloid leukemia K562 cells. Leuk Res 1999;23(12):1113-19 155. Wetzler M, Estrov Z, Talpaz M, Kim KJ, Alphonso M, Srinivasan R, Kurzrock R. Leukemia inhibitory factor in long-term adherent layer cultures: increased levels of bioactive protein in leukemia and modulation by IL-4, ILand Cancer Res 1994; 54:1837-42 156. Maekawa T, Metcalf D, Gearing DP. Enhanced suppression of human myeloid

1. LIF and cancer metastasis leukemic cell lines by combinations of IL-6,

LIF, GM-CSF and G-CSF. Int J Cancer

1990;45(2):353-58

Yamamoto-Yamaguchi Y, Tomida M, 157. Hozumi M. Prolongation by differentiationstimulating factor/leukemia inhibitory factor of the survival time of mice implanted with mouse myeloid leukemia cells. Leuk Res 1992;16(10):1025-29 Godard A, Heymann D, Raher S, Anegon I, 158. Peyrat MA, Le Mauff B, Mouray E, Gregoire M, Virdee K, Soulillou JP et al. High and low affinity receptors for human interleukin for DA cells/leukemia inhibitory factor on human cells. Molecular characterization and cellular distribution. J Biol Chem 1992;267(36):3214-22 Umemiya-Okada T, Natazuka T, Matsui T, 159. Ito M, Taniguchi T, Nakao Y. Expression and regulation of the leukemia inhibitory factor/D factor gene in human T-cell leukemia virus type 1 infected T-cell lines. Cancer Res 1992;52(24):6961-65 Lal RB, Rudolph D, Buckner C, Pardi D, 160. Hooper WC. Infection with human Tlymphotropic viruses leads to constitutive expression of leukemia inhibitory factor and interleukin-6. Blood 1993;81(7):1827-32 Gu ZJ, Zhang XG, Hallet MM, Lu ZY, 161. Wijdenes J, Rossi JF, Klein B. A ciliary neurotrophic factor-sensitive human myeloma cell line. Exp Hematol 1996; 24:1195-00 Portier M, Zhang XG, Ursule E, Lees D, 162. Jourdan M, Bataille R, Klein B. Cytokine gene expression in human multiple myeloma. Br J Haematol 1993; 85:514-20 Nishimoto N, Ogata A, Shima Y, Tani Y, 163. Ogawa H, Nakagawa M, Sugiyama H, Yoshizaki K, Kishimoto T. Oncostain M, leukemia inhibitory factor, and interleukin 6 induce the proliferation of human plasmacytoma cells via the common signal transducer, gp130. J Exp Med 1994; 179(4): 1343-47 Zhang XG, Gu JJ, Lu ZY, Yasukawa K, 164. Yancopoulos GD, Turner K, Shoyab M, Taga T, Kishimoto T, Bataille R. et al. Ciliary neurotropic factor, interleukin 11, leukemia inhibitory factor, and oncostatin M are growth factors for human myeloma cell lines using the interleukin 6 signal transducer gp130. Institute for Molecular Genetics, CNRS BP5051, Montepellier, France. Marusic A, Kalinowski JF, Jastrzebski S, 165. Lorenzo JA. Production of leukemia inhibitory factor mRNA and protein by malignant and immortalized bone cells. J Bone Miner Res 1993;8(5):617-24 Gouin F, Heymann D, Raher S, De Groote 166. D, Passuti N, Daculsi G, Godard A. Increased

23 levels of leukaemia inhibitory factor (LIF) in urine and tissue culture supernatant from human primary bone tumors. Cytokine 1998;10(2):11014 167. Soueidan A, Gan OL, Gouin F, Godard A, Heymann D, Jacques Y, Daculsi G. Culturing of cells from giant cell tumour of bone on natural and synthetic calcified substrata: the effect of leukaemia inhibitory factor and vitamin D3 on the resorbing activity of osteoclast-like cells. Virchows Archiv 1995;426(5):469-77 168. Gouin F, Couillaud S, Cottrel M, Godard A, Passuti N, Heymann D. Presence of leukaemia inhibitory factor (LIF) and LIF-receptor chain (gp190) in osteoclast-like cells cultured from human giant cell tumour of bone. Ultrastructural distribution. Cytokine 1999;11(4):282-89 169. Estrov Z, Samal B, Lapushin R, Kellokumpu-Lehitinen P, Sahin AA, Kurzrock R, Talpaz M, Aggarwal BB. Leukemia inhibitory factor binds to human breast cancer cells and stimulates their proliferation. J Interferon Cytokine Res 1995;15(10):905-13 170. Kellokumpu-Lehtinen P, Talpaz M, Harris D, Van Q, Kurzrock R, Estrov Z. Leukemiainhibitory factor stimulates breast, kidney and prostate cancer cell proliferation by paracrine and autocrine pathways. Int J Cancer 1996;66(4):515-19 Liu J, Hadjokas N, Mosley B, Estrov Z, 171. Spence MJ, Vestal RE. Oncostatin M-spccific receptor expression and function in regulating cell proliferation of normal and malignant mammary epithelial. Cytokine 1998;10(4):29502 172. Douglas AM, Goss GA, Sutherland RL, Hilton DJ, Berndt MC, Nicola NA, Begley CG. Expression and function of members of the cytokine receptor superfamily on breast cancer cells. Oncogene 1997;14(6):661-69 Dhingra K, Sahin A, Emami K, Hortobagyi 173. GN, Estrov Z. Expression of leukemia inhibitory factor and its receptor in breast cancer: a potential autocrine and paracrine growth regulatory mechanism. Breast Cancer Res Treat 1998;48(2):165-74 174. Crichton MB, Nichols JE, Zhao Y, Bulun SE, Simpson ER. Expression of transcripts of interleukin-6 and related cytokines by human breast tumors, breast cancer cells, and adipose stromal cells. Cell Endocrinol 1996;120(2):215 175. Tatoud R, Desgrandchamps F, Gegeorges A, Thomas F. Peptide growth factors in the prostate. Pathol Biol 1993 ;41:731-40 176. Mori M, Yamaguchi K, Honda S, Nagasaki K, Ueda M, Abe O, Abe K. Cancer cachexia syndrome developed in nude mice bearing

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melanoma cells producing leukemia-inhibitory factor. Cancer Res 1991;51(24):6656-59 177. Paglia D, Oran A, Lu C, Kerbel RS, Sauder DN, McKenzie RC. Expression of leukemia inhibitory factor and interleukin-11 by human melanoma cell lines: LIF, IL-6, and IL-11 are not coregulated. J Interferon Cytokine Res 1995;15(5):455-60 178. Heymann D, Godard A, Raher S, Ringeard S, Lassort D, Blanchard F, Harb J. Human interleukin for DA cells/leukemia inhibitory factor and oncostatin M enhance membrane expression of intercellular adhesion molecule-1 on melanoma cells but not the shedding of its soluble form. Cytokine 1995;7(2):111 179. Kamohara H, Sakamoto K, Ishiko T, Mita S, Masuda Y, Abe T, Ogawa M. Human carcinoma cell lines produce biologically active leukemia inhibitory factor (LIF). Res Commun Mol Pathol Pharmacol 1994 180. Baumann H, Ziegler SF, Mosley B, Morella KK, Pajovic S, Gearing DP. Reconstitution of the response to leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor in hepatoma cells. J Biol Chem 1993;268(12):8414-17 181. Hermanns HM, Radtke S, Haan C, SchmitzVan de Leur H, Tavernier J, Heinrich PC, Behrmann I. Contributions of leukemia inhibitory factor receptor and oncostatin M receptor to signal transduction in heterodimeric complexes with glycoprotein 130. J Immunol 1999;163(12):6651-58 182. Guimbaud R, Abitbol V, Bertrand V, Quartier G, Chauvelot-Moachon L, Giroud J, Couturier D, Chaussade DC. Leukemia inhibitory factor involvement in human ulcerative colitis and its potential role in malignant course. Eur Cytokine Netw 1998;9(4):607-12 183. Halfter H, Kremerskothen J, Weber J, Hacker-Klom U, Barnekow A, Ringelstein EB, Stogbauer F. Growth inhibition of newly established human glioma cell lines by leukemia inhibitory factor. J Neuro Oncol 1998, 39(1):118 184. Halfter H, Lotfi R, Westermann R, Young P, Ringelstein EB, Stogbauer FT. Inhibition of growth and induction of differentiation of glioma cell lines by oncostatin M (OSM). Growth Factors 1998;15(2):135-47 185. Liu J, Li H, Moreau JF. Expression of LIF as autocrinal growth factor in human medulloblastomas. Chung Hua Ping Li Hsueh Tsa Chih 1996;25(l):24-26 186. Liu J, Li H, Hamou MF. Inhibitory effect of antisense LIF oligonucleotide on the outgrowth

Chapter 1 of human medulloblastoma cells. Chung Hua Ping Li Hsueh Tsa Chih 1996;25(3): 132-34 187. Fu J, Zheng J, Fang W, Wu B. Effect of interleukin-6 on the growth of human lung cancer cell line. Chin Med J 1998;111(3):265-68 188. Kajimura N, Iseki H, Tanaka R, Ohue C, Otsubo K, Gyoutoku M, Sasaki K, Akiyama Y, Yamaguchi K. Toxohormones responsible for cancer cachexia syndrome in nude mice bearing human cancer cell lines. Cancer Chemother Pharmacol 1996;38 Suppl:S48-52 189. Bamberger AM, Jenatschke S, Erdmann I, Schulte HM. Progestin-dependent stimulation of the human leukemia inhibitory factor promoter in SKUT-1B uterine tumor cells. J Reprod Immunol 1997;33(3):189-01 190. Tomida M. Presence of mRNAs encoding the soluble D-factor/LIF receptor in human choriocarcinoma cells and production of the soluble receptor. Biochem Biophys Res Commun 1997;232(2):427-31 191. Voyle RB, Haines BP, Pera MF, Forrest R, Rathjen PD. Human germ cell tumor cell lines express novel leukemia inhibitory factor transcripts encoding differentially localized proteins. Exp Cell Res 1999;249(2): 199-11 192. Shabo Y, Lotem J, Rubinstein M, Revel M, Clark SC, Wolf SF, Kamen R, Sachs L. The myeloid blood cell differentiation-inducing protein MG1-2A is interleukin-6. Blood 1988; 72:2070-73 193. Keller JR, Gooya JM, Ruscetti FW. Direct synergistic effects of leukemia inhibitory factor on hematopoietic progenitor cell growth: comparison with other hematopoietins that use the gp130 receptor subunit. Blood 1996;88(3):863-69 194. Heymann D, Gouin F, Guicheux J, Munevar JC, Godard A, Daculsi G. Upmodulation of multinucleated cell formation in long-term human bone marrow cultures by leukaemia inhibitory factor. Cytokine 1997;9(l):46-52 195. Zucman J, Delattre O, Desmaze C, Plougastel B, Joubert I, Melot T, Peter M, De Jong P, Rouleau G, Aurias A. et al. Cloning and characterization of the Ewing’s sarcoma and peripheral neuroepithelioma t(11;22) translocation breakpoints. Genes Chromosome Cancer 1992;5(4):271-77 196. Mansi JL, Berger U, McDonnell T, Pople A, Rayter Z, Gazet JC, Coombes RC. The fate of bone marrow micrometastases in patients with primary breast cancer. J Clin Oncol 1989;7:44549 197. Akatsu T, Ono K, Katayama Y, Tamura T, Nishikawa M, Kugai N, Yamamoto M, Nagata N. The mouse mammary tumor cell line, MMT060562, produces prostaglandin E2 and

1. LIF and cancer metastasis leukemia inhibitory factor and supports osteoclast formation in vitro via a stromal celldependent pathway. J Bone Miner Res 1998;13(3):400-08 198. Bamberger AM, Thuneke I, Schulte HM. Differential regulation of the human ‘leukemia inhibitory factor’ (LIF) promoter in T47D and MDA-MB231 breast cancer cells. Breast Cancer Res Treat 1998;47(2):153-61 Bard JB, Ross AS. LIF, the ES-cell 199. inhibition factor, reversibly blocks nephrogenesis in cultured mouse kidney rudiments. Development 1991;113(l):193-98 Hartner A, Sterzel BR, Reindi N, Hocke 200. GM, Fey GH, Gopplet-Struebe M. Cytokineinduced expression of leukemia inhibitory factor in renal mesangial cells. Kidney Int 1994;45:1562-71 Gleave ME, Hsieh JT, von Eschenbach AC, 201. Chung LW. Prostate and bone fibroblasts induce human prostate cancer growth in vivo: implications for bidirectional tumor-stromal cell implication in prostate carcinoma growth and metastasis. J Urol 1992;147:1151-59 202. Hirano T, Matsuda T, Nakajima K. Signal transduction through gp130 that is shared among the receptors for the interleukin 6 related subfamily. Stem Cells 1994;12:262-77 Chung TD, Yu JJ, Spiotto MY, Bartkowski 203. M, Simons JW. Characterization of the role of IL-6 in the progression of prostate cancer. Prostate 1999;38:199-207 Mori S, Murakami-Mori K, Bonavida B. 204. Oncostatin M (OM) promotes the growth of DU 145 human prostate cancer cells, but not PC-3 or LNCaP, through the signaling of the OM

25 specific receptor. Anticancer Res 1999;19:10115 205. Heymann D, Blanchard F, Raher S, De Groote D, Godard A. Modulation of LIF expression in human melanoma cells by oncostatin M. Immunol Lett 1995;46(3):245-51 206. Schrell UM, Koch HU, Marschalek R, Schrauzer T, Anders M, Adams E, Fahlbusch R. Formation of autocrine loops in human cerebral meningioma tissue by leukemia inhibitor factor, interleukin-6, and oncostatin M: inhibition of meningioma cell growth in vitro by recombinant oncostatin M. J Neurosurg 1998,88(3):541-48 207. Knight DA, Lydell CP, Zhou D, Weir TD, Schellenberg R, Bai TR. Leukemia inhibitory factor (LIF) and LIF receptor in human lung. Distribution and regulation of LIF release. Am J Respir Cell Mol Biol 1999;20(4);834-41 208. Freidin M, Kessler JA. Cytokine regulation of substance P expression in sympathetic neurons. Proc Natl Acad Sci U S A 1991;88(8):3200-03 209. Murphy M, Reid K, Hilton DJ, Bartlett PF. Generation of sensory neurones is stimulated by leukaemia inhibitory factor. Proc Natl Acad Sci USA 1991;88:3498-01 210. Yamakuni H, Minami M, Satoh M. Localisation of mRNA for leukaemia inhibitory factor receptor in the adult brain. J Neuroimmunol 1996;70:45-43 211. Cheema SS, Richards L, Murphy M, Bartlett PF. Leukemia inhibitory factor prevents the death of axotomised sensory neurones in the dorsal root ganglion of the neonatal rat. J Neurosci Res 1994;37:213-18

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Chapter 2 INTERLEUKIN-2 AND ITS RECEPTORS IN HUMAN SOLID TUMOURS: IMMUNOBIOLOGY AND CLINICAL SIGNIFICANCE 1

Theresa L. Whiteside, 2Torsten E. Reichert, and 3Qing Ping Dou.

1

University of Pittsburgh Cancer Institute, Pittsburgh, 2 University of Mainz, 3 Mainz, Germany and H. Lee Moffitt Cancer Center & Research Institute, Tampa, USA

Key words:

Interleukin-2 (IL-2), IL-2R, tumour growth, carcinomas, cell cycle arrest

Abstract:

Human carcinomas were found to express IL-2 R and to produce, but not to secrete, IL-2. Intermediate affinity detected on the surface and in the cytoplasm of carcinoma cells binds exogenous IL-2 at the nanomolar or micromolar concentrations and mediates cell cycle arrest (CCA) possibly through the upregulation of the CDK inhibitor expression. In contrast, is modestly expressed on the cell surface, and it may be involved in the intracrine pathway of delivering endogenous IL-2 to the cell surface. IL-2 is a growth factor for human carcinomas, and as it binds to the high-affinity IL-2R, it promotes cellular proliferation by suppressing expression of It also protects tumour cells from apoptosis. The presence in carcinomas and in normal tissue cells of two IL-2/IL-2R pathways regulating cellular growth and survival is a novel finding. Both the endogenous and exogenous IL2/IL2R pathways could become therapeutic targets in the future and could be explored to obtain insights into the mechanisms of tumour growth control as well as to modulate its sensitivity to other therapies.

1. INTRODUCTION Human cancer cells are known to express receptors for hematopoietic growth factors and cytokines (1-3). Exposure of these cells to cognate ligands leads to positive or negative receptormediated regulation of cell growth. Several years ago, we reported the presence of functional receptors for IL-2 (IL-2R) on human carcinoma cell lines and tumours in situ (4). Since then, others

have described expression of IL-2R on a variety of human normal and malignant cells and have demonstrated that binding of IL-2 to these receptors has significant and varied consequences in several types of non-hematopoietic cells (5-11). The biological and physiologic significance of IL-2R expression on these tissue cells remains poorly understood and controversial. However, it is important to note that binding of exogenous IL-2 to 27

W.G. Jiang et al. (eds.), Growth Factors and their Receptors in Cancer Metastasis, 27–50. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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human non-lymphoid tumour cells has been reported to alter expression of cell surface molecules involved in cell-to-cell interactions, increase sensitivity of tumour cells to cytostatic effects of other cytokines and drugs or to cytotoxicity mediated by immune effector cells. Furthermore, IL-2 is now known to either impair or promote growth of a variety of cells, including human tumour cells (1214). IL-2, also known as the T-cell growth factor (TCGF), is one of the first cytokines to be purified and cloned (15, 16). It is a 15 kDa glycoprotein encoded by a single gene on human chromosome 4 (17). It is considered to be a potent modulator of lymphocyte functions and to play a major role in the immune response by mediating activation, differentiation and growth of immune cells (17, 18). IL-2 acts through a receptor complex, containing at least three distinct chains, and which is found on all classes of lymphocytes and monocytes (18, 19). The IL-2R chain (p55) is the low-affinity while the IL-2R receptor chain function chain and the IL-2R together as the intermediate-affinity The non-covalent receptors association of the IL-2R a with the and chains on the cell surface results in expression of a high-affinity receptor IL-2-induced stimulation and proliferation of lymphocytes is mediated through the IL-2R and chains, since the α chain itself lacks the cytoplasmic domain necessary for signal transduction. The chain has been shown to be responsible for internalization of the IL2/IL-2R complex in addition to its signaltransducing function (20). Although IL-2 has been generally perceived as the key cytokine in the immune response, it is actually not essential to cellular immune functions, because "knock out" (KO)

Chapter 2 mice, lacking the ability to produce it, develop a normal immune system and are not immunodeficient, readily clear viruses such as LCMV or vaccinia and reject allotransplants (21). On the other hand, the chain is a critical element, as alterations in its gene cause X-linked severe combined immunodeficiency (22). IL-2 has been extensively used in cancer therapy, either as a locoregionally - or systemically - administered single agent or in combination with other biologicals (23-25). Antitumour effects of IL-2 have been universally attributed to its ability to up-regulate activities of immune cells (17). The published evidence that IL-2 can have direct antitumour effects (26, 27) or that it is produced by non-hematopoietic cells (5-11) has been largely ignored. More recently, the view of IL-2 as a "T-cell growth factor" has undergone a change, brought about by the realization that the IL-2/IL-2R pathway is involved in the regulation of both cell proliferation and cell death (14, 28). Experiments in mice have indicated that IL-2 can protect immune cells from apoptosis, but at the same time, it can potentiate death of these cells via, e.g., the Fas-mediated pathway (i.e., "activation-induced cell death" = AICD) under conditions of antigen-driven rapid proliferation (14). In light of these findings, the observation that the IL-2/IL2R pathway is ubiquitously expressed in human tissue cells and that it is upregulated in carcinoma cells take on a new significance. The possibility has to be considered that, similar to its effects in hematopoietic cells, the presence of the IL-2/IL-2R pathway in carcinomas might be linked to the susceptibility of these cells to apoptosis. Indeed, the growth of tumours is known to depend not only on cellular proliferation but also on the rate of cell death. This implies that effects of

2. IL-2 and IL-2R in solid tumours endogenous and/or exogenous IL-2 on ILtumour cells might influence tumour growth as well as tumour death. The therapeutic implication of this type of mechanism(s) is great, particularly since IL-2 has been established and approved as a therapeutic cytokine in some types of cancer, specifically, melanoma and renal cell carcinoma (29, 30). In the following narrative, we will first review evidence for the presence of functional IL-2R on human carcinomas. We will then consider experiments in support of the observation that endogenous, tumour-derived IL-2 functions as a growth factor and an apoptosis protection factor in tumour cells, while exogenously-delivered IL-2 has an entirely opposite effect, as it inhibits tumour cell growth and increases tumour cell susceptibility to immune cells or other cytokines. The mechanisms that might be responsible for these seemingly contradictory results will be considered. Finally, we will discuss the importance of the IL-2/IL-2R pathway for tumour therapy with IL-2. Overall, we expect to be able to convince the reader that the IL2/IL-2R pathway plays a significant role in the regulation of growth of human carcinomas and to provide some insights into the mechanisms responsible for growth or death of tumour cells as a result of perturbations of this pathway. 2. IL-2R ON CARCINOMAS 2.1 Expression of IL-2R on human tumours Receptors for various growth factors and cytokines are known to be expressed on human cancer cells. Ample evidence has accumulated for the crucial role of

29 these receptors, e.g., the epidermal growth factor receptor (EGFR) or insulin-like growth factor receptors (IGFR), in the development of cancer (1, 3, 31). Exposure of cells expressing these receptors to cognate factors leads to regulation of cell growth. The initial observation in our laboratory that recombinant IL-2 inhibited growth of tumour cells involved ex vivo exposure of head and neck cancer (HNC) cell lines and gastric carcinoma cell lines to various concentrations of the cytokine (Figure 1). To explain these results, we postulated that IL-2R were present on the tumour cells. Indeed, using monoclonal antibodies (Abs) specific for the IL-2R chain, we were able to confirm that nearly all of tumour cells in suspensions prepared from cellular monolayers expressed the chain of IL-2R on the cell surface when tested by flow cytometry (4, 26). The mean fluorescence intensity (MFI) for expression on the cell surface varied considerably among the tumour cell lines. Since human carcinomas grow as tight monolayers, it was possible that the dissociation solution or trypsin solution used for the preparation of tumour cell suspensions influenced the level of IL-2R expression. However, immunostaining of intact monolayers and of tumour biopsies confirmed the ubiquitous, albeit variable, expression of on the carcinoma cells/specimens examined. In contrast to the ubiquitous expression of the chain, the chain of IL-2R was either very weakly expressed or not detectable on the cell surface of chain expression carcinomas, and the was observed to be highly variable, ranging from 40 to 50% of tumour cells by flow cytometry (32).

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2. IL-2 and IL-2R in solid tumours To establish that IL-2R expressed on carcinomas were able to bind the ligand, IL-2, competitive binding studies with I25 I-labeled IL-2 were performed, using the PCI-1 cell line (4). Saturable binding of the radiolabeled ligand to the tumour cells was shown to be completely blocked by mAbs to the IL-2 binding site on the IL-2R chain but not by mAbs to other epitopes on this chain or by isotype control Abs (4, 33). By Scatchard analysis, PCI-1 tumour cells were found to express 13,000 of the intermediate affinity IL-2R and 300 of the high affinity IL-2R (14). Crosslinking of the receptors labeled with using 2 mM disuccinimidyl suberate (DSS) followed by SDSpolyacrylamide gel electrophoresis under reducing conditions, indicated the presence of a doublet corresponding to peptides with the molecular weights of 66 and 55 kDa (4). Thus, both and peptides were expressed on PCI-1 cells, although the chain had a smaller molecular weight than the expected 70-75 kDa, presumably due to the extent of its glycosylation. On the other hand, a possibility emerged that the on tumour cells was not identical to that expressed on hematopoietic cells, such as lymphocytes. The presence of mRNAs for all three IL-2R chains in carcinoma cells was confirmed by RT-PCR or RNase protection assays (32, 34) By flow cytometry performed with permeabilized cells, we compared intracytoplasmic expression of and chains in tumour cells and human lymphocytes (i.e., YT cell line or fresh NK cells). As shown in Table 1, the chain was detectable in nearly all carcinoma cells, the chain in about half of them and the chain in a very small proportion (l%-3%) of the cells.

31 The Scatchard analyses performed previously (4) indicated that these tumour cells expressed a small number (e.g., about 300) of chain-containing receptors on the cell surface. We, therefore, interpreted our combined intracytoplasmic and surface staining flow cytometry data to mean that a small number of chains were present on the surface of carcinoma cells. However, the level of expression of these chains was often at the borderline of the detection level for flow cytometry. On the other hand, abundant mRNA for the chain was always detectable in these cells (4, 26) and suggested post-transcriptional regulation of the α chain protein. Although our initial experiments were performed using squamous carcinoma of the head and neck (SCCHN) cell lines, subsequently obtained evidence indicated that expression of these IL-2R chains was detectable in human gastric and renal cell carcinoma cell lines (26). Surface expression of IL-2R was associated with significant inhibition of in vitro proliferation in these lines (26, 27). Normal epithelial cells, e.g., keratinocytes, in primary cultures were also shown to express IL-2R mRNA and protein. However, binding of exogenous IL-2 to IL-2R on keratinocytes did not result in a negative growth signal (26). These studies have led to a conclusion that functional IL-2Rs are ubiquitously expressed on human carcinomas as well as normal epithelial cells in culture, and that their function might be modified in cancer cells relative to that in normal lymphoid cells. Consistently observed expression of the intermediate-affinity IL-2R on HNC cell lines prompted us to examine frozen sections of tumour-involved as well as tumour- free adjacent oral mucosa of the oropharynx and of laryngeal tumours for the presence of IL-2R and localization of

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IL-2R+ tumour or normal cells in tissues. A clear pattern emerged from these immunoperoxidase studies as follows: a) expression was documented in all tumour tissues examined (n=27) as well as in tumour-uninvolved oral mucosa ( n= 11) in the suprabasal location (see Figure 2a and b); b) the chain was not detectable in normal or tumour tissues by immunoperoxidase staining; c) expression of the chain was strongest in the basal epithelial layer both in normal oral

Chapter 2 mucosa and in tumour tissues. (Figure 2c and d). Furthermore, it appeared that poorly differentiated SCCHN tumours showed stronger staining for IL-2R than well differentiated tumours (33). The results of immunohistologic analyses for IL-2R in tissues appeared to correlate with those obtained for IL-2 expression (see below), although additional studies will be necessary to obtain statistically significant correlations.

2. IL-2 and IL-2R in solid tumours 2.2. Growth inhibition induced in carcinomas by exogenous IL-2 The detection of IL-2R in tumour and normal epithelial cells raised the possibility that a functional IL-2/IL-2R pathway might exist in tumour or tissue cells of the non-hematopoietic origin. Therefore, tumour cells were incubated in the presence of various concentrations of recombinant IL-2 to evaluate its effect on proliferation of these targets (Figure 3). Using 4-day colorimetric MTT assays (35), 4-day incorporation assays or cell counts (36), we were able to demonstrate that exogenous IL-2 at the concentrations consistently nM or inhibited growth of carcinoma cell lines. These effects were particularly pronounced in tumour cells cultured in the presence of low concentrations of fetal calf serum (i.e., 1% v/v). Furthermore, these inhibitory effects were IL-2 dosedependent (Figure 3). Growth inhibition induced by exogenous IL-2 in tumour cells could have resulted from the cell cycle arrest (CCA) or apoptosis. To distinguish between these two mechanisms, SCC cells inhibited in growth after 3-day culture in the presence of exogenous IL-2 (22nM) were compared with control tumour cells cultured in tissue culture medium (TCM) alone for the presence of DNA fragmentation, using flow cytometry TUNEL assays. No evidence for apoptosis was ever observed in SCC cells cultured in the presence of growth-inhibitory concentrations of IL-2. An alternative mechanism of CCA was, therefore, investigated by flow cytometry analyses of the cell cycle in tumour cells incubated in the presence of 22nM of IL-2 for 3 days. The tumour cells were labeled with propidium iodide (PI), and the proportion of cells in the S and G2/M phases of the cell cycle were quantitated. As shown in Table 2, the

33 fraction of tumour cells in the phase was significantly increased, while that in the S phase was decreased in cells incubated with the growth inhibitory doses of exogenous IL-2. It must be emphasized that although these experiments were not performed with synchronized cultures, the fraction of phase tumour cells arrested in the of the cell cycle was significant in cultures incubated with exogenous IL-2 at the concentrations >22nM. These ex vivo experiments, repeated with various carcinoma cell lines, indicated that exogenous IL-2 used at the relatively high concentration of 22nM or greater inhibited tumour cell growth by inducing the CCA. Furthermore, earlier immunotherapy experiments performed with IL-2 in nude mice bearing established human carcinoma xenografts demonstrated that either local or systemic (iv) delivery of this cytokine (e.g., 6000 IU/mL injected peritumourally once a day for 2 weeks) consistently resulted in a significant reduction of the tumour size (26, 27). In view of the fact that >22nM concentrations of exogenous IL-2 were necessary to induce the CCA in carcinoma cells, it was assumed that the effect was mediated via the intermediate IL-2R, i.e., . This hypothesis was consistent with considerable levels of expression on carcinomas and very low levels of IL2R expression, as detected by flow cytometry or immunostaining on frozen tissue sections. To investigate whether expression of the high affinity IL2R in carcinoma cells would result in greater sensitivity to IL-2 and more pronounced CCA, we transduced the gene into gastric carcinoma, RCC and SCCHN cell lines, using lipofection and the p55CDM 8Neo expression vector (34).

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The transduced and selected tumour cells expressed substantially higher levels on the cell surface than of IL-2R parental or LacZ control cells (Figure 4a). Growth of transduced PCI-1 cell clones (SCCHN) selected for high levels of the IL-2R chain expression and cultured in the presence of rIL-2 over a wide range of concentrations was comparable to that of the controls (Fig 4b;ref 32). Thus, increased expression of and the increased number of binding sites on the transduced HNC cells were not accompanied by their greater sensitivity to growth inhibitory effects of IL-2 (Figure 4b). On the other hand, when the IL-2R chain was transduced into PCI-1 cells, based on the observation that mRNA and was protein expression of demonstrated to be lower in these cells than that in control lymphoid cells (26), the transduced cell line became significantly more sensitive to growth inhibition mediated by exogenous IL-2 than parental or LacZ-transduced controls (Figure 4b). These experiments demonstrated that transfer of the chain gene into HNC cells increased the number of the IL-2 binding sites on the tumour cell surface (32) as well as tumour cell sensitivity to growth inhibition by exogenous IL-2 These observations were confirmed using another of our SCCHN cell lines (PCI-13) transduced with the IL2Ra or gene (data not shown). Taken together, the IL-2R gene transduction data indicated that in HNC cells, the complex (intermediate affinity receptor) was the functional receptor responsible for the observed growth inhibitory effects of exogenous IL-2.

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In contrast to the results described above for SCCHN, both HR (gastric carcinoma) and a RCC tumour cell line chain gene transduced with the and found to express high-affinity IL-2R by flow cytometry (Fig 4a) became significantly more sensitive to growth inhibitory effects of exogenous IL-2 than parental or LacZ transduced cells (Figure 4b). When HR cells transduced with the IL-2R chain gene were compared to parental HR cells for growth in tissue culture medium not containing any exogenous IL-2, they were found to proliferate significantly better. These data suggested that expression of the IL-2R chain on HR was important for growth of these cells even in the absence of exogenous IL-2 (Figure 5). In experiments designed to confirm

the essential role of

in tumour cell growth, the HR cell line was transduced with pCEP4p70R, containing antisense IL-2R cDNA (34). Expression of ILon transduced and control (LacZ transduced) HR cells was assessed by flow cytometry and found to be absent (32). Growth of these tumour cells transduced with antisense was significantly inhibited (p<0.0001) relative to the sense-treated controls. The addition of exogenous IL-2 to the cultures had no effect on the transduced cells. This experiment showed that: (a) in the absence of expression, HR cells were not sensitive to exogenous IL-2 and (b) HR proliferated less cells devoid of well than the parental cells, which were Positive. Hence, expression of was found to be necessary for proliferation of HR cells in the absence of exogenous IL-2.

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To further verify that tumour cell growth depended on expression of the chain on the cell surface, various tumour cells were incubated in the presence of mAbs to the IL-2R. As shown in Figure 6, mAbs to the IL-2 binding site on the chain or TU27) were able to significantly block growth of SCCHN cell lines. However, neither isotype control Abs nor mAbs to other epitopes on the or Ab 341) were chain (e.g., inhibitory (36). Thus, our experiments showed that both the ligand, IL-2, and mAbs to the IL-2 binding site on the chain induced growth inhibition in tumour cells. When the mAb chain, to the IL-2 binding site on the TU27, was added in excess to tumour cells incubated with increasing rIL-2 concentrations, it partly abrogated growth inhibition by preventing the binding of the exogenous ligand to the chain of IL-2R (32). In aggregate, these experiments showed that negative signaling induced in tumour cells by exogenous IL-2 was dependent on the availability of the free ligand at sufficiently high concentrations to cross-link the receptors and of the IL-2 binding sites on the chain of the receptor complex. The mechanisms of the CCA induced by exogenous IL-2 in human carcinomas were next investigated by probing expression of the cell cycle regulatory proteins, specifically, cyclindependent kinase inhibitors p27 and p21, in carcinoma cells incubated in the presence of exogenous IL-2. Using Western blots and RT-PCR, we observed that in unsynchronized tumour cells (PCI – 13) treated with exogenous IL-2 and inhibited in growth, as measured in MTT assays, the levels of p27 and mRNA for p27, respectively, were increased relative to controls not treated with IL-2. In the presence of neutralizing Ab to IL-2, p27

Chapter 2 mRNA and protein levels were not increased. IL-2 treatment had no such effects on p21 expression. In PCI – 13 cells synchronized by serum starvation or by the treatment with aphidicolin, exogenous IL-2 was shown to inhibit the G1 to S transition. Our preliminary data suggest that the observed increase in p27 levels is due to its enhanced synthesis and not to the inhibition of its degradation (G. Gao, J. Stanson, T. L. Whiteside and Q. P. Dou, unpublished data). 2.3 Molecular analysis of the chain in human carcinomas In view of the evidence reviewed above that exogenous IL-2 at nM or greater concentrations inhibited growth of carcinoma cells by inducing CCA, we considered a possibility that the expressed on carcinomas and responsible for negative signaling was different from that expressed on lymphoid cells. To investigate this possibility, mRNA for the chain was isolated, reversetranscribed, amplified, and compared in carcinomas and lymphoid cells (37). Using RT-PCR with pairs of sense-antisense oligonucleotide primers specific for the various regions of extracellular, transmembrane and intracellular domains of the chain, we amplified mRNA obtained from three human carcinoma cell lines and human lymphoid cells (YT and NK cells) as controls. The list of sense and antisense primers used for this analysis is extensive and can be found in the reference 54. The identity of the amplicons was confirmed by Southern analysis with the cDNA probe coding for the entire span of the chain (37). The message for all three domains (extracellular, transmembrane and intracytoplasmic) of the chain was found to be identical in tumour cells and in normal lymphoid cells used as controls. In

2. IL-2 and IL-2R in solid tumours addition, genomic DNA obtained from the tumour cell lines was sequenced to examine the possibility that a mutation is present in the gene coding for the intracellular chain domain. No mutations or deletions were detected. Also, by Western blot and northern analyses, no differences between chain in tumours vs. that expressed in lymphoid cells were demonstrable (37). The results were consistent with our other observations, indicating that the same IL2/IL-2R pathway is operative in human carcinomas and in normal epithelial or lymphoid cells. 3.

IL-2 IN HUMAN CARCINOMAS

3.1 Discovery of endogenous IL-2 in human carcinomas While performing immunoperoxidase (IP) staining on carcinoma cell lines and tissue sections of human tumours, we observed what appeared to be specific although weak staining for IL-2 in these cells. Upon more careful examination, all carcinoma cells grown in chamber slides were found to constitutively express IL-2 in the absence of any exogenous IL-2 in TCM (Figure 7). These cells showed a characteristic-staining pattern, with immunoreactive IL-2 localized to a circumscribed area of the cytoplasm, corresponding to the Golgi zone (Figure 7a-c). Immunostaining with FITC-labeled Abs to the Golgi complex and PE-and PE labeled Abs toIL-2 demonstrated colocalization of these two cellular targets (Figure 7c). PHA-stimulated Jurkat cells or Con-A-activated normal T lymphocytes used as controls also showed the same staining pattern (36, 38). Normal human keratinocytes were positive for IL-2, but stained weakly compared to tumour cells or activated T cells (36, 38).

39 While IL-2 protein was detectable by immunostaining in permeabilized tumour cells, it was not detectable in cellular supernatant of carcinoma cells tested by ELISA or in biologic assays, using an IL2-dependent CTLL line. Minimal levels of IL-2 were shown to be present in these supernatants (39, 40). However, carcinoma cells disrupted by cycles of rapid freezing and thawing in the presence of protease inhibitors yielded cytosols containing 4060 of IL-2, as determined in IL-2-specific ELISA (32). Western blots of tumour cell lysates confirmed the presence of IL-2 protein in carcinoma cells (Figure 8). These data indicated that tumour cells produced endogenous IL-2, which was either not secreted or was secreted only at levels not detectable by immunoassays. At the same time, the presence of IL-2 on the cell surface of variable proportions of tumour cells was confirmed by flow cytometry (32, 38). We interpret this finding as evidence for existence of an autocrine IL-2 pathway in carcinoma cells. Endogenous IL-2 is transported from the cytokine to the cell surface, where it occupies IL-2R and signals via the component of the high affinity IL-2R. To confirm the presence of mRNA for endogenous IL-2 in tumour cells, RT-PCR was performed, followed by Southern blots with IL-2 cDNA. In tumour cell lines and lymphoid cells used as positive controls, mRNA for IL-2 was consistently detected, although semiquantitative analyses showed that the number of copies of the transcript was considerably lower in tumour cells than that in Jurkat T cells (38). By in situ hybridization (ISH) for IL2 mRNA, we have also shown that our cell lines were positive for its expression (Figure 9; ref 32).

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IL-2 expression in tissue biopsies of OSCC tumour cells and normal oral mucosa was evaluated next by immunohistology (33). IL-2 Protein was found to be expressed throughout the tumour tissue, although in normal oral mucosa, IL-2 protein (or mRNA for IL-2 by ISH) was localized to the basal epithelial layer (33). 3.2 Transduction of carcinoma cells with the IL-2 gene Carcinoma cells (PCI-50, PCI-1 and HR) were transduced with the IL-2 gene, using a retroviral vector, selected for neomycin resistance and evaluated for the ability to produce and secrete biologicallyactive IL-2 (39, 40). These transduced tumour cells were shown to secrete cells between 10 and 30 ng of /48h (39, 40). Importantly, growth of these transduced tumour cells in culture was significantly improved over that of the parental cell lines or control tumour cells transduced with the LacZ gene (Figure 10). The results suggested that these IL2R+ carcinoma cells utilized the secreted IL-2 for growth. As expected, Abs to IL-2 used at the concentrations of 25 and 50 ug/ml inhibited in part proliferation of the IL-2- secreting, transduced tumour cells (36). In addition, these cells were found to be significantly less susceptible to apoptosis mediated in vitro by IL-2 activated human effector cells than the parental or LacZ control tumour cells (39). However, these seemingly advantageous properties of transduced tumour cells were not useful for the tumour growing in vivo: secreted IL-2 facilitated tumour cell destruction and/or regression of metastases by inducing accumulation in situ of antitumour effector cells (NK cells and

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macrophages) in the xenograft carcinoma models established in nude mice (39, 40). 3.3 The role of endogenous IL-2 in carcinoma cell cycle progression We have observed that in monolayers of tumour cells and in human tumour tissues, IL-2 was especially strongly expressed in dividing cells (Figure 11). To examine the possibility that endogenous IL-2 is involved in the regulation of tumour cell division, we examined its expression as well as expression of its receptors in various phases of the cell cycle in tumour cell lines. By flow cytometry and immunostaining, expression of these proteins was found to be induced in the S phase, and significantly up-regulated in the phase of the cell cycle (Figure 12). The level of mRNA for IL-2 was 5 to 10 fold higher in the M-phase than in the phase, as shown by quantitative competitive RT-PCR. When tumour cell lysates were examined in Western blots, we found that expression of the cyclindependent kinase (CDK) inhibitor nearly absent in the was high in the S phase, and reappeared in the phase. In contrast, expression of p21 remained relatively unchanged during the cell cycle (38). Our results indicate that IL-2 and the 1L-2R complex behave like cell-cycle-related proteins in human carcinoma cells. Analogous to its function in T cells, endogenous IL-2 is important in CDK regulating expression of the inhibitor and thus controlling cell cycle progression of tumour cells. Additional studies showed that growth of carcinoma cell lines was inhibited by the well known immunosuppressive agents, cyclosporin A, FK506 and rapamycin, similar to the effect of these drugs on the IL-2/IL-2R pathway in lymphoid cells (36).

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3.4 Correlation of IL-2 expression with cellular proliferation in SCCHN To demonstrate that expression of IL-2 in carcinoma cells in tissue correlated with that of the proliferation-associated Ki-67 antigen, immunohistology for these proteins was performed in 34 tissue samples obtained from patients with oral carcinoma (33). Expression of IL-2 and Ki-67 in these tissues was correlated to the histological grade of the carcinomas (33). The strongest IL-2 expression was seen in tumour cells undergoing mitosis, identified by double staining with the antibody to Ki67 protein, a marker of cellular proliferation. In the tumour tissue, the highest level of co-expression of IL-2 and Ki-67 was observed in poorly differentiated carcinomas, with the labeling index (LI) of 67.2% for IL-2 and 68.8% for Ki-67. Well-differentiated carcinomas showed a significantly lower expression of both proteins (LI = 35.0 % for IL-2 and 26.5% for Ki-67). The correlation between the labeling indices was statistically significant (R = 0.747, p < 0.001). These results demonstrate that IL-2 expression in SCCHN is strongly associated with cellular proliferation, and that endogenous IL-2 might function as a growth factor for these carcinomas (33).

data indicated that IL-2 produced by tumour cells and precipitated by the IL-2specific Abs had the same electrophoretic mobility under reducing conditions as lymphoid cell-derived IL-2. The specificity of the precipitating Ab for IL-2 was confirmed by absorption with rIL-2, which almost completely eliminated the ability of this Ab to precipitate IL-2 in cellular extracts (36).

3.5 Immunoprecipitation of endogenous IL-2 in carcinoma cells In view of the data indicating that the IL-2/IL-2R pathway was active in human carcinomas and that endogenous IL-2 was involved in tumour cell proliferation, we next wished to compare endogenous IL-2 in carcinomas to that expressed in human lymphocytes. Therefore, we performed metabolic labeling with 35[S]-methionine followed by immunoprecipitation of IL-2 with specific Abs in cell lysates of SCC cell lines and Western blotting (36). Our

4. TWO IL-2 PATHWAYS IN CARCINOMA CELLS The opposite effects of endogenous and exogenous IL-2 in carcinoma cells deserve special attention. While the endogenous IL-2 at pM concentrations benefits the tumour cell, enhancing its proliferation, the exogenous ligand, working at nM or concentrations, disrupts the cell cycle by increasing expression of p27, a CDK inhibitor, and arresting the cell cycle in the G1/S phase of growth. As suggested by the schema presented in Figure 13,

3.6 Endogenous IL-2 is essential for SCC growth If endogenous IL-2 is required for carcinoma growth, then interference with its expression should inhibit growth. We generated IL-2 specific antisense and sense oligonucleotide phosphorothioates and, using DOTAP as a carrier, delivered them to tumour cells, attempting to disrupt the endogenous pathway. This strategy resulted in a transient loss of IL-2 protein and mRNA expression as well as significant inhibition of tumour cell growth (36). These results, together with the data indicating that endogenous IL-2 regulates expression of provide compelling evidence that in tumour cells, similar to lymphocytes, endogenous IL-2 is involved in the regulation of cellular division and proliferation (37).

2. IL-2 and IL-2R in solid tumours exogenous IL-2 at nM or concentrations binds to intermediate affinity IL2R and, since substantial numbers of these receptors are present on the tumour cell surface (4, 26), the ligand may be able to cross-link them. As a result, a negative signal is delivered to the cell, and CCA leads to growth inhibition. In contrast, endogenous IL-2 may interact with the IL2R chains present in the cytoplasm, as it leaves the Golgi. The IL-2/IL-2R complexes are either transported to the cell surface and/or to the nucleus. Signaling associated with this intracrine IL-2 utilization leads to cellular proliferation (Figure 13). Our data indicate that two IL-2 pathways might exist in carcinoma cells: an exocrine pathway resulting in the growth inhibition and an intracrine pathway promoting tumour growth. This implies that exogenous IL2 internalized by the tumour cell and endogenous IL2 synthesized by it would be found in distinct cellular compartments: in endosomal vesicles and in the Golgi, respectively. Indeed, as

45 shown in Figure internalized exogenous (labeled) IL-2 is detected in the cytoplasm of tumour cells and is distributed similarly to IL-2R or chains and In contrast, (Figure 14 endogenous IL-2 was localized in the Golgi by confocal microscopy (Figure Furthermore, double staining for ILand the endocytic compartment marker, rab7, followed by confocal microscopy showed that rab7 and IL chain co-localized to late endosomes in Together, PCI-1 tumour cells (Fig 15 these observations suggest that internalized IL-2 and the and chains of IL-2R are sorted toward the degradation pathway, similar to the intracellular fate of these chains in T lymphocytes, as described in the literature (41). The α chain of the IL-2R has been reported to recycle to the plasma membrane in T lymphocytes (41), and our preliminary evidence suggests that its fate is similar in carcinoma cells.

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2. IL-2 and IL-2R in solid tumours The presence of both the receptor chains and IL-2 in the cytoplasm of tumour cells, as demonstrated by immunostaining with specific Abs and confocal microscopy (Figures 14 and 15), and of receptor-bound IL-2 detectable by flow cytometry on the cell surface, as previously described (32, 36, 38), suggests that endogenous IL-2 might bind to the receptor inside the cell, and that the IL-2 receptor complex is then transported to the cell surface. As indicated earlier, no IL-2 is secreted by tumour cells, as none can be detected even in concentrated supernatants of these cells. The existence of interactions between the endogenous and exogenous IL-2 pathways is possible and is under investigation in our laboratories. These interactions might be regulated at the level of the receptor (Figure 13) both at the cell surface and inside the cell. One possibility is that exogenous IL-2 interferes with the cell cycle progression and overrides the endogenous IL-2 pathway at the level of p27 CDK inhibitor. It is also possible that the competition for the available IL-2R exists between exogenous and endogenous IL-2. These and other mechanisms are currently under study in hope of elucidating the complexity of interactions between the two IL-2 pathways. 5. SUMMARY AND CONCLUSIONS Our studies and those of others (11, 25) indicated that IL-2R and endogenous IL-2 are ubiquitously expressed in human carcinomas both in culture and in situ. Data available from various laboratories provide evidence for the presence of IL-2R on melanoma cell lines and other tumour cell lines. Furthermore, the IL-2/IL-2R pathway appears to be operative in normal non-hematopoietic tissues, e.g., fibroblasts and keratinocytes (5, 26). Thus, this pathway appears to be active in vivo and in

47 vitro in the regulation of growth in a broad range of tumour/tissue cells. Endogenous IL-2 produced by tumour cells in culture, in the absence of any exogenous IL-2, behaved like a cell-cycle associated protein and was shown to promote cell cycle progression. Similar to its role in lymphocytes, IL-2 produced in tumour cells and shown to be localized to the Golgi complex (36, 38) promoted their growth as evidenced by: a) increased expression of both IL-2 and IL-2R (at the protein and mRNA levels) in mitotic tumour cells (36, 38); b) an association between increased IL-2 expression and a decreased level of p27 CDK inhibitor in pre-mitotic cells (38); c) inhibition of IL-2 production by IL-2 specific antisense oligonucleotide, which not only resulted in the inhibition of tumour growth but led to its apoptosis (36). Thus, in carcinoma cells, endogenous IL-2 behaves like a growth-promoting factor, and it also protects tumour cells from apoptosis (36). In our studies, IL-2 emerges as a growth regulatory hormone not only for hematopoietic but all cells, which is capable of regulating tumour cell proliferation as well as its death. This concept is entirely consistent with the role played by IL-2 in the hematopoietic system (42). The evidence for the presence of the endogenous or intracrine/autocrine IL-2 pathway in human carcinomas and its involvement in tumour cell survival has important biological and therapeutic implications. The possibility exists for modulating or disrupting the intracrine/ autocrine pathway by various strategies, so that it no longer benefits the tumour. Such strategies might involve the use of exogenous IL-2 at the high ligand/receptor molecular ratios to induce a negative growth signal in tumour cells or the application of IL-2-specific antisense

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oligonucleotides to induce the cell cycle arrest. Alternatively, it is possible to envision elimination of tumour cells by targeted delivery of, e.g., IL-2 fused with a diphtheria toxin (43). Upon IL-2 mediated internalization and endocytosis of such modified IL-2, IL-2R-positive tumour cells are selectively killed (43). It has been observed in various animal models of tumour growth that delivery of exogenous IL-2 exerts an inhibitory effect on tumour growth, although in most instances, this has been interpreted as an immunologic effects of IL-2 up-regulating the host

Chapter 2 antitumour defenses (17). We suggest that beyond activation of immune mechanisms, therapeutically-delivered IL-2 directly inhibits tumour growth possibly via competing with endogenous IL-2 for IL2R on tumour cells or other as yet unknown mechanisms. It is also likely, and already demonstrated in animal tumour models, that exogenous IL-2 makes tumour cells more sensitive to radiotherapy (44). This possibility deserves close attention as it has an important and widely applicable therapeutic potential.

References 1.

2.

3. 4.

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Salomon DS, Brandt R, Ciardello R, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995; 19:183-232 Waldmann TA, White JD, Goldman CK. The interleukin-2 receptor: a target for monoclonal antibody treatment of human T-cell lymphotropic virus I-induced adult T-cell leukemia. Blood 1993; 82:1701-1712 Baserga R. The IGF-I receptor: a key to tumour growth? Cancer Res 1995; 55:249-252 Weidman E, Sacchi M, Plaisance S, Heo DS, Yasamura S, Lin WC, Johnson JT, Herberman RB, Azzarone B, Whiteside TL. Receptors for interleukin-2 on human squamous cell carcinoma cell lines and tumour in situ. Cancer Res. 1992; 149:340-349 Plaisance S, Rubenstein E, Alileche A, Sahraoui Y, Krief P, Augery-Bourget Y, Jasmin C, Suarez H, Azzarone B. Expression of the interleukin-2 receptor on human fibroblasts and its biological significance. Int Immunol 1992; 4:739-746 Rimoldi D, Salvi S, Hartmann F, Schreyer M, Blum S, Zografos L, Plaissance S, Azzarone B, Carrel S. Expression of IL-2 receptors in human melanoma cells. Anticancer Res 1993; 13:555-564 Plaisance S, Rubenstein E, Alileche A, Benoit P, Jasmin C, Azzarone B. The IL-2 receptor present on human embryonic fibroblasts is functional in the absence of P64/IL-2R gamma chain. Int Immunol 1993; 843-848 Saneto RP, Altman A, Knobler RL, Johnson HM, De Vellis Y. Interleukin-2 mediates the

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inhibition of oligodendrocyte progenitor cell proliferation in vitro. PNAS USA 1986; 83:9221-9225 Arzt E, Stelzer G, Renner U, Lange M, Muller OA, Stalla GK. Interleukin-2 and interleukin-2 receptors on intestinal epithelial cells. J Clin Invest 1992; 90:1944-1955 Ciacci C, Mahida YR, Dignass A, Koizuma M, Podolsky DK. Functional interleukin-2 receptors on intestinal epithelial cells. J Clin Invest 1993; 92:527-532 McMillan DN, Kernohan NM, FlettME, Heys SD, Deehan DJ, Sewell HF, Walker F, Eremin O. Interleukin-2 receptor expression and interleukin-2 localization in human solid tumour cells in situ and in vitro: evidence for a direct role in the regulation of tumour cell proliferation. Int J Cancer 1995; 60:766-772 Alileche A, Plaisance S, Han DS, Rubinstein E, Mingari C, Bellomo R, Jasmin C, Azzarone B. Human melanoma cell line M14 secretes a functional interleukin-2. Oncogene 1993; 8:1791-1796 Azzarone B, Pottin-Clemenceau C, Krief P, Rubinstein E, Jasmin C, Scudeletti M, Indiveri F. Are interleukin-2 and interleukin-15 tumour promoting factors for human non-hematopoietic cells? Eur Cytokine Newt 1996; 7:27-35 Van Parejis L, Ibraghimov A, Abbas AK. The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance. Immunity 1996; 4:321-328 Morgan DA, Ruscetti FW, Gallo RC. Selective in vitro growth of T lymphocytes from normal

2. IL-2 and IL-2R in solid tumours human bone marrow. Science 1976; 193:10071008 16. Robb RJ, Kitny RM, Chowhry V. Purification and partial sequence analysis of human T-cell growth factor. PNAS USA 1993; 80:59905994 17. Waldmann TA. The interleukin-2 receptor. J Biol Chem 1991; 266:2681-2684 The IL-2/IL-2 18. Taniguchi T, Minami Y. receptor system: a current overview. Cell 1993; 73:5-8 19. Minami Y, Kono T, Miyazaki T, Taniguchi T. The IL-2 receptor complex its structure, function and target genes. Ann Rev Immunol 1993; 11:245-267 20. Takeshita T, Ohtani K, Asao H, Kumaki S, Nakamura M, Sugamura K. An associated molecule, p64, with IL-2 receptor chain. Its possible involvement in the formation of the functional intermediate affinity IL-2 receptor complex. J Immunol 1992; 148:2154-2158 21. Kindig T, Schorle H, Backman M, Hengartner H, Zinkernagel R, Morak I. Immune responses in IL-2 deficient mice. Science 1993; 262:1059-1061 Sever 22. Voss SD, Hong R, Sondel PM. combined immune deficiency, interleukin-2 and the IL-2 receptor. Experiments of nature continue to point the way. Blood 1994; 83:626-635. 23. Sznol M, Parkinson DR. Clinical applications of IL-2. Oncology 1994; 8:61-71 24. Sondel PM, Kohler PC, Hank JA, Moore KH, Rosenthal NS, Sosman JA, Beckhofer R, Storer B. Clinical and immunological effects of recombinant interleukin-2 given by repetitive weekly cycles to patients with cancer. Cancer Res 1988; 48:2561-2567 25. Rosenberg SA. Perspectives on the use of interleukin-2 in cancer treatment. Cancer J Sci Am 1997; 3:52-56 26. Yasumura S, Lin WC, Weidmann E, Hebda P, Whiteside TL. Expression of interleukin-2 receptors on human carcinoma cell lines and tumour growth inhibition by interleukin-2. Int J Cancer 1994; 59:225-234 27. Rabinowich H, Vitolo D, Altarac S, Herberman RB, Whiteside TL. Role of cytokines in the adoptive immunotherapy of an experimental model of human head and neck cancer by human IL-2-activated natural killer cells. J Immunol 1992; 4:739-746 28. Lenardo M, Ka-Ming Chan F, Hornung F, McFarland H, Siegel R, Wang J, Zheng L. “Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment.” In Annual Review of Immunology, Vol 17, William E. Paul, C.

49 Garrison Fathman, Laurie H. Glimcher, Eds. Palo Alto, CA: Annual Reviews, 1999. 29. Atkins MB. Interleukin-2 in metastatic melanoma: establishing a role. Cancer J Sci Am 1997; 3:57-58 30 . Figlin R, Gitlitz B, Franklin J, Dorey F, et al. Interleukin-2-based immunotherapy for the treatment of metastatic renal carcinoma: an analysis of 203 consecutively treated patients. Cancer J Sci Am 1997; 3:592-597 31. Resnicoff M, Burgand J-L, Rotman H, Abraham D, Baserga R. Correlation between apoptosis, tumourgenesis and levels of insulinlike growth factor I receptors. Cancer Res 1995; 55:3739-3741 32. Lin W-C, Yasamura S, Suminami Y, Sung MW, Nagashima S, Stanson J, Whiteside TL. Consitutitve production of IL-2 by human carcinoma cells, expression of IL-2 receptor, and tumour growth. J Immunol 1995; 155:4805-4816 33. Reichert TE, Watkins S, Stanson J, Johnson JT, Whiteside TL. Endogeneous IL-2 in cancer cells – a marker of cellular proliferation. J. Histochem.Cytochem.1998; 46:603 -611 34. Lin W-C, Yasamura S, Whiteside TL. Transfer of interleukin-2 receptor genes into squamous cell carcinoma: Modification of tumour cell growth. Arch Otolaryngol Head Neck Surg 1993; 119:1229-1235 35. Heo DS, Park J-G, Hata K, Day R, Herberman RB, Whiteside TL. Evaluation of tetrazoliumbased semi-automatic colorimetric assay for measurement of antitumour cytotoxicity. Cancer Res 1990; 50:3681-3690 36. Reichert TE, Kashii Y, Stanson J, Zeevi A, Whiteside TL. The role of endogenous interleukin-2 (IL-2) in proliferation of human carcinoma cell lines. Br J Cancer 1999; 81(5):822-831 37. Suminami K, Kashii Y, Law JC, Lin W-C, Stanson J, Reichert TE, Rabinowich H, Whiteside TL. Molecular analysis of the IL-2 receptor chain gene expressed in human tumour cells. Oncogene 1998;16: 1309-1317 38. Reichert TE, Nagashima S, Kashii Y, Stanson J, Dou QP, Whiteside TL. Interleukin-2 expression in human carcinoma cell lines and its role in cell cycle progression. Oncogene 2000;19:514-525 39. Nagashima S, Reichert TE, Kashii Y, Suminami Y, Suzuki T, Whiteside TL. In vitro characteristics of human squamous cell carcinoma of the head and neck cells engineered to secrete interleukin-2. Cancer Gene Therapy 1997;4:366-376 40. Nagashima S, Kashii Y, Reichert TE, Suminami Y, Suxuki T, Whiteside TL. Human

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Die and let live: eliminating dangerous lymphocytes. Cell 1996; 84:655657 43. Duvic M, Gather J, Maize J, Frankel AE. IL-2 diphtheria fusion toxin produces clinical responses in tumour stage cutaneous Tcell lymphoma. Am J Hematol 1998; 58:87-90 44. Lee J, Moran JP, Fenton BM, Koch CJ, Frelinger JG, Keng PC, Lord EM. Alteration of tumour response to radiation by interleukin-2 gene transfer. Br J Cancer 2000; 82:937

Chapter 3 INTERLEUKIN-8 AND ANGIOGENESIS

Tracey A. Martin Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, UK

Key words:

Angiogenesis, Interleukin-8

Abstract:

Interleukin-8 (IL-8) is an angiogenic CXC chemokine produced by a variety of cell types. Although initially described as a chemokine for neutrophils, IL-8 has become known as a potent angiogenic factor, involved in normal physiological processes such as wound healing, and abnormal processes such as cancer metastasis. IL-8 is secreted by numerous solid tumour types and associated inflammatory cells, and has been shown to exert a potent angiogenic effect via paracrine and autocrine routes in tumourigenesis, and as such represents an valid opportunity for intervention in cancer metastasis.

1. ANGIOGENESIS AND METASTASIS Angiogenesis is the complex process of the generation of new blood vessels from pre-existing vessels. In normal physiological events, angiogenesis plays a vital part, including embryonic development, wound healing and endrometrial proliferation (1). During such processes, angiogenesis is highly regulated and is only turned on for brief periods, before complete inhibition (2). Angiogenesis can however, occur under abnormal, pathological conditions such as arthritis, retinopathy and cancer metastasis. Under these conditions, the angiogenic process is persistent and unregulated. Growth and development of a tumour requires transport of nutrients to and

removal of waste products from the tumour site (1). Local diffusion will suffice for tumours up to 2mm in diameter, but for tumours to continue to grow, a connection must be made to the blood supply. The process of creating such a stable blood source is mediated by many angiogenic factors (Figure. 1). Tumours must then continually stimulate the growth of new capillary blood vessels for continued growth. The blood vessels within the tumour can then provide a route for detached tumour cells to enter the circulatory system and metastasize to distant sites (2, 3). The capillary vessels formed are composed of endothelial cells and pericytes. For angiogenesis to occur, endothelial cells must be stimulated by angiogenic factors. 51

W.G. Jiang et al. (eds.), Growth Factors and their Receptors in Cancer Metastasis, 51–65. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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The tumour cells themselves do not have to be the only source of angiogenic signals: associated inflammatory cells, such as macrophages may be recruited and activated to produce angiogenic activity (1,2). There are many different growth factors, chemokines and cytokines that have been shown to exert an angiogenic effect, including basic fibroblast growth factor (bFGF), hepatocyte growth factor/scatter factor (HGF/SF), vascular endothelial growth factor (VEGF), PDGF, , IL-1 and IL-8. Growth at the tumour site (both primary and secondary loci) requires that angiogenesis be initialised. Activation is achieved by a change in the balance of inhibitory and stimulatory factors in favour of stimulation (4). Increased levels of angiogenic factors can induce tumour angiogenesis, with decreased levels of angiogenic inhibitors by cancer cells, vascular endothelial cells & other stromal cells(5). As most tumour cells are surrounded by stroma, interaction between the stroma and the malignant cells are extremely important in the development of tumour angiogenesis (5). 2. INTERLEUKIN-8: CHEMOKINE

A

CXC

2.1 CXC Chemokines Chemokines are cytokines that were originally identified as exhibiting chemotactic activity toward specific types of leukocytes. They are low molecular weight proteins - 8 to 12 kDa- with a basic nature and an affinity for heparin (6). Chemokines have molecular identity in the conservation of four cysteine residues which are essential for their tertiary structure, with disulfide bridges forming between either cysteines 1 and 3, or between cysteines 2 and 4 at the

terminus (7, 8). The family members include CXC, CC, C and CX3C chemokines, of which CXC and CC are by far the largest and well-defined groups (6, 9). In CXC chemokines the first two cysteines are separated by a nonconserved amino acid residue (the CXC cysteine motif) and mainly interact with neutrophils (8, 10). These cytokines in their monomer forms are around 10 kDa and appear to have pro-inflammatory and reparative activities (10). CXC chemokines are produced as precursor molecules containing a signal sequence of between 17 and 34 amino acids, which after cleavage produce a mature protein of 70 to 103 amino acids (8). CXC chemokines can be divided into two categories, depending whether or not they have the Glu-Leu-Arg (ELR) motif in front of the first cysteine residue (7, 8). Those that have the Glu-Leu-Arg (ELR) motif are predominantly angiogenic; those without are generally angiostatic (8, 10, 11, 12), Table 1, suggesting that CXC chemokines may function in regulating neovascularisation (13). They are nonglycosylated and the genes for all CXC chemokines (except for one, SDF-1) are located on chromosome 4q 12-21 (8, 14). CXC chemokines have between 20 and 50% homology at the amino acid level. Chemokines can be produced by many different cell types after stimulation with endogenous and/or exogenous inducers (8). As mentioned previously, the balance between angiogenic and angiostatic factors at a tumour site is critical in regulating the angiogenic status of the local milieu. There has been increasing evidence to suggest that CXC ELR+ chemokines and their receptors play an important role in upsetting the normal balance in solid tumours (11).

3. IL-8 and angiogenesis

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2.2 Interleukin-8 Interleukin-8 (IL-8) is a member of the CXC chemokine family, and has now been demonstrated as a potent angiogenic factor (7, 13). IL-8 was initially described as an agent promoting chemotaxis and activation of neutrophils produced by stimulated peripheral blood monocytes. It was not until 1992 that IL-8 was described as an angiogenic factor that could induce in vitro endothelial cell chemotaxis and proliferative activity and in vivo angiogenic activity in the absence of an inflammatory response (11, 15, 16). Unlike other CXC chemokines, which are only produced by a small number of cell types, production of IL-8 is found in numerous leukocyte cell types (NK cells, myeloid precursors, neutrophils, eosonophils, mast cells, etc.) and by various tissue cells, such as fibroblasts, epithelial cells, stromal cells and endothelial cells (8, 12), Table 2. In addition, some tumour cells also produce IL-8.

Chapter 3

IL-8 functions as a potent chemoattractant for neutrophils, leukocytes and keratinocytes, also affecting neutrophils by mobilisation and up-regulating cell adhesion receptors integrins), causing neutrophils (such as and leukocytes to accumulate at sites of injury, increasing vascular permeability and destroying synovial membranes (14). IL-8 is also able to increase the chemotaxis and proliferation of human vascular cells, leading to increased angiogenesis (13, 14, 17). The angiogenic activity of IL-8 is believed to be equivalent on a molar basis to other inducers of angiogenesis, such as bFGF and VEGF (10, 13), see Table 2. The precursor form of IL-8 consists of 99 amino acids with a signal sequence of 22 amino acids that is cleaved to generate a 77 residue non-glycosylated mature protein of approximately 8 kDa (12, 18), or a 79 amino acid form originating from cleavage within the signal sequence. A number of N-terminal variants of human IL-8 have been described; 69, 70, 71, 72 or 77 amino acids that are yielded

3. IL-8 and angiogenesis following processing at the terminus, depending on cell type and condition (19). The truncation of IL-8 forms are due to proteases released by the IL-8 secreting cells themselves, or from accessory cells (8). The two major forms are the 72 and 77 amino acid forms; the 72 amino acid form is highly active and produced by monocytes/macrophages, whereas the 77 amino acid intact form is predominantly produced by endothelial (endothelialderived IL-8), fibroblast and human glioblastoma cells (8, 12, 14). Thrombin is able to cleave the 77 amino acid form to the more active 72 amino acid form (14). 2.3 Interleukin-8 receptor The specific effects that CXC chemokines have on target cells is believed to be mediated by their receptors (CXCR1 – 5) (9), which are dimeric glycoproteins consisting that are members of the rhodopsin superfamily of Gprotein-coupled receptors containing seven transmembrane domains, with the NH2-terminus and COOH-terminus located extracellularly and intracellularly respectively. There are at least two high affinity receptors for IL-8: one with a high affinity for IL-8 (only low affinity for other CXC chemokines), the type-1 receptor (IL-8RA or CXCR1, 44-59 kDa) and the type-2 receptor (IL-8RB or CXCR2, 67-70 kDa) which has high affinity for IL-8, and NAP1, ENA-78, MGSA and certain CC chemokines (8, 14). Another receptor has been shown to bind promiscuously to both CXC and CC chemokines, the Duffy antigen receptor for chemokines, DARC (9). CXCR1 and CXCR2 map to human chromosome 2q35 and share a 77 % homology at the amino acid level.

55 Evidences suggests that the ELR motif present on chemokines such as IL-8 is important for binding of the chemokine to the two IL-8 receptors, which are found on endothelial cells (9). In addition the two disulfide bridges, the COOH-terminal (to CXCR2) and the Tyr-13/Lys-15 residues (CXCR1) are also important for high affinity binding (20, 21). It is generally accepted that the main endothelial cell receptor for IL-8 is CXCR2 (22, 23, 24). 3. ROLE OF IL-8 IN ANGIOGENESIS

The observation that CXC ELR+ chemokines are angiogenic suggests that they may play a role in facilitating tumourigenesis. Numerous cancer cell types have now been observed to produce IL-8, which can be shown to be the source of angiogenic promotion by the tumour cell (6). However, although IL-8 has been shown to be an angiogenic factor, until recently has the biological consequences of increased IL-8 expression in metastasis has been unclear (25). When examining the effect IL-8 has on angiogenesis in cancer, one must consider the source of this chemokine: the tumour itself, endothelial cells and associated cells e.g. macrophages. 3.1 IL-8: expression by tumour cells As mentioned previously, evidence is accumulating to propose IL-8 as a potent angiogenic factor involved in tumour angiogenesis and hence metastasis. Within the last five years, the number of papers ascribing a tumourigenic function to IL-8 has grown enormously, showing that this chemokine is present and active in a number of tumour types.

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3. IL-8 and angiogenesis Growth and metastasis of a cancer is directly correlated with tumour angiogenesis. Small tumours (3-4 mm in diameter) have been shown to express more IL-8 (and bFGF) that large tumours (greater than 10 mm in diameter which express higher levels of VEGF), where it is mostly expressed at the periphery of the tumour, where the highest cell division occurs (26). Kumar et al. also found that that sparse tumour cultures had a higher level of IL-8 than confluent cultures, but that expression was not reduced when growing the cancer cells on confluent monolayers of normal cells. This suggests that expression of different angiogenic factors in tumour cells can be regulated by their proximity to other tumour cells, or host cells (26). IL-8 produced by human colon cancer cells lines equal an increased metastatic behaviour (27) and shown to be present as both mRNA and protein in human colorectal cells in vivo (28). IL-8 is secreted by a number of human colorectal carcinoma cell lines; the protein has been detected in 74% of samples (in the cell cytoplasm), with infiltrating leukocytes, endothelial cells and fibroblast-like cells also expressing IL-8 (29). IL-8 has also been shown to act as an autocrine growth factor in colon carcinoma cell lines, supporting the suggestion the a similar autocrine loop could also operate in vivo (30). Although IL-8 does not act as an autocrine growth factor in human nonsmall cell carcinoma (NSCLC) proliferation, IL-8 produced by the NSCLC shows significant angiogenic activity. IL-8 neutralising antibodies were able to reduce the primary tumour size and decrease spontaneous metastasis to the lung in transplanted SCID mice, via this angiogenic effect (13, 31).

57 Head and neck squamous cell carcinomas produce IL-8 that can be detected in the serum of patients (32), with a positive correlation between high levels of IL-8 and increased aggressiveness of the disease in patients which is controlled by angiogenic factors expressed by the tumours such as IL-8 (33). Human ovarian carcinomas introduced into nude mice express IL-8; with a direct correlation between the level of IL-8 expressed and decreased chance of survival of the mouse (17). Highly metastatic human prostate cancer cells constitutively express high levels of IL-8 at the prostate site in nude mice (34). Human prostate cancer cell lines such as PC-3 constitutively produce IL-8, which increases angiogenic activity of these cells (35). IL-8 over-expression equals an increased metastatic type in prostate cancer cell lines by induction of MMP-9 expression. Antisense IL-8 decreases tumour-induced vascularity, resulting in a decrease in tumourigenesis and metastasis. The IL-8 thus is able to regulate angiogenesis in prostate cancers (36). Such a phenomenon is also observed in human transitional cell carcinoma (37). Human gastric carcinomas express IL8 resulting in increased tumour vascularity and disease progression. IL-8 expressed by transfected human TMK-1 gastric carcinoma cells produces an increased vascular neoplasma number (38). Gastric carcinoma cell lines also express the receptors for IL-8, suggesting that IL-8 plays a crucial role in the progressive growth of human gastric carcinoma by both autocrine and paracrine mechanisms (39).

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IL-8 may actively modulate the effect of chemotherapy through its effects on tumour vascular endothelium in some ovarian cancer cell lines (40). Increased IL-8 expression from human ovarian cancer implanted into nude mice peritoneal cavity correlates with an increase in cancer progression (17). In addition, hypoxia of these carcinoma cells results in a higher level of IL-8 (41). Conversely however, human ovarian cancers in vivo can have reduced tumourigenicity due to neutrophil infiltration from expression of IL-8 induced by the chemotherapeutic, Paclitaxel from the tumour (42) Constitutive and inducible IL-8 expression by hypoxia and acidosis renders human pancreatic cancer cells more tumuorigenic and metastatic, as the increased IL-8 levels result in increased angiogenesis (43). This was also shown by orthotopic implantation of human pancreatic cancer cells into nude mice pancreas, where hypoxia and acidosis contributed to over-expression of IL-8 resulting in increased angiogenesis and aggressiveness of the tumour (43). IL-8 (and act on the human pancreatic cancer cell line Capan-1 as a growth factor in an autocrine manner through the CRX2 receptor (44). In pancreatic cancers, chemokines such as IL-8 may also contribute to the accumulation of tumour associated immune cells (45). In a study by Miller et al. (46), IL-8 is also found in human breast cancer tissue, where all tumour cells tested also expressed the IL-8 receptors IL-8RA and IL-8RB (CXCR1 and CXCR2 respectively), whereas only 50% of benign cell expressed either receptor. The majority of small vessel endothelial cells expressed RA and RB, with the large vessel endothelial cells expressing mostly RB. Such evidence suggests that tumour

Chapter 3 and vascular endothelial cells express the IL-8 receptors and are likely to play a role in regulating the tumour and endothelial cell activation, which will then control proliferation, angiogenesis and metastasis in human breast cancer. UV-B irradiated human cutaneous melanoma cells have an increased IL-8 and increased angiogenesis by virtue of the increased levels of IL-8 (47). IL-8 expressed by human melanoma cells correlates with metastatic potential via an increase in angiogenesis (48). Human melanoma cells in nude mice also show increased expression of IL-8 after irradiation with UVB, with the increased IL-8 corresponding to increased tumour growth and metastasis (25). This study showed that transfection of non-metastatic melanoma cells (IL-8 negative) with the IL-8 gene caused an increase in tumourigenicity and metastatic potential in nude mice. IL-8 transfected cells also showed increased expression of active MMP-2, correlating with increased invasiveness. Activation of MMP-2 by IL8 can enhance invasion of the host stroma by tumour cells, resulting in increased angiogenesis. The environment could also be a major factor in regulation of IL-8 expression as this study also shows that hypoxia increases IL-8 expression (25). It has also been demonstrated that IL-8 produced by cultured melanoma cells acts as an essential autocrine growth factor (49). Accumulating evidence in melanoma research suggests that IL-8 is only present in malignant melanoma, and as such can be used to distinguish between the malignant and benign forms (25, 49). An increased IL-8 expression in primary melanoma indicates high metastatic potential. IL-8 is also one of the cytokines/chemokines produced by choriocarcinoma cell line such as BeWo,

3. IL-8 and angiogenesis and may be involved in the modulation of proliferation and angiogenesis of such tumours (50). Increased IL-8 levels are found in uterine cervical cancers, together with an increased microvessel count and increased macrophage infiltration. The higher the IL-8 levels, the lower the survival rate of the patients (51). 3.2 IL-8: expression by tumourassociated cells Macrophages have a wide range of functions, and have been suggested as a major force in tumour angiogenesis (5) as they produce a number of angiogenic cytokines and growth factors, especially IL-8. They are able to modulate events in the extracellular matrix with the secretion of matrix-degrading and modulating enzymes. It has been shown that there is a correlation between increased macrophage index, malignancy and high vascular grade in malignant melanomas, where activated macrophages are involved in neovascularisation in human malignant melanoma (5). IL-8 produced by macrophages associated with cervical cancers produce increased angiogenesis, which correlates with decreased survival rates of patients (51). Monocytes from metastatic breast cancer release greater amounts of IL-8 than monocytes from non-metastatic breast cancer and healthy donors – all age matched (52). The expression of IL-8 by small cell lung carcinoma is induced by expression from the stromal cells of surrounding it (53). Numerous studies have shown that endothelial cells themselves are able to produce IL-8 under

59 normal conditions (54), levels of which can be markedly increased on treatment with a diverse range of substances such as Calcitonin-gene-related-peptides, oxidised low density lipoproteins and thrombin (54, 55, 56, 57, 58, 59, 60, 61). 3.3 IL-8: paracrine and autocrine functions? From the evidence presented above, it can be concluded that the effect of IL-8 on tumour angiogenesis can occur in both a paracrine and autocrine fashion. Tumour cells, endothelial cells, inflammatory cells and stromal cells are all able to express varying levels of IL-8, and also in some cases express an IL-8 receptor. This especially true for endothelial cells and some of the tumours discussed. The relationships between these cell types and their expression of IL-8 is shown in Fig. 2, illustrating the complexity involved when endeavouring to select a strategy for inhibiting IL-8 function as an angiogenic factor. 4. INHIBITION OF IL-8 PROMOTED ANGIOGENESIS The discovery of molecular mediators of angiogenesis and understanding the relationships between these angiogenic factors is critical to developing successful strategies to either enhance or inhibit angiogenesis. In addition, as antiangiogenic therapies should only stop new endothelial growth, but not attack healthy vessels, there would be no harm done to blood vessels serving normal tissues, except in menstruation and wound healing (3).

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3. IL-8 and angiogenesis Natural inhibitors of IL-8 exist such as the ELR- CXC chemokines IP-10 and MIG which are able to prevent the angiogenic effect of IL-8 (11). In studies concerning inhibition of IL-8 as an angiogenic factor, various strategies have been used. Lithium (salt) was found to down-regulate the IL-8 expression of monocytes from metastatic breast cancers (52). Paclitaxel, a chemotherapeutic agent, however, effected a 5-fold increase in IL8 expression in human lung cancer cell lines (62). Angiostatin has been used as an inhibitor of angiogenesis; the conclusion being that regulation of tumour angiogenesis is dependant on multiple factors, including IL-8, but that inhibition of angiogenesis for therapeutic purposes should not concentrate on a single factor (63). Therapy with chimerised monoclonal antibody to epidermal growth factor receptor (EGFR mAb C225) of human transitional call carcinoma of the bladder in athymic nude mice, resulted in significantly lower levels of IL-8 (VEGf and bFGF), and involution of blood vessels (64), the significant anti-tumour effect was mediated in part by inhibition of angiogenesis. Interestingly, IL-8 itself induces apoptosis in leukemic cells, in vitro and in vivo, where the interaction between endothelial cells and leukemic cells cause induction of apoptosis through the release of endothelial IL-8 (65). They also used a pentapetide of IL-8: NH2-terminal region of endothelial IL-8, Ala-Val-Leu-Pro-Arg (AVLPR) induced apoptosis in leukemic cell lines and inhibited the growth of cell tumour masses. 5. CONCLUSION: IL-8 AND TUMOUR ANGIOGENESIS For tumour angiogenesis to take place, there are a number of prerequisites:

61 (1) Cytokines, chemokines and growth factors from tumour cells and associated cells such as inflammatory cells must interact with appropriate receptors on the endothelial cells; the endothelial cells then become activated into an angiogenic mode. (2) The endothelial cells must then undergo proliferation and migration, facilitated by the production of proteinases, such as MMP’s to degrade the surrounding extracellular matrix and change the expression of cell adhesion molecules on the endothelial cells (such as VEcadherin, integrins, CD44). Loss of cell-cell and cell-matrix adhesion allows the cells to become motile. (3) Lumen are formed by the endothelial cells (angiogenesis), following reorganisation and renewed expression of cell-cell adhesion molecules, and the tumour becomes connected to the blood supply. (4) Tumour cell with metastatic behaviour can then metastasise by invading the stroma, intravasating the newly formed blood supply, and after circulating in the blood system, extravasate to form a secondary loci. How does IL-8 cause angiogenesis? (1) IL-8 can be produced by tumour cells, and associated cells, such as macrophages (often recruited by tumour cells), endothelial cells themselves and surrounding stromal cell types (see Table 2). In addition, IL-8 can be induced by a number of other factors, from all these cell types (Table 2). (2) IL-8 induces proliferation of endothelial cells and is able to promote motility/migration of these cells as endothelial cells have now

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been shown to express IL-8 receptors (11, 13). (3) IL-8 is able to promote angiogenesis by inducing endothelial cells to become angiogenic, and by activating or causing secretion of matrix metalloproteinases (MMP’s) (2, 29). (4) Numerous studies have indicated that IL-8 is directly involved in angiogenesis and hence metastasis of

Chapter 3 a number of human tumours (see section 3.1). In conclusion, Interleukin-8 is a chemokine that exerts a potent angiogenic effect via paracrine and autocrine routes in tumourigenesis, and as such represents an opportunity for intervention in tumour metastasis.

References 1 2 3 4

5

6 7

8

9

10

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interleukin-8 function identified by design of analogs and CXC chemokine hybrids. J Biol Chem 1994; 269:16075-16081 Schraufstatter IU, Ma M, Oades ZG, Barrit DS, Chchrane CG. The role of and of interleukin-8 in the high affinity interaction with the interleukin-8 receptor type A. J Biol Chem 1995; 270:10428-10431 Schonbeck U, Brandt E, Petersen F, Flad H, Loppnow H. IL-8 specifically binds to endothelial but not smooth muscle cells. J Immunol 1995; 154:2375-2385 Nanney LB, Mueller SG, Bueno R, Peiper SC, Richmond A. Distributions of melanoma growth stimulatory activity or growth-related gene and the interleukin-8 receptor type B in human wound repair. Am J Pathol 1995; 147:1248-1260 Luan J, Shattuck-Brandt R, Haghnegahdar H, Owen JD, Strieter R, Burdick M, Nirodi C, Beauchamp D, Newsom K, Richmond A. Mechanism and biological significance of constitutive expression of MGSA/GRO chemokines in malignant melanoma minor progression. J Leukoc Biol 1997; 62:588-597 Bar-Eli M. Role of interleukin-8 in tumour growth and metastasis of human melanoma. Pathobiol 1999; 67(1):12-18 Kumar R, Kuniyasu H, Bucana CD, Wilson MR, Fidler IJ. Spatial and temporal expression of angiogenic molecules during tumour growth and progression. Oncol Res 1998; 10(6):301-311 Kitadai Y, Bucana CD, Ellis LM, Anzai H, Tahara E, Fidler IJ. In-situ messenger-RNA hybridisation technique for analysis of metastasis-related genes in human coloncarcinoma cells. Amer J Pathol 1995; 147(5):1238-1247 Brew R, Erikson JS, West DC, Flanagan BF, Christman SE. Interleukin-8 as a growth factor for human colorectal carcinoma cells in vitro. Immunol 1996, 89: ORV12-ORV12 Brew R, Southern SA, Flanagan BF, McDIcken IW, Christmas SE. Detection of IL-8 mRNA and protein in human colorectal carcinoma cells. Eur J Can 1996; 32A(12):2142-2147 Brew R, Erikson JS, West DC, Kinsella AR, Slavin J, Christmas SE. Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro. Cytokine 2000; 12(1):78-85 Arenberg DA, Keane MP, DiGiovine B, Kunkel SL, Morris SB, Xue YY, Burdick MD, Glass MC, Iannettoni MD, Strieter RM. Epithelial-neutrophil activating peptide (ENA-78) is an important angiogenic factor

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in non-small cell lung cancer. J Clin Invest 1998, 102:(3)465-472 Chen Z, Malhotra PS, Thomas GR, Ondrey PG, Duffey DC, Smith CW, Enamorado N, Yeh NT, Kroog GS, Rudy S, McCullagh L, Mousa S, Quezada M, Herscher LL, Van Waes C. Clin Can Res 1999; 5(6): 1369-1379 Eisma RJ, Spiro JD, Kreutzer DL. Role of angiogenic factors: Coexpression of interleukin-8 and vascular endothelial growth factor in patients with head and neck squamous carcinoma. Laryngoscope 1999; 109(5):687-693 Greene GF, Kitadai Y, Pettaway CA, vonEschenbach AC, Bucana CD, Fidler IJ. Correlation of metastasis-related gene expression with metastatic potential in human prostate cancer cells implanted in nude mice using in situ messenger RNA hybridisation technique. Amer J Pathol 1997; 150(5):15711582 Moore BB, Arenberg DA, Stoy K, Morgan T, Addison CL, Morris SB, Glass M, Wilke C, Xue YY, Sitterding S, Kunkel SL, Burdick MD, Strieter RM. Distinct CXC chemokines mediate tumourigenicity of prostate cancer cells. Amer J Pathol 1999; 154(5):1503-1512 Inoue K, Slaton JW, Eve BY, Kim SJ, Perrotte P, Balbay MD, Yano S, Bar-Eli M, Radinsky R, Pettaway CA, Dinney CPN. Interleukin-8 expression regulates tumourigenicity and metastases in androgenindependent prostate cancer. Clin Can Res 2000; 6(5):2104-2119 Inoue K, Slaton JW, Kim SJ, Perrotte P, Eve BY, Bar-Eli M, Radinsky R, Dinney CPN. Interleukin-8 expression regulates tumourigenicity and metastasis in human bladder cancer. Can Res 2000; 60(8):22902299 Kitadai Y, Takahashi Y, Haruma K, Kaka K, Sumii K, Yokozaki H, Yasui W, Mukaida N, Ohmoto Y, Kajiyama G, Fidler IJ, Tahara E. Transfection of interleukin-8 increases angiogenesis and tumourigenesis of human gastric carcinoma cells in nude mice. Brit J Can 1999; 81(4):647-653 Kitadai Y, Haruma K, Mukaida N, Ohmoto Y, Matsutani N, Yasui W, Yamamoto S, Sumii K, Kajiyama G, Fidler IJ, Tahara E. Regulation of disease-progression genes in human gastric carcinoma cells by interleukin8. Clin Can Res 2000; 6(7):2735-2740 Penson RT, Kronish K, Duan Z, Feller A, Stark P, Cook SE, Duska LR, Fuller AJ, Goodman AK, Nikurai N, MacNeill KM, Matulonis UA, Preffer FI, Seiden MV. IL-2, IL-6, IL-8, MCP-1, Cytokines

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interleukin-8 related angiogenesis in uterine cervical cancers. Can Res 2000; 60(10):26322635 Meredino RA, Arena A, Gangemi S, Ruello A, Losi E, Bene A, D’Ambrosio FP. In vitro interleukin-8 production by monocytes treated with lithium chloride from breast cancer patients. Tumouri 2000; 86(2): 149-152 Anderson IC, Mari SE, Broderick, RJ, Mari BP, Shipp MA. The angiogenic factor interleukin-8 is induced in non-small cell lung cancer/pulmonary fibroblast co-cultures. Can Res 2000; 60(2):269-272 Ansel JA, Song I, Harten B, Kramp JM, Armstrong CA, Bunnett NW. Calcitonin gene related peptide induction of interleukin-8 in human microvascular endothelial cells. Regul Peptid 1996; 64(1):4 Claise C, Edeas M, Chalas J, Cox A, Abella A, Capel L, Lindenbaum A. Oxidised lowdensity lipoprotein induces the production of interleukin-8 by endothelial cells. FEBS Letts 1996 398(2):223-227 Al-Okla S, Chatenay-Rivauday C, Klein J-P, Wachsmann D. Involvement of integrins in interleukin-8 production induced by oral viridans streptococcal protein I/Iif in cultured endothelial cells. Cellul Micro 1999; 1(2):157-168 Ueno A, Murakami K, Yamanouchi K, Watanabe M, Kondo T. Thrombin stimulates production of ilterleukin-8 in human umbilical vein endothelial cells. Immunol 1996; 88(1):76-81 Volk T, Hensel M, Mading K, Egerer K, Kox WJ. Intracellular dependence of nitric oxide mediated enhancement of interleukin-8 secretion in human endothelial cells. FEBS Letts 1997; 415(2): 169-172 Sano H, Nakagawa N, Chiba R, Kurasawa K, Saito Y, Iwamoto I. Cross-linking of intracellular adhesion molecule-1 induces interleukin-8 and RANTES production through the activation of MAP kinases in human vascular endothelial cells. Biochem Biophys Res Comms 1998; 250(3):694-698 Eberl T, Amberger A, Herold M, Hengster P, Steuer W, Hochleitner BW, Gnaiger E, Margreiter R. Expression of stress proteins, adhesion molecules and interleukin-8 in endothelial cells after preservation and reoxygenation. Cryiobiol 1999; 38(2):106118 Burns MJ, Sellati TJ, Teng EI, Furie MB. Production of interleukin-8 (IL-8) by cultured endothelial cells in response to Borrelia burgdorferi occurs independently of secreted [corrected] Il-1 and tumour necrosis factor

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alpha and is required for subsequent transendothelial migration of neutrophils. Infect Immun 1997; 64(4): 1217-1222 Collins TS, Lee LF, Ting JPY. Paclitaxel upregulates interleukin-8 synthesis in human lung carcinoma through an NF-kappa B- and AP-1-dependent mechanism. Can Immun Immuno 2000; 49(2):78-84 Westphal JR, Van’t Hullenaar R, Peek, R, Willems RW, Crickard K, Crickard U, Askaa J, Clemmensen I, Ruiter DJ, De Waal RMW. Angiogenic balance in human melanoma: Expression of VEGf, bFGF, IL-8, PDGF and angiostatin in relation to vascular density of xenografts in vivo. Int J Can 2000; 86(6):768776

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Perotte P, 64 Matsumoto T, Inoue K, Kuniyasu H, Eve BY, Hicklin DJ, Radinsky R, Dinney CPN. Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clin Can Res 1999; 5(2):257-265 Terui Y, Tomizuka H, Mishima Y, Ikeda M, Kasahara T, Uwai M, Mori M, Itoh T, Tanaka M, Yamada M, Shimamura S, Ishizaka Y, Ozawa K, Hatake K. NH2-terminal pentapeptide of endothelial interleukin-8 is responsible for the induction of apoptosis in leukemic cells and has an antitumour effect in vivo. Can Res 1999; 59(22):5651-5655

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Chapter 4 THE ROLE OF INTERLEUKIN-11 IN THE FORMATION OF BONE METASTASES

Naoya Fujita and Takashi Tsuruo Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan

Key words:

Interleukin-11, Bone metastasis, Breast tumours, Osteoclast

Abstract:

IL-11 has various biological functions, such as being an inducer of acute-phase proteins and of antigen-specific antibody responses. It also stimulates erythrocytopoiesis, adipocyte differentiation, and neuronal development. Furthermore, IL-11 possesses the ability to induce osteoclast formation, a function seen in many osteotropic factors. Since IL-11 neutralization can suppress osteoclast development induced by these osteotropic factors, IL-11 may play an important role in regulating bone remodeling. In the course of molecular analysis of bone metastasis, we found that IL-11 production in both osteoblasts and endothelial cells in bone was promoted by interaction with tumour cells. Because the neutralization of IL-11 bioactivities abrogated the tumour-induced bone resorption, IL-11 might be involved in enlarging bone metastatic foci.

1. THE BIOLOGICAL FUNCTIONS OF INTERLEUKIN-11 Interleukin-11 (IL-11) is a cytokine with a wide spectrum of biological functions in the lymphopoietic, hematopoietic, neuronal, and osteoclastic systems. It is known to be secreted from bone marrow stromal cells, articular chondrocytes, synoviocytes, lung fibroblasts, osteoblasts, and endothelial cells in bone. Unlike other cytokines associated with hematopoiesis, IL-11 is not produced from monocytes or lymphocytes. Because IL-11 and IL-6 share a common subunit receptor,

gp130 in signal transduction, many of their biological functions overlap, although the amino acid sturucture of IL11 is unique. Like IL-6, IL-11 plays an important role in the bone metabolism, and its neutralization could suppress osteoclast development induced by several osteotropic factors.

1.1 The Role of Interleukin-11 and Its Receptor in the Regulations of Hematopoietic Systems IL-11 was originally identified as a 23kDa multifunctional cytokine secreted from a bone marrow-derived stromal cell 67

W.G. Jiang et al. (eds.), Growth Factors and their Receptors in Cancer Metastasis, 67–78. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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line, and it showed the ability to induce proliferation of the IL-6-dependent plasmacytoma cell line T1165 (1). The analysis of cDNA encoding IL-11 revealed that it contained 597 nucleotides encoding 199 amino acids (1). The human IL-11 gene was mapped on chromosome 19 at band 19q13.3-q13.4 (2). The chromosomal location of the murine IL-11 gene was assigned to chromosome 7 (3). IL-11 has various biological functions, such as being an inducer of acute-phase protein and of antibody secretion. It also acts as a thrombopoietic factor and a competence factor for hematopoietic stem cells (4). The IL-11 receptor is composed of a chain (ILspecificity-determining and of gp130 (5,6,7). IL-11 binds first to its specific low-affinity and then to two subunits of gp!30 to generate a high-affinity complex (6,8). IL-11 utilizes gp130 as a signal transducer. Because gp130 is a common subunit of IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM), and ciliary neutrophic factor (CNTF) receptors (9), along with IL-11, have common biological activities (Figure 1) (8,10). 1.2 The Role of Interleukin-11 in Bone Metabolism Bone is a dynamic organ that undergoes formation and resorption throughout life. In this remodeling process, osteoblasts participate in bone formation, and osteoclasts mediate bone resorption (11). Alterations in the balance between these activities are believed to play an important role in osteolytic and osteoblastic disorders. Several cytokines

Chapter 4 and hormones regulate the activities of osteoblasts and osteoclasts (12). Interferons (IFNs) suppress bone resorption both by inhibiting the formation and activity of osteoclasts and by stimulating osteoblast-mediated bone formation (12). However, transforming growth colonystimulating factors (CSFs), interleukin-1 (IL-1), tumour necrosis factors (TNFs), and interleukin (IL)-6 stimulate bone resorption by enhancing the formation and activity of osteoclasts (12,13,14,15,16,17). Recent studies have suggested that IL-11 is an important factor that regulates bone remodelling (4,16,17,18,19). Like IL-6, IL-11 is associated with bone resorption because it induces osteoclastogenesis (16). and suppresses the activity of osteoblasts (20). However, unlike IL-11, IL-6, itself, cannot promote osteoclast formation in vitro under physiological conditions. A soluble receptor is indispensable for IL-6 to exhibit osteoclastogenesis (16). In another experiment, IL-11 was associated with the osteoclastogenesis in ovariectomized mice in the same way as in sham–operated mice (4). IL-6 seemed to be involved in osteoclast formation only in pathological states (i.e., without estrogen) because the neutralizing antibody to IL-6 inhibited the increase of osteoclasts in ovariectomized mice but not in sham–operated mice in vivo (15). IL-11 is known to be secreted from osteoblasts, fibroblasts, and endothelial cells (17,18,21,22,23). These observations indicate that IL-11 plays an important, general role in osteoclast formation.

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4. IL-11 and bone metastasis 2. THE ROLE OF IL-11 IN TUMOUR METASTASIS The molecular analysis of osteolytic bone metastasis revealed that tumour cells enhanced osteoblast and endothelial cell IL-11 production in bone. Furthermore, some tumour cells secreted IL-11 by themselves. Since the neutralization of IL-11 suppressed the tumour-mediated bone resorption, IL-11 might participate in the osteoclastmediated enlargement of bone metastatic foci. 2.1 Induction of Tumour-Induced

Interleukin-11 Production in

Osteoblasts

Human melanoma (A375M) and human breast cancer (MDA-MB-231) cells formed osteolytic bone metastasis in vivo when these tumour cells were injected into the left ventricles of BALB/c nude mice (Figure 2) (17). These tumour cells promoted bone resorption in the in vitro neonatal murine calvaria organ culture system by indirectly stimulating the production of a bone resorption-inducing factor (or factors) from human osteoblast-like Saos2 cells. In the course of examining the factors, we found that Saos-2 cells, in response to the interaction with tumour cells, produced a factor that promoted the proliferation of T10 cells. Because the T10 cell growth was IL-6 and IL-11 dependent (24), we checked the concentrations of both in the Saos-2– conditioned medium. We found that this conditioned medium stimulated the production of IL-11, but not IL-6, from Saos-2 cells. Furthermore, we found that a specific anti–IL-11, but not anti–IL-6, antibody could neutralize the abilities to induce T10 proliferation and to promote bone resorption (Figure 3) (17). Thus, tumour cells in bone metastatic foci

71 promote osteolysis by indirectly promoting IL-11 secretion in osteoblasts. Bone releases growth factors, stored in the matrix, during resorption. Thus, excessive bone resorption might provide a favorable microenvironment for metastatic cancer cells to proliferate. Sasaki et al. have reported that a specific inhibitor of osteolytic bone resorption could suppress the progression of established osteolytic lesions (25). These results suggest that IL-11 is also associated with the proliferation of tumour cells by enhancing the release of growth factors and cytokines from the bone matrix. 2.2 Bone-Derived Endothelial Cells Produce Interleukin-11 in Response to Several Osteotropic Factors The histological study of bone metastasis has revealed that most metastatic foci occur near the epiphyseal plate where microvasculatures are abundant (25). Some osteolytic bone diseases are also known to be associated with excessive angiogenesis in the bone (26,27). Thus, we established three different bone-derived endothelial cells (BDECs) from the knee joint, bone marrow, and cortical bone of the femur (23). The established BDECs cells secreted a bone resorption–inducing factor (or factors) that promotes calcium release in vitro from neonatal murine calvaria when the BDECs were treated with several inflammatory cytokines and the conditioned medium of tumour cells. The secreted bone resorption–inducing factor was identified as IL-11 (23). Because the production of IL-11 was not observed in the mouse brain–derived endothelial cell line MBEC4 and the mouse aortic endothelial cell line MAEC3, bone endothelial cells possessed the unique ability to produce IL-11.

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4. IL-11 and bone metastasis These findings indicate that BDECs are also involved in bone resorption by producing the osteoclast-regulating cytokine IL-11. 2.3 The Possible Mechanisms of Interleukin-11–Mediated Bone Resorption Prostaglandins (PGs) are abundantly produced from cells of the osteoblast lineage and are complex regulators of bone metabolism. Many osteoclast- and osteoblast-regulating hormones and cytokines, such as PTH, TGFs, IL-1, TNFs, and FGFs, are known to affect PG production from bone (28,35). Among the PGs, PGE2 is a potent stimulator of bone resorption (36,38). Two types of cyclooxygenases (prostaglandin G/H synthetases) are associated with PG production. Cyclooxygenase-1 (COX-1) is an enzyme expressed constitutively in many tissues. In contrast, the expression of COX-2 is induced by appropriate stimuli in fibroblasts, endothelial cells, macrophages, and osteoblastic cells (39,42). is acutely increased in inflammatory conditions and could result from the rapid and transient increase in COX-2 expression. We found that adding IL-11 promoted production from calvaria in culture medium (43). This finding was consistent with Girasole’s report that IL-11 promoted the development of osteoclasts through PG synthesis (4). We examined the effects of a nonselective COX1/COX-2 inhibitor, indomethacin, or selective COX-2 inhibitors, NS-398 and dexamethasone, on bone resorption of calvaria. All COX inhibitors suppressed IL-11-mediated osteoclast increase and calcium release from calvaria (Figure 4) (43). These results indicate that is the important mediator of IL-11 in boneresorption activity.

75 The interaction between osteoblasts/stromal cells and osteoclast progenitor cells is essential for osteoclastogenesis (44). Recently, several groups have independently isolated the TNF ligand family member ODF/OPGL that is expressed in osteoblasts/stromal cells and is involved in osteoclastogenesis (45,46). ODF/OPGL was identical to TRANCE/RANKL, which was cloned as a factor regulating the functions of T and dendritic cells (47,48). Because the ODF/OPGL gene expression in mouse primary osteoblasts is upregulated by IL11 and (46), ODF/OPGL expression might also be involved in the IL-11– mediated formation of bone metastasis. 3. CONCLUSIONS Tumour-host interaction is known to be involved in the metastasis formation. Our recent findings indicate that both osteoblasts and bone endothelial cells are involved in resorption at the sites of bone metastasis (17,23,43). We have reported that IL-11 produced from osteoblasts and bone endothelial cells is the factor associated with bone resorption (17,23). Although many cytokines and hormones were reported to be involved in resorption, neutralization of IL-11 partly decreased osteoclast development induced by these factors (4). Thus, IL-11 could be the main factor involved in osteoclastogenesis and bone resorption at the site of bone metastasis. Several reports have also clarified the notion that some cancer cells secrete IL-11 by themselves (17,49). According to our observations (43), COX2 inhibitors can suppress IL-11-mediated bone resorption. These results suggest that therapies aimed at IL-11 and COX-2 could prove most successful in preventing bone metastasis.

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Chapter 5 THERAPEUTIC POTENTIAL OF ADENOVIRUS

MEDIATED INTERLEUKIN-12 GENE THERAPY

FOR PROSTATE CANCER

Shin Ebara,1 Yasutomo Nasu,1 Takefumi Satoh,1 Satoru Shimura,1 Chris H. Bangma,1 Gerald W. Hull,1 Mark A. McCurdy,1 Jianxiang Wang,1 Guang Yang,1 Terry L. Timme,1 and Timothy C. Thompson1,2,3 Scott Department of Urology,1 Department of Molecular and Cellular Biology,2 and Department of Radiology,3 Baylor College of Medicine, Houston, Texas, USA

Key words:

Interleukin-12; prostate cancer; orthotopic prostate cancer model; in situ gene

therapy; metastasis

Abstract:

Prostate cancer is characterized by extreme heterogeneity and multifocality of the primary tumour. The available clinical, pathological and molecular data suggest a lack of substantial clonal expansion at the primary site, yet metastatic progression of the disease often proceeds in an unpredictable and clinically undetectable fashion. Clinical and experimental data suggest that primary prostate cancer tumour cells can seed from relatively small tumour foci at the primary site. Overall, this unique biological pattern of progression presents unique and challenging problems regarding the detection and treatment of the disease. In general, currently used potentially curative therapies involve a single cytoablative modality (radical prostatectomy or radiation therapy) and are exclusively directed at the malignant cells within the prostate gland. At present the widespread use of these treatments has not resulted in substantial reduction in mortality from prostate cancer. Gene therapy used alone or as an adjuvant approach could, at least conceptually, provide a rational solution for the prostate cancer dilemma. With gene therapy protocols designed to stimulate antitumour immunity, it may be possible to treat localized and systemic disease effectively and simultaneously. Our previous preclinical and clinical studies have (Herpes Simplex focused on the use of adenoviral vector-mediated Virus thymidine kinase + ganciclovir) in situ gene therapy for prostate cancer. More 79

W.G. Jiang et al. (eds.), Growth Factors and their Receptors in Cancer Metastasis, 79–91. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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recently, specific immunomodulatory genes, such as interleukin-12 (IL-12), have been tested using adenoviral vector-mediated in situ and gene-modified cell based vaccine protocols. The results of these preclinical studies are promising and demonstrate the possibility of effectively generating cytotoxic activities in localized prostate cancers through the recruitment of activated immunocytes while generating systemic anti-tumour immunity that results in antimetastatic activities. We discuss the potential of IL-12 gene therapy for prostate cancer in this review.

1. INTRODUCTION Prostate cancer is the most commonly diagnosed internal malignancy and the leading cause of cancer death in US men (1). Although the early detection of prostate cancer has shifted the stage at the time of diagnosis favorably toward locally confined disease, up to 10% of men with initially diagnosed cancers have distant metastases (2). In men who are treated by surgical removal of the prostate, 20%– 57% develop an elevation of prostatespecific antigen (PSA) following surgery, suggesting the presence of micrometastasis in men with histologically confined disease (3, 4). Patients with occult or clinically evident metastatic disease are managed with palliative hormone therapies and there are no documented curative therapies available. Therefore, there is clearly a need for more effective therapeutic approaches for the treatment of prostate cancer. Tumour immunology represents one of the more promising areas of preclinical/clinical investigation for prostate cancer and other malignancies. The immune system is uniquely suited to generate specific antitumour activities, and specific therapeutic strategies can be adapted to various clinical scenarios including targeting residual tumour cells that may persist following localized therapy for prostate cancer that often lead to disease recurrence (5). However, the development of tumour immunology is limited in many ways by the development of certain characteristics in malignant cells during tumour progression (Table-1).

Although the obstacles to successful immunotherapy are formidable, new technological approaches, such as the manipulation of specific immune cell populations and the efficient transfer and expression of therapeutic genes have driven recent advances in the field. One of the most active immunomodulatory cytokines that has been considered as a candidate for cancer gene therapy is IL12. IL-12 has been shown to suppress the development and progression of numerous malignancies through various, pleiotropic activities (6-13). We have previously demonstrated that introduction of IL-12 via an adenoviral vector directly into orthotopic mouse prostate cancer can not only have local therapeutic effects but also have a therapeutic impact on systemic metastatic disease (13). In this review, we discuss the potential role of IL-12 gene therapy in the treatment of prostate cancer. 2. BIOLOGICAL EFFECTS OF IL-12 IL-12 is a heterodimeric protein composed of two disulfide-linked subunits of 35 and 40 kD (14-16). It is predominantly secreted by activated antigen presenting cells (APCs), including monocytes, macrophages, B-cells, and dendritic cells. IL-12 interacts with specific cell receptors, which in turn can activate gene expression through the Stat4 signal transduction pathway [reviewed in (14)]. The effects of IL-12 play an important role in initiating and orchestrating an immune response directed by central lymphoid effector

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cells, including natural killer (NK) cells, lymphokine-activated killer (LAK) cells, and both CD4-positive and CD8-positive T cells (see Figure-1). Under the stimulation of IL-12, CD4-positive T cells differentiate toward Th1 cells and are inhibited from differentiation into Th2 cells, a critical step in the determination of a cell versus humoral mediated immune response, respectively (15-18). The Th2 response predominantly results in the generation of anti-tumour antibodies, which have been shown to have therapeutic effects under some conditions (14). However, many consider the generation of a Th1 response and the subsequent induction of cytolytic CD8positive T cells as a critical determinant in the generation of anti-tumour immunity (19-21). CD8-positive cytotoxic Tlymphocytes (CTL) can kill tumour cells directly via specificity for antigens on the surface of tumour cells within the context of MHC class 1 presentation. On the other hand, the activation of NK/LAK cells can produce anti-tumour cytotoxic activities against tumour cells that fail to express at least one of the MHC class 1 molecules. The production of cytokines by NK cells and T cells ultimately results not only in their self stimulation but also the further stimulation of APCs and other non-immune cells, including hematopoietic cells, endothelial cells, and tumour cells (see Figure-1). Although there are many molecules generated by lymphoid effector cells in response to IL-12 that can result in downstream secondary cellular response, two important cytokines relevant to the development of antitumour immunity are and Recent studies indicate that is upregulated in T-cells in response to a specific T box transcription factor, T-bet (22). The activity of T-bet and the secondary effects

Chapter 5 of are major determinants in the differentiation of uncommitted CD4positive T cell to Th1 cells. Furthermore, T-bet activity was shown to repress the Th2 phenotype (22). can have dramatic effects in producing activated cytotoxic macrophages, which in turn can produce tertiary anti-tumour factors, such as nitric oxide (23-25). Additional effects of on endothelial cells can have dramatic effects on the development of antitumour immunity through the induction of adhesion molecules that regulate the type and levels of tumour infiltrating lymphocytes (8, 20, 25-27). Interestingly, also produces direct effects on tumour cells that can affect the course of tumour progression. Many of these effects involve the activities of interferoninducible chemokines that, in turn, regulate the migration of specific immunocytes and suppress angiogenesis through paracrine activities (10, 20, 2830). Recent studies have shown that can concurrently upregulate p21 (31) and downregulate neu/HER-2 (32) in prostate cancer cells resulting in antiproliferative effects. T cell derived also plays an important role in conjunction with in activating macrophages and modulating the activities of other hematopoietic cell populations (33, 34). Once activated, macrophages produce that can have profound effects on the endothelium resulting in increased fluid and immunocyte entry into tissues [reviewed in (35)]. The induction of tertiary effectors in these cells by TNFcan also affect immunocyte migration and angiogenic activities (36, 37). The direct effects of on various tumour cell populations including prostate cancer have been well documented [reviewed in (38)]. Although some prostate cancer cells

5. IL-12 gene therapy in cancer are insensitive to the effects of others undergo apoptosis through welldefined, caspase-mediated events (39-41). We have recently conducted an extensive analysis of macrophage infiltration and activities in prostate cancer (42) and found that the density of macrophages (CD68+ cells) in radical prostatectomy specimens is an independent predictor for disease-free survival after surgery. Further, immunohistochemical analysis of cytotoxicity-related biomarkers in stromaassociated mononuclear cells indicated reduced functional activities in highly aggressive prostate cancer compared to less aggressive disease. This suggests that specific macrophage activities (e.g., phagocytosis and proteolysis) may provide a bridge between local cytotoxicity and systemic antitumour activities that normally affects prostate cancer progression and ultimately disease free survival. The correspondence of these potential endogenous protective effects and the induction of macrophage activities by IL-12 are noteworthy (see Figure 2). Thus the extraordinary capacity of IL-12 to generate secondary and tertiary cascades of anti-tumour activities that are self-sustaining and result in extensive antitumour effects make IL-12 a highly attractive candidate for various gene therapy protocols.

3. ADENOVIRAL VECTORMEDIATED IN SITU GENE THERAPY 3.1 Local Anti-Tumour Effects In previous studies we have used the RM-9 prostate cancer model for preclinical studies (13, 43, 44). Our protocol for testing in situ gene therapy involves inoculation of tumour cells

83 orthotopically and their subsequent local growth and metastatic progression. This mouse model is suitable for studying prostate cancer, as like human prostate cancer, it develops via the stimulation of relevant oncogenic pathways; it highly resembles human prostate cancer morphologically and through the expression of specific molecular markers; it has widespread metastatic activities; and it is of low intrinsic immunogenicity. Upon inoculation of RM-9 tumour cells directly within the prostate and subsequent injection of adenoviral vectors expressing IL-12 (AdmIL-12), various determinants of efficacy of this gene therapy protocol were evaluated. Initially, we observed significant growth suppression (>50% reduction of tumour weight) compared to controls. In an effort to determine potential mechanisms for this response, specific immune cell populations were assayed either biochemically or directly through quantitative immunohistochemical staining. We observed extensive immune cell activation following injection of AdmIL-12 and ultimately concluded from this study that the local anti-tumour activities likely resulted from: 1) enhanced NK lysis during the first 7 days following virus injection, 2) enhanced macrophage activity such as NOS activation, and 3) support of cytokine production from and possible cytolytic activities of CD4-positive and/or CD8positive T cells within the local tumour tissue (13). The observation of multiple immunocyte activities that potentially could develop into a systemic anti-tumour immune response involving the generation of memory T cells was evident, and the results of analysis of distant antimetastatic activity in response to local injection of AdmIL-12 further supported this notion.

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5. IL-12 gene therapy in cancer

3.2 In Situ IL-12 Gene Therapy Effects on Distant Metastases In this initial study, we further evaluated the potential for in situ IL-12 gene therapy to affect distant metastatic disease using two different approaches. As RM-9 cells metastasize spontaneously from the orthotopic site, we initially evaluated the effects of this gene therapy protocol on the extent of spontaneous metastasis to lymph nodes. The results indicated that localized gene therapy could significantly suppress the incidence of spontaneous lymph node metastasis. Further studies also demonstrated that in situ IL-12 gene therapy could suppress the formation of preestablished lung metastases following injection of RM-9 cells into the tail vein (13). These results clearly indicated that our therapeutic strategy was not only capable of generating localized cytotoxic response through specific effector cells but also of generating a systemic response was generated that impacted on metastatic disease (see Figure-1).

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Specific NK depletion analysis demonstrated that NK cells were predominantly responsible for the antimetastatic effects of locally administered AdmIL-12 in preestablished RM-9 lung metastases. However, they also indicated that other cell types were likely involved. Recent studies concerning the relationship between IL-12 efficacy and NK cell versus T cells may explain in part the potent initial NK activities both in the local tumour as well as in distant metastases but also the later increase in CD8 infiltration that we observed in this study. IL-12 has been reported to induce an NK-mediated cytolytic phase, followed by a T cell phase that is characterized by CTL activities (15, 16, 45, 46). Although interrelationships between the NK phase and CTL phase are poorly understood, it is well established that the generation of a Th1 response is required for the CTL phase. We observed (13) maximal NK activity at day 1 after AdmIL-12 injection but increased

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infiltration of both CD4-positive and CD8-positive T cells 7 days following virus injection. The tumour-infiltrating CD4 and CD8 T cell populations subsequently decreased over time, and it is not clear whether the infiltrating CD4 cells were of the Th1 phenotype (13). However, the immune cells infiltrate that we observed in the local tumour following in situ IL-12 gene therapy may have relevance to the antimetastatic activities that it generated. Since NK depletion did not completely eliminate the IL-12 antimetastatic activities, we strongly suspect that CD4-/CD8-positive T cells are involved. We further suspect that the development of CD4 Th1 activities and potentially macrophage activities may serve to bridge the initial NK response to more potent and long-lasting antitumour effects that could significantly impact on metastatic disease (see Figure-2). Further studies are in progress to test these concepts and develop further therapeutic approaches, based on the results. 3.3 Gene Modified Cell-Based Vaccines The use of adenoviral vector mediated IL-12 gene therapy is not restricted to intratumoural vector injections, i.e., in situ gene therapy approaches. Indeed, the combination of an adenovirus delivery system together with remarkable immune inductive activities of IL-12 represents a highly versatile combination for use in various cell-based vaccine strategies. In particular for prostate cancer where multiple opportunities for neoadjuvant and adjuvant approaches exist vaccine therapies using adenovirus-mediated IL12 gene transfer may provide unique opportunities. For example, it is conceivable that tumour biopsies could be utilized for the recovery of viable cells into which IL-12 could be transduced using adenoviral vectors. In recent

Chapter 5 studies, we have demonstrated that adenoviral vectors could be used to effectively transfer IL-12 into tumour cells and following irradiation and subsequent subcutaneous implantation, this vaccine was capable of protecting a significant percentage of animals from later tumour challenge using RM-9 cells (44). Other studies have demonstrated the feasibility of using adenoviral vectors to transduce genes into dendritic cells (47, 48), and it was recently demonstrated that dendritic cells transduced with retroviral vectors expressing IL-12 have local and systemic antitumour activities when injected directly into various rodent tumours (49). Therefore, the potential of adenovirus-mediated IL-12 cell based vaccine strategies are numerous and various preclinical studies have demonstrated not only their feasibility but the therapeutic potential of this highly versatile gene therapy approach. 4. IL-12 TOXICITY Recent studies have indicated that the administration of recombinant IL-12 may present a difficult challenge in regard to its association with life-threatening toxicities. Indeed, because of the difficulties in controlling the systemic levels and the widespread systemic effects of recombinant IL-12 on various cell types, previous studies on IL-12 in humans have documented that lifethreatening myelosuppression and splenomegaly can occur (19, 50-52). Therefore, because of these considerations, we and others have considered local delivery of potent cytokines such as IL-12 by using gene therapy protocols to be preferable, in that they may provide sufficiently effective local concentrations of the cytokine without generating toxic systemic levels that are difficult to control.

5. IL-12 gene therapy in cancer In our previous study (13) we evaluated serum levels of IL-12 following orthotopic injection of AdmIL-12. The results indicated that serum concentrations of IL-12 were increased during a period of 10 days after initiation of the treatment and with maximal levels occurring the first day following vector injection. These levels led to enlargement of the spleen after a lag time of several days, as the maximum spleen size was observed on day 7 following treatment (13). This splenomegaly was reversible, since the gradual decrease in serum IL-12 strongly correlated with a return to normal spleen size. Further studies are indicated regarding the adenoviral vector delivery of IL-12 compared to systemic administration. Our studies indicated that adenoviral-vector-delivered IL-12 that had both local and antimetastatic effects was relatively safe and did not result in limiting systemic toxicities. 5. COMBINATION THERAPIES Although we are optimistic regarding the therapeutic potential of adenoviraldelivered in situ IL-12 gene therapy for prostate cancer, we consider this approach to be only another step in the ultimate generation of an optimized therapeutic protocol. The next step forward that we and others have considered is combining IL-12 gene transduction with other costimulatory molecules such as B7-1. This approach has been tested in various other systems and has indicated that the addition of this potent co-stimulatory molecule can modify the effector cell response to IL-12 and, in some cases, result in enhanced therapeutic activity (26, 44, 53). As discussed above IL-12 alone or together with co-stimulating genes could be used in various cell–based vaccine strategies that involve tumour cells and/or antigen presenting cells.

87 An additional approach would be to combine suicide gene therapy together with IL-12 or IL-12+B7. This therapeutic approach also may afford may specific advantages, as be effective at maximizing tumour antigen presentation to effector cells and in conjunction with IL-12, which could mature the effector cell responses and lead to highly synergistic activities (5). At our institution we now have the extensive experience with adenoviralvector-delivered gene therapy in ongoing clinical trials. The addition of adenoviral-vector-delivered IL-12 or IL-12+B7 could be integrated both into our preclinical and clinical programs in an effort to take the next step toward successfully treating both localized and systemic metastatic disease in prostate cancer. Hopefully, these new novel approaches together with currently available radical prostatectomy and radiation will make dramatic improvements in survival with this devastating disease. 6. SUMMARY The lack of effective therapy for locally invasive and metastatic prostate cancer dictates the necessity for intensive focus on the development of novel and effective therapeutic approaches for this important malignancy. The notion of initiating active and persistent antitumour immunity through molecular gene transfer approaches offers a viable avenue of investigation for addressing this problem. The cytokine IL-12 is remarkable and unique in its capacity to induce widespread multilevel cascades of cellular and gene activity that can contribute to the development of a specific antitumour immune response. The use of adenoviral vector gene transfer systems together with IL-12 offers a highly flexible and efficient

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avenue for initiating the potentially therapeutic activities yet limiting the toxicities associated with IL-12. For prostate cancer this strategy can involve in situ-based gene therapy approaches and various cell based vaccine protocols. With continued preclinical studies and clinical trials in this area it seems likely that immunomodulatory gene therapy will make a significant contribution to the

Chapter 5 effective treatment of prostate cancer in the future. Acknowledgements The work in the authors’ laboratory was supported by grants from CaP CURE and NIH (CA50588, CA68814 and SPORE P50-58204). We are grateful to Dr. Joann Trial for critical review of the manuscript.

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a peritumoural stromal reaction in the induction of T-cell migration responsible for interleukin12-induced tumour regression. Cancer Res 1999; 59:1531-8 10. Boggio, K., Di Carlo, E., Rovero, S., Cavallo, F., Quaglino, E., Lollini, P. L., Nanni, P., Nicoletti, G., Wolf, S., Musiani, P., and Forni, G. Ability of systemic interleukin-12 to hamper progressive stages of mammary carcinogenesis in HER2/neu transgenic mice. Cancer Res 2000; 60:359-64 11. Nanni, P., Rossi, I., De Giovanni, C., Landuzzi, L., Nicoletti, G., Stoppacciaro, A., Parenza, M., Colombo, M. P., and Lollini, P. L. Interleukin 12 gene therapy of MHC-negative murine melanoma metastases. Cancer Res 1998; 58:1225-30 12. Colombo, M. P., Vagliani, M., Spreaflco, F., Parenza, M., Chiodoni, C., Melani, C., and Stoppacciaro, A. Amount of interleukin 12 available at the tumour site is critical for tumour regression. Cancer Res 1996; 56:2531-4 13. Nasu, Y., Bangma, C. H., Hull, G. W., Lee, H. M., Hu, J., Wang, J., McCurdy, M. A., Shimura, S., Yang, G., Timme, T. L., and Thompson, T. C. Adenovirus-mediated interleukin-12 gene therapy for prostate cancer: suppression of orthotopic tumour growth and pre-established lung metastases in an orthotopic model. Gene Ther 1999; 6:338-49 14. Gately, M. K., Renzetti, L. M., Magram, J., Stern, A. S., Adorini, L., Gubler, U., and Presky, D. H. The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annu Rev Immunol 1998; 16:495521 15. Scott, P. IL-12: initiation cytokine for cellmediated immunity. Science 1993; 260:496-7 16. Gately, M. K. Interleukin-12: a recently discovered cytokine with potential for enhancing cell-mediated immune responses to tumours. Cancer Invest 1993; 11:500-6

5. IL-12 gene therapy in cancer 17. Wu, C. Y., Demeure, C., Kiniwa, M., Gately, M., and Delespesse, G. IL-12 induces the production of IFN-gamma by neonatal human CD4 T cells. J Immunol 1993; 151:1938-49 18. Stern, A. S., Podlaski, F. J., Hulmes, J. D., Pan, Y. C., Quinn, P. M., Wolitzky, A. G., Familletti, P. C., Stremlo, D. L., Truitt, T., Chizzonite, R., and Gately, M. K. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human Blymphoblastoid cells. Proc Natl Acad Sci U S A 1990; 87:6808-12 19. Brunda, M. J., Luistro, L., Warrier, R. R., Wright, R. B., Hubbard, B. R., Murphy, M., Wolf, S. F., and Gately, M. K. Antitumour and antimetastatic activity of interleukin 12 against murine tumours. J Exp Med 1993; 178:1223-30 20. Zou, J. P., Yamamoto, N., Fujii, T., Takenaka, H., Kobayashi, M., Herrmann, S. H., Wolf, S. F., Fujiwara, H., and Hamaoka, T. Systemic administration of rIL-12 induces complete tumour regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-gamma production by antitumour T cells. Int Immunol 1995; 7:1135-45 21. Boggio, K., Nicoletti, G., Di Carlo, E., Cavallo, F., Landuzzi, L., Melani, C., Giovarelli, M., Rossi, I., Nanni, P., De Giovanni, C., Bouchard, P., Wolf, S., Modesti, A., Musiani, P., Lollini, P. L., Colombo, M. P., and Forni, G. Interleukin 12-mediated prevention of spontaneous mammary adenocarcinomas in two lines of Her2/neu transgenic mice. J Exp Med 1998; 188:589-96 22. Szabo, S. J., Kim, S. T., Costa, G. L., Zhang, X., Fathman, C. G., and Glimcher, L. H. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 2000; 100:655-69 23. MacMicking, J., Xie, Q. W., and Nathan, C. Nitric oxide and macrophage function. Annu Rev Immunol 1997; 15:323-50 24. Michel, T. and Feron, O. Nitric oxide synthases: which, where, how, and why? J Clin Invest 1997; 100:2146-52 25. Cavallo, F., Di Carlo, E., Butera, M., Verrua, R., Colombo, M. P., Musiani, P., and Forni, G. Immune events associated with the cure of established tumours and spontaneous metastases by local and systemic interleukin 12. Cancer Res 1999; 59:414-21 26. Joki, T., Kikuchi, T., Akasaki, Y., Saitoh, S., Abe, T., and Ohno, T. Induction of effective antitumour immunity in a mouse brain tumour model using B7-1 (CD80) and intercellular adhesive molecule 1 (ICAM-1; CD54) transfection and recombinant interleukin 12. Int J Cancer 1999; 82:714-20

89 27. Colombo, M. P., Lombardi, L., Melani, C., Parenza, M., Baroni, C., Ruco, L., and Stoppacciaro, A. Hypoxic tumour cell death and modulation of endothelial adhesion molecules in the regression of granulocyte colony-stimulating factor-transduced tumours. Am J Pathol 1996; 148:473-83 28. Tannenbaum, C. S., Wicker, N., Armstrong, D., Tubbs, R., Finke, J., Bukowski, R. M., and Hamilton, T. A. Cytokine and chemokine expression in tumours of mice receiving systemic therapy with IL-12. J Immunol 1996; 156:693-9 29. Duda, D. G., Sunamura, M., Lozonschi, L., Kodama, T., Egawa, S., Matsumoto, G., Shimamura, H., Shibuya, K., Takeda, K., and Matsuno, S. Direct in vitro evidence and in vivo analysis of the antiangiogenesis effects of interleukin 12. Cancer Res 2000; 60:1111-6 30. Sgadari, C., Angiolillo, A. L., and Tosato, G. Inhibition of angiogenesis by interleukin-12 is mediated by the interferon-inducible protein 10. Blood 1996; 87:3877-82 31. Hobeika, A. C., Etienne, W., Cruz, P. E., Subramaniam, P. S., and Johnson, H. M. IFNgamma induction of p21WAF1 in prostate cancer cells: role in cell cycle, alteration of phenotype and invasive potential. Int J Cancer 1998; 77:138-45 32. Kominsky, S. L., Hobeika, A. C., Lake, F. A., Torres, B. A., and Johnson, H. M. Downregulation of neu/HER-2 by interferon-gamma in prostate cancer cells. Cancer Res 2000; 60:39048 33. Munoz-Fernandez, M. A., Fernandez, M. A., and Fresno, M. Synergism between tumour necrosis factor-alpha and interferon-gamma on macrophage activation for the killing of intracellular Trypanosoma cruzi through a nitric oxide-dependent mechanism. Eur J Immunol 1992; 22:301-7 34. Trinchieri, G. Proinflammatory and immunoregulatory functions of interleukin-12. Int Rev Immunol 1998; 16:365-96 35. Vassalli, P. The pathophysiology of tumour necrosis factors. Annu Rev Immunol 1992; 10:411-52 36. Fajardo, L. F., Kwan, H. H., Kowalski, J., Prionas, S. D., and Allison, A. C. Dual role of tumour necrosis factor-alpha in angiogenesis. Am J Pathol 1992; 140:539-44 37. Downey, G. P. Mechanisms of leukocyte motility and chemotaxis. Curr Opin Immunol 1994; 6:113-24 38. Wong, G. H. W., Vehar, G., and Kaspar, R. L. Apoptosis and Cancer. In: S. J. Martin (ed.), pp. 245-257: Karger Landes System, 1997.

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39. Sherwood, E. R., Ford, T. R., Lee, C., and Kozlowski, J. M. Therapeutic efficacy of recombinant tumour necrosis factor a in an experimental model of human prostatic carcinoma. J. Biol. Response Modif. 1998; 9:4452 40. Rokhlin, O. W., Gudkov, A. V., Kwek, S., Glover, R. A., Gewies, A. S., and Cohen, M. B. p53 is involved in tumour necrosis factor-alphainduced apoptosis in the human prostatic carcinoma cell line LNCaP. Oncogene 2000; 19:1959-68 41. Kimura, K., Bowen, C., Spiegel, S., and Gelmann, E. P. Tumour necrosis factor-alpha sensitizes prostate cancer cells to gammairradiation-induced apoptosis. Cancer Res 1999; 59:1606-14 42. Shimura, S., Yang, G., Wheeler, T. W., Frolov, A., and Thompson, T. C. Reduced Infiltration of Tumour-Associated Macrophages in Human Prostate Cancer: Association with Cancer Progression. Cancer Res 2000; 60: 5857-5861 43. Baley, P. A., Yoshida, K., Qian, W., Sehgal, I., and Thompson, T. C. Progression to androgen insensitivity in a novel in vitro mouse model for prostate cancer. J Steroid Biochem Mol Biol 1995; 52:403-13 44. Hull, G. W., McCurdy, M. A., Nasu, Y., Bangma, C. H., Shimura, S., Lee, H.-M., Wang, J., Albani, J., Ebara, S., Sato, T., Timme, T. L., and Thompson, T. C. Prostate cancer gene therapy: Comparison of adenovirus mediated expression of interleukin-12 with interleukin-12 plus B7-1 for in situ gene therapy and genemodified cell-based vaccines. Clin. Cancer Res. 2000; 6:4101-4109 45. Brunda, M. J. Interleukin-12. J Leukoc Biol 1994; 55:280-8 46. Banks, R. E., Patel, P. M., and Selby, P. J. Interleukin 12: a new clinical player in cytokine therapy [editorial]. Br J Cancer 1995; 71:655-9 47. Arthur, J. F., Butterfield, L. H., Roth, M. D., Bui, L. A., Kiertscher, S. M., Lau, R., Dubinett, S., Glaspy, J., McBride, W. H., and Economou, J. S. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther 1997; 4:17-25 48. Zhong, L., Granelli-Piperno, A., Choi, Y., and Steinman, R. M. Recombinant adenovirus is an efficient and non-perturbing genetic vector for human dendritic cells. Eur J Immunol 1999; 29:964-72 49. Nishioka, Y., Hirao, M., Robbins, P. D., Lotze, M. T., and Tahara, H. Induction of systemic and therapeutic antitumour immunity using intratumoural injection of dendritic cells genetically modified to express interleukin 12. Cancer Res 1999; 59:4035-41

Chapter 5 50. Orange, J. S., Salazar-Mather, T. P., Opal, S. M., Spencer, R. L., Miller, A. H., McEwen, B. S., and Biron, C. A. Mechanism of interleukin 12mediated toxicities during experimental viral infections: role of tumour necrosis factor and glucocorticoids. J Exp Med 1995; 181:901-14 51. Lamont, A. G. and Adorini, L. IL-12: a key cytokine in immune regulation. Immunol Today 1996; 17:214-7 52. Leonard, J. P., Sherman, M. L., Fisher, G. L., Buchanan, L. J., Larsen, G., Atkins, M. B., Sosman, J. A., Dutcher, J. P., Vogelzang, N. J., and Ryan, J. L. Effects of single-dose interleukin-12 exposure on interleukin-12associated toxicity and interferon- gamma production. Blood. 1997; 90:2541-8 53. Putzer, B. M., Hitt, M., Muller, W. J., Emtage, P., Gauldie, J., and Graham, F. L. Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumour regression. Proc Natl Acad Sci USA 1997; 94:10889-10894 54. Ferrone, S. and Marincola, F. M. Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance. Immunol Today 1995; 16:487-94 55. Garrido, F., Ruiz-Cabello, F., Cabrera, T., PerezVillar, J. J., Lopez-Botet, M., Duggan-Keen, M., and Stern, P. L. Implications for immunosurveillance of altered HLA class I phenotypes in human tumours. Immunol Today 1997; 18:89-95 56. Nakazaki, Y., Tani, K., Lin, Z. T., Sumimoto, H., Hibino, H., Tanabe, T., Wu, M. S., Izawa, K., Hase, H., Takahashi, S., Tojo, A., Azuma, M., Hamada, H., Mori, S., and Asano, S. Vaccine effect of granulocyte-macrophage colony-stimulating factor or CD80 genetransduced murine hematopoietic tumour cells and their cooperative enhancement of antitumour immunity. Gene Ther 1998; 5:1355-62 57. Sehgal, I., Baley, P. A., and Thompson, T. C. Transforming growth factor betal stimulates contrasting responses in metastatic versus primary mouse prostate cancer-derived cell lines in vitro. Cancer Res 1996; 56:3359-65 58. Fakhrai, H., Dorigo, O., Shawler, D. L., Lin, H., Mercola, D., Black, K. L., Royston, I., and Sobol, R. E. Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy. Proc Natl Acad Sci U S A 1996; 93:2909-14 59. Finke, J. H., Zea, A. H., Stanley, J., Longo, D. L., Mizoguchi, H., Tubbs, R. R., Wiltrout, R. H., O’Shea, J. J., Kudoh, S., Klein, E., Bukowski, R. M., and Ochoa, A. C. Loss of T-cell receptor zeta chain and p561ck in T-cells infiltrating

5. IL-12 gene therapy in cancer human renal cell carcinoma. Cancer Res 1993; 53:5613-6 60. Alexander, J. P., Kudoh, S., Melsop, K. A., Hamilton, T. A., Edinger, M G., Tubbs, R. R., Sica, D., Tuason, L., Klein, E,, Bukowski, R. M., and Finke, J. H. T-cells infiltrating renal cell carcinoma display a poor proliferative response even though they can produce interleukin 2 and express interleukin 2 receptors. Cancer Res 1993; 53:1380-7

91 61. Sturmhoefel, K., Lee, K., Gray, G. S., Thomas, J., Zollner, R., O’Toole, M., Swiniarski, H., Dorner, A., and Wolf, S. F. Potent activity of soluble B7-IgG fusion proteins in therapy of established tumours and as vaccine adjuvant. Cancer Res 1999; 59:4964-72 62. Lee, H. M., Timme, T. L., and Thompson, T. C. Resistance to lysis by cytotoxic T cells: a dominant effect in metastatic mouse prostate cancer cells. Cancer Res 2000; 60:1927-33

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Chapter 6 FIBROBLAST GROWTH FACTORS AND THEIR RECEPTORS IN METASTASES OF PROSTATE AND OTHER UROLOGICAL CANCERS

Zoran Culig, Marcus V. Cronauer, Alfred Hobisch, Georg Bartsch, and Helmut Klocker Department of Urology, University of Innsbruck, Austria

Key words:

Acid and basic fibroblast growth factor, prostate cancer, bladder cancer, renal cell carcinoma, invasion, metastasis, angiogenesis

Abstract:

Theraputical options for advanced carcinoma of the prostate, bladder or kidney are limited. Therefore it is important to understand their invasion and metastasis, processes in which fibroblast growth factors play an important role. Basic fibroblast growth factor (bFGF) is expressed in androgen-insensitive prostate cancer cell lines PC-3 and DU-145 and in some clinical specimens. During progression of prostate cancer, the expression of the FGF receptor 2 isoform IIIb, which preferentially binds keratinocyte growth factor (KGF) decreases and the expression of the isoform IIIc, which preferentially binds bFGF increases. A similar phenomenon was observed in bladder cancer. Several FGFs are proposed to act as andromedins, proteins that mediate the effects of androgens in target tissues: FGF-7 (KGF), FGF-8 and FGF-10. In prostate and bladder cancer, FGFs regulate tumour metastases by induction of the matrix metalloproteinase promatrilysin. Matrix metalloproteinases degrade extracellular matrix proteins and their expression is elevated in prostate cancer cells. bFGF is strongly expressed in invasive bladder cancers in which it promotes angiogenesis. bFGF levels in bladder cancer are down-regulated by administration of interferon-alpha. bFGF and aFGF are frequently elevated in urine of bladder and renal cancer patients. There is a strong association between urinary bFGF and clinical parameters in bladder cancer. In renal cancer, it was shown that the transfection of bFGF cDNA leads to an increased invasiveness and formation of metastatic nodules.

93 W.G. Jiang et al. (eds.), Growth Factors and their Receptors in Cancer Metastasis, 93–106. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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1. FIBROBLAST GROWTH FACTOR FAMILY – STRUCTURE, FUNCTION AND SIGNAL TRANSDUCTION A common characteristics of members of the fibroblast growth factor (FGF) family is that they bind to heparin and this binding protects them from degradation by proteases, high temperature or low pH. Basic FGF (bFGF), a single-chain protein of 146 amino acids with a molecular mass of 16 500 Da, was purified from pituitary and brain extracts as a protein with strong mitogenic activity for fibroblasts (1,2). Bigger forms of bFGF which are translation products of alternative initiation sites have been isolated from various tissues. The existence of a second mitogen for fibroblasts distinct from bFGF was detected in the same tissues and the factor was named aFGF because of its acidic isoelectric point (3). aFGF is a 140-amino acid protein with a molecular mass of 15 500 Da. aFGF and bFGF are homologous proteins with about 55% sequence identity. The genomic organization of the human aFGF and bFGF is similar. The coding sequence consists of three exons, which are interrupted by two introns (4). The aFGF single-copy gene is located on chromosome 5 and the bFGF gene is situated on chromosome 4q26-27. Four members of the FGF family (FGFs 3-6) are oncogene products. FGF-7 was found to be a growth factor for keratinocytes and was therefore termed keratinocyte growth factor (KGF). Newly discovered FGFs are FGF-8, -9 and –10 (5-7). The FGF receptor (FGFR) family consists of four genes which exhibit structural heterogeneity; FGFR-1 (the fig gene product), FGFR-2 (the bek gene product), FGFR-3 and FGFR-4 (8). Both aFGF and bFGF lack a signal peptide consensus sequence and there are several hypotheses to explain their secretion from cells. The

Chapter 6 mechanism of growth factor secretion involves cell lysis or the damage of plasma membrane. Alternative release mechanisms may involve an ATP-driven peptide pump or a formation of a complex between FGF and and a carrier protein. FGFs are synthesized by many cell types – fibroblasts, endothelial, smooth muscle, granulosa and adrenocortical cells and astrocytes. Due to the presence of the respective receptors, autocrine proliferation loops exist. FGF production was observed in a number of tumours, such as central nervous system ones, leukemias and liver tumours. FGFs effects in target cells include mitogenesis, stimulation of motility and migration and synthesis of specific cellular proteins. Various signal transduction pathways are initiated after binding of FGFs to their receptors; induction of the protooncogenes fos and myc, stimulation of the adenylate cyclase, breakdown of phosphatidyl inositides and the generation of the second messenger diacylglycerol, induction of the protein kinase C or mitogen-activated protein kinase pathways and elevation of intracellular calcium were described (914). One of the most important functions of FGFs is induction of new blood vessel growth which occurs during early stages of tumour development. Formation of capillary blood vessels consists of endothelial cell proliferation, the sprouting of new capillaries, endothelial cell migration and the breakdown of extracellular matrix surrounding capillaries. FGFs stimulate these activities and also stimulate the proliferation of endothelial cells from blood vessels (15). Extracellular matrix molecules, such as laminin and collagens, send signals for capillary tubular formation, elongation and differentiation. Blood vessel formation is regulated by interplay between bFGF and transforming growth

6. FGF and cancer metastasis factor-ß (TGF-ß). The extracellular matrix molecule urokinase-type plasminogen activator (uPA), which is up-regulated by bFGF, induces TGF-ß activity. TGF-ß displays a biphasic effect on FGF-induced endothelial cell proliferation; at low concentrations it acts in cooperation with bFGF whereas at high doses it inhibits bFGF-induced proliferation (16). 2. THE ROLE OF FIBROBLAST GROWTH FACTORS IN PROSTATE CANCER Prostate cancer, which is the most commonly diagnosed malignant tumour in the Western world, could be cured with radical prostatectomy in its early stages. All other prostate tumours need to be treated with androgen ablation therapy. This therapy is only palliative and nearly all tumours progress to the therapyrefractory stage. Various experimental treatments for advanced disease have been tried with little success. Progression of prostate cancer involves alterations in expression and function of several positive and negative growth factors and their receptors, including those of the FGF family. The three peptides, aFGF, bFGF and, more recently, FGF-8 have been intensively investigated in prostate tumours with regard of their expression, interaction with the respective receptors, effects on growth in vivo and in vitro and regulation of angiogenesis and invasion. Importance of fibroblast-derived growth factors for prostate cancer was recognized in series of studies in which formation of LNCaP tumours in vivo was studied (17,18). The tumours were consistently induced when androgensensitive LNCaP cells were coinoculated with non-tumourigenic bone, rat

95 urogenital system mesenchymal and prostate fibroblasts. Coinoculation of LNCaP cells with a matrix absorbed with bFGF also resulted with induction of tumours (17). bFGF was shown to be a potent mitogen for LNCaP cells. However, the mitogenic effect of fibroblast-conditioned media was not eliminated by anti-bFGF antibodies thus indicating that other soluble factors contribute to interactions between fibroblasts and cancer cells. Early studies on FGFs in carcinoma of the prostate were focused on their expression in rat Dunning tumours. In the slow-growing and androgen-responsive cell line R3327 aFGF is predominantly produced whereas in the metastatic cell line AT-3 both aFGF and bFGF are expressed (19). Immunoreactivity for aFGF was not detected in stromal cells and was weak in both basal and luminal cells in the normal tissue. In normal prostate, strong staining for FGFR-1 was noted in stromal and endothelial cells of blood vesels as well as in basal cells (2022). Similarly, FGFR-2 was detected in endothelial and basal cells (22) whereas its appearance in normal epithelium is a matter of debate (22,23). In the Dunning tumour system progression is associated with changes in the expression of the FGFR-2 gene (24). In early stages, the predominant receptor form is the exon IIIb-isoform which preferentially binds stroma-derived keratinocyte growth factor (KGF) whereas during tumour progression the IIIc form which has a high affinity for bFGF is expressed. This change in receptor expression is associated with progression from a mixed stromalepithelial to a stromal independent

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6. FGF and cancer metastasis phenotype, a phenomenon that was observed in several studies on human prostate cancer (25,26). Tumour progression is further characterized with the development of the bFGF autocrine loop, activation of the FGFR-1 and enhanced expression of FGF-3 and -5. Splicing of the FGF-R2 was also investigated in the human cell lines LNCaP, PC-3 and DU-145. That study revealed a similar correlation between FGFR expression and malignant phenotype. DU-145 cells, which exhibit a more aggressive growth express the IIIc isoform whereas the IIIb isoform was found in LNCaP cells (27). Androgensensitive xenografts DUKAP-1 and –2 also displayed IIIb isoform expression and the IIIc isoform was found in the androgen-independent tumour DU 9479. Taken together, these results consistently show that the loss of FGFR-2 IIIb isoform might be a common event in prostate cancer progression. There was the lack of observable FGFR-2 protein in PC-3 cells (27). In one of initial studies on bFGF in prostate cancer cell lines high amounts of mRNA for the FGFR in PC-3 cells were reported. The cells did not respond to exogenous bFGF, most probably because of an autocrine production of the growth factor (28). An information as to functional significance of the two receptors in prostate, FGFR-1 and FGFR2, was obtained recently (29,30). Those researchers showed that nearly all rat prostate malignant cells express the R1 whereas the expression of the R2 was reduced. Following transfection into nonmalignant cells, FGFR-1 kinase accelerated progression to the malignant phenotype and promoted a mitogenic response. In contrast, reexpression of the FGFR-2 IIIb led to the re-establishement

97 of stromal dependency of a tumour and to epithelial differentiation. In clinical specimens derived from prostate cancer tissue both FGF receptors were detected with tendency towards a stronger expression in cancers with a higher Gleason grade (22) (Figure 1). These results were obtained by immunohistochemistry and, in some specimens, confirmed by Western blot. In this context, it is important to know that an increased expression of the FGFR-1 correlates with advanced tumour stage in head and neck squamous carcinomas and in non-small cell lung carcinoma (31,32). However, an information regarding expression of FGFR isoforms in clinical material is not available because of the lack of respective antibodies. In human prostate cancer cell lines, bFGF expression was consistently demonstrated in androgen-insensitive PC3 and DU-145 cells whereas no bFGF was detected in LNCaP cells (28,33,34). Since bFGF lacks its signaling sequence it is poorly secreted into the supernatants (33). There is a little information about the regulation of bFGF in prostate cancers available. In PC-3 cells down-regulation of bFGF is induced by interferons alpha and beta (35). The cell lines LNCaP and DU-145 respond to exogenous bFGF (28). Concentration of bFGF in carcinomatous tissue was measured by ELISA and it was found that the mean concentration in prostate cancers was markedly increased relative to controls (22). Immunohistochemical studies, however, revealed the expression of bFGF only in stromal cells. No immunoreactivity was seen in the neoplastic epithelium even in those samples in which high bFGF concentration was measured by ELISA.

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6. FGF and cancer metastasis These findings differ from those reported by Cronauer et al. and Dorkin et al. (33,36). Cronauer and associates found differences in bFGF-staining between healthy epithelium and cancer tissue; staining was more intense in the malignant tissue. Elevated bFGF values were measured in sera of men with prostate cancer but no correlation with clinical stage, Gleason score or prostate volume was found (33,37). On the basis of our immunohistochemical results, we have postulated that the source of elevated bFGF are tumour cells themselves. The importance of FGFs for prostate tumourigenesis has been studied in the PNT1a cell line transfected with bFGF cDNA (38). This transfection led to the acquisition of anchorage-independent growth that was not seen in the parental cells. bFGF injected orthotopically into the rat ventral prostate, preferentially stimulated growth of the epithelial compartment (39). In addition to aFGF and bFGF, the expression of FGF-8 in prostate cancer has become a subject of investigation. FGF-8 was first identified as a protein that mediates androgen-induced growth of the mouse mammary Shionogi carcinoma cell line SC-3 (40). It was shown that application of the neutralizing monoclonal antibody against FGF-8 abolishes effect of androgen on cell growth. FGF-8 is overexpressed in malignant prostatic epithelium and its expression correlated with Gleason score and clinical stage (36) (41). The mechanisms of regulation of FGF-8 in advanced carcinoma of the prostate have not been investigated but they may involve non-steroidal activation of the AR. It became clear that AR activity is modulated by a number of peptide hormones in ligand-independent and synergistic manner (for review see (42)). In addition to bFGF and FGF-8, a

99 high percentage of clinical cancers (86.1%) express aFGF (36,40). The correlation between expression of aFGF and FGF-8 was statistically significant. In the study by Wang and associates, a correlation between the expression of the androgen receptor and FGF-8 was found, consistent with findings previously reported by Tanaka et al. (40,43). The most important fibroblast growth factor produced by prostatic stromal cells is KGF which is, in contrast to bFGF, efficiently secreted. Based on tissue and organ culture studies, it was proposed that KGF and FGF-10 act as andromedins, mediators of of androgen-induced growth of epithelial cells (44,45). There is a consensus that KGF is expressed in stromal tissue whereas its receptor was detected exclusively in the epithelium (46). However, there is no supportive evidence that KGF expression decreases following castration and therefore its role in vivo is not clear at present (47). FGF-9, which was recently reported to be expressed in stromal cells, is mitogenic for both stromal and epithelial cells, but its alterations were not found in tumouradjacent stroma. In addition, tumour cells themselves do not express FGF-9 (48). An important mechanism by which FGFs regulate prostate tumour metastases is induction of promatrilysin, which belongs to matrix metalloproteinase (MMP) enzymes (Fig. 2) (49). MMPs are enzymes which degrade extracellular matrix proteins and their expression is elevated in prostate cancer cells (50). aFGF is a potent inducer of promatrilysin even at low concentrations. Similar effects were observed also with higher concentrations of bFGF, FGF-8 and -9 (49). A possible reason for the observed strong effect of aFGF is its high affinity for all FGF receptors. Induction of promatrilysin by FGFs was observed in

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cancer cells but not in normal prostatic epithelial cells. A direct effect of bFGF on rat prostate cancer cell motility was demonstrated (51). bFGF utilizes different signaling pathways in various cell lines; these pathways include activation of mitogenactivated protein kinase or protein kinase C signal transduction cascades and elevation of intracellular calcium. Involvement of protein kinase C alpha and epsilon in bFGF signaling was demonstrated in rat prostate cancer cells (52). In those cells, application of aFGF and bFGF antisense oligonucleotides specifically inhibited proliferation (52). We have investigated bFGF signal transduction in connection with androgen receptor protein down-regulation by bFGF in LNCaP cells (53). It was investigated whether this down-regulation is mediated by elevation in intracellular calcium. In LNCaP cells, addition of calcium ionophore leads to reduction of expression of the androgen receptor (54). However, we have demonstrated that bFGF does not increase calcium levels in LNCaP cells (53). It is interesting to note that bFGF in LNCaP causes both stimulation of proliferation and inhibition of androgen receptor expression. 3. FIBROBLAST GROWTH FACTORS AND BLADDER CANCER Bladder cancer is the second most common malignancy of the genitourinary tract. If the metastases are present, the prognosis is poor. Therefore, there is a need to investigate the mechanisms of metastasis and invasion in that disease. In this context, expression and function of FGFs has been studied. In normal bladder, strong bFGF staining was observed in the basal lamina of the transitional epithelium and smooth

Chapter 6 muscle of the bladder wall. In a high percentage of superficial bladder cancer tissue bFGF could not be detected (55). In contrast, bFGF mRNA and protein expression are high in invasive tumours (56). In these cancers bFGF contributes to the degradation of extracellular matrix and angiogenesis and could therefore be considered therapeutical target. The proposed mechanism involves invasion of the basement membrane by tumoursecreted enzymes such as urokinase, cathepsin D and heparanase, involvement of the muscle layers of the bladder and release of bFGF from intracellular stores. bFGF that is released stimulates angiogenesis and is detected in blood vessels. In bladder cancer bFGF also upregulates the expression of MMPs (57) thus facilitating metastasis. There was no significant difference in proliferation and cell motility between parental and bFGF cDNA-transfected cells. However, the production and activity of MMP-2 and -9 were considerably higher in the cells expressing bFGF cDNA and were reduced in the cells treated with bFGF antisense oligonucleotides. In parallel, in vitro metastatic potential increased in the cells expressing bFGF. In concordance with those data, De Boer et al. reported only a minor effect of bFGF on growth of a human transitional cell cancer cell line and similar data were obtained with organoid-like primary cultures (58). A clinical study showed that the levels of MMP-2 and –9 are higher in invasive bladder tumours than in superficial ones (59). NBT-II cells transfected with aFGF cDNA were not more tumourigenic than control cells but these tumours were highly vascularized with a high density of enlarged vessels (60). Those findings also indicate that strong angiogenesis is not sufficient to accelerate tumourigenesis.

6. FGF and cancer metastasis Those aFGF expressing cells give rise to rapidly growing well-vascularized tumours (61). It also promoted motility of bladder cancer cells (62). It was recently postulated that nuclear bFGF confers metastatic properties on rat bladder carcinoma cells by a mechanism which does not involve FGF receptor (63). Systemic administration of interferonalpha inhibits the expression of bFGF mRNA and protein by human transitional cell cancer. This down-regulation is associated with inhibition of angiogenesis and tumour growth in vivo (64). Human umbilical vein endothelial cells, in medium containing interferon-alpha, showed a significant inhibition of branching. Daily application of interferonalpha in bladder cancers implanted in nude mice inhibited tumour growth, vascularization and expression of bFGF and MMP-9 (65). Bladder cancer cell lines strongly induced endothelial cell migration in the in vitro assay (66) whereas normal urethral cells were antiangiogenic. Antiangiogenic effect of bladder cell lines was abolished with neutralizing antibodies to vascular endothelial growth factor and bFGF. Thrombospondin-1 was identified as a compound mainly responsible for antiangiogenic effect of conditioned media from normal bladder cells. Secretion of thrombospondin-1 was downregulated by bladder cancer cells. KGF stimulated proliferation of bladder cancer cells and the expression of its receptor was reported (67). Progression of bladder tumours is associated with a decreased expression of the receptor IIIb, as revealed in clinical specimens (68). Thus, there is a similar trend in prostate and bladder cancer regarding expression of the FGFR-2 IIIb. In bladder cancer, there is a strong association between urinary bFGF and

101 clinical parameters (69,70). It was found that FGF-like activity measured in urine samples correlates with tumour volume (71). Urinary bFGF was elevated in 67% of locally active bladder cancer patients, a percentage that was higher than that in several other malignancies (kidney, prostate, breast) (72). The source of the elevated bFGF are most probably proliferating capillary endothelial cells. aFGF was also detected in urine of bladder cancer patients and its expression correlated with the stage of the disease (73). During embryogenesis and neoplastic transformation, bladder epithelium changes its state of differentiation. This epithelium-to-mesenchyme differentiation is mediated by aFGF and not by related FGFs (74). The changes in plasticity of bladder NBT-II cells are monitored by immunohistochemical staining of the desmosomal protein desmoglein. aFGF treatment causes disappearance of desmoglein. 4. EXPRESSION AND FUNCTION OF FIBROBLAST GROWTH FACTORS IN RENAL CANCER Renal cell carcinomas are characterized by hypervascularity, rapid metastasizing and poor prognosis. Therapeutical options for this malignancy are very limited. There was an interesting observation that human omental adipose tissue bFGF demonstrates greater angiogenic and mitogenic activity than either benign or cancerous tissue bFGF (75). It is known that renal cancer correlates with obesity, in particular in females (76). bFGF cDNA-transfected renal cancer cell lines were more invasive than controls (77). Those cell lines also show an increased MMP-2 production and formed more than 10 times as many metastatic nodules in lungs as non-transfected or

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control cells. In that study, endogenous expression of bFGF fails to stimulate cell proliferation. bFGF was detected in conditioned media from the RC29 renal cancer cells which also respond to the exogenously added growth factor (78). Thus, at least some renal cancers might be stimulated by bFGF in an autocrine fashion. In human renal cancer cell cultures bFGF is inversely regulated to cell density (79). The expression levels of bFGF in renal cancer mRNA and protein were higher than those measured in normal tissue (80). High bFGF levels were measured in kidney tumour tissue and in urine. The expression of bFGF in renal cell carcinoma and urinary bFGF inversely

Chapter 6 correlated with patient survival (72,81). bFGF expression in the tumours is transient and dependent on the organ environment (82). Renal cell cancer metastatic cells were injected into the kidney or subcutis of nude mice. High vascularization was observed only in tumours injected into the kidney and urine of these animals contained higher bFGF levels. The studies summarized in the present review show that FGFs are important for progression of urological cancers. A number of experimental therapies targeting angiogenesis is being developed and it is expected that these treatments could be evaluated in near future.

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demonstrates the presence of basic fibroblast growth factor. J Urol 1993; 150:997-1001. 79. Singh RK, Llansa N, Bucana CD, Sanchez R, Koura A, Fidler IJ. Cell density-dependent regulation of basic fibroblast growth factor expression in human renal cell carcinoma cells. Cell Growth Differ 1996;7:397-404. 80. Eguchi J, Nomata K, Kanda S, Igawa T, Taide M, Koga S, Matsuya F, Kanetake H, Saito Y. Gene expression and immunohistochemical localization of basic fibroblast growth factor in renal cell carcinoma. Biochem Biophys Res Commun 1992;183:937-944.

Chapter 6 81. Nanus DM, Schmitz-Drager BJ, Motzer RJ, Lee AC, Vlamis V, Cordon-Cardo C, Albino AP, Reuter VE. Expression of basic fibroblast growth factor in primary human renal tumours: correlation with poor survival. J Natl Cancer Inst 1994;85:1597-1599. 82. Singh RK, Bucana CD, Gutman M, Fan D, Wilson MR, Fidler IJ. Organ site-dependent expression of basic fibroblast growth factor in human renal cell carcinoma cells. Am J Pathology 1994;145:365-374.

Chapter 7 INSULIN-LIKE GROWTH FACTOR AXIS ELEMENTS IN BREAST CANCER PROGRESSION

Emilia Mira, Rosa Ana Lacalle, Carlos Martínez-A. and Santos Mañes Department of Immunology and Oncology, Centro National de BiotecnologíaCSIC, Campus de Cantoblanco, Universidad Autónoma de Madrid, Madrid, Spain

Key words: Insulin-like growth factor, chemotaxis, cell polarization, tumour invasion, breast cancer Abstract:

Insulin-like growth factors (IGF) exhibit very potent mitogenic activity, promote cell survival, and have insulin-like functions essential for embryogenesis and postnatal growth physiology. Attention has recently focused on the role of IGF in neoplastic transformation, growth, and dissemination in several cancer types, including human breast cancer. Neoplastic cells are characterized by relative growth autonomy, a consequence of the constitutive expression of IGF and their receptors involved in autocrine loops. This chapter will focus on the molecular mechanisms underlying IGF action in tumourigenesis, in particular its chemoattractant activity and its relevance in tumour motility, both of which lead to invasion. Several of the IGF-induced cellular changes will be highlighted, such as cell polarizatiom, adhesion and detachment, as well as proteolysis induction. Finally, we will summarize the significance of IGF system components as prognostic markers in human breast cancer, and discuss the possible therapeutic considerations encompassed by these factors.

active oncogenes and the loss of specific tumour suppressor genes. In addition to these genetic factors, however, tumour growth is controlled by hormonal cues that also regulate cell proliferation in nonpathological conditions. The steroid hormone estrogen, an important hormonal stimulus for breast development, is also one of the most potent mitogens for breast

1. OVERVIEW Progression from a benign, noninvasive in situ carcinoma to a malignant, invasive breast carcinoma is a complex multistage process, in which uncontrolled cell proliferation is a hallmark of the disease. This progression is believed to involve a number of genetic events, including activation of dominant107

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cancer cells. Expression of the mediator of estrogen-induced proliferation, the estrogen receptor, is lost in advanced breast carcinomas, suggesting that other tumour-produced factors provide these cells with a hormone-independent proliferation status. Over the past two decades, considerable evidence has accumulated indicating that a family of growth factors, the insulin-like growth factors I and II (IGF-I and IGF-II), are critical elements in maintaining and supporting the progression of breast cancer cells. IGF-I and IGF-II are members of a multifunctional family of proteins that elicit diverse effects in a variety of biological processes in cell culture systems and have a broad range of functions in the embryo, fetus and adult. The name reflects their deep structural relationships with insulin and emphasizes their growth-promoting activity; nonetheless, IGF are more pleiotropic in their biological activities than insulin. In fact, the IGF were discovered as three entirely independent activities: the sulfation factor activity (SFA), later called somatomedin, as it mediates the message of growth hormone (GH) stimulating somatic growth in vivo (1), the nonsuppressible insulin-like activity (NSILA), since their hypoglycemic activity could not be abrogate with antiinsulin antibodies (2), and the multiplication-stimulating activity (MSA) as they promoted cell replication in vitro (3). Although initially IGF were considered as endocrine hormones, in the 70’s was observed that specific cell lines produced their own IGF and, hence, they may also act as autocrine/paracrine factors. The interplay between autocrine and endocrine roles of IGF may be of pivotal importance in the incidence and progression of different human cancers.

Chapter 7 All the IGF effects, both growthpromoting and metabolic, are mediated through interaction of these molecules with specific cellular receptors. Both IGFI and IGF-II bind to the insulin (IR), type I IGF (IGF-1R) and type II IGF receptors (IGF-2R) with differential affinity. The growth-promoting activities are mediated by the IGF-1R, a receptor tyrosine kinase that also binds to insulin, whereas both the IGF-1R and the IR probably mediate the metabolic effects. This promiscuity between ligands and receptors is only disrupted by the IGF-2R that binds specifically IGF-II. This receptor appears to function by sequestering IGF-II binding to the IGF-1R or the IR and, hence, must be considered an antagonist of the IGF-II function. Another relevant step in understanding IGF biological activity was the finding that these polypeptides do not exist in serum or in other body fluids in the free form; instead, IGF are bound to specific carrier proteins, termed IGFBP (IGF binding protein). At present, seven distinct IGFBP have been identified and cloned in humans, some of which are expressed by specific cell types or under determined circumstances. These IGFBP increase IGF half-life in the vasculature, but their precise function as regulators of IGF bioavailability at the cellular level remains unclear. Initial observations indicated that IGF forms bound to IGFBP do not readily interact with the IGF receptors, explaining why the high IGF concentrations in human serum do not cause hypoglycemia. Even though different experimental evidences argue to an inhibitory role of IGFBP, it is becoming evident that IGFBP are also required for the proper IGF biological activity at the cell level. Indeed, enhancement of the IGF-induced proliferative activity by IGFBP in different cell types has been reported.

7. IGF in breast cancer progression Different evidences suggest that this enhancing effect may be explained by the limited proteolysis of IGFBP at or in close proximity to the cell membrane. The proteolytic cleavage of IGFBP would lead to a decreased affinity for IGF and to the controlled release of the growth factor in the pericellular enviroment. We must thus consider the IGF axis composed of IGF ligands, IGF receptors, IGFBP, and IGFBP proteases. 2. INSULIN-LIKE GROWTH FACTOR AXIS COMPONENTS 2.1

Structure of IGF-I and IGF-II

Mature IGF-I and IGF-II are singlechain polypeptides of 70 and 67 amino acids, respectively, with 62% overall sequence identity (4-5). Due to structural identity with insulin, the IGF polypeptide chain has been divided into four domains arranged as B-C-A-D. IGF A- and Bdomains have 45% sequence identity with insulin A- and B-chain; however, the connecting peptide C is shorter than the proinsulin C-chain, and the carboxylterminal D-domain extension is exclusive to IGF. Another parallel between IGF and insulin structure is the presence of three intrachain disulphide bonds arranged in the same disposition as in insulin, i.e., two connecting B- and A-domains and one intra-A-domain (6). Moreover, the IGF are synthesized as preproproteins with signal peptides of about 25 amino acids at the N-terminus of the B-domain, and further extensions of 35-85 residues at the C-terminus of the D-domain, termed the E-peptide (7). Although the signal peptide and E-domain are deleted sequentially by post-translational processing before secretion, the presence of different IGF-II proforms has been reported in serum (8). Interestingly, these “large” IGF-II forms have the same mitogenic activity as the

109 processed species (8-10). In Northern hybridization studies, cDNA probes detect several IGF-I mRNA species, of which the predominant forms appear to be 7.0-8.0, 4.6-4.7, 1.7-2.1 and 1.0-1.2 kilobases (kb) in length. Similarly, IGF-II mRNA species of 3.4-4.0, 2.2, 1.61.75 and 1.1-1.2 kb have been described (11-12). These multiple mRNA arise from alternative splicing, since IGF genes have a discontinuous structure; IGF-I gene contains five exons that span at least 45 kb (13), whereas IGF-II consists of seven exons spanning more than 16 kb (14). The physiological significance of these different splicing forms nonetheless remains unknown. As mentioned above, IGF interact with four different molecular species: the IGF-1R, the IGF-2R, the IR and the IGFBP. The IGF domains involved in these interactions have been defined mostly by homologous scanning mutagenesis, replacing IGF-I domains with those corresponding to homologous areas of insulin (15). With this approach, it was found that IGF-I residues 1-3 and 49-51 are important for IGFBP and IGF2R binding, whereas amino acids 21, 23, 24, 44 and tyrosines 31 and 60 are required for binding to the IGF-1R (1522). Extrapolation of these amino acids on a partially-resolved three-dimensional structure of IGF-I (23) indicates that the IGFBP and the IGF-1R binding surfaces are on opposite sides of the IGF-I molecule, suggesting that IGFBP-IGF complexes may bind to the IGF-1R on the cell surface. However, these ternary complexes have not been demonstrated to date. Most studies indicate that IGFBP abrogates IGF-I biological activity; and this inhibition is not observed for IGF-I mutants with low affinity for IGFBP (24). Structure-function analysis with a panel of 28 monoclonal antibodies covering the

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entire exposed IGF-I surface indicated an overlap of the IGF-I domains involved in IGF-1R and IGFBP binding (25). These results are compatible, since scanning mutagenesis provides direct information about the residues involved in ligandreceptor interaction, but not about the steric hindrance produced by each receptor. Even though IGF amino acids interacting with IGFBP and IGF-1R are on distinct sides of the molecule, the IGFBP and IGF-1R footprints thus overlap on the IGF-I surface. 2.2. IGF binding proteins (IGFBP) and IGFBP proteases Circulating IGF are tightly bound in serum and in the extracellular milieu to soluble receptors termed IGFBP. Seven members of the IGFBP family have been cloned so far (26). IGFBP-1 to -6 are structurally related proteins of 216-289 amino acids, with highly conserved cysteine-rich N- and C-terminal domains involved in ligand binding; the domains are linked by a central portion that is very dissimilar among each IGFBP. These six IGFBP bind with high affinity to both IGF-I and -II, but do not bind insulin (27). Conversely, IGFBP-7/mac25 binds to insulin with high affinity and with very low affinity to IGF-I or IGF-I (28). Most IGF-I and IGF-II is found in circulation as 50 and 150 kDa complexes with IGFBP (29-30). These complexes prolong IGF half-life in circulation; indeed, injection of radioactively-labeled IGF-I in rats showed that the 150 kDa form remained in the bloodstream for 3-4 h, compared with 10 minutes recorded for unbound IGF (31). The 150 kDa form, which is the main IGF reservoir in serum (32), is a ternary complex formed by IGF, IGFBP-3 and a glycoprotein termed acidlabile subunit (ALS) (33-34). The presence of ALS in the 150 kDa complex

Chapter 7 impedes its crossing the endothelial barrier, increasing IGF half-life in the bloodstream (35). Other IGFBP in serum, such as IGFBP-1, -2 and -4, are able to traverse the vasculature, and hence transport IGF from the circulation to peripheral tissues (27, 36). The endocrine effects of IGF on somatic growth or tumourigenesis may therefore be regulated by controlling its availability through the formation of IGF complexes with specific IGFBP. In addition to their role as IGF reservoirs and carrier proteins in the circulation, IGFBP act as inhibitors (3739) or enhancers (40-41) of IGF biological activity at cell level. The inhibitory effects of IGFBP have been attributed to competitive scavenging of IGF peptides away from the IGF-1R (4243). The enhancer mechanism is poorly understood, and usually involves binding of the proteolytic cleavage of membranebound IGF/IGFBP complex, which in turn results in a decrease in IGFBP binding affinity (44-45). Several authors have suggested that the IGFBP enhancing effect is mediated by the sequestering of IGF from the extracellular milieu, which prevents the negative IGF-1R feedback that occurs at high IGF concentrations (41, 46-47). Alternatively, IGFBP may facilitate IGF binding to IGF-1R by anchoring the ligands in close proximity to their cell receptors. In fact, binding of the IGF/IGFBP complex to the cell surface is critical in the IGFBP enhancing effect. IGFBP-3 blocks cell growth if added simultaneously with IGF-I, but increases IGF-I-induced mitogenesis if added prior to IGF-I stimulation (48). In addition to IGFBP binding to the cell membrane, other processes such as proteolytic cleavage of IGFBP, should account to enhance IGF activity. Several proteases, including serine proteases,

7. IGF in breast cancer progression cathepsins and matrix metalloproteinases (MMP), are reported to use IGFBP as substrates. In all cases, this proteolytic cleavage results in a drastic decrease in IGFBP affinity for IGF and in the controlled release of the growth factors (49-52). Furthermore, is reported that IGF-I upregulates MMP-2 synthesis in carcinoma cells (53), establishing a positive feedback loop between IGF activity and the IGFBP proteolytic activities. Strikingly, some of the proteases acting on IGFBP are also associated to the cell surface (54-56); this explains the observation that factors that increase IGFBP association to the plasma membrane also contribute to IGFBPenhanced IGF activity (57). Several recent lines of evidence in various cell systems have suggested that the IGFBP, especially IGFBP-3, may have more active, IGF-independent roles in cell growth regulation. In support of this hypothesis, high affinity IGFBP-3 binding to the surface of various cell types and IGF-independent direct inhibition of monolayer growth have been shown; both are presumably induced by specific interaction with cell membrane proteins that function as an IGFBP-3 receptor (5859). Interest in IGFBP has increased since it was found that IGFBP-3 could act as an antineoplastic agent. IGFBP-3 inhibits cell proliferation in fibroblasts that express or lack the IGF-1R (60-61), indicating that IGFBP-3-mediated growth suppression is independent of IGF-1R action, IGFBP-3 has also been identified as a p53-regulated target gene (62). Taken together, these data suggest that the IGFBP-3 gene may be a tumour suppressor. The newest member of the family, IGFBP-7, is also suggested to be a tumour suppressor binding protein. The IGFBP-7/mac25 cDNA was originally cloned from leptomeningial cells and subsequently

111 reisolated by differential display as a sequence expressed preferentially in senescent human mammary epithelial cells (63). mac25 mRNA is detectable in a wide range of normal human tissues, with decreased expression in breast, prostate, colon, and lung cancer cell lines, suggesting that IGFBP-7 may function as a growth-suppressing factor (26, 63). 2.3. The IGF receptors There are at least three receptors in the IGF axis, including the insulin receptor (IR), the type-1 IGF receptor (IGF-1R) and the type-2 IGF receptor (IGF-2R). The IGF-1R is the most active in terms of cellular proliferation, and crossreacts with all three ligands; it shares several functional and structural characteristics with the IR, but the IGF-1R subunit is ten-fold more mitogenic than the IR (64). Supraphysiological insulin concentrations also activate the IGF-1R, and the mitogenic effects of insulin at microgram concentrations are probably exerted through binding to the IGF-1R (65). The IGF-1R belongs to the tyrosine kinase receptor family (66) and its amino acid sequence is 70% identical to that of the IR (67). It is a heterotetrameric glycoprotein composed of two ligandbinding subunits, entirely extracellular, and two transmembrane subunits linked by disulfide bonds, which have the enzymatic activity (68-69). The receptor is synthesized as a single preprotein of 1,367 amino acids that is cleaved to generate two half receptors, which are joined by disulfide bonds between the a subunits to form the mature receptor. The IGF-1R binds to all three ligands with distinct affinities; IGF-I and IGF-II bind to the receptor at nanomolar concentrations, whereas insulin binds with 100-fold lower affinity. Ligand binding to subunits triggers autophosphorylathe

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tion of subunits (70-71) by an intramolecular trans mechanism similar to that used by other receptors (72-73). The IGF-2R is a single chain, membrane-spanning glycoprotein that is identical to the cation-independent mannose-6-phosphate receptor (74). The mature human receptor consists of a large extracellular domain and a short intracytoplasmic domain (75). The extracellular domain contains the ligandbinding domain (mannose-6-phosphate and IGF-II) and comprises 15 repeated domains. The intracellular portion has no tyrosine kinase activity and has been implicated mainly in trafficking among different subcellular compartments. The function of IGF-2R remains puzzling. It does not stimulate cell proliferation; in fact, IGF-2R gene deletion causes body weight increase in mice, and blocking of IGF-II binding to the receptor does not alter its mitogenicity (76-77). This supports the argument that the IGF-2R is a specific downregulator of IGF-II (the receptor binds neither IGF-I nor insulin). IGF availability may thus be regulated in two ways, through IGF-2R, which controls IGF-II levels, and through IGFBP, which may serve as storage sites for both IGF-I and IGF-II ligands (insulin does not bind to IGFBP). An additional receptor in the IGF axis must be considered: the insulin-receptor related receptor (IRRR), which has substantial identity with the IGF-1R and the IR (78). In chimeric constructs with IR or IGF-1R subunits, the IRRR tyrosine kinase domain was shown to be mitogenic, but the ligand involved in the activation of this receptor has not yet been identified. In fact, serum does not activate the IRRR (79), suggesting that the IRRR may be activated by an unknown ligand through a strictly autocrine mechanism.

Chapter 7 3. ON THE PHYSIOLOGICAL ROLE OF IGF-I AND IGF-II

IGF-1R are present in a wide variety of cell types and mediate most in vitro IGF-I and IGF-II effects, as well as insulin effects when this hormone is present at sufficiently high concentrations. In general, the IGF effects are either acute anabolic effects on protein and carbohydrate metabolism, or longer-term effects on cell replication and differentiation. There are excellent reviews surveying the effects of IGF in multiple specific cell types, as well as its role as a mediator of GH-induced somatic growth (8, 27, 80). We will focus only on the mitogenic and anti-apoptotic activities of IGF, since the balance between these two processes regulates the growth of any tissue or tumour cell. 3.1.

Mitogenic effects

The IGF molecules appear to regulate cell proliferation in both epithelial and mesenchymal tissues. They act as a nontissue-specific permissive mitogen, required for optimal proliferative responses to highly tissue-specific trophic factors such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) or thyroid-stimulating hormone (TSH). They consequently perform the function of a general “progression factor” for cells previously stimulated by a more specific “competent” factor (81). This has been formally demonstrated using cells derived from mice with a targeted disruption of the IGF-1R genes (IGFThese cells do not grow in serumfree medium supplemented with growth factors that sustain the same type of cells derived from wild type littermates (82). These cells do proliferate in medium containing 10% fetal calf serum, although the doubling time is much

7. IGF in breast cancer progression slower than for wild type fibroblasts (109 h and 44 h, respectively). IGF-I mitogenic activity has also been reported in vivo. Pygmies and Laron-type dwarfism are the consequence of low IGF-I levels (8). The correlation between IGF-I levels and body size also extends to dogs and mice. Transgenic mice overexpressing the IGF-I gene confirm the importance of the IGF axis in body growth. GH overexpression causes increased growth in transgenic mice (83). Although circulating GH levels at birth were ten-fold higher, accelerated growth was not evident until two weeks of age, at which time serum IGF-I was elevated compared with control mice. Transgenic mice expressing human IGF-I in liver and other tissues also showed enhanced growth, although to a lesser extent than that observed in GH transgenic mice (84). This may reflect the fact that GH also upregulates other components of the IGF axis, as IGFBP-3 (8). Further in vivo evidence indicating the essential role of the IGF axis in cell proliferation and survival has been elucidated using gene knockout (KO) techniques (Table I). Mice lacking IGF-II were only 60% the size of their wild-type littermates at birth, but a normal postnatal growth rate was observed (85). Equivalent impairment in fetal growth was also observed in homo- and heterozygote mice inheriting a paternal mutant IGF-II allele (86), indicating paternal imprinting of the IGF-II gene. Deletion of IGF-2R genes showed that, despite the puzzling signaling pattern, this receptor is critical

113 for fetal growth in rodents. In mice, IGF2R maps to an imprinted locus termed Tassociated maternal effect (Tme), which is essential for viability. Mice lacking IGF2R die of major cardiac anomalies in the perinatal period; they are 125-130% the size of their wild-type littermates (77, 87) and show up to a 2.6-fold increase in circulating IGF-II levels (77), indicating again that IGF-2R does not mediate IGFII-induced growth effects. Mice lacking IGF-I have markedly diminished postnatal growth as well as fetal retardation (88-90). IGF-I KO mice showed reduced viability, with perinatal death attributed to muscular hypoplasia and decreased lung maturation. The dwarfism observed in IGF-I KO mice is increased in the double IGF-I/IGF-IIdeficient mice, with 30% of wild-type size at birth. Deletion of the IGF-1R also results in postnatal death due to muscular hypoplasia (88-89). Deletion of early signaling molecules downstream of the IGF-1R also result in growth impairment. KO mice for the insulin receptor substrate-1 (IRS-1) show a 40% reduction in postnatal growth (91). Interestingly, deletion of IRS-2, an IRS-1 homologue, does not affect prenatal and postnatal growth (92), indicating that IRS-1 is the main element in IGF-1 R-mediated somatic growth. These results indicate that expression of both IGF ligands and receptors is required for normal embryonic growth, and highlight the potential of the IGF system in promoting cell proliferation and survival in many different tissues.

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Anti-apoptotic effects

The protective effect of the activated IGF-1R on cell survival has been known for some years, mainly in the central nervous system (CNS), where IGF-I inhibits low potassium-induced apoptosis in cerebellar granule neurons (93). Other reports have indicated the role of IGF-I as an anti-apoptotic agent. For example, IGF-I inhibits apoptosis induced following IL-3 withdrawal in pro-B cells (25), probably via a mechanism involving receptor-induced phosphatidyl inositol-3 kinase (PI-3K) activity (unpublished observations); it also protects cells from cmyc-induced apoptosis (94). All of these reports deal with the protective effects of the ligands, but it has become apparent that the receptor is the limiting factor. The

Chapter 7

protective effect of IGF-I following growth factor withdrawal, observed in IL3-dependent B cell lines (which usually express a high IGF-1R receptor number), is lost in other B cell types in which this receptor is absent or present at low levels (unpublished observations). An increase in IGF-1R receptor number may rescue cells from various apoptotic agents, including FAS (95). This is also true in vivo, where a decrease in IGF-1R using an antisense strategy or the use of a dominant negative receptor give rise to massive apoptosis of tumour cells (see below). IGF-1R apparently achieves its protective effect by interfering with ICE and ICElike proteins (96), which form the most commonly used apoptotic pathway. A specific role has recently been reported for the transcription factor nuclear

7. IGF in breast cancer progression in mediating IGF-I survival effects (9798). 4. IGF-1R-MEDIATED SIGNALING TRANSDUCTION PATHWAYS 4.1. Initiation of the IGF-1R-triggered signaling pathways

As mentioned above, IGF-2R activation appears not to mediate IGF-II biological activities. We will therefore assume that IGF-I and IGF-II mediate most of their cellular activities through activation of the IGF-1R. Activation by ligand binding causes rapid tyrosine phosphorylation of the IGF-1R and the subsequent recruitment and phosphorylation of insulin receptor substrates (IRS) (99). Four members of the IRS family have been cloned in mammals (100-102): IRS-1 and IRS-2 are widely expressed, IRS-3 is restricted to adipose tissue, and liver; and IRS-4 is expressed in thymus, brain and kidney. Targeted disruption of each IRS gene in mice suggests that IRS-1 and IRS-2 coordinate essential effects of IGF mitogenic and metabolic activities (92, 103-104). In contrast, disruption of IRS-3 and IRS-4 genes results only mild effects, suggesting that they have redundant roles in IGF action (105-106). IRS proteins have a conserved Nterminus composed of adjacent pleckstrin homology and phosphotyrosine binding domains that mediate IRS association to the activated IGF-1R. The C-terminus is tyrosine phosphorylated at multiple sites, creating docking motifs for proteins with src homology 2 (SH2) domains. A wide diversity of signaling proteins containing SH2 domains bind to distinct IRS docking sites, based both on recognition of the phosphorylated tyrosine and on the

115 sequence surrounding this modified tyrosine (100), thus triggering different cell responses (Fig. 1). Despite their high sequence similarity, IRS proteins are an important site of signal redundancy and diversity. In cell-based assays, IRS-1 and IRS-3 activate PI-3K more strongly than IRS-2, whereas IRS-4 barely activates PI3K (101, 107). IRS-2 nonetheless plays a major role in PI-3K activation in murine liver cells (92); IRS proteins may therefore coordinate distinct IGFsignaling pathways in a cell type-specific manner. Concurring with this, it has been reported that myoblast and adipocyte proliferation use mainly the p21 ras/mitogen-activated protein (MAP) kinase pathway, whereas breast tumours and brain capillary cells proliferate in response to signals by the pathway (108). 4.2 Cross-communication between the IGF-1R and other cell receptors

Although IRS proteins were originally described as specific signaling molecules for the IR and IGF-1R, recent reports have found them at the crossroads of several intracellular pathways. Indeed, IRS-1 appears essential for IL-4 stimulation of hematopoietic cells (109-110), for signaling by the GH receptor and interferon (111), interacts with the JAK family of transducing molecules (112), with certain integrins (113) and with G protein-coupled receptors (114). The central role of IRS proteins in various signaling pathways suggests intensive crosstalk between IGF and other transducing systems. This crosscommunication is essential for several IGF-I-induced cell activities, as discussed below.

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Chapter 7

7. IGF in breast cancer progression 4.2.1. IGF-1R/estrogen receptor crosstalk: a mitogenic connection IGF function is regulated by other factors important in breast cancer development, such as estrogens. These hormones are required for the development of female secondary sexual characteristics, including mammary glands. Estrogens are potent mitogens for a number of breast cancer cells in vitro, and transcriptional upregulation of the estrogen receptor (ER) is one the earliest and most common defects associated with breast tumourigenesis (115). The IGF-1R and the ER are usually co-expressed, and the two signaling systems are engaged in complex functional crosstalk that controls cell proliferation (116). Estrogens and IGF synergistically stimulate proliferation in most ER-positive breast cancer cell lines; this synergism is achieved in part since ER upregulates IGF-1R and IRS-1 levels (117-118). Simultaneously, ER downregulates expression of IGF antagonists such as IGFBP-1 and IGF-2R (119-120). ER therefore increases IGF1R-mediated signaling by upregulating IGF-1R and IRS levels; this finally results in enhanced tyrosine phosphorylation and MAPK activation after ligand binding, leading to an increase in cell proliferation. IGF signaling may also regulate ERinduced cell proliferation through a mechanism involving the transcriptional activation of endogenous ER in an estrogen-independent manner (121-122). Inhibition of IGF-I signaling by addition of IGFBP produces a decrease in the expression of ER-inducible genes (123), indicating that a basal level of IGF-1R activation is required for maximal ER activity. This interplay between IGF and ER signaling has also been shown in vivo. Indeed, upregulation of IRS-1 expression was observed after estrogen stimulation of MCF-7 cells grown as xenografts in

117 athymic mice; conversely, estrogen withdrawal resulted in a dramatic decrease in tumour growth as well as much lower IRS-1 expression in the tumour cells (118). In ER-positive cells, therefore, both estrogen and IGF regulate proliferation in an interdependent manner, such that the blockage of one of these pathways results in inhibition of the other. This is of the utmost relevance, since the major treatment modality for ER-positive breast cancers is the use of estrogen antagonists such as tamoxifen. Antiestrogen inhibits IGF-I-stimulated cell proliferation (123). Its mechanism of interaction with IGF signaling has yet to be defined, but antiestrogens decrease circulating IGF-I levels and increase IGFBP-3 expression (123-124). It has been also suggested that antiestrogens may modulate the activity of phosphatases that shut down IGF-1R signaling (116). IGF/ER crosstalk in ER-positive cells may also sensitize the cells to the action of other growth factors such as transforming a growth growth factor inhibitor in normal and ER-positive tumour cells, that nonetheless contributes to tumour progression in ER-negative cells (125). The mechanism of this differential activity in ERpositive and -negative cells is not understood, although it is clear that it occurs in conjunction with changes in gene expression. Indeed, ER-positive cells do not produce IGF in an autocrine manner and activation of the IGF pathway hence depends on the exogenous growth factor supplement. Conversely, ERnegative cells usually show aberrant expression of IGF, which is the primary stimulus leading to their proliferation (discussed below). expression in ER-positive is tightly regulated by both IGF and ER, whereas this control is lost in

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Chapter 7

ER-negative cells (125). Breast tumour progression thus depends on crosstalk between stimulatory and inhibitory factors that regulate cell proliferation. 4.2.2.IGF/chemokine invasive connection

crosstalk:

an

One important biological function of IGF-I as well as of other growth factors is to induce cell invasion. As will be discussed’ in detail later, achieving this invasive ability requires extreme rearrangements of cell structure that culminate in the acquisition of a motile, polarized cell phenotype. It has been established in different cell types that cell polarization requires trimeric protein signals. Activation of G proteins releases two potential downstream signaling molecules, a GTP-bound subunit that interacts directly with effectors, and a subunit that regulates an array of free targets, including ion channels and enzymes (126). dimers mediate cell polarization in mammalian cells (127, itself is not required in 128), whereas this process (129). In fact, we found that IGF-I-induced cell chemotaxis of the breast adenocarcinoma MCF-7 is inhibited by treatment of the cells with pertussis toxin, which specifically prevents activation and hence, release. The convergence of G-protein-coupled receptors (GPCR) and receptor tyrosine kinase (RTK) signaling pathways is supported by observations that the receptors of PDGF, PDGFR (130), epidermal growth factor, EGFR(131), and IGF-1R (132) are tyrosine phosphorylated after GPCR activation. Transactivation of GPCR signaling by IGF-I, although reported (133-135), remains a matter of controversy (136-138). Indeed, pertussis toxin is reported to inhibit both the IGF-Iinduced opening of a calcium-permeable

cation channel and MAPK activation in rat cerebellar granule neurons and NG108 neuronal cells (134-135). Furthermore, MAPK activation by IGF-I was inhibited in fibroblasts expressing subunit binding proteins (133). The question arises as to the mechanisms by which IGF-I, which signals through a receptor tyrosine kinase, triggers these G protein-mediated events. As mentioned earlier, an association has been suggested between IRS proteins with proteins following IGF-I stimulation (114). This interaction may occur through the pleckstrin homology domain in the subunits (139). It has likewise been to reported that activated IR recruits the signaling complex, in a manner similar to classical GPCR (140). Based on coprecipitation experiments, it has been suggested that heterotrimeric proteins may associate constitutively with the IGF1R (141); the authors proposed that IGF1R activation upon IGF-I binding leads to release of the subunit as consequence activation. Although these reports of point to an intracellular connection between IGF-1R and trimeric proteins, the physical association between these molecules does not explain how G proteins are activated. In fact, seems to be phosphorylation of independent of IGF-I stimulation (141), suggesting that activation does not occur through tyrosine phosphorylation. Moreover, there is no evidence for the mechanism by which the IGF-IR induces subunit release from the complex. We found that IGF-I stimulation of the ER-positive human breast adenocarcinoma MCF-7 cell line induces the specific transactivation of the coupled chemokine receptor CCR5, triggering its tyrosine phosphorylation and protein recruitment (142). This

7. IGF in breast cancer progression transactivation occurs via a mechanism involving upregulation of gene expression and secretion of RANTES, the natural CCR5 ligand. Neutralizing anti-RANTES antibodies or drugs that block protein secretion abrogate the IGF-I-induced CCR5 transactivation, indicating that an extracellular step is required to achieve chemokine receptor activation. Our data indicate that IGF-I-induced chemokine receptor activation involves two transmembrane signaling events: first, IGF-I-mediated IGF-1R activation induces synthesis of RANTES, which is secreted to the extracellular milieu, and second, extracellular RANTES binds to and activates CCR5, which recruits trimeric and promotes activation and release. A similar mechanism was recently demonstrated in RTK transactivation by GPCR receptors. Indeed, GPCR-induced EGFR transactivation is mediated by a paracrine loop involving EGF precursor release from the membrane (143), suggesting that this extracellular cross-communication between RTK and GPCR may operate in both directions. The chemokines belong to a superfamily of low molecular weight chemotactic proteins implicated mainly in leukocyte activation and chemotaxis (144). The IGF-I-induced CCR5 transactivation observed thus provides a new mechanism that links chronic inflammation and tumourigenesis (145). The constitutive IGF-I secretion observed in invasive ER-negative breast tumour cells may increase proinflammatory chemokine concentrations in the environment, providing the cells with a built-in invasive capacity. In fact, prevention of chemokine receptor activation by drugs such as pertussis toxin, by neutralizing anti-chemokine receptor antibodies, or by transdominant

119 negative chemokine receptor mutant overexpression, abolishes IGF-I-induced MCF-7 cell chemotaxis in vitro (142). Using the RANTES antagonist AOPRANTES (146-147) and the dominant negative CCR5 mutant (148150), we observed that chemokine signals do not promote cell motility but, rather are required for IGF-I-induced cell polarization (unpublished results). These results stress again the absolute requirement for heterotrimeric activation, probably through mediated signaling, to achieve the polarized motile cell phenotype. The cross-communication between IGF-1R and chemokine receptors is also important for in vivo tumour invasion; indeed, RANTES gene expression is upregulated in invasive human breast carcinomas (151-152). Our preliminary results from intravasation assays in chicken chorioallantoic membrane (CAM) (153) using highly invasive (ER-negative) and non-invasive (ER-positive) tumour cells concur with these data. The intravasation process constitutes an important event in tumour dissemination and is related to the invasive ability of the tumour (154). Overexpression of the transdominant negative mutant in ER-negative tumour cells, which produce large amounts of IGF-I in an autocrine fashion (155), significantly decreased intravasation capacity of the cells. Conversely, overexpression of RANTES in the low invasive MCF-7 cell line resulted in an increased intravasation capacity of these cells. Together, these results suggest that IGF-chemokine crosstalk may have a fundamental role in tumour progression, probably by modulating the invasiveness of tumour cells.

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5. ROLE OF THE IGF AXIS IN TUMOUR FORMATION AND GROWTH It has been clear for many decades that the phenomenon called “cancer” is in some way related to defects in the mechanisms that control normal cell proliferation. The use of gene transfer (transfection) allowed the transmission of the cancerous state from one cell to another using DNA as the vehicle, indicating that distinct cell determinants of transformation, called oncogenes, resided in tumour cell DNA (156-158). In the ensuing years, evidence accumulated indicating that cell transformation paralleled genetic defects leading to the constitutive activation of oncogenes or the constitutive downregulation (or loss of function) of tumour suppressor genes. Nonetheless, cell transfection of oncogenes in vitro suggested that this relationship was not linear. For instance, ras-transformed 3T3 fibroblasts require progression factors found in serum to form colonies in soft agar. These progression factors were present in the serum of normal rats, but not in that derived from hypophysectomized rats; the factors were identified as IGF (159). Several retroviruses containing v-ras (vH-ras, v-K-ras) and v-mos oncogenes or growth factor receptor-derived oncogenes (v-erbB, v-fms) were shown to replace the epidermal keratinocyte proliferative requirement for EGF, a growth factor necessary for these cells in culture. Nevertheless, none of these oncogenes permitted keratinocyte growth in defined medium without IGF, indicating that IGF1R signals are important for cell proliferation independently of oncogene activation (160).

Chapter 7 Further evidence was obtained in vivo. Activation of H-ras is a frequent event for tumour initiation in thyroid follicular cells (161-162); however, in vivo induction of H-ras mutations by thyroid injection of retroviruses carrying a mutated ras, yields formation of few tumours. If the mitogenic activity of the gland is increased by stimulation with thyroidstimulating hormone besides with the introduction of ras mutations, the number of thyroid tumours is greatly enhanced (161, 163). These observations were explained by assuming that initial mutations leading to oncogene activation are not sufficient to induce cell proliferation at low primary mitogen levels. If clonal expansion of the oncogene-transformed cell occurs due to the influence of a physiological stimulus, however, new mutations may then take place, leading to a selective advantage for tumour growth. Later evidence indicated that growth factor signals, in particular those from the IGF-1R, are required for efficient tumour implantation and progression. The concept that IGF is a second signal in cell transformation has become stronger. This evidence will be discussed in this section, together with epidemiological data in humans that support this view. 5.1. IGF-1R signals are necessary for cell transformation 5.1.1.IGF-1R is required for cell transformation in vitro and in vivo

It has long been known that overexpression and/or constitutive activation of the IGF-1R leads to liganddependent neoplastic transformation (164), i.e., ability to form colonies in soft

7. IGF in breast cancer progression

agar and/or to produce tumours following injection into nude mice. Transformation can, however, be considered a common outcome of gene product overexpression; overexpression of many growth factor receptors, such as PDGF, EOF, FGF, insulin and CSF-1, as well as protooncogenes, activated oncogenes, signal transducing molecules, and glycolytic enzymes, results in cell transformation. Two findings distinguish IGF-1R. First, cells (derived from mice with a targeted IGF-1R gene disruption) are refractory to transformation by certain viral and cellular oncogenes that readily transform mouse embryo fibroblasts expressing IGF-1R. These oncogenes include the SV40 large T antigen (82), activated ras, or a combination of ras and T antigen (165), bovine papilloma virus E5 protein (166), or growth factor receptor overexpression (167). Second, the transformed phenotype of tumour cell lines can be reversed to a non-transformed

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phenotype by decreasing IGF-1R number or by interfering with its function. Several approaches have been used, including antisense expression plasmids or oligonucleotides of IGF-II (168), IGF-I (169-170) or IGF-1R (171-177), antibodies to the IGF-1R (178-179), and dominant negative mutants of the IGF-1R (180-181). Cells from a highly metastatic human breast cancer line (182) or a murine carcinoma (183) carrying an antisense IGF-1R construct show a delay in tumour formation and a reduction in metastatic capacity. Inhibition of IGF-1R in metastatic breast cancer cells using a dominant negative mutant prevented metastasis to the lungs, liver and lymph nodes when cells were injected into the mammary fat pad (184); all these findings are summarized in Table II. The list is incomplete, but clearly shows that IGF-1R targeting can reverse the transformed phenotype in several tumour types of human and rodent origin.

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5.1.2. IGF-1R domains involved cel l transformation The introduction of modified IGF-1R into cells has provided information on essential domains and critical amino acids involved in different aspects of receptor function, including cell transformation. Mutants of both the and have mutants been reported. The provided information about the IGF-1R residues involved in ligand specificity. This section will concentrate on IGF-1R which mutated in the intracellular furnish some information on the IGF-1R domains involved in the recruitment of cell signaling molecules and the engagement of the IGF-1R in cell transformation. The intracellular kinase domains of IGF-1R and IR are 84% identical and activate mostly the same signal intermediates (IRS-1, Shc, etc.). Despite this considerable structural identity, analysis of chimeric IGF-1R/IR receptors in fibroblasts revealed that the is ten times more potent IGF-1R than that of IR in promoting DNA synthesis (64). Conversely, metabolic effects such as glucose transport are exerted with the same potency. However, a chimeric IGF-1R containing the last 112 amino acids of the insulin receptor in place of the 121 homologous residues showed only a modest increase in glycogen synthesis, PI-3K activity, and MAP kinase activity compared with the wild type IGF1R; DNA synthesis was nevertheless unaltered (185). This experiment suggests that the mitogenic activity does not reside in the C-terminal part of the IGF-1R βchain. Analysis of deletion and substitution yielded new mutations within the information on IGF-1R function. A point mutation at lysine 1003 (the ATP binding

Chapter 7 site) results in an IGF-1R that has essentially lost its function (73). Replacement of tyrosines 1131, 1135 and 1136 (the tyrosine kinase domain) by phenylalanine results in a markedly reduced level of autophosphorylation and in the abolition of both mitogenicity and transforming activity (186). Mutation of tyrosine 1136 leads to reduced DNA synthesis, whereas cells expressing IGF1R mutated in tyrosines 1131 and 1135 can still replicate (187). In contrast, substitution of any of these residues blocks colony formation in soft agar. These results demonstrate that each tyrosine in this cluster is not equivalent, and indicate that a fully functional receptor is required for anchorage-independent growth, but not for mitogenesis. Other mutations affect only selected receptor functions. For instance, tyrosine 950 is essential for binding and phosphorylation of IRS-1 (99), although it does not influence autophosphorylation or ligand-dependent internalization. This mutation abolishes mitogenic and transforming activities, but still protects murine hematopoietic cells from apoptosis induced by IL-3 withdrawal (188). The conclusions drawn from the many mutants tested are that: (i) IGF-1R mitogenicity and transforming activity are located in different domains (189-191), indicating that there is at least one pathway for transformation that is additional to and distinct from the mitogenic pathway; (ii) the IGF-1R transforming domain is located between residues 1245 and 1310 (191), and (iii) tyrosine 1251 and the serines at 1280-1283 are extremely important for IGF-1R-induced cell transformation (190).

7. IGF in breast cancer progression 5.1.3. IGF-1R interaction with oncogenes and tumour suppressor genes The results described above provide compelling evidence that the IGF-1R or signals provided by this receptor are required for transformation by different oncogenes. It has also been demonstrated that IGF-1R can interact directly with oncogenes such as src. In cells transfected with temperature-sensitive v-src, the IGF1R is rapidly phosphorylated on tyrosine at permissive temperatures. Indeed, srcstimulated phosphorylation correlated with receptor activation, even in the absence of IGF-I (192). v-src is the only oncogene cells (165), able to transform probably due to its ability to interact with the IGF-1R signaling machinery, such as IRS proteins. A further corollary may be added, since oncogenes were observed to upregulate and tumour suppressors to downregulate several components of the IGF system (for an extensive discussion with examples of other receptors and oncogenes see ref. (193). Several reports have documented that transfection of active oncogenes such as H-ras (194-196), SV40 large T antigen (197) or c-myb (198) upregulate the autocrine or paracrine production of IGF-I and/or IGF-II through a transcriptional mechanism. Conversely, tumour suppressor genes such as WT1 (199-200) and (201) cause a decrease in receptor number at the transcriptional level. In addition to the control of IGF-1R levels, also regulates the expression of other IGF components. Wild-type p53 represses IGF-II and IR promoter transcription (202) and induces IGFBP-3 expression (62), which antagonizes the effects of IGF-I and -II. This is of utmost relevance, since about 30% of breast tumours show allelic loss in the gene

123 region, presumably exposing a single mutated allele that most often encodes a missense protein with altered or absent transactivating capacity (203). Loss of repression following mutations of may thus result IGF-1R and IGF-II overexpression (200). The fact that oncogenes and anti-oncogenes modify components of the IGF system is extremely interesting, as this suggests that they act through the regulation of growth factors and their receptors. Although signals from the IGF-1R are required for cell transformation, the IGF1R cannot be considered an oncogene. It is intriguing to ask whether other IGF components may be tumour suppressors. As discussed above, several lines of evidence in various cell systems suggest that IGFBP-3 and IGFBP-7/mac25 may be considered tumour suppressor factors (26, 58-59, 63). The case of IGFBP-3 is of special interest, since its growth suppressive activity appears to be independent of the IGF-1R activation by the IGF-I or IGF-II ligands (60-61). It has also been suggested that IGFBPassociated proteins, such as the ALS (see section 1.2), may have a fundamental role in controlling IGF activity in peripheral tissues. Certain IGF-II-secreting tumours, such as fibrosarcomas, rhabdosarcomas and leiomyosarcomas, produce severe tumour-associated hypoglycemia (204). Nonetheless, total IGF-I or IGF-II serum levels in these patients are not elevated and may even be reduced as compared with normal individuals (205). The hypoglycemic patients show marked abnormalities in serum IGFBP composition (205); the ternary complex ALS/IGFBP-3/IGF-I or IGF-II is virtually absent from the circulation of these individuals, even though IGFBP-3 is present (206). As mentioned earlier, this ternary complex may prevent the passage

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of IGFBP/IGF complexes through the capillary membrane (27, 35-36). In the absence of ALS, IGFBP-3/IGF complexes may thus have greater access to peripheral targets. This mechanism to promote nonpancreatic tumour-induced hypoglycemia may also account for increased IGFinduced tumour cell proliferation. Several reports have indicated that IGF-2R may potentially be a tumour suppressor for liver cancer. IGF-2R expression is reduced in hepatocellular carcinomas (207), and loss of heterozygosity at the human IGF-2R locus has been found in approximately 70% of liver cancers (207). Tumours with inactivating mutations in the IGF-2R gene grew more rapidly than those with an intact IGF-2R allele, again suggesting that the IGF-2R may be implicated in the clearance and degradation of the IGF-II molecule. Recent observations indicate that IGF-2R levels are significantly lower in breast cancer cells than in normal epithelial cells (208), and downregulation of this receptor resulted in an increased tumour growth rate in vivo (209). All together, these results suggest that the IGF-2R may act as a putative tumour suppressor gene for liver and breast tumours, possibly by decreasing IGF-II biological activity through its binding to the IGF-1R (210). Nonetheless, these observations will require substantiation and development of experimental models to test the hypothesis directly. 5.2. IGF autocrine loops and tumour growth

Although the liver is the major synthesis site for the systemic circulating IGF-I pool in adulthood, there is also significant growth factor production by both epithelial and stromal cells (211). Many tissue-specific hormones, such as estrogen, adrenocorticotrophic hormone

Chapter 7 (ACTH), luteinizing hormone (LH) or thyroid-stimulating hormone (TSH), appear to induce or modulate local IGF-I expression in their target tissues (8). These findings promoted a shift in understanding of the role of IGF-I in growth regulation from an endocrine to a paracrine/autocrine mode. This autocrine/paracrine circuit is operative in many tissues of very diverse origin. In evolutionary terms, the nontissue-specific mitogenic activity of IGF constitutes a primitive mechanism providing independent regulation of many tissue types in higher organisms. This system has probably been maintained since it provides a mechanism that allows integration of multiple mitogenic signals to coordinate the final somatic growth of a tissue. Thus, for example, growing epithelial cells produce a non-specific secondary mitogen, such as IGF-I, which in turn stimulates the associated stromal elements that were not influenced by the tissue-specific primary mitogen. The IGF system is therefore justified in terms of cell-cell communication, which cannot be achieved by a convergence of intracellular signaling pathways. The IGF loop is thus a system for paracrine, rather than autocrine stimulation, that operates not only in a conventional heterotypic role coordinating stroma with epithelia, but also in a homotypic mode to coordinate the growth of the epithelium itself. Many tumour cell lines produce elevated levels of IGF-I and/or IGF-II (212). Proliferation of these cells is partially inhibited by interfering with the IGF/IGF receptor interaction (213-215), suggesting a role for the IGF autocrine loop in tumour cell growth. Using an antiIGF-I mAb that recognizes the IGF-I/IGF1R complex, we formally demonstrated the existence of this IGF autocrine loop, at least in human prostatic adenocarcinomas

7. IGF in breast cancer progression (155). Increased IGF secretion in tumours is not a consequence of a primary abnormality of the IGF genes themselves since IGF are not oncogenes, but represents constitutive activation as a result of inappropriate intracellular signal activation. In correlation with this, as indicated in the previous section, some oncogenes such as the SV40 large T antigen or c-myb modify IGF ligand expression at the transcriptional level. Interestingly, this relationship also works in the opposite direction, as IGF-I may also regulate the expression of some oncogenes (216). Many tumour cell lines are characterized by relative growth autonomy, hypothesized to be a consequence of the constitutive expression of growth factors and their receptors. During physiological growth, part of the normal cell response to the primary mitogen involves the autocrine induction of a secondary non-tissue-specific mitogen, such as IGF-I and/or IGF-II. In tumour cells, transformation by an oncogene produces constitutive activation of IGF secretion to the extracellular milieu. Expression of this autocrine loop in tumour cells is thus independent of, or independent of an increase in, primary mitogen stimulation. The obvious consequence of this abnormal activation is autonomous proliferation of the tumour. It must nonetheless be remembered that the IGF loop works not only in a homotypic mode, coordinating the growth of the tumour itself, but that it also operates in a heterotypic mode, coordinating tumour and stroma. In fact, IGF-I is also expressed in stromal zones of cancer tissues (217). Furthermore, breast cancers are infiltrated with IGF-IIexpressing stromal cells (218) and this stromal-derived growth factor production has been implicated in protecting pre-

125 neoplastic breast cancer cells from apoptosis (219). The IGF autocrine/paracrine loops may therefore coordinate different functions in tumour progression in a manner similar to which they coordinate the somatic growth of a tissue throughout fetal and childhood development. Another level of complexity is added, since IGF autocrine loops do not involve only one or two ligands (IGF-I and IGF-II) and one receptor (the IGF-1R), but are regulated by other components of the IGF axis. We will return to reveal the true complexity of this system. 5.3. Role of the IGF axis in the andtumour immune response

The ability of tumours to produce and respond to IGF-I may also modulate the immune response to the the tumour. The evasion of a host immune response to transformed cells is also pivotal in the development of established tumours. Several reports have described that the abolition of the IGF loop in tumour cells promotes a host anti-tumour immune response. This was first observed using rat C6 glioblastoma cells that express IGF-I and form rapidly-growing tumours in syngeneic animals. Subcutaneous injection of C6 cells transfected with an episomebased vector encoding anti-sense IGF-I cDNA caused the regression of established brain glioblastomas, although the antisense-bearing cells were injected in a distal site (169); wild type cells do not confer this resistance. These antitumour lioma-specific effects result from a gglioma-specific immune response involving lymphocytes (169), which is restricted to the cell type carrying the antisense cDNA (220). Similar results have been obtained using an antisense RNA for the IGF-1R (172). This is not particularly surprising, except that the animals become resistant to

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challenge with wild type C6 cells even when they are pretreated with unrelated tumour cells, including tumour cells of other species (221). The mechanisms underlying this non-classical immune response are still unclear and require further investigation. 5.4. IGF as the second signal in tumourigenesis

Taking together all the observations discussed above, it becomes clear that the IGF axis is fundamental in the establishment and progression of tumours. This leads to the concept that IGF axis components may be a second signal in cell transformation (Fig. 2). As discussed above, oncogene-induced transformation is

Chapter 7 a highly inefficient process except when a simultaneous mitogenic stimulus is provided (163). Indeed, transgenic mice expressing the SV40 large T antigen in all Langerhans islets develop solid tumours in only 1-2% of them. If the SV40 large T antigen transgenic mice are crossed with IGF-II KO mice, the percentage of transformed islets as well as tumour malignancy is severely reduced (168). In addition, the tumours developed in SV40 large T antigen/ IGF-II KO mice showed a larger number of apoptotic cells than in SV40 large T antigen-expressing mice, indicating that IGF-II signals are required for the hyperproliferation and establishment of pancreatic tumours.

7. IGF in breast cancer progression In terms of autocrine/paracrine loops described in the previous section, the process leading to tumour formation is the final result of a homotypic cooperative loop (222). According to this, malignant transformation due to oncogene expression in a single cell would eventually result in the increased expression of a progression factor (IGF-I for instance); nevertheless, this mitogen will not reach the critical concentration required to promote proliferation of the transformed cell. If the oncogene transformation is followed by stimulation with a primary mitogen, however, both the oncogene-expressing cell and all neighboring untransformed cells will increase expression of the progression factor, which may now reach the threshold required to promote proliferation. At the end of this proliferation period for both cell types, the tumour would reach sufficient mass to maintain IGF-I above the minimal level required for cell proliferation; tumour growth would hence be independent of primary mitogen stimulation. Concurring with this hypothesis, growth of mammary tumour transplants is drastically reduced in the lit/lit mouse strain, which have extremely low circulating growth hormone (GH) and low IGF-I levels, compared with those transplanted in normal mice (223). Further, transgenic mice overexpressing IGF-II develop a diverse spectrum of tumours, including mammary cancer, at a higher frequency than control mice with normal IGF-II levels (224-225). Transgenic mice in which IGF-I expression was targeted specifically to the basal layer of epidermis likewise showed increased spontaneous tumour promotion (226). Epidemiological data indicate that cooperative homotypic IGF loops may

127 operate in promoting major human cancers. Acromegalic individuals have elevated circulating IGF-I levels and experience a high incidence of prostate and colorectal tumours (227). A weak, although consistent association has been reported between final adult height (indicated by elevated GH/IGF-I serum levels) and breast (228), prostate (229), colorectal (230), and hematopoietic (231) cancers. Two recent studies showed strong association between circulating IGF-I levels and the risk of breast and prostate cancers (232-233). The association between risk of prostate cancer and IGF-I was stronger in men over 60 years of age, when androgens may have less influence (233). This indicates that IGF-I acts not merely as a surrogate of steroid status but that it probably regulates steroid activity, as discussed above for estrogen. 5.5. Implications of the IGF axis in breast cancer incidence Experimental evidence and epidemiological data support the association of IGF in breast cancer development, and that intervention to reduce IGF levels may decrease proliferation of breast neoplasms. In breast cancer prevention strategies, IGF family members might therefore be considered potential therapeutic targets. Nonetheless, there is disagreement about the value of IGF as a prognostic marker, which will be summarized here. 5.5.1. Prognostic value of IGF and IGFBP The association between serum IGF-I levels and cancer risk has been suggested in several studies (234). Higher serum IGF-I levels are reported in breast cancer patients than in normal age-matched controls, and are associated with a poor prognosis (235). Stromal IGF-II levels are

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above normal in 50% of invasive breast cancer cases, which correlates with increased cell proliferation (236). For breast cancer, IGF-I association was not apparent in postmenopausal women, but strong associations were observed for premenopausal cases (237) (reviewed in (238). Furthermore, in recent epidemiologic studies, relatively high plasma IGF-I and low IGFBP3 levels have been independently associated with greater risk of breast cancer among premenopausal women (232). According to this study, the relationship between IGF-I and risk of breast cancer may be greater than that of other established breast cancer risk factors, with the exception of a family history of breast cancer or a high density mammographic profile. IGF-I concentrations may be particularly relevant to breast cancer risk in premenopausal women, since estradiol enhances IGF-I action in the breast (239). Although an increase in serum IGF-I levels in breast cancer patients has been documented, causality has not been established. In a multivariate analysis, the conventional prognostic factors were superior to IGF-I expression in predicting disease outcome, although IGF-I expression in tumour stroma showed some independent prognostic significance in early phases of the disease (240). This may indicate that IGF-I in tumour stroma acts as a paracrine growth promoter for breast cancer cells, increasing tumour malignancy. These studies were nonetheless potentially limited by their retrospective design. Plasma IGF-I levels were evaluated after diagnosis in these cases, so that an effect of the tumour on IGF-I levels was not ruled out. In support of this possibility, surgical removal of the tumour lowers circulating IGF-I levels (241). When measured in tumour cytosol, IGF-I showed no correlation with other

Chapter 7 IGF family members (IGF-II, IGFBP-1, or IGFBP-3) or with other prognostic markers such as ER or nodal status. The Plasma IGF-I level did not correlate with better or worse short-term survival of patients at an advanced stage of breast cancer (242). In contrast, IGF-II expression correlated positively with IGFBP-1 and -3, and high IGF-II expression correlated weakly with poor prognostic indicators (high p53, low ER, and low cathepsin D). Immunohistochemical measurement of IGF-II showed a weak inverse relationship with poor prognostic features (tumour grade, S-phase, DNA ploidy) (243). Although in vitro data suggest that IGF expression would be a marker of poor prognosis in breast tumours, the very limited clinical evidence to date do not strongly support this. Breast tumours express IGFBP-2 through -6. In non-invasive breast cancer patients, ER-positive tumours expressed high IGFBP-4 and -5, whereas ERnegative tumours expressed high IGFBP-3 levels (244). IGFBP-4 was correlated with poor prognostic factors and was an independent prognostic marker when patients were sorted by tumour size; patients with large tumours (>2cm) and low IGFBP-4 expression had improved disease-free survival compared to patients with high IGFBP-4 expression (244). Contradictory results have been reported between IGFBP-3 expression and breast cancer prognosis. Serum IGFBP-3 levels have been found to correlate negatively with cancer risk (245). Although no significant association was found between IGFBP-3 and breast cancer recurrence, survival analysis indicated that risk of death was strikingly increased in patients with higher IGFBP-3 levels (246). IGFBP-3 was associated with poor prognostic features (low ER, high S-

7. IGF in breast cancer progression phase, tumour size and aneuploid tumours), although IGFBP-3 levels were not independent factors in multivariate analysis (247-248). Breast cancer patients with elevated levels of the IGFBP proteinase PSA have a better prognosis in multivariate analysis, and PSA levels fall in the transition from benign to malignant breast disease (249-250). The proteolytic activity of PSA on IGFBP-3 may account for a decrease in IGFBP-3 levels and thus an improved prognosis. 5.5.2. Prognostic value of IGF-1R

Despite the impressive evidence supporting a role for the IGF-1R in experimental carcinogenesis, there are only correlative data with regard to the importance of this receptor in human cancers. Early studies indicated that a percentage of primary breast tumours expressed IGF-1R (50-93%), and that expression correlated positively with ER expression (251-253). Nonetheless, IGF1R expression predicts a better prognosis both in relapse-free survival and overall survival of breast cancer patients (254255). Taken as an independent factor, the IGF-1R has been reported as a positive, a negative or an insignificant marker of disease-free survival (253, 256-257), indicating the contradictions in the literature. Indeed, long-term survival has been inversely correlated with IGF-1R levels (257), whereas more recently IGF-1R has been associated with a decline in shortterm survival but without predictive value for long-term survival (258). Although IGF-1R expression appears to be enhanced in breast cancers compared with adjacent normal tissue, no studies have yet been performed that directly implicate the IGF-1R in initiation or propagation of human cancers (80, 259-260). Elevated IGF-1R expression was also associated

129 with “in-breast” cancer recurrence after breast irradiation (258). IRS-1 expression did not correlate with other known prognostic factors, but elevated expression did forecast a shorter disease-free survival period in patients with small tumours (<2 cm) (248). High IRS-1 expression has been correlated with early disease recurrence in ER-positive human primary breast tumours (118). IGF-1R expression was likewise associated with ER expression; it is an adverse prognostic marker in an ERnegative subgroup but a favorable prognostic marker in an ER-positive subgroup (257, 261). ER status was prognostic for disease-free survival when IGF-1R was positive, but not when it was negative. The category may have a favorable prognosis, perhaps because IGF-1R expression provides a marker of intact ER function. The association of the IGF-1R with prognostic parameters other than ER status are still insufficiently documented. A recent study assessed IGF-1R status in two patient subgroups, representing either a low risk (ER-positive, plasminogen receptor-positive, low mitotic index, diploid) or a high risk (ER-negative, plasminogen receptor-negative, high mitotic index, aneuploid) population. This analysis established a highly significant correlation between IGF-1R and improved prognosis (255). In breast tumours, the IGF-1R, even when highly overexpressed, is co-expressed with the ER, and the occurrence of the ER-negative/IGF-1Rpositive phenotype is rare (262). The favorable prognosis provided by high IGF-1R expression is in conflict with the majority of the in vitro studies supporting a role for IGF-1R in cell transformation and malignancy. One may speculate that more aggresive tumours usually express high IGF levels, which

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may in turn downregulate IGF-1R expression. High IGF-1R expression may thus reflect the lack of an operative IGF autrocrine loop, whereas low IGF-1R levels would indicate that the receptor is autocrinously activated; tumour growth would thus be independent of the environment and have a worse prognosis. On the other hand, the prognostic significance of IGF-1R may be similar to that of ER, whose expression reflects well-differentiated tumours with a favorable prognosis; IGF-1R expression thus identifies a relatively welldifferentiated tumour that requires IGF for proliferation. The role of the IGF-1R in malignant progression of human breast cancer thus remains obscure. 5.5.3. IGF axis-based clinical intervention and therapy for breast cancer Epidemiological data are therefore insufficiently conclusive to establish whether increased plasma IGF-I concentration is the cause or the consequence of breast cancer. Even so, the determination of serum IGF-I concentration may be of clinical interest. If augmented serum IGF-I is the outcome of a breast tumour, its measurement could permit monitoring of the effectiveness of anti-tumour therapy. If IGF-I concentration is associated with increased breast cancer risk, strategies to lower IGFI plasma levels would be advantageous in breast cancer therapy. It is well established that the decrease in serum estrogen levels with age partially protects against occurrence of hormonesensitive cancers (263). As commented above, antiestrogenic drugs such as tamoxifen reduce serum IGF-I in breast cancer patients (264-266). Tamoxifeninduced reduction in IGF-I levels involves both suppression of pituitary growth

Chapter 7 hormone output and growth hormoneindependent mechanisms (267-268). IGF-I bioactivity is reduced both by downregulation of IGF-I and IGF-1R gene expression and by upregulation of IGFBP expression (269). Again, whether or not tamoxifen-induced decline in circulating IGF-I levels contributes to the efficacy of antiestrogens against these tumours is not yet clear (124). Further research is needed to determine whether antiestrogens will be of specific value in risk reduction for women with high IGF-I levels (260). Retinoids have also been shown to suppress growth and prevent development of breast cancer in animals. These agents suppress tumourigenesis in carcinogentreated rats and in transgenic mice, and inhibit the growth of transplanted breast tumours (270). The mechanism by which retinoids inhibit breast cancer progression has not been completely elucidated; however, retinoids have been shown to affect multiple signal transduction pathways, including IGF-, TGFβ-, and AP-1-dependent pathways (271). Retinoids, TGFβ, and vitamin D analogs also upregulate IGFBP expression, an action that correlates with lower IGF-I bioactivity in certain physiological contexts (272-274). Evidence from several sources suggests that manipulating IGF-I may also reduce cancer incidence successfully. A variety of treatments have been proposed to decrease systemic IGF-I bioactivity, including the use of recombinant IGF binding proteins, somatostatin analogs or antagonists for GH releasing hormone or GH. Of this panel, somatostatin analogs appear to be the most important approach. Treatment of postmenopausal breast cancer cohorts with a combination of tamoxifen and the somastatin analog octreotide is reported to be more effective than tamoxifen alone (275-277).

7. IGF in breast cancer progression Interestingly, octreotide plus tamoxifen treatment also resulted in a more potent suppressor effect of circulating IGF-I levels than either agent alone, achieving serum levels nearly 50% lower than those measured before treatment (278). Nevertheless, one study reports that somatostatin does not increase the antitumour effect of tamoxifen (279). At present, the major questions relate less to the challenges of optimizing pharmaceutical approaches to suppression of the GH/IGF axis, than to the uncertainty as to the proportion of human breast cancers that are indeed dependent on endocrine IGF-I bioactivity. Until this is established, cautions should be used with therapies such as GH replacement that increase IGF-I levels, based on the putative role of GH/IGF axis in cancer incidence. Treatment of patients with GH must evaluate both lifetime exposure to high IGF-I concentrations and the magnitude of serum IGF-I elevation, in an attempt to maintain IGF-I within normal age-related ranges in children as well as in adults (280-281). 6. ROLE OF THE IGF AXIS IN TUMOUR INVASION The acquisition of the ability by neoplastic cells to violate surrounding tissues and matrix barriers defines tumour progression to an invasive and metastatic stage (282). This step is critical in tumour progression, as localized primary cancers are largely curable, whereas invasive and metastatic tumours are usually lethal. Tumour cell invasiveness is marked by cellular transition to a mesenchymal phenotype (283). These dedifferentiated cells lose attachment to the primary tumour mass and migrate through the surrounding matrix and/or stroma. It is becoming evident that cell motility is a rate-limiting step in tumour invasion. Cell

131 motility is nonetheless a process with a central role in many physiological situations, such as host immune responses, angiogenesis, wound healing, embryogenesis and neuronal patterning, among others. The question thus arises as to the differential cell properties that distinguish physiological and malignant invasion. Cell migration switches “off” and “on” based on quantitative differences in molecular components such as adhesion receptors, extracellular matrix ligands, affinity of membrane-bound chemoattractant receptors, proteases, cytoskeletal linking proteins, and certain signaling molecules (284). All these checkpoints have important roles in tightly controlled physiological events. Indeed, the mechanisms used by a tumour cell to invade tissues are biochemically indistinguishable from those used by nonmalignant cells under physiological conditions (282). Invading tumour cells appear to have lost the control mechanisms for a pre-existing physiological program of invasion, which prevents normal cells from inappropriate invasion of neighboring tissues. A major goal is thus to understand what signals are constitutively activated or unrestrained in tumour invasion. IGF-I increases the migration of several cell types, such as T lymphocytes, bronchial epithelium, endothelial, and smooth muscle as well as tumour cells, including breast carcinomas (56, 285291). IGF-I-triggered signaling pathways are deregulated in tumour cells, as they produce this growth factor in an autocrine manner. In this section, we will focus on the IGF-I-triggered molecular mechanisms that lead to cell migration and may explain deregulatory phenomena in tumour invasion.

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Chapter 7

7. IGF in breast cancer progression 6.1. Cell movement: the interplay between adhesion and detachment Cell movement across a twodimensional substrate can be viewed as a continuous, dynamic interplay of attachment at the cell front and deadhesion at the rear cell edge, combined with cell traction machinery that pulls the net cell body forward (Fig. 3). All of these steps form a cycle necessary for migration, with no obvious starting point. The earliest detectable “motile” morphological change induced by IGF-I and other growth factors is redistribution of the actin cytoskeleton associated with the formation of membrane ruffles (292), rapidly-moving membrane protrusions that form the cellular lamella (293). Ruffling is followed by protrusion of membranes from the ventral surface of the lamella, forming the lamellipodia (294). Cell adhesion to the extracellular matrix stabilizes the newlyformed cellular extensions, allowing the cell to exert force against the substrate. Adhesion is therefore an important step in the acquisition of motile potential. Among the cell adhesion receptors, a family of heterodimeric transmembrane receptors known as integrins is central to cell migration, as they “integrate” ligand interaction through ECM and cytoskeletal signals. This is possible since integrins can signal through the cell membrane in either direction; the extracellular binding activity of integrins is regulated from the within the cell (inside-out signaling) through clustering and, simultaneously, ECM binding elicits signals that are transmitted into the cell (outside-in signaling)(295298). Several pieces of evidence suggest that integrin-cytoskeleton interactions have a major role in mediating cell migration as well as tumour invasion and metastasis (299-301). Indeed, tumour cells overexpressing integrin migrate in

133 vitro and form metastases in vivo with no exogenous supply of cytokines (302). Although integrin-mediated adhesion is necessary for cell motility, it is not in itself sufficient. For instance, cells expressing integrin require a tyrosine kinase-mediated signaling event for motility on vitronectin (303). Much current evidence indicates that several growth factors cooperate with integrins to promote cell migration in vitro and in vivo (56, 290, 304). Indeed, signaling molecules activated by the IGF-1R associate to integrin and to the focal adhesion kinase (FAK)(113, 305), a kinase activated after integrin activation (304), which may explain the adhesionindependent growth observed in tumourigenesis (306). The dissemination of several malignant tumour cells in vivo is also dependent on cooperation between and IGF-I (291). Further, IGF-Iinduced cell migration is achieved only when the integrin receptor is activated (288), and antibodies that block integrin binding to the ECM substrate result in inhibition of the IGF-I chemotactic effect (56, 290). The chemotactic response is thus the sum of the signaling generated both by integrin-mediated cell attachment and by the IGF-1R. It is intriguing to ask how this growth factor-integrin cooperation occurs. Overlapping has been described between integrin and growth factor signaling (307308); for instance, growth factor-induced PI-3K activation depends on cellular adhesion (309-310). Cellular adhesion also regulates signaling through the MAPK pathway by modulating and MAPK activation (311). Nonetheless, crosstalk among these signaling pathways is not always unidirectional; indeed, IGF-I induces FAK phosphorylation in unattached cells, but triggers FAK dephosphorylation in attached cells (288,

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312-313). To induce migration, IGF must activate signals both to promote integrin-mediated adhesion and to suppress integrin activation. IGF-I stimulation of MCF-7 cells increases and adhesion to vitronectin and type IV collagen, respectively, in a time- and dosedependent manner, although IGF-Ienhanced cell adhesion proceeds through a mechanism independent of FAK activation (313-314). As mentioned above, integrins may propagate conformational changes from their cytoplasmic domains to enhance receptor avidity. Specific mutations in the IGF-1R are reported to disrupt the actin cytoskeleton and the cellular localization of vinculin, a protein implicated in control of adhesion by other growth factors (315-317). Our results in MCF-7 cells suggest that IGF-I increases cell adhesion by inside-out integrinmediated signaling, although the molecules involved in such integrin activation remain elusive (313). Growth factors suppress integrin activation through Ras/Raf-induced MARK activation (318) or by regulating the phosphorylation status of FAK through recruitment and activation of the tyrosine phosphatase SHP-2 (313, 319). In the case of MCF-7, IGF-I induces the specific dephosphorylation of FAK through SHP-2 recruitment and activation. SHP-2mediated integrin suppression appears to be independent of both MAPK and PI3K signaling pathways (313). Interestingly, cells derived from embryos with targeted gene deletions of FAK (FAK-/-) and SHP2 (SHP-2-/-) show identical phenotypes, with an increased number of focal adhesions and reduced spreading and motility on diverse ECM substrates (304, 320). Therefore, IGF-1R activates integrin, probably acting through cytoskeletal

Chapter 7 proteins such as vincunn. As a consequence of cell adhesion, FAK autophosphorylation takes place and focal adhesions are assembled, involving other proteins such as paxillin. Activated IGF1R subsequently associates to integrins at the focal adhesion contacts (321); it then recruits SHP-2, which turns off FAK at a particular focal adhesion contact and the cell is locally detached. 6.2. Spatial and functional asymmetry in response to IGF-I

cell

Since cell adhesion and detachment occurs at opposite cell edges, the moving cell must obviously acquire and maintain spatial and functional asymmetry, a process called polarization. This asymmetry develops between two opposite cell edges; one becomes the leading edge, exhibiting protrusion, and the other becomes the rear and undergoes retraction. As discussed earlier, evidence suggests dimers initiate the engagement of that the machinery needed for acquisition of spatial and functional asymmetry during dimers are necessary for chemotaxis. chemokine-induced chemotaxis in mammalian cells (127-128) and to trigger localized leading edge activation of chemotactic signals in D. discoideum dimers have (322-323). Moreover, been identified as the primary signaling molecules in pheromone-induced chemotropism in yeast (324), a process morphologically similar to chemoattractant-induced polarization. The acquisition of front-rear polarity is controlled by external directional signals that generate a series of repeated excitation and adaptation events (325). Molecular rearrangement ensues, leading to the spatial asymmetries involved in migration, such as integrin-cytoskeleton linkage as well as forward redistribution of integrin adhesion receptors and of chemosensory

7. IGF in breast cancer progression receptors (326-328). Whether or not chemoattractant receptors also redistribute to the leading cell edge during chemotaxis is the subject of debate. Whereas some authors report asymmetric membrane distribution of different chemoattractant receptors (329-331), including several chemokine receptors (332-333), the use of GFP-tagged receptors leads to the conclusion that there is no enrichment of chemoattractant receptors at the leading edge in moving cells (334-335). Experimental evidence in our model system indicates that forward redistribution of chemoattractant receptors may be a mechanism to position a cell guidance system in specific areas of moving cells. We used the criterion that focal adhesion formation occurs preferentially at the cell front (336-337) to determine unequivocally the leading edge of migrating MCF-7 cells. Resting MCF-7 cells show a rounded morphology over which focal adhesion are uniformly distributed. Shortly after IGF-I stimulation (5-10 min), cell spreading increases and the cell issues a variable number of pseudopodia and lamellipodia, which have a large number of focal adhesions. One extension eventually predominates and forms the leading edge. At longer stimulation times, both the leading edge, rich in focal contacts, and a trailing tail with few or no focal adhesions, are clearly distinguished. IGF-I stimulation of MCF-7 breast carcinoma cells induces CCR5 redistribution to the leading edge, whereas IGF-1R remains evenly distributed on the cell surface (338). This concurs with the IGF-I-induced upregulation of RANTES, the CCR5 ligand, which activates CCR5 in an autocrine manner. In fact, prevention of CCR5 activation by neutralizing antibodies or by overexpressing the transdominant negative receptor mutant abolishes IGF-I-induced MCF-7

135 cell asymmetries involved in migration (142). These data thus suggest that IGF-Iinduced chemokine receptor transactivation selectively localizes the signal(s) for directed movement, providing a mechanism to engage the chemotactic machinery at the leading cell edge. Evidence is accumulating to indicate that autocrine secretion of chemokines is a primary response to growth factor stimulation. Different growth factors stimulate distinct chemokine arrays, which appear to be more dependent on cell type than on the growth factor stimulus. IGF-I stimulation of MCF-7 cells induces RANTES secretion; similarly, IGF-I stimulation of DU-145 prostate carcinoma cells stimulates secretion of RANTES and IL-8 (unpublished results). Chemokine secretion occurs in response to other growth factors; for instance, bFGF stimulation of bovine aortic endothelial cells (BAEC) induces secretion of the βchemokine monocyte chemoattractant protein (MCP)-l. Both FGF and PDGF receptor activation in fibroblasts upregulates gene expression of the JE chemokine (339), the murine homolog of human MCP-1. IL-2 stimulation induces secretion several CC-chemokines, including RANTES, in natural killer cells (340), or of MCP-1 in fibroblasts (341). One of the first effects following growth factor stimulation appears to be chemokine secretion to the extracellular milieu. It is important to observe that chemokine secretion is probably not necessary in growth factor-induced cell motility. Cell polarization signals are therefore independent of those triggering cell motility, and asymmetric protein redistribution is hence not the consequence of cell movement. This concurs with data showing that immobilized amoebae retain the capacity to redistribute signaling

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molecules involved in directional sensing (322, 325). One may then speculate that chemokine signals induce the cell asymmetries required for persistent directional movement, whereas growth factors provide signals required for chemokinesis by modulating FAK turnover and integrin-dependent adhesion. Optimal cell chemotaxis is thus the result of crosstalk between these two pathways, explaining the synergy observed when these two classes of chemoattractant are used in combination. Certainly, IGF-I and chemokines synergize to elicit a chemotactic response in most tumour cell lines studied (142) and unpublished observations), and this synergistic effect resembles those observed between autotaxin and IGF-I in melanoma cells (136, 342). The cooperation between tyrosine kinase and G protein-coupled receptors in cell migration thus appears to be a general phenomenon, reported for many other chemokine and cytokine receptors (343). 6.3. Structures asymmetry

involved

in

cell

Lamellipod extension is the most obvious process in the achievement of cell asymmetry, since integrins, chemokine receptors and signaling molecules involved in cell polarization accumulate in this extension. Nonetheless, the signals that determine the formation of the leading lamella in a specific area of the cell are largely unknown. Since activation chemokine receptors or trimeric Gproteins correlates with acquisition of a migrating phenotype, the simplest interpretation is that signals from these molecules determine the plasma membrane domain that will become the leading edge. Nonetheless, asymmetric chemokine receptor redistribution in leukocytes appears to be independent of

the polarization-inducing agent used (331332). These reports suggest that cell asymmetry is probably not a consequence of chemoattractant receptor distribution, but rather thatprotein redistribution is the final result of the polarization process. Cell polarization may thus be the conclusive reflection of complex mechanisms that establish and maintain functionally specialized domains in the plasma membrane and cytoplasm during cell migration. Migrating cells are not the only cells that segregate proteins into specific plasma membrane regions. In polarized cell types such as neurons or epithelial cells, distinct plasma membrane domains (apical and basolateral compartments for epithelia, and axon and dendrites for neurons) differ considerably in lipid and protein composition (344-345). It is proposed that membrane proteins are selectively delivered to specialized cell surfaces in polarized neurons and epithelial cells by cosorting with glycosphingolipids in vesicular carriers (345-349). In this model, specific membrane proteins cluster with glycosphingolipids and cholesterolenriched membrane rafts in the trans-Golgi network (TGN), leading to protein-lipid complexes (350). In nonpolarized cells such as BHK, CHO or 3T3 fibroblasts, several membrane proteins are also delivered with glycosphingolipids in vesicular carriers from the TGN to the cell surface (351-353). Since these proteinlipid complexes are conserved on the surface of living cells (354-355), it may be assumed that the initial raft/non-raft protein segregation has functional significance once cells have acquired a polarized phenotype. Indeed, membrane compartmentalization between rafts and non-rafts is required for efficient T cell activation (356-357), and T lymphocyte costimulation is mediated by

7. IGF in breast cancer progression reorganization of membrane raft microdomains (358). Several recent lines of evidence suggest that segregation between raft and non-raft proteins in unstimulated cells is a critical factor in distributing specialized molecules to specific locations during cell migration (338). Chemokine receptors associate with membrane raft microdomains, and receptor asymmetry parallels the preferential leading edge redistribution of other raft-linked molecules, including GM1, glycosylphosphatydil inositol-anchored green fluorescent protein (GFP-GPI), and ephrinB1; conversely, proteins not located in rafts are distributed homogeneously over the cell surface. Moreover, modification of proteins in such a way that they do not associate with rafts inhibits their asymmetric redistribution in migrating cells. Whereas raft-associated ephrinB 1wt preferentially decorates the leading edge, the non-raft associated mutant (359) is distributed homogeneously in the plasma membrane of polarized cells. Therefore, raft association seems to be a pivotal determinant for the redistribution of proteins to the leading edge of migrating cells. Furthermore, this asymmetric redistribution of proteins is not restricted to molecules with functional significance in cell motility or signaling, since the raftassociated GFP-GPI protein preferentially accumulates in the leading lamella of migrating MCF-7 cells. Since cell asymmetry appears to be dependent on protein association with rafts, an obvious question is whether functional rafts are required to achieve a polarized cell phenotype during migration. Raft structure, and hence function, can be disrupted by sequestering cholesterol from the membrane with the drug cyclodextrin (353). Raft disruption by

137 chemical depletion of membrane cholesterol impedes IGF-I-induced cell polarization and inhibits cell chemotaxis, which can be restored by cholesterol replenishment (338). Cholesterol depletion does not, however, inhibit IGF-1Rmediated early signaling, such as IGF-1R autophosphorylation and IRS-1 recruitment, and these cells retain the capacity to emit pseudopods after IGF-I stimulation. These results indicate that raft integrity is necessary for the acquisition of front-rear polarity and thus for cell motility. Since cholesterol depletion treatment abolishes the association of CCR5 with raft membrane microdomains (338), these findings suggest that chemoattractants engage all the signaling machinery necessary to induce cell polarization, using the lipid rafts as platforms. 6.4. Proteolytic activity is involved in tumour migration

Growth regulatory polypeptides such as IGF are implicated in the control of pericellular proteolysis. Several growth factors can regulate the amount and composition of the extracellular matrix and this, in turn, may affect interaction with and/or activation of growth factors by the pericellular matrix structures. A regulatory loop is thus formed in which active growth factors affect the secretion of proteolytic enzymes and, consequently, the concentrations of active ligands (360). A large number of proteases have been cloned from breast carcinoma samples and/or are described to be overexpressed in breast cancer (361). These proteases belong to several groups, including MMP, members of the urokinase-type plasminogen activator system, and cathepsins. Even the digestive enzyme pepsinogen C has been described in breast tumours, and its production is associated

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with lesions of favorable evolution (362). The serine protease prostate-specific antigen (PSA) produced by breast carcinomas is also a marker of favorable clinical prognosis (363). The classical concept of protease activity in tumour invasion as a simple proteolytic “opening” of the ECM has evolved concurrently with data indicating that transmigration through the ECM requires little or no proteolytic remodeling (364-366). Nonetheless, a positive correlation between protease activity and tumour invasion may be clearly established even in migration assays using a two-dimensional substratum (56). In the next sections, we will discuss the relevance of tumour proteases, with attention to their classical role as ECM proteolytic enzymes as well as their emerging role as activators of ECM-linked signals (51). 6.4.1. Matrix metalloproteinases (MMP)

Tumoural dissemination has been correlated with MMP due to their proteolytic activity on ECM, a crucial event in tumour cell migration (367). Several MMP have been cloned from breast tumours, including stromelysin-3, collagenase-3, and the membrane-type matrix metalloproteinase 4 (368-370). More recently, the release release of the soluble form of growth factors by MMP has also been described (371), as has MMP activity on IGFBP proteolysis (51). These data highlight the relevance of this proteinase family in sustaining an invasive phenotype. For example, the spontaneous development of premalignant and malignant lesions is increased in the mammary glands of transgenic mice that express an autoactivating form of MMP-3 compared with their nontransgenic littermates (372).

Chapter 7 Of these MMP, MMP-9 is abundantly expressed in a variety of human cancers, suggesting its involvement in human tumour dissemination (373). Direct evidence of its role in tumour progression is derived from transfection experiments in which the MMP-9 gene in non-metastatic cells endowed them with the ability to metastasize (374). The mechanism mediating the MCF-7 migratory response to IGF-I through the ECM component vitronectin implicates the proteolytic activity of MMP-9 (56). We have identified MMP-9 and its proteolytic activity on the MCF-7 cell surface, and shown that the MMP inhibitor BB-94 specifically inhibits MCF-7 cell migration (56). More important, IGF-I regulates MMP activity as this growth factor induces a transient increase in MMP-9 cell surface-associated proteolysis that correlates with MCF-7 cell invasion. The IGF-induced upregulation of MMP has been also extended to endothelial cells, in which a direct effect of IGF-II on MMP-2 levels was reported (375). The enzymatic activities of MMP are inhibited by the tissue inhibitors of metalloproteinases (TIMP), a family of specific, naturally-occurring MMP inhibitors (376). Transgenic TIMP-1 overexpression inhibits SV40 T antigeninduced hepatocyte hyperplasia and liver tumourigenesis by inhibiting MMPmediated IGFBP-3 proteolysis; this leads to elevated IGFBP-3 and reduced IGF-II levels in hepatic tissues (377). TIMP-2 forms a complex with latent pro-MMP2 and inhibits the active form of both MMP2 and MMP-9. TIMP-2 inhibits in vitro tumour cell invasion, and it has been suggested that a balance between TIMP and activated gelatinases may determine the extent of ECM degradation and remodeling during tumour cell invasion (378). Surprisingly, despite its MMP

7. IGF in breast cancer progression inhibitory activity, breast carcinomas have elevated TIMP-2 levels, usually localized in the tumour stroma (379). A positive correlation has also been described between TIMP-2 stromal immunostaining and tumour recurrence (380). Stromal TIMP-2 may exert other effects on cells, related to its growth-promoting activity, with a role in breast cancer progression (381). Adhesion to the ECM and proteolysis of its components are necessary for tumour invasion (55, 382-383). A functional link between adhesion and proteolysis has been shown in melanoma cells, in which MMP2 is induced and cellular invasion increased after vitronectin ligation to (384). In MCF-7 cells, IGF-I-induced migration is effected by precise regulation of adhesion and proteolysis via integrin and MMP-9, respectively. MMP-9 must be localized and active on the cell surface, where membrane association may affect its activity, as has also been suggested for MMP-2 (384). MMP proteolytic activity on integrin ligands including vitronectin has also been reported (385). This degradation may represent another regulatory level of the integrin activation state. Furthermore, the IGF-I concentration leading to maximum migration in the MCF-7 model does not alter levels, but higher doses, associated with a lack of MCF-7 migration, produce an increase in integrin levels (56). Changes in the levels or activation state of integrin in response to IGF-I must modify cell spreading, adhesion and detachment, which may in turn affect MMP activity, leading to an invasive phenotype. These findings emphasize cell surface proteases as suitable targets for the prevention of cell dissemination, particularly in the case of tumour metastasis.

139 6.4.2. The plasminogen/plasmin system

The urokinase-type plasminogen activator system is an important constituent in the process leading to metastasis (386). It comprises at least four proteins, urokinase-type plasminogen activator (uPA), its membrane-bound receptor (uPAR), and two plasminogen activator inhibitors (PAI-1 and PAI-2). A plasminogen-deficient mouse strain was obtained and crossed with a transgenic mouse carrying the polyoma middle-T oncogene under control of the mouse mammary tumour virus (MMTV) promoter (387). The number of metastases was markedly inhibited in transgenic mice lacking plasminogen gene. In contrast, primary tumour growth was independent of the plasminogen status of the host animal (388). In breast cancer, high expression levels of uPA and uPAR is strongly associated with recurrence, decreased survival, and a poor prognosis (389-390). Tumours, including breast cancers, frequently contain areas with significant hypoxia (391); this decrease in oxygen tension induces uPAR expression by uPAR mRNA stabilization and transcriptional activation of the gene (392). uPA mRNA is expressed by myofibroblasts surrounding the tumour cells in human ductal breast carcinoma, whereas the cancer cells themselves normally lack detectable uPA mRNA (393). uPAR is expressed in tumour-infiltrating macrophages, but in 80-90% of the cases the tumour cells lack detectable uPAR (393). In squamous cell carcinoma of the breast, however, uPA mRNA was expressed by the cancer cells and was absent from stromal cells (394). In the human breast cancer cell line MDA-MB231, IGF-I does not increase MMP activity, but uPA mRNA and protein

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levels were induced in a time-dependent manner (395). Surprisingly, PAI-1 levels are increased in a highly metastatic MCF-7 subline compared to the parental line (396); the inhibitory activity of PAI-1 on uPA cannot explain this phenotype but in addition, PAI-1 competes with uPAR binding to vitronectin and interferes with integrin-mediated cell adhesion (397). 6.4.3. Cathepsins

Cathepsin B is a lysosomal cysteine protease secreted in a significantly higher amount from malignant breast tumours than from normal tissue explants (398). Compared to benign fibroadenomas, breast carcinomas show increased secretion of cathepsin B (399). Cathepsin D is also a lysosomal aspartyl endoproteinase associated to increased risk of metatastasis (400-401). When secreted in excess as in tumour tissues, cathepsin D interferes with IGF-II signaling by displacing its binding to IGF-2R. In turn, abnormally high IGFII levels may alter the homeostasis of cathepsin D, leading to overexpression of this protease. Secretion of procathepsin B may also indicate saturation of the mannose 6-phosphate pathway, leading to dumping of the lysosomal enzyme to the secretory pathway (402). The extracellular pH in tumours is generally more acidic that in the corresponding normal tissues (403), and both secreted pro-cathepsin-D and -B may be activated extracellularly in sufficiently acidic milieu, producing the active forms. 7. ROLE OF THE IGF AXIS IN TUMOUR-INDUCED ANGIOGENESIS According to the initial studies by Folkman (404), tumours are unable to grow, yet remain viable in the absence of neovascularization. Tumour production of a diffusible factor resulted in the shift of

Chapter 7 nearby vascular endothelial cells from a resting to an activated state. This shift results in new vessel formation and subsequent vascularization of the tumour, which then expands and forms metastases. A growing body of clinical and experimental data supports this hypothesis, and it is now recognized that tumourassociated angiogenesis is one of the rate limiting steps for tumour progression (405). In this section, we will review experimental evidence supporting a role for IGF axis elements in this process. 7.1. Positive and negative regulation of the angiogenic process

Spontaneously arising human and animal tumours are not usually angiogenic at the beginning of their development. During the prevascular phase of solid tumours, little or no angiogenic factor is released from the transformed cells. The absence of neovascularization severely restricts the size of the tumour mass beyond a few cubic millimeters, however, although the cells proliferate at a rate equivalent to that of vascularized tumours. These unvascularized primary tumours and micrometastases are usually maintained as small, dormant tissue nodules whose volume is held constant by a balance between cell proliferation and cell death (406). Neovascularization permits rapid tumour expansion (407) by a dual action: the oxygen supply provided by blood perfusion of the tumour, and the release factors, such as IGF-I and IGF-II, produced by the new capillary endothelial cells (408-409). These factors may decrease the apoptotic rate of tumour cells, shifting the balance of proliferation versus cell death. Accordingly, specific inhibitors of angiogenesis achieve not only cessation of further tumour neovascularization and growth, but also decrease tumour size and re-induce tumour dormancy by increasing

7. IGF in breast cancer progression the tumour cell apoptosis rate (410). The tumour-secreted factors responsible for inducing the “switch” from unvascularized to pro-angiogenic phenotype are not completely understood. Experiments in transgenic mice bearing spontaneous tumours indicate that only a subset of cells, as few as 4-10%, become angiogenic, and this subpopulation of cells confers increased tumourogenicity on the whole tumour (407, 411-412). In a transgenic mouse tumour model containing the genome of the bovine papilloma virus type 1, the switch to the angiogenic phenotype appeared to be associated with the ability to export basic FGF from the cell (411). Angiogenesis depends on a net balance of positive and negative factors released from the tumour cells per se, from cells recruited to the area, or mobilized from extracellular matrix (413-414). An extensive list of endogenous proangiogenic polypeptides has been described, including basic- and acidic and tumour FGF, angiogenin, necrosis factor vascular endothelial growth factor (VEGF), platelet-derived endothelial growth factor (PD-ECGF), granulocyte-colonystimulating factor (G-CSF), interleukin-8 (IL-8), hepatocyte growth factor/scatter factor (HGF/SF), and IGF-I and -II. Conversely, a variety of naturally-occuring agents such as thrombospondin, angiostatin, platelet factor-4, etc., specifically inhibit the process of angiogenesis and arrest tumour growth in vivo (415-416). Pro-angiogenic factors exert their activity by triggering the proliferation, migration, and differentiation of endothelial cells from capillaries near the tumour (417). The angiogenesis process also requires direct interaction between endothelial cells and the surrounding

141 matrix. The integrin is selectively expressed on growing neovasculature, but not on quiescent blood vessels; specific signals enhance the in vivo survival of vascular endothelial cells (418). Furthermore, binds to activated MMP-2 at the cell surface of endothelial cells and invasive melanoma cells in vivo, facilitating cell-mediated collagen degradation (55). Migration of endothelial cells into tissue ECM is facilitated by proteases such as MMP. These enzymes, secreted by the same cells that produce angiogenic factors, are responsible for the breakdown of tissue matrix surrounding the growing vessels as well as for releasing ECM-anchored growth factors. It integrin may thus appears that function in cooperation with MMP-2, and probably other ECM-linked factors, to promote cell adhesion and migration, resulting in directed endothelial and tumour cell invasion. The increased production of positive angiogenic factors is necessary, but not sufficient for the angiogenic phenotype, as negative regulators must also be decreased. Thrombospondin-1 (TSP-1), the first angiogenesis inhibitor described, is constitutively produced by normal cells but its expression is downregulated in tumours (419). In human fibroblasts, this inhibitor is under control of the p53 tumour suppresor gene, and its production decreases when p53 is mutated or deleted (420). Angiostatin, another inhibitor of angiogenesis, functions in the opposite manner, as its expression is downmodulated by removal of the primary tumour. Angiostatin accumulates in the circulation in the presence of a growing primary tumour and disappears when the tumour is removed (421). This inhibitor suppresses angiogenesis in remote metastases; when its levels are diminished, intense angiogenesis and rapid metastatic

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growth. The mechanisms leading to active proangiogenic activity usually involve alterations of tumour suppresor genes, with a subsequent decrease in levels of an angiogenesis inhibitor. Indeed, loss of p53 gene function is associated with reduced TSP-1 expression and the susequent increase in pro-angiogenic factors (420). It has furthermore been shown that in vitro restoration of p53 function in human glioblastoma cells resulted in the secretion of a new, potent angiogenesis inhibitor termed glioma-derived angiogenesis inhibitory factor (422). Since p53 regulates expression of several IGF axis components (62), it is tempting to speculate that part of the anti-angiogenic activity of this tumour suppressor is mediated by regulation of IGF activity. In fact, as will be discussed in the next section, IGF also has an important role in the neovascularization process. 7.2. Positive regulation of expression in tumour hypoxia

IGF

Human solid tumours, even those <1 cm in diameter, frequently contain areas of significant hypoxia (391, 423). Hypoxia in solid tumours is a major problem for radio- and chemotherapy, as it accelerates malignant progression and metastasis (424). Recent studies show that this decrease in oxygen tension detected in the central region of a tumour can be the leading cause of angiogenesis, as it activates expression of several hypoxiainducible angiogenic factors such as VEGF (425-426), (427), Interleukin (IL)-6 (428), IL-8 (429) and acidic/basic FGF (430). Induction of these genes is mediated by a common basic helix-loop-helix (bHLH)-PAS transcription complex formed by the hypoxia-inducible and another bHLH-PAS, the aryl hydrocarbon

nuclear translocator (ARNT). Hypoxic stress induces all of these genes by elevating the level. In the case of VEGF, mRNA stabilization under hypoxic conditions is also a significant component in regulating the resulting protein levels (431). is overexpressed in human breast, colon, prostate and lung cancer (432), and abrogation of expression reduces rates of xenograft growth and vascularization (433-434). It is reported that IGF and IGFBP expression is transcriptionally upregulated in hypoxia (435-438). Hypoxia-induced IGFBP-1 in hepatoma cells was mediated by an HRE containing a putative site, providing a precedent for direct regulation (435). Likewise, seems to be required for maximal expression of IGF-II, IGFBP-2 and IGFBP-3 mRNA (439), although several authors failed to find hypoxia response element sequences in their promoter regions (436). Hypoxiainduced IGF-II expression in HepG2 cells is reportedly due to the enhanced activity of Egrl, a zinc finger-containing transcription factor, on the IGF-II P3 promoter (440-441). The Egr-1 binding site (EBS) in this promoter is essential for the transcriptional regulation of IGF-II under hypoxic conditions, as Egr-1 transcripts were also upregulated during hypoxia (441). Direct effects have been described of IGF-II on angiogenesis in both CAM (436, 440) and rat cornea models (442). Indeed, IGF-II may induce angiogenesis by stimulating migration and morphological differentiation of endothelial cells (375). Despite this direct IGF angiogenic activity, most reports favor an indirect effect by induction of pro-angiogenic genes, such as VEGF (443). IGF-I and IGF-II stimulation increase VEGF mRNA and protein levels in a time-dependent manner in endometrial adenocarcinoma (444) and in human

7. IGF in breast cancer progression hepatoma cells (436), respectively. This effect has been explained as the reciprocal and IGF positive regulation of gene expression. Exposure of cells to insulin, IGF-I or IGF-II results in protein expression, induction of even in IGF-1R deficient cells (439). In addition to IGF-1R, IR or IGF-2R activation also induces expression (445). Insulin-IGF-induced expression of genes containing the hypoxia response element (HRE) may also occur by stabilization of the transcription complex in various cell types (445). Nonetheless, the road connecting the IGF signaling pathway and the transcriptional activity probably is not direct. The tumour suppressor PTEN is reported to regulate hypoxia- and IGF-Iinduced angiogenic gene expression by regulating Akt activation and transcriptional activity in glioblastomaderived cell lines (446). Loss of PTEN during malignant progression contributes to tumour expansion by inducing regulated genes following hypoxia or growth factor stimulation. The increased expression of IGF-I and/or IGF-II observed in transformed cells may therefore contribute fundamentally to the promotion of deleterious tumour neovascularization. This pro-angiogenic tumour phenotype may be achieved indirectly by increasing expression of positive angiogenic factors, by increasing expression and/or activity of MMP, or by affecting the migration and differentiation of endothelial cells. Measurement of serum levels of proangiogenic factors, such as VEGF, has been shown useful to monitor tumour status and prognosis (447). There are nevertheless no data on VEGF levels in patients receiving therapy that decreases IGF-I levels. The relationship between

143 IGF and upregulation of pro-angiogenic peptides opens an interesting, unexplored avenue for clinical intervention by combining anti-angiogenic agents with treatments that affect IGF biological activity. 8. MOVING TOWARD METASTASIS The fundamental difference between normal and tumour cells lies in their regulation. Deregulated proliferation, invasiveness and pro-angiogenic activity characterize tumour cells in advanced stages, although this ominous phenotype is not observed at initial stages. In situ breast carcinoma, for instance, can be viewed as the result of the clonal expansion of hyperproliferating cells that permeate the ductal system (448). This stage of cancer may be benign, as the tumour cells are unvascularized and separated from stromal lymphatics by a basement membrane barrier. The ductal lesion may even regress if progression to invasive cancer does not occur. In other cases, additional progression events take place in a few neoplastic cells, which gain invasive capacity (449). Tumour-induced vascularization occurs in parallel with this transition to invasiveness and provides a vascular entry portal for dissemination, which may precede evident primary tumour outgrowth. The clonal expansion of these invasive cells generates a metastatic breast carcinoma with a poor prognosis. The identification of factors influencing the malignant evolution of incipient in situ carcinoma is thus a critical turning point in impeding tumour progression. Tumour progression depends on complex interactions between host and tumour-produced factors. This process is slow; the average transition period from non-invasive hyperplastic breast lesion to invasive breast carcinoma has been

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estimated at 6 years (448). During this time, several host factors instill genetic modifications that foster tumour progression. In the case of breast carcinoma, host estrogens may increase genetic instability directly, and/or indirectly by regulating IGF-I availability. Indeed, early efforts to control breast cancer included either ovariectomy or hypophysectomy, in an attempt to decrease steroids or IGF-I levels in the tumour environment. These efforts, now replaced by antiestrogenics or somatostatin analogs, have beneficial although mostly transient effects. The reason for this failure is that clinical manifestations of the disease occur at late stages, when the tumour has acquired invasive capacity and most tumour cells secrete constitutively growth factors that influence their growth in an autocrine manner. In this chapter, we have reviewed evidence supporting a role for the IGF family members in tumour growth, neovascularization and invasion. New efforts must thus be oriented toward the early detection of in situ carcinomas. New epidemiological data suggesting that high IGF-I levels may be a risk factor for breast and other human cancers may thus be of interest. Data are not yet sufficient, however, and much work is required to establish a firm correlation that nonetheless seems clear from carcinogenesis studies in animal models. On the other hand, new therapeutical approaches must be designed to target growth factors acting in autocrine/paracrine loops. Again, carcinogenesis in animals indicates that strategies that target IGF axis elements may prevent tumour formation or arrest tumour evolution, although these strategies in humans have been only partially explored. In addition, although IGF-I levels are elevated in breast cancer patients compared to normal women, IGF-I serum

Chapter 7 concentration does not identify patients with better or worse short-term survival (242). This may indicate that autocrine/paracrine, rather than endocrine IGF-I, is the most important growth factor source in promoting tumour progression. If this is the case, intereference with endocrine IGF-I levels may have a major impact early in cancer development during the pre-diagnostic phase, whereas advanced carcinomas would be independent of host IGF-I. Development of new strategies to target IGF autocrine loops in tumours must be based on an understanding of the molecular mechanisms that regulate IGF-I activity. How an IGF paracrine loop works in vivo is largely unknown, however, and only modest information has been gained with cultured cells. Our experience in the prostate adenocarcinoma cell line DU-145, which may be comparable to the most pernicious ER-negative breast tumours, suggests that the IGF autocrine loop in tumour cells is more complex than ligandreceptor interaction, due to concurrent IGFBP secretion in great excess over IGF ligands. DU-145 expresses IGF-1R and secretes IGF-I, IGF-II, and at least three different IGFBP (IGFBP-2, -3 and -4); it proliferates in serum-free medium with no exogenous growth factor supply. The addition of IGFBP-3 to DU-145 inhibits serum-free growth of this cell line; however, mixtures of antagonist anti-IGF-I and anti-IGF-II antibodies do not block the IGF-I autocrine mitogenic effect (155). These results suggest that IGFBP-3 regulates IGF bioavailability by mechanisms other than the simple scavenging of IGF ligands from the IGF1R. IGFBP-3 is proteolyzed in DU-145conditioned medium, and we found that the protease responsible for this degradation is MMP-9. MMP-9 control of DU-145 serum-free growth is critical, as

7. IGF in breast cancer progression addition of non-toxic concentrations of MMP inhibitors such as BB-94 or TIMP2, or overexpression of MMP-9 antisense cDNA, block 60-80% of DNA synthesis as well as IGF-1R-mediated signaling (155). A scenario can be envisioned in which autocrine IGF-I and IGF-II are bound to IGFBP, which regulates growth factor liberation to the IGF-1R through MMP-9 proteolysis. The active MMP-9 form is localized on the DU-145 cell membrane, making it feasible that membrane-bound MMP-9 could act as an IGFBP-3 receptor that permits delivery of small doses of IGF in close proximity to the IGF-1R (Fig. 4). Optimal DU-145 growth is thus the result of a balance in autocrine secretion of IGF ligands, IGFBP and MMP levels. Our observations and those of others (450) indicate that IGFBP-3 secretion is needed for maximum IGF-induced proliferation in tumour cells, mirroring some epidemiological data in breast cancer patients. As mentioned, high IGFBP-3 tumour secretion has been negatively correlated with ER and status; hence, IGFBP-3 overexpression has been associated with a poor prognosis in breast cancer (451). A similar situation is described for prostate cancer. Low IGFBP3 levels have nonetheless been correlated with high breast cancer risk in other studies (238). These data may indicate a different role for IGFBP-3 in regulating IGF activity, as a function of tumour stage. High IGFBP-3 levels in preneoplastic states might act as a negative regulator of IGF activity by impeding IGF/IGF-1R interaction, since IGFBP-3 protease levels are low in these pathologies. Increased IGFBP-3 protease levels in advanced carcinomas may facilitate controlled IGF release, enhancing its signaling by a still poorly understood mechanism. IGFBP-3 may therefore be a switch that turns off

145 tumour formation or turns on tumour progression as a function of IGFBP-3 protease levels. Nonetheless, epidemiological data supporting this model are still insufficently conclusive. The growth- and survival-promoting effects of IGF-I may be caused by direct interaction of the IGF-1R with appropriate signaling pathways, although crosstalk with ER-mediated signaling is also evident in breast cells. Other IGF-I-mediated functions, such as angiogenesis and invasion, nevertheless require active extracellular cross-communication through other molecules. In the case of tumour invasion, IGF-I directly controls signals required to activate biophysical processes associated to cell motility, such cell adhesion and detachment from the ECM substrate. But IGF-I also regulates other molecules, including proteases and chemokines that signal through G proteincoupled receptors, which are also necessary for cell movement (Fig. 4). This regulation occurs in both neoplasic and untransformed cells (56, 142). In fact, the IGF-I/chemokine network described here may elucidate the fine regulation and coordination of physiological cell invasion. Pathological invasion presumably subverts this mechanism, however, since tumour cells constitutively express autocrine growth factors and chemokines, which may provide them with a built-in metastatic capacity. Initial experiments in vivo have shown that when this network is disturbed, for instance by inhibition of chemokine receptor signals, tumour cell invasion is effectively abolished. We thus hypothesize that growth factors and chemokines integrate an autocrine/paracrine loop through which both neoplastic and non-malignant cells effect invasion.

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7. IGF in breast cancer progression We therefore propose that a deregulated IGF autocrine loop is a key factor in tumour progression. These autocrine/paracrine loops implicate not only IGF ligands and IGF-1R, but also IGFBP and proteases that specifically cleave IGFBP. Binding of these proteases to the cell surface may thus provide a mechanism that coordinates IGFBP proteolysis with increased IGF availability in close proximity to the IGF-1R. This would enhance IGF-mediated biological effects, such as cell proliferation, invasion, and angiogenesis, all of which are critical events in tumour progression.

147 Acknowledgements

We are indebted to Concepción Gómez-Moutón, who has contributed decisively to much of the work from our laboratory that is included in this review, and to Catherine Mark for editorial assistance. We apologize to any authors whose work has not been cited due to space limitations. Work in our laboratory is supported by grants from the CICyT, the Dirección General de Investigación Científica, the European Union/FEDER, and the Comunidad Autónoma de Madrid. The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (CSIC) and by the Pharmacia Corporation.

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Chapter 8 THE ROLE OF PLATELET DERIVED GROWTH FACTOR (PDGF) AND ITS RECEPTORS IN CANCER AND METASTASIS

Sara Weiss Feigelson, Cheryl Fitzer-Attas, Lea Eisenbach Weizmann Institute of Science, Department of Immunology, Rehovot, Israel

Key words: PDGF, signal transduction, integrins, angiogenesis, therapy Abstract:

Platelet derived growth factor (PDGF) ligands and receptors are frequently overexpressed or exclusively expressed in many diverse tumors, compared with their non-malignant counterparts. This tumor specific expression often has diagnostic value and prognostic significance. There is now accumulating evidence that this PDGF expression is functionally relevant. It has been demonstrated that the constitutive activation of PDGF receptors transforms cells and directly leads to the development and progression of tumors. PDGF ligands stimulate tumor cell growth by both autocrine and paracrine mechansims via intacellular signal transduction pathways which are well elucidated. PDGF crosstalk with integrins has also been proposed. Additionally, PDGF has been implicated in several steps of the metastatic cascade and in angiogenesis. The improved understanding of the signaling and oncogenicity of PDGF has enabled researchers to develop new anti-cancer strategies and therapies.

chains, each amino acids in length with amino acid identity, which form disulfide-linked dimers. All three dimeric forms of PDGF exist naturally and their assembly appears to be a random process (1-3). PDGF dimers stimulate the growth and motility of several cell types,

1. PDGF AND ITS RECEPTORS Platelet-derived growth factor (PDGF) was originally isolated from human platelets, but it is now known to be produced by many different cell types, and in response to various external stimuli. PDGF consists of both A and B 167

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including fibroblasts, smooth muscle cells, endothelial cells, and neurons (4). In vivo, PDGF was shown to have roles in embryonic development (5-8), wound healing (9), the regulation of blood vessel tonus (10-12), and platelet aggregation (13,14). Importantly, PDGF is involved in the progression of human disorders which involve excessive cell proliferation, including certain cancers, artherosclerosis, and fibrotic conditions (4). When expression of PDGF and its receptors in different tissues was analyzed, it was found that sometimes ligand and receptors are expressed in the same cells, while sometimes they are found in adjacent cell layers, thus suggesting that both autocrine and paracrine growth pathways are involved (15). Very recently, a new protease-activated form of PDGF, PDGFC, was identified, which appears to have a unique role in kidney development (16). Homodimeric (AA or BB) and heterodimeric (AB) isoforms of PDGF exert their effects on target cells by specifically binding to two structurally related receptors, termed and The and receptors for PDGF each contain five extracellular immunoglobulin-like domains, of which the three outermost are most important for ligand binding. The subunit can bind both A and B chains and therefore all three dimers of PDGF; the subunit, however, can only bind PDGF-B chains with high affinity. As with the growth factor, all three receptor combinations are found in nature. In fact, the different isoforms of PDGF dictate the formation of homodimeric or heterodimeric complexes as a result of ligand-induced receptor dimerization. The receptor-ligand complexes are then further stabilized by direct interactions between the fourth Ig domain of the two receptors (17,18). Even in the absence of ligand, low affinity

Chapter 8 interactions between receptors exist, and overexpression of receptors appears to be sufficient for their activation (19). Each receptor type also possesses an intracellular tyrosine kinase domain, which is divided into two parts by kinase insert sequences characteristic of this receptor family. Once receptors are dimerized, the close proximity of their kinase domains allows for autophosphorylation in trans between the two receptor subunits. Autophosphorylation on tyrosine residues within the receptor’s kinase domain serves to further activate its catalytic activity (20,21). Homologous tyrosine residues in other tyrosine kinase receptors also appear to have important roles in kinase activation (22-24). Autophosphorylation on tyrosine residues outside the kinase domain initiates a signal transduction cascade, as outlined below. 2. PDGF RECEPTOR SIGNALING Intracellular signal transduction is the product of interactions between molecules of different enzymatic pathways, often leading to changes in their conformation, subcellular localization, or enzymatic activity. These molecular interactions are mediated by various domains that specifically recognize certain amino acid residues and/or their modifications. For example, Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains recognize phosphorylated tyrosine residues situated in specific environments. SH3 domains recognize proline-rich areas, pleckstin homology (PH) domains interact with membrane phospholipids, and PDZ domains detect a C-terminal valine and its upstream sequences (25). For PDGF receptors, there are many autophosphorylation sites in addition to those located within the receptor kinase

8. PDGF and its receptor in cancer metastasis domains. In the well-mapped ß-receptor, 11/15 intracellular tyrosine residues undergo autophosphorylation. The main purpose of this activity is to provide phosphotyrosine docking sites for downstream signaling molecules, in particular those containing SH2 and PTB domains. For SH2 domains at least, it is well established that the amino acids 3-6 positions downstream of the phosphotyrosine residue dictate the specificity of binding (26), and in fact more than 10 different SH2-containing molecules are known to bind to different autophosphorylation sites within the and ß-receptors. Most of these molecules can bind to both receptor types, yet there are also examples of SH2 domain proteins that bind to only one or the other. 2.1. Signal transduction molecules with enzymatic activity Enzymes that were shown to bind to PDGF receptor autophosphorylation sites include phosphatidylinositol 3’ kinase (PI3-K), phospholipase intracellular tyrosine kinases (Src, Fer), the tyrosine phosphatase SHP-2, and the GTPase activating protein GAP. PI3-K is an enzyme that phosphorylates phosphoinositides in their 3’position. In consists of a p110 catalytic subunit in complex with a p85 regulatory subunit. p85 is the phosphotyosinebinding molecule, with two SH2 domains that interact with the specific sequence pYXXM (pY denotes the phosphorylated tyrosine residue) (26). The pYXXM motif is present twice in both PDGF α- and ßreceptors, and was indeed shown to be important for binding of PI3-K. PI3-K, after interacting with the receptor, becomes phosphorylated by its kinase on a specific tyrosine residue, and also undergoes a conformational change. This

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latter event increases the enzymatic activity of the associated catalytic subunit (27-29). The product of PI3-K, continues the signal transduction chain by binding to the PH domain of the serine/threonine kinase Akt/PKB (30). Additional kinases such as certain PKC family members, p60 S6 kinase, c-Jun N-terminal kinase (JNK), and small GTPases of the Rho family are additional candidates for downstream effector molecules of PI3-K signaling. The signaling pathways of PI3-K are important for PDGF-stimulated actin reorganization and cell movement, cell growth, and inhibition of apoptosis (31), although the precise role of the individual effector molecules in each of these responses is still unclear. Another enzyme with a lipid substrate is whose resulting products inositol(1,4,5)trisphosphate and diacylglycerol are themselves second messengers, leading to mobilization of from intracellular stores and activation of the enzyme PKC, contains two SH2, respectively. one SH3, and one PH domain, allowing for multiple interactions with other proteins. Upon receptor autophosphorylation, can bind to two phosphotyrosines within the correct motif in either of the PDGF receptor subunits (32-35); this association (36,37) and the subsequent phosphorylation of (38) are important for its activation. Interestingly, another mode of regulation for activation is the binding of its PH domain to the product of PI3-K, thereby anchoring it in the cell membrane (39). This mechanism demonstrates the importance of crosstalk between two signaling pathways involved in phophoinositide metabolism. As a result of

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mobilization and PKC activation, also has a role in the activation of the exchanger (40). In certain cell is apparently involved in types PDGF-stimulated cell growth and motility (41). The Src family of intracellular tyrosine kinases is characterized by the presence of one SH2, one SH3 domain, an N-terminal myristylation site, and a regulatory tyrosine residue in the Cterminus. This tyrosine, when phosphorylated, shuts off the kinase domain via an intramolecular interaction with the SH2 domain. Members of the Src family bind to phosphotyrosine residues in the PDGF or ß-receptors (42,43), resulting in phosphorylation of additional tyrosine residues within Src SH2 and SH3 domains, displacement of the intramolecular inhibition, and activation of Src kinase (44,45). Upon PDGF binding, another widely expressed intracellular tyrosine kinase named c-Fer also becomes physically associated with and phosphorylated by the PDGF ßreceptor (46). This molecule belongs to a family of kinases that also contain an SH2 domain, but does not have any of the other regulatory domains characteristic of the Src family. While Src appears to be important for the mitogenic response of PDGF, the role of c-Fer is not known. Two signaling molecules that appear to have a negative effect on PDGF signaling are SHP-2 and GAP. SHP-2 is a ubiquitously expressed tyrosine phosphatase with two SH2 domains. As with the kinase molecules, binding of SHP-2 SH2 domains to receptor autophosphorylation sites results in an increase in its phosphatase activity (47). Interestingly, full activation requires the simultaneous interaction of both of the SHP-2 SH2 domains. Once activated, this enzyme can dephosphorylate the receptor

Chapter 8 and its substrates, leading to negative signals (48). However, in certain situations SHP-2 can act as an adaptor for Grb2/Sos, activate Ras and the MAP kinase pathway, and thereby have a positive role in signaling through the PDGF receptor (49,50). The GTPase activating enzyme, GAP, also has two SH2 domains and was been shown to bind to a phosphotyrosine residue in the PDGF ß-receptor only. It is unclear if the subsequent phosphorylation of GAP affects its activity, yet by converting RasGTP to Ras-GDP, GAP deactivates Ras and may lead to a lowered mitogenic signal (51). 2.2. Signal transduction molecules with adaptor function Adaptor molecules such as Grb2, Grb7, Grb10, Grbl4, Shc, Nck, and Crk do not possess an enzymatic activity of their own, but instead link the receptor with downstream enzymes and their pathways. The best-studied of these molecules, Grb2, consists of one SH2 domain and two SH3 domains. Grb2 either binds directly to the pYXNX motif in PDGF receptors, or indirectly via another adaptor She or the phosphatase SHP-2, after these molecules have bound to the receptor and are phosphorylated in a pYXNX motif of their own (52-54). In either case, since Grb2 forms a complex with the Ras nucleotide exchange factors Sos1, its association with the receptor allows for the formation of active RasGTP molecules at the plasma membrane. The pathway leading from Ras-GTP involves the activation of the MAP kinase cascade and the regulation of specific transcription factors implicated in cell growth, differentiation, and migration. Interestingly, however, mitogenic receptor in signaling via the

8. PDGF and its receptor in cancer metastasis endothelial cells is not dependent on direct binding of Src (55). 2.3. Signal transducers and activators of transcription (STAT) STATs are a family of signal transducers that translocate into the nucleus to initiate gene transcription. This translocation occurs only when the STAT is in dimeric form as a result of the SH2 domain of one STAT molecule binding to a phosphotyrosine a second STAT molecule, and vice versa (56). Stat1, 3, 5, and 6 can bind activated PDGF receptors and are subsequently phosphorylated, allowing for their homoor heterodimerization (57-59). While STAT molecules are essential for signal transduction downstream of cytokine receptors, their functional significance in PDGF signaling is still unclear. 2.4. Cross-talk with integrins

Cooperation between PDGF and integrin signaling has been proposed based on the following observations: a) a fraction of phosphorylated PDGF-ß receptors forms a complex with integrins, increasing the biological activity of PDGF (60) b) stimulation of Bl integrins on fibroblasts results in the phosphorylation of PDGF ß-receptors, independent of PDGF itself (61) and c) PDGF can stimulate the synthesis of collagen binding integrins, and this correlates with an enhanced migratory response to extracellular matrix proteins (62,63). Biologically, the cross-talk between PDGF and integrin signaling makes sense as most cells that respond to PDGF are in fact anchorage-dependent, requiring contacts with matrix molecules in the environment. Integrin binding to

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these extracellular matrix molecules allows for the formation of focal adhesions, the gathering of signaling proteins at the cytoplasmic tails of the integrins, and enhanced growth-factor mediated responses (64,65). 2.5. Signal transduction and cellular responses The cellular responses mediated by the two PDGF receptor types are overlapping, but not identical, as unique responses to each receptor type have also been reported. Thus, both receptors can mediate mitogenicity, chemotaxis, and mobilization. However, differences appear with regard to actin reorganization. Ligation of both receptors will result in edge ruffling and the loss of stress fibers, yet circular ruffling only occurs upon stimulation through the ß-receptors (66). Growth factor binding to the ß receptor can also inhibit apoptosis, whereas activation of the a receptor in some cell types mediates an inhibitory chemotactic (67-69). A fascinating aspect of PDGF signaling is the induction of stimulatory and inhibitory signals in parallel, as seen by the simultaneous positive and negative effects on Ras after binding of Grb2/Sos1 or GAP, respectively; the physiological outcome of growth factor stimulation depends on the balance between these signals. Interestingly, however, as a result of significant overlap between the different pathways described above, none of these circuits is solely responsible for any one PDGF-stimulated event (70). Furthermore, a single signaling pathway can also give rise to more than one cellular response, depending on the potency or duration of the signal (71).

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3. PDGF IN CANCER PDGF and its receptors have been proposed to play an important role in tumorigenesis. Perhaps the earliest linkage of PDGF to cancer was the discovery that one of its chains, PDGF B, was homologous to the v-sis oncogene of simian sarcoma virus (SSV) (72). Since then, there have been several demonstrations of the transforming activity of v-sis in fibroblasts and the inhibition of v-sis-mediated transformation by antiPDGF antibodies (73). PDGF has been further implicated in cancer since both of its chains as well as both its receptors have been so frequently found either exclusively expressed or overexpressed in many tumor cell lines and primary tumor tissue compared to nonmalignant cells. Often, neighboring non-tumor cells produce the PDGF ligand for which the tumor cells express the receptor, thus making a paracrine mode of regulation possible. In other cases, the PDGF receptor-expressing tumor is stimulated by its own secreted PDGF ligands in an autocrine fashion.

3.1 PDGF expression and prognostic significance in cancer PDGF ligands and their receptors are expressed in many diverse tumors and/or in the tumor milieu. The production of PDGF and expression of its cognate receptor type has been particularly well established in tumor cells of neuronal origin. The expression of PDGF and PDGF (74) as well as the coexpression of PDGFB chain and PDGF . (75) were found in gliomas of various degrees of malignancy. Neurofibrosarcoma-derived Schwann cells overexpress PDGF receptors and are induced to proliferate by PDGF-BB (76). More recent in situ studies of human gliomas show overexpression of receptors in glioma cells of high-grade tumors. In a few cases, this overexpression

Chapter 8 is caused by receptor amplification. Since high-grade glioma cells also express the PDGF A-chain, it is assumed that an autocrine activation of the leads to the proliferation of these glioma cells in vivo (77). Several other human brain tumors, such as meningiomas, astrocytomas, medulloblastomas, ependymomas, and oligodendrogliomas, were also found to express the mRNA for the PDGF subunits and their receptors (78). Sometimes the in particular, either PDGF exclusively or in combination with the is implicated in tumor PDGF cells of neuronal derivation, such as in medulloblastomas and related childhood primitive neuroectodermal tumors (PNET) (79). In contrast, it is specifically the ßreceptor subunit of PDGF and its corresponding ligand (PDGF-BB) which are coordinately expressed in fresh surgical isolates of human meningioma, suggesting that activated PDGF might contribute to the pathology of this common brain neoplasm (80). PDGF-A, PDGF-B, and PDGF expression are also elevated in human malignant astrocytomas (81). Interestingly, one group found gene amplification as well as an exon deletion coding for a portion of the extracellular region of the PDGF in a primary brain tumor of glial origin (82). It is also well established that PDGF and its receptors are expressed in mesothelioma cells. The receptors display differential expression in these cells; Human malignant mesothelioma cell lines express PDGF whereas cultured normal mesothelial cells express predominantly PDGF (83). With regard to the ligand it is more a matter of all or nothing; while PDGF is not regularly expressed in mesenchymal tissues, PDGF-B mRNA and protein are often found in tumors derived from these

8. PDGF and its receptor in cancer metastasis tissues (84). In support of the in vitro findings, elevated expression of PDGF-B mRNA was also found in tumor specimens of patients with malignant mesothelioma (MM) (85). Yet PDGF expression is by no means limited to brain tumors and mesothelioma cells. PDGF was detected early on in human melanoma cell lines (86), breast cancer cells (87), and human osteosarcomas (88,89). Elevated levels of PDGF were also reported in patients with head and neck cancer (90). Expression of were found in PDGF and the neuroendocrine tumors of the digestive system (91). PDGF-A and genes may be preferentially turned on in epithelial and stromal prostate tumor cells (92). Induction of PDGF A and B chains and over-expression of their receptors was also demonstrated in human pancreatic cancer (93), and PDGF have been found in situ in AIDS-related Kaposi sarcoma (KS) cells, some of which also express PDGF-A and PDGF-B (94). In some tumors, the PDGF receptor protein is abnormally expressed, either as a slightly different protein, or as the normal protein expressed at abnormally high levels. For instance, researchers identified a developmentally regulated expression of two novel PDGF transcripts in human teratocarcinoma cells. These cells express a unique mRNA species, resulting from alternative promoter usage and alternative splicing of the human PDGF receptor gene and only do so while the cells are in an undifferentiated state (95). Neoplastic cytotrophoblasts are an example of cells which maintain an abnormally high level of PDGF expression, suggesting that a deregulated PDGF autostimulatory loop is involved in the genesis of human choriocarcinoma (96). Occasionally, the expression levels of PDGF ligand and/or receptors in the tumor

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correspond to the severity of the malignancy, such as in human soft tissue sarcomas, where a strong correlation was found between PDGF-B expression and increasing malignant tumor grade (97). Similarly, expression of PDGF-AB or PDGF-BB in blast cells play an important role in the pathogenesis of marrow fibrosis associated with accelerated and blastic phase of CML (98). PDGF expression can also be an important factor in assessing a cancer patient’s clinical outcome. Indeed, the concomitant expression of PDGF and PDGF in malignant ovarian tumor cells, suggestive of an autocrine mechanism, is related to progression of the tumor and has prognostic significance (99). Similarly in breast cancer, where PDGFBB is secreted by the carcinoma cells, it was found that those patients with elevated plasma levels of PDGF had a significantly lower response rate to chemotherapy as well as significantly shorter duration of survival (100). However unlike in ovarian cancer, in breast cancer a paracrine stimulation seems likely since the PDGF βreceptors found in malignant breast tissue are localized to the periepithelial stroma surrounding, but not on the carcinoma cells themselves (101). PDGF is also a prognostic indicator in lung cancer (102). Lung carcinoma cell lines are well known to express and secrete both PDGF-A and PDGF-B/sis genes (103) and also express functional PDGF-β receptors (104). Additionally, the in vivo expression of PDGF and PDGF proteins were found in malignant cells of primary human lung carcinomas, suggestive of autocrine self-stimulation and unregulated growth of lung cancer tumor cells (105). PDGF expression can also be a prognostic parameter for pulmonary adenocarcinoma (106). Recently, a

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prognostic value was found for the expression of PDGF-A in gastric carcinoma, with a shorter overall survival rate in patients who are PDGF-A positive (107).

3.2. PDGF function in cancer Over the years, it became increasingly clear that the expression of PDGF and its receptors in tumor cells was not merely incidental to but rather critical for the tumor cell development and proliferation. For instance, PDGF-AB and -BB could stimulate the growth of meningiomas, which was reduced by PDGF-neutralizing antibodies (108). So too many tumor cells, expressing both PDGF receptors and ligands, were shown to have chronically activated cell cycles. Exposure of these cells to suramin, an agent which interferes with ligand-receptor interactions at the cell surface, as well as PDGF-neutralizing antibodies, led to a marked reduction in DNA synthesis (109). A demonstration of the tumor promoting and maintaining property of a PDGF-B autocrine system was shown in human glioblastoma cells, which express PDGF and continuously secrete small amounts of PDGF-B/c-sis. When PDGF-B/v-sis was overexpressed in these cells, the cells greatly increased their proliferation rate (110). It was also demonstrated that such PDGF-dependent mitogenic processes could be oncogenic; Indeed, the v-sis oncogene, PDGF-B, could activate PDGF and intracellularly and initiate cellular transformation (111). Although it was originally thought that this was an exclusive ability of PDGF-BB, it was later shown that both PDGF B and A homodimers could transform murine fibroblasts depending on the genetic background of the cell (46).

Chapter 8 PDGF receptors were also implicated in cellular transformation in the absence of ligand. The bovine papillomavirus E5 protein constitutively activates and forms a stable complex with the PDGF ß-receptor It was shown that when the E5 protein and PDGF ß-receptor are coexpressed, the sustained proliferative signal results in fibroblast transformation (112). Known oncogenes were also demonstrated to be linked to this process. The v-fps oncogene was shown to constitutively activate PDGF receptors resulting in a sustained proliferative signal and fibroblast transformation (113). Similarly, oncogenic cbl enhanced the tyrosine kinase signaling of PDGF which becomes hyperphosphorylated and constitutively complexed with a number of SH2 domaincontaining proteins (114). It was also shown that the oncogenicity of the fusion protein in TEL/PDGF patients with chronic myelomonocytic leukemia (CMML) is likely due to its ability to induce a constitutively active PDGF ß-receptor, with a specific requirement for myc (115). Still, these results collectively were only correlative: that is, PDGF or oncogene- activated PDGF receptors had the ability to transform cells, and many tumors expressed PDGF and its receptors. However, direct evidence of PDGF involvement in actual neoplastic transformation of human tumors was demonstrated in a fusion protein found in the infiltrating skin cancer Dermatofibrosarcoma protuberans (DFSP). Expression of the fusion protein, consisting of the collagen type I alpha 1 (COLIA1) gene fused to the platelet-derived growth factor (PDGF) B-chain gene, which is processed to PDGF-BB, led to morphological transformation and increased growth rate of fibroblast cells, consistent with an autocrine mechanism of

8. PDGF and its receptor in cancer metastasis activating the endogenous PDGF receptor (116). Of course, some caution need be taken in interpreting all the in vitro results. For example, although it was found that sarcoma tumor cells are stimulated by PDGF-AB in vitro, such tumor-bearing mice, which expressed PDGF receptors, did not display any tumor growth in vivo in response to PDFG-AB. It is possible that in this case, as the authors suggest, the in vivo milieu or tumor growth pattern render the tumors less susceptible to exogenously administered PDGF-AB (117). On the other hand, many of the in vitro findings were later substantiated by in vivo experiments. Indeed, a murine retrovirus coding for the PDGF B-chain was shown to directly induce brain tumors in mice. These mice coexpressed PDGF B-chain mRNA, again suggesting an and autocrine mechanism of transformation as an early event in oncogenesis (118). Furthermore, when PDGF-B/v-sis was overexpressed in human glioblastoma cells, these ordinarily nontumorigenic cells developed tumors and metastasized in vivo (110). So too, the fibroblast transforming COLIA1/PDGFB-expressing cells described above also generated tumors after subcutaneous injection into nude mice (119). Another interesting function ascribed to PDGF in cancer is that as a chemoattractant. One group found that malignant mesothelioma cells express the PDGF and respond to PDGFBB in mediating integrin–dependent migration (120). PDGF has also been functionally implicated with unique roles in particular cancers. For example, the chromosomal translocation that leads to the oncogenic fusion of Ewing-sarcoma protein (EWS) to the Wilms tumour suppressor and transcriptional represser (WT1) in Desmoplastic small round-cell

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tumor (DSRT), results in the induction of PDGF-A. This in turn contributes to the characteristic reactive fibrosis associated with the tumor (121). One study even suggests that PDGF may be implicated in smoking-induced cervical cancer. Nicotine postpones the degradation and thus increases the accumulation of PDGF-AA in the cytoplasm and nucleus of cervical cancer SiHa cells, which may contribute to nicotine-induced carcinogenesis (122).

4. PDGF AND PDGF RECEPTORS IN METASTASIS The development of metastasis depends on complex molecular events governed by the tumor cells and the microenviroment. Tumor cells detach from the solid mass, degrade extracellular matrix, aggregate with platelets, escape from apoptosis in the circulation, adhere to endothelial cells, extravasate from the circulation at target sites, and induce angiogenesis. Interestingly, PDGF and PDGF receptors, expressed by tumor cells or the microenviroment were implicated in many of these processes. Attachment of cells to neighboring cells is mediated by intercellular adhesion, formed particularly by E-cadherin/catenin complexes (123). PGDF-ß receptor was shown to bind to the adhesion molecule integrin upon stimulation with the growth factor, with several signaling molecules found in the same complex, pointing to the involvement of PDGF in adhesion processes (60). PDGF was also shown to upregulate the expression of CD44, a hyaluronan binding molecule which was shown to be overexpressed in highly metastatic variants of the rat breast carcinoma MLT3 relative to low metastatic variants. Overexpression of CD44 variants was also demonstrated in advanced human breast carcinoma and in other tumors (124).

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Invasion of tumor cells into blood vessels and lymphatics is mainly regulated by proteolysis of extracellular matrix, mediated by metalloproteinases, heparanase and plasminogen activators (125). Stromelysin-1, a matrix metalloproteinase is induced by PDGF in Ras-Raf dependent and PKC dependent mechanisms. The PDGF-responsive element (SPRE) binds two types of AP1 molecules and a specific SPBP transcription factor (126). Thus PDGF also influences the invasion of tumor cells. After proteolysis of the extracellular matrix and basal membranes, the active locomotion of tumor cells is the next step in the metastatic cascade. Motility factors such as AMF, HGF/SF and MSF, secreted by the tumor cells or by normal cells, control directional migration. Paracrine stimulation of cell motility has been described for many growth factors, including PDGF. Chemotactic responses to PDGF were found in a large panel of melanoma cell lines and in some carcinoma cell lines. Yet, no correlation with metastasis was observed (127). Evading tumor cells, especially of epithelial and endothelial origin, may undergo anoikis in the circulation as a result of loss of integrin-mediated cellmatrix contact or cell-cell contact (65). In addition, circulating cells are more vulnerable to attack by cells of the immune system (128). It is well established that platelets often form aggregates with circulating tumor cells. Various functions were attributed to these aggregates, including protection from the immune system, protection from apoptosis by producing PDGF that serves as a survival factor, and enhancment of adhesion to vascular endothelium (129,130). Circulating tumor cells are retained in the capillaries of target organs through interaction with endothelial cells.

Chapter 8 Selectins, RGD binding integrins and mucins were shown to participate in these interactions (131-134). The Eicosanoid 12(S)-HETE (12(S)hydroperoxyeicosatetraenoic acid), a metabolite of integrin arachidonic acid, upregulates expression and promotes tumor cell adhesion to the vascular endothelium (135). It is unknown whether PDGF ßreceptor associated with the integrin promotes adhesion (60). After extravasation into the target organs, the nested cells can proliferate to form 1-2 avascular micrometastases. Micrometastases may stay dormant as long as the proliferation rate equals the rate of apoptosis (136), while the development of overt metastases requires the supply of oxygen and nutrients through newly formed blood vessels. Angiogenesis consists of sequential processes that involve proliferation of endothelial cells, migration and penetration in host stroma and expansion and morphogenesis that produce capillaries. These newly formed capillaries differ in permeability, stability and growth regulation compared to normal blood vessels (137). The angiogenic switch is mediated by a variety of promoters and suppressors released by the tumor cells and by host cells. Angiogenic factors released by the tumor cells stimulate the growth of endothelial cells and the invasion of host cells like macrophages, which in turn stimulate factors and receptors that promote, by paracrine mechanisms, the proliferation of tumor cells (138). The most potent endothelial growth factor, VEGF, is induced in tumor cells by PDGF, basic FGF, and IL-6. Activated Raf, Ras and Src as well as loss of suppressor genes like p53 correlate with increased VEGF secretion from tumor cells (139). Hypoxia is another mediator of VEGF secretion that acts through induction

8. PDGF and its receptor in cancer metastasis of a hypoxia inducible factor- l(HIF-1), that binds to the promoter of VEGF and increases its transcription (140). PDGF not only activates VEGF secretion from tumor cells, but also activates the secretion of VEGF from endothelial cells expressing PDGF receptor (141). Moreover, VEGF and PDGF-A levels correlate with microvascular density and metastasis in advanced breast cancer (142). PDGF, however, also mediates direct effects in angiogenesis, as a chemotactic factor to capillary endothelial cells that express PDGF ß-receptors (143). Thus PDGF and PDGF receptors have dual functions in tumor related angiogenesis. Many types of human tumors express PDGF and PDGF receptors. As described above, in some cases expression of PDGF or PDGF receptors or both is correlated with a high tumor grade or with poor survival. In murine tumors, we have shown that high metastatic clones of 3LL Lewis lung carcinoma and T10 sarcoma, but not low metastatic variants of the same (144). tumors, express PDGF These receptors were shown to be functionally intact and stimulated by PDGF-AA, PDGF-BB and by lung conditioned medium but not by liver or kidney conditioned medium (145). These data suggested that the interaction of PDGF-like factors in the lungs with metastatic tumor cells expressing PDGF receptors contributes through a paracrine mechanism to the metastatic phenotype. A causal relationship between PDGF receptor expression and metastasis was shown in the 3LL model, since transfection of low metastatic cells by PDGF resulted in metastatic phenotypes of tumor cells and transfection of high metastatic cells with a truncated kinase domain, negative dominant variant receptor resulted in low metastasizing cells (146). Thus PDGF and PDGF receptors play various

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roles in various stages of tumorigenesis and through the metastatic cascade.

5. CLINICAL STRATEGIES Perhaps the most straightforward means of demonstrating that PDGF plays an important role in the development and/or maintenance of cancer and metastasis is by blocking the function of PDGF in an attempt to reduce tumor growth or metastatic spread. Many of the same assays used to test if PDGF has functional relevance in a particular cancer may also have clinical, therapeutic applications. Since many studies suggested that tumor progression was PDGF dependent, it was hypothesized that tumor growth could be attenuated by therapeutic targeting directed against the PDGF ligand. One approach taken was the use of antisense RNA. In principle, antisense PDGF oligodeoxynucleotides can be taken up in vivo by tumor cells and selectively block gene expression. Indeed, the introduction of PDGF-A chain mRNAspecific antisense molecules into human malignant melanoma cell lines in vitro resulted in growth inhibition of the cells (147). Similarly, exposure of human glioma cell lines to antisense PDGF-B mRNA inhibited cell proliferation in a time- and dose-dependent manner (148). The use of antisense technology as a form of treatment is attractive since the antisense oligodeoxynucleotides are easy to produce, can be systemically administered or microinfused for specificity, and to date there are few reported adverse effects (149). Another strategy has been to inhibit PDGF activity using specific neutralizing antibodies. PDGF neutralizing antibodies suppressed the mitogenic effect of PDGFenriched wound fluid on DNA synthesis in glioma cells (150), reduced the growth of meningioma cultures (108), and reversed

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the transformed phenotype of glioblastoma cells (151). Some groups introduced mutants of PDGF ligands into cells to functionally block PDGF binding and PDGF-induced mitogenic activity. Expression of a nonreceptor-binding mutant of PDGF-A into csis-expressing fibroblast cells significantly inhibited the transformed phenotype of these cells. The investigators could show that the PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF-B chain by forming low-affinity heterodimers with wild-type PDGF chains (152). Concurrently, another group showed that dominant-negative mutants of the PDGF ligand could break the autocrine loop and revert the phenotype of BALB/c 3T3 cells transformed by the PDGF-A or PDGF-B (c-sis) gene and also revert the transformed phenotype of human astrocytoma cell lines (153). Applied in vivo, a receptor bindingdeficient mutant of the platelet-derived growth factor A-chain increased the survival of hamsters implanted intracerebrally with a highly invasive glioblastoma cell line (154). Another strategy undertaken to determine whether ligand stimulation of PDGF receptors contributes to tumor proliferation was to interfere with PDGF receptor signaling in an attempt to reduce tumor growth. This anti-receptor strategy is perhaps more promising than targeting PDGF ligands in terms of widespread therapy, since many tumors have constitutively activated PDGF receptors which are not dependent on PDGF ligand stimulation (see oncogenic signaling above). Since normal signaling through the PDGF receptors is activated by ligandinduced dimerization, the introduction of mutant receptors that are kinase deficient but still dimerization competent is a powerful strategy to study the importance

Chapter 8 of PDGF receptors in tumor development and progression. Indeed, a truncated PGDF-ß receptor was introduced into rat glioma cells and the PDGF-mediated signaling and subsequent cell growth were significantly reduced. Furthermore, the ability of cells expressing the truncated receptor to grow as xenografts in nude mice was significantly impaired (155). Other approaches have been taken to inhibit the proliferative and transforming effects of PDGF indirectly, without targeting the growth factor ligand itself. It has been suggested to targert c-myc in treating CMLL patients, since myc was demonstrated to be required for the oncogenic function of its PDGF ß-receptor fusion protein (see above) (115). Others could inhibit PDGF receptor signaling through chemical means, such as the perturbation of the PDGF receptor signaling by dibutyryl-cAMP in human astrocytoma cells (156). Still others experimented with cytostatic drugs such as suramin, a compound well known to inhibit the binding of growth factors to their receptors (157). Suramin could inhibit the growth of malignant fibrous histiocytomas, cells dependent on PDGF growth factors for their proliferation (158). Addition of suramin also inhibited the proliferation of human glioblastoma cells in culture (110) and nearly abolished PDGF-B-induced cell proliferation in human cerebral meningioma (159). Another compound, carboxymethyl benzylamide dextran (CMDB7), was demonstrated to block the binding of PDGF-BB to its receptor. CMDB7 could inhibit the growth of a highly tumorigenic human breast cancer cells in vitro, and its injection could suppress tumor growth of its xenografts in nude mice (160). Some of the more recent therapeutic strategies have focused on inhibiting the tyrosine kinase phosphorylation of PDGF

8. PDGF and its receptor in cancer metastasis receptors by various chemical agents including green tea compounds (catechins possessing the gallate group in their chemical structure) (161) and CGP57148B (119). A novel class of tyrosine kinase blockers represented by the tyrphostins AG1295 and AG1296 can inhibit selectively the platelet-derived growth factor (PDGF) receptor kinase and the PDGF-dependent DNA synthesis in certain cells. Treatment by AG1296 reverses the transformed phenotype of sis-transfected NIH 3T3 (162). Another small organic molecule, N-(4-(trifluoromethyl)phenyl)5methylisoxazole-4-carboxamide (SU101, leflunomide), inhibited PDGF-mediated signaling events, including receptor tyrosine phosphorylation, DNA synthesis, cell cycle progression, and cell proliferation in vitro and tumor growth in vivo (163). Perhaps SU101, with its limited in vivo results, is indeed one of the more promising therapies produced thus

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far, as its use in patients with advanced solid tumors has already entered phase I clinical trials (164).

6. CONCLUDING REMARKS Platelet derived growth factors and their receptors are involved in both development and disease and are expressed on many diverse tissues and tumors. Regulated PDGF receptor stimulation, by both autocrine and paracrine mechanisms, can induce normal cellular growth. However, receptor and ligand overexpression, or chronic ligandindependent receptor activation can trigger a signal transduction pathway which leads to cell transformation, tumor growth, and cancer metastasis. In these instances, attempts to interfere with PDGF receptor ligand-binding and/or signaling should provide new forms of cancer treatments and therapies.

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152. Vassbotn FS, Andersso M, Westermark B, Heldin CH, Ostman A. Reversion of autocrine transformation by a dominant negative plateletderived growth factor mutant. Mol Cell Biol 1993; 13: 4066-76 153. Shamah SM, Stiles CD, Guha A. Dominantnegative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells. Mol Cell Bio 1993; 13: 720312 154. Kaetzel DM, Reid JD, Pedigo N, Zimmer SG, Boghaert ER. A dominant-negative mutant of the platelet-derived growth factor A-chain increases survival of hamsters implanted intracerebrally with the highly invasive CxT24-neo3 glioblastoma cell. J Neurooncol 1998; 39: 33-46 155. Strawn LM, Mann E, Elliger SS, Chu LM, Germain LL, Niederfellner G, Ullrich A, Shawver LK. Inhibition of glioma cell growth by a truncated platelet-derived growth factor-beta receptor. J Biol Chem 1994; 269: 21215-22 156. Tsai CH, Hung LM, Chen JK. Perturbation of the platelet-derived growth factor receptor signaling by dibutyryl-cAMP in human astrocytoma cells. J Cell Physiol 1995; 164: 108 157. Stein CA, LaRocca RV, Thomas R, McAtee N, Myers CE. Suramin: an anticancer drug with a unique mechanism of action. J Clin Oncol 1989; 7: 499-508 158. Abdiu A, Larsson SE, Wasteson A, Walz TM. Suramin blocks growth-stimulatory effects of platelet-derived growth factor on malignant fibrous histiocytomas in vitro. Cancer Lett 1999; 146: 189-94 159. Schrell UM, Gauer S, Kiesewetter F, Bickel A, Hren J, Adams EF, Fahlbusch R. Inhibition of

Chapter 8 proliferation of human cerebral meningioma cells by suramin: effects on cell growth, cell cycle phases, extracellular growth factors, and PDGFBB autocrine growth loop. J Neurosurg 1995; 82: 600-7 160. Bagheri-Yarmand R, Kourbali Y, Morere JF, Jozefonvicz J, Crepin M. Inhibition of MCF-7ras tumor growth by carboxymethyl benzylamide dextran: blockage of the paracrine effect and receptor binding of transforming growth factor beta1 and platelet-derived growth factor-BB. Cell Growth Differ 1998; 9: 497-5044 161. Sachinidis A, Seul C, Seewald S, Ahn H, Ko Y, Vetter H. Green tea compounds inhibit tyrosine phosphorylation of PDGF beta-receptor and transformation of A172 human glioblastoma. FEBS Lett 2000; 471: 51-5 162. Kovalenko M, Gazit A, Bohmer A, Rorsman C, Ronnstrand L, Heldin CH, Waltenberger J, Bohmer FD, Levitzki A. Selective platelet-derived growth factor receptor kinase blockers reverse sistransformation. Cancer Res 1994; 54: 6106-14 163. Shawver LK, Schwartz DP, Mann E, Chen H, Tsai J, Chu L, Taylorson L, Longhi M, Meredith S, Germain L, Jacobs JS, Tang C, Ullrich A, Berens ME, Hersh E, McMahon G, Hirth KP, Powell TJ. Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5methylisoxazole-4-carboxamide. Clin Cancer Res 1997; 3: 1167-77 164. Eckhardt SG, Rizzo J, Sweeney KR, Cropp G, Baker SD, Kraynak MA, Kuhn JG, VillalonaCalero MA, Hammond L, Weiss G, Thurman A, Smith L, Drengler R, Eckardt JR, Moczygemba J, Hannah AL, Von Hoff DD, Rowinsky EK. Phase I and pharmacologic study of the tyrosine kinase inhibitor SU101 in patients with advanced solid tumors. J Clin Oncol 1999; 17: 1095-104

Chapter 9 RECEPTOR SIGNALING IN CANCER AND METASTASIS

Martin Oft

UCSF Cancer Center. University of California, San Francisco, CA 94143-0128,

USA

Key words:

Transforming growthfactor ß, epithelial-mesenchymal transition, EMT, invasion,

metastasis, angiogenesis.

Abstract:

Progression of epithelial tumor cells to the invasive and metastatic phenotype requires increased cellular plasticity of the tumor cell. This plasticity allows tumor cells to invade locally and into neighboring organs, penetrate blood vessels, and metastasize to distant organs. Metastasizing tumor cells show increased migratory capacity and altered matrix degradation and deposition. A partial loss of epithelial cell fate differentiation and a gain of mesenchymal characteristics has been described as epithelial-mesenchymal transition (EMT) of tumor cells. EMT allows epithelial derived tumor cells, similar to mesenchymal cells, to migrate on and invade in mesenchymal extra-cellular matrix (ECM) of the tumor stroma and into blood vessels, and metastasize. plays a complex role during the development of carcinoma formation and cancer progression. While untransformed cells and early steps of tumor formation are often inhibited in proliferation by later stages of tumor progression are positively regulated by Accordingly, elements of signal transduction are mutated in some tumor types while signaling is maintained and activated in the majority of other tumors. positively regulates many aspects of tumor progression such as extracellular matrix regulation, cell invasion, angiogenesis, immune suppression and, most notably, EMT of tumor cells. EMT induced by has been found to be essential for tumor invasion metastasis. Accordingly, clinical evidence reveals as a major risk factor for tumor progression. Here I review both signaling in tumor suppression as well as the increasing evidence linking to tumor progression, invasion and metastasis.

187 W.G. Jiang et al. (eds.), Growth Factors and their Receptors in Cancer Metastasis, 187–222. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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1. INTRODUCTION Transforming growth factor plays a central role in the signaling network that controls morphogenesis, growth and cell differentiation in multicellular organisms. The different members of this pleiotropic family of growth and differentiation factors seem to regulate many processes in human disease and, in particular, tumor development. Our understanding of how initiated signals are mediated has increased dramatically in the last fifteen years. Firstly, the prototype of this still constantly growing family, was identified and cloned (1). Secondly, the receptors for family receptors were identified by expression cloning from mammalian tissue culture (2-7). Thirdly, genetic screens in Drosophila melanogaster, Caenorhabditis elegans, and Xenopous laevis as well as studies of mammalian tumor have contributed a whole family of regulated transcription factors known as Smad proteins (8-11). These transcription factors in turn interact with many crucial components of the transcriptional machinery to either activate or inhibit transcription in a dependent manner (for review see (12-14). While the knowledge of Smad protein involvement in human cancer is still relatively small, the our knowledge in the potential role of in tumor development and progression has increased enormously in the last decade. Nevertheless, there is a broad field of partially conflicting being a theories, ranging from tumor suppressor to as a major tumor-promoting factor leading to tumor progression and metastasis. This review summarizes the scientific basis of both extreme viewpoints and what is known

Chapter 9 about the involvement of signaling in tumor development and metastasis. 2. THE BASICS SIGNALING 2.1.

OF

receptor signaling The family of growth factors consists of more than thirty members in humans alone (15, 16). They cluster in two major groups, the group composed of both the bone morphogenetic proteins (BMP) and growth and differentiation factors (GDFs), and the group formed by the Activins, and Nodals. The two groups differ in their use of transmembrane receptors and the subsequent activation of the transcriptional mediators (for recent reviews see (13, 14, 17)). The mechanism by which the ligand-induced signal is transduced, is, however, highly conserved (Figure 1 for It consists of two transmembrane receptors, the receptor type I and II, which both have serine threonine kinase activity. Upon ligand binding, both receptors form a complex with the ligand and the type II receptor phophorylates and thereby activates the type I receptor (3, 5, 18-20). The type I receptor subsequently phosphorylates and activates a transcription factor of the Smad family (21-25). The family of Smad transcription factors is divided into three classes: receptor regulated Smads, CoSmads and inhibitory Smads. Receptor regulated Smads (R-Smads) are directly phosphorylated by the ligand stimulated type I receptors, and mediate specificity between the different subgroups of ligand receptor complexes. BMP receptors activate the R-Smads 1,5, and 8, while receptors for Activin and phosphorylate Smad2 and Smad3.

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An exception to this rule is ALK1, which binds and transmits mediated signals, but activates Smad5 (26, 27). Phosphorylation of the R-Smad proteins by the receptor occurs on two serine residues (SSV/MS - motif) at the extreme C-terminus of the molecule (13, 14). The FYVE domain protein SARA (Smad anchor for receptor activation) mediates the interaction of Smad2 and Smad3 with the receptor by targeting Smad proteins to the correct subcellular localization and facilitating direct interaction between Smads and receptor. The FYVE domain binds phospholipid and localizes SARA to phospholipid-containing membrane compartments, presumably in the cell membrane (28). In the non-activated state, Smad proteins exist as monomers. Upon receptor-mediated phosphorylation Smad2 forms predominantly dimers and trimers, either homomeric or heteromeric complexes, with Smad4 (29). Smad4, the Co-smad, forms heteromeric complexes with all known R-Smads and is an induced essential mediator of transcription (14). Both the R-Smad homomer and the R-Smad - Co-Smad heteromer accumulate in the nucleus. The mechanism of nuclear translocation is at present unknown. In the absence of a clear nuclear import signal it is most likely that the Smads are transported to the nucleus by a co-shuttle mechanism with one of the many transcriptional partners found to form complexes with Smad proteins. Two inhibitory Smad proteins have been found to date, Smad6 and Smad7 (30-33). Smad6 inhibits the phosphorylation of Smad2 and Smad 1(32). In addition, Smad6 competes with Smad4 for binding to the receptoractivated Smad1 (34) and inhibits BMP1 signaling in Xenopous embryos receptor (34). Smad7 binds to the

complex similar to R-Smads, it lacks, however, the activated phosphorylation sites and is therefore not phosphorylated by and released as the R-Smads, but stays associated with the receptor, competitively inhibiting access of R-Smads to the receptor (33). In addition to direct Smad-induced activates a Map transcription, kinase cascade by activation of a MapKKK, activated kinase (TAK1). TAK1 activation contributes to induced transcription (35). In vitro TAK becomes activated by both the addition of or BMP. During embryonic development in the frog, however, active TAK induces a BMP-like phenotype, suggesting that TAK1 mainly contributes to BMP-induced transcription (36). Recently it has been found that induces phosphorylation and activation of of protein phosphatase the subunit 2A(37). Upon induced activation PP2A binds to its target molecules, such as the and induces their dephosphorylation (38). normally phosphorylates the ribosomal S6-protein, inducing the translation of mRNAs essential for Gl-S progression (39, 40). Inactivation of by competes with its activation by growth factors or transforming oncogenes via the PI3kinase pathway (38). 2.2.

Smad regulation in the nucleus The induced and Smadmediated transcription involves binding of the Mad homology domain 1 (MH1) of Smads to the Smad Binding Element (SBE, found in many promoters (41 -43). Although the affinity of the Smads or the isolated MH1 domain to a SBE element is relatively low (44), Smad proteins bind to

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a variety of transcriptional co-factors which might mediate high affinity binding to DNA or stabilize the DNA contact. The DNA recognition motifs of those transcriptional co-factors are often in close proximity or even partially overlapping with SBEs. These co-factors fall in several categories (see Table 1): DNA binding transcription factors, general transcription activator or histone acetylases, and transcriptional repressors attracting histone deacetylases (HDAC) to the promotor elements (14, 45). Many of these interacting molecules have an established role in cell transformation, tumorigenesis and metastasis. Others play an important role during embryonic development. The transcriptional modulation of Smad binding promoters is not only regulated by the presence and activation of the particular Smad proteins but also by the competition of different interacting proteins and their state of activity. I summarize in the following the most important Smad - cofactor interactions (see also Table 1). The molecular interaction observed so far link various signaling pathways to signaling. They might explain much of the variability observed in the response to in different cell types or in the response of transformed cells to compared to their non-transformed progenitors (46). It is too early, however, to judge the role of the individual transcriptional complexes in tumor progression and metastasis. Similarly, it is at present unclear if the plethora of interacting factors compete for Smad binding or if modifications of R-Smad determine the specificity in the selection of the binding partners. Transcription factors of the AP1 family like c-jun or c-fos, bind to Smad3 and to elements in -induced promoters like the plasminogen activator

191 inhibitor1 (PAI1) or the collagenase1 promoters. By stabilization of the DNA binding complex and attraction of general transcription factors they co-operate with activated SmadS to induce transcription in a dependent fashion (47, 48). Interaction of AP-1 transcription factors with signal transduction has been suggested from genetic studies in the fruitfly (49, 50). Most importantly, AP-1 activity has also been linked to regulation of cellular motility and to tumor invasion (51, 52). Activation of c-fos induces tumor progression from a polarized epithelial cell type to an invasive, mesenchymal cell type (53). The Glucocorticoid receptor (GR) induces differentiation of epithelial cells and transrepresses AP-1 family members (54). Similarly it is now reported that the GR binds the C-terminus of Smad3 and Smad4 and inhibits the transactivation function of both (55). Like and the Smad proteins, transcription factor family is intimately involved in the control of apoptosis as well as cellular interacts transformation(56, 57). directly with SmadS and induces transcription as exemplified on the junB promoter (58). This finding sheds a new and different light on earlier reports that induces the expression of the inhibitory Smad7 and thereby inhibits signaling (59). It is well established that the up-regulation of Smad7 in response to constitutes an autoregulatory feedback loop limiting -induced transcription (33). In the new scenario could be an essential co-factor for the and Smad3-mediated expression of Smad7. It is tempting to speculate that promoters involved in apoptosis are activation. co-stimulated by

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Forkhead transcription factors such as FAST1/2 interact with Smad-induced transcription and are essential coactivators on certain promoters, presumably during embryonic development (60-62). Smad proteins also form complexes with the general transcription activator CBP/p300 (63-66). Transcriptional repressors, like SnoN and Ski, silence transcription by interaction with Smad3, presumably by attraction of Histone deacetylases (HDAC) to the SBE (67-71). Upon stimulation, SnoN is transiently destabilized, but becomes upregulated within several hours of induction (68, 70). Transcriptional repressors such as SnoN, Ski or Evi1, have been characterized as oncogenes, mainly in hematopoietic tumors and in the transformation of hemotopoietic stem cells (72-75). The homeodomain protein TGIF binds

Smad3, recruits HDACs to Smad binding

promoter elements, and thereby represses

responsive promoters (76). 2.3. in cell cycle control Inhibition of cell cycle progression is one of the important functions of on cells in tissue culture and has many implications for tumorigenesis. Many primary cells are highly sensitive to cell cycle inhibition and apoptosis induction Most tumor cells, and also

by untransformed, immortalized cell lines

show a relatively modest response to the

cytokine, suggesting a selection for

modifications in the mechanisms of

growth inhibition. Since

the loss of cell cycle regulation is

presumably an early event in

carcinogenesis (77), I describe the main

mechanisms of

cell cycle

arrest and recommend recent

193 comprehensive reviews for further information (15, 16, 78). inhibits the Gl-S progression of epithelial, haematopoietic, endothelial, neural, and to a certain extent mesenchymal cells. The most common mechanism involves the transcriptional up-regulation of the cdk4/6 inhibitor (79). binds to cdk4, releasing cyclinD and the cdk inhibitor thus inactivating the cdk4/6 complex. in turn, binds to cdk2/cyclinE complexes and inhibits their activity toward the product of the retinoblastoma gene, RB (80). reduces the Alternatively expression of the tyrosine phosphatase cdc25A. Cdc25A induces dephosphorylation of cdk4/6 late in Gland is essential for G1-S progression induces the (81). Additionally translational suppression of cdk4 expression in a p53-dependent manner (82). Independent of Smad-induced transcription, induces cell cycle arrest in epithelial cells by activation of protein phosphatase 2A, and inhibition of (38). 3.

IN HUMAN TUMORS

3.1. overexpression is associated with tumor progression It has been noted since the cloning of the first family member, that many tumor cell lines express much than their higher levels of untransformed counterparts (1). These early results have been extensively confirmed by many researchers and in many different tumor types. Advanced cases of breast cancer, for example, express higher levels of expression is here a risk factor that is independent of age, stage, nodal status, or estrogen receptor status (83) High

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expression was linked to lymph node metastasis in advanced human breast cancer (84). In a rat prostate cancer model it has been observed that expression of is confined to the stromal compartment in early stages while later in tumor progression the tumor cells express high levels of especially in lymph node metastases (85). In human breast expression in the carcinomas, primary tumors was elevated, particularly at the site of invasion, the invasion front. Moreover lymph node metastases showed, in all cases studied, a much higher intraand extracellular level of (86). in colon cancer Expression of correlated with disease progression to metastasis. Patients with high levels of expression in the primary tumor had an 18-fold higher risk to experience recurrences than patients with low expression patterns (87). Interestingly several studies have used the serum levels of to determine clinical prognosis. In both hepatocellular carcinomas (HCC), and in colon cancer the serum levels of are significantly elevated. Moreover the levels correlate with clinical prognosis, in that higher levels are associated with the more progressed disease (88-90). In contrast, inflammatory diseases like chronic hepatitis or cirrhosis led to only a modest increase of serum levels. Similarly elevated serum levels of have been associated with invasive prostate cancer (91). Taken together most clinical and experimental studies find a clear association of high expression and activity levels of with an increased risk of tumor progression and metastasis. The important question arising from these association is about the target cell of the vast amount of activated present in the invasive tumors? Most cells of the tumor

Chapter 9 environment, such as fibroblasts, endothelial cells, and cells of the immune system, are affected by (see: on the tumor Paracrine effects of stroma). It might therefore be tempting to speculate that tumor cells, once having acquired complete resistance to are selected for their expression of Optimization of the stromal environment, the attraction of angiogenesis and repression of the immune response against the neoplasm would confer a significant growth advantage in vivo. 3.2. receptor mutation in human cancer inhibits cell cycle progression of epithelial cells in Gl . Resistance to signaling or mutation of the pathway should therefore confer a strong selective advantage to tumor cells. Consistent with this notion the type

II receptor

has been found to be

mutated in a subset of familial colon tumors, the hereditary non polyposis colon cancer entity (HNPCC) (92-94). Tumors arrise in those patients due to a DNA mismatch repair defect which causes microsatellite instability (MSI), nucleotide additions or deletions in simple repeated sequences, or microsatellites, which occur randomly throughout the genome. HNPCC tumors arise as a consequence of inherited mutations of the DNA mismatch repair (MMR) genes hMSH2 and hMLH1 (95). This type of genomic instability occurs also in sporadic cases of cancer due to defects in the same DNA mismatch repair genes. The TbRII contains a sequence of 10 consecutive adenine residues in the extracellular domain and is therefore a common target for mutations through MSI. Surprisingly, however, mutations in TbRII seem to be limited to colon cancers in HNPCC patients. The

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majority of HNPCC colon tumors and sporadic colon carcinomas occurring due to microsatellite instability (MSI) harbor mutations in the TbRII gene. Carcinomas of the endometrium, the lung, the pancreas, the small bowel, the ovary or the urinary tract do not seem to select for mutations in TbRII, even if they arise from the same DNA repair defect (96-98). Interestingly colon tumors with MSI have a significantly better prognosis compared to other colon tumor entities (99-102). The presence or absence microsatellite instability does not, however, affect the prognosis in other tumor types such as endometrial cancer (103) or is even associated with increased malignancy and shortened survival rate in breast cancer and small cell lung cancer (104-106). Regardless of the vast amount of other mutations occurring in MSI tumors, loss of the function appears to be a ratelimiting step in growth of colon tumors. Reintroduction of wildtype into MSI colon cancer or a gastric cancer cell line reduced the ability of those cancer cells to form tumors in nude mice, and restored sensitivity to the growth inhibitory effects of (107, 108). Upon the reintroduction of wildtype in HNPCC tumor cells, the cells became sensitive to growth arrest and apoptosis, they gained however migratory and invasive properties (109). Interestingly, one study found inactivating mutations in receptor type I in 6% of primary carcinomas of the breast, but showed a dramatically increased frequency of the mutations (41%) in lymph node metastases of the same tumors (110). The subsequently performed analysis of similar tumor tissues could not, however,

195 confirm the initial findings (111). TbRI mutations were, as well, infrequently observed in pancreatic and biliary adenocarcinomas (112). It is thus far unclear whether the loss of receptor function occurs early or late during tumor development. Further analysis will be necessary to investigate at which step in tumor progression mutations receptors confer a selective of advantage to the growing tumor. From the data described here it appears that the loss receptors gives colon cancer of cells a selective growth advantage early in tumorigenesis. This loss of signaling is, however, detrimental for further tumor progression and metastasis. 3.3. Smad mutations in human cancer signaling appears however to be more often targeted by mutations in the transcription factors, the Smad genes. Smad4, previously Dpc4 (deleted in pancreatic cancer) has been identified as a tumor suppressor gene on human chromosome 18q21(9). In this study Smad4 suffered bi-allelic loss in one third of all pancreatic tumors, either by homozygous deletions or heterozygous losses and point mutations of the second allele. Interestingly Smad4 mutations were also found to be associated with tumor progression in colon cancer. Whereas colon adenomas showed no mutations in Smad4, carcinomatous lesions harbored a mutation in Smad4 in 10% of the cases studied. Most strikingly, distant metastases had mutations in 30% of the cases (113). These data clearly link the loss of Smad4 to tumor progression and metastasis. Germline mutations in Smad4 seem to be responsible for a subset of juvenile polyposis syndromes, an autosomal dominant syndrome leading to hamartomatous polyps and gastrointestinal cancer (114). The

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attenuation of signaling induced by Smad4 appears to occur specifically in pancreatic tumors, gastric and less frequently in colon tumors. Smad4 has in many studies been shown to be an essential component of transcription (reviewed in (13, 16, 115). Surprisingly, however, tumors which show homozygous loss of Smad4 were found to be fully responsive to induced effects, such as transcription of target genes and cell cycle arrest in response to (116). This might be due either to the presence of a second homologue of Smad4, or the formation of transcriptionally active homoheterotrimers of Smad2 and Smad 3 (without the contribution of Smad4). There is indeed evidence for the existence of a Smad4 homologue termed in Xenopus (117). A human homologue has, however, not yet been found. The above evidences show that the deletion of Smad4 in pancreatic tumors confers a significant growth advantage to tumor cells in vivo. It might not, however, completely abrogate all signals. The loss of Smad4 might selectively repress the transcription of genes involved in growth inhibition by and thereby confer resistance to in vivo, potentially even without affecting the tumorpromoting effects of Other Smad genes are mutated much less frequently in human tumors. Initial results indicated several mutations in Smad2 in colorectal cancer (21). In addition, Smad2 is lost as part of the tumor suppressor locus on chromosome 18q21. The second allele however seems to remain non-mutated in most cases. Moreover, extensive search for mutations in other Smad proteins in different tumor types including gastric carcinomas has revealed no evidence for frequent

Chapter 9 mutations in either Smad2 or Smad3 (30, 118-121). 4. PARACRINE EFFECTS OF ON THE TUMOR STROMA 4.1.

induces angiogenesis The induction of the growth of new blood vessels to support the growing tumor is a crucial step in the progression toward the invasive and metastasizing tumor phenotype (122). Tumor-associated microvessels are often immature and highly fenestrated. As a consequence increased filtration of serum macromolecules in the tumor bed is observed, changing the growth factor supply and the composition of the ECM in the tumor stroma. Moreover, tumor cell shedding or active intravasation may be largely facilitated by the immature blood vessels. Upon sub-cutaneous injection purified induces the attraction of fibroblast and endothelial cells and a very rapid formation of granulation tissue (123). Dependent on its concentration, inhibits endothelial cell proliferation in the cell culture dish. However, under organotypic culture conditions, such as in three-dimensional matrigels induces the formation of vessel sprouting and acts thereby as an angiogenic factor (124). Interestingly, similar concentrations inhibit endothelial cell of proliferation in conventional twodimensional cultures, but fail to do so in the three dimensional gel systems (125). which is often found overexpressed in epithelial tumors, is a potent inducer of vessel sprouting in vitro, whereas shows a reduced potency in similar assays (126). Bovine aortic smooth muscle cell migration is also induced by but not by

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(126). In contrast and exert similar effects and both stimulate migration of endothelial and vascular smooth muscle cells (127). In three-dimensional fibrin gels induces three-dimensional tube formation of microvascular endothelial cells in vitro. This stimulation of in vitroangiogenesis is tightly dependent on the concentration of the cytokine, as at 1ng/ml functions as a migration and tube-inducing factor, whereas 5ng/ml of inhibit tube formation (128, 129). Similarly when is added to organotypic endothelial culture in collagen gels, it induces dose dependent invasion of the endothelial cells into the gel matrix (130). Potentially even more important for the process of intravasation and extravasation of metastasizing tumor cells, is the observation that induces the opening of endothelial fenestrae (131). The induction of leaking blood vessels often associated with tumors has been postulated to provide crucial components to the tumor stroma, such as fibrin and blood clots, and to facilitate tumor growth and metastatic spreading (122). Increased permeability of the endothelial vessel surface may increase the supply of growth factors and oxygen tumor, and to the facilitate migration of inflammatory as well as tumor cells through the fenestrated endothelium. Interestingly, the phenotype of knockout animals for and different receptor support an essential role for in angiogenesis. Mice deficient die early in embryogenesis in around 8.5 d.p.c. (132). The primary defects lie in the extra-embryonic tissue namely in the extra-embryonic mesoderm, but affect both the yolk sac vasculature and haemapoiesis (132, 133). Endothelial

197 cell differentiation appears most defective. The extraembryonic mesoderm defective in signaling fails to provide the structural requirements for proper vessel development (133). The phenotype of the -/- embryos is not fully penetrant, as is expected for a growth factor having several closely related family members with similar expression patterns. -/Consequently only 50% of the embryos die at this early stage due to the defect in the yolk sac angiogenesis (132). Canonically, binds to the activin receptor-like kinase 5 (ALK5) and the signal is transmitted by activation of the transcription factors Smad2 and Smad3 (13). It comes therefore as a surprise that mice deficient for activin receptor-like kinase 1 (ALK1) are in certain aspects of vascular development a phenocopy of -/- animals (26, 134). Mutations in ALK1 are also observed in patients with hereditary hemorrhagic telangiectasia (HHT), a disease commonly associated with type III receptors. mutations in the Oh and colleagues show in addition that ALK1 inhibits ALK5 signaling, and might thereby act as a repressor of induced Smad2/3 signaling (26). Some of the function of signaling on tumor progression has been associated with a direct or indirect link to directly angiogenesis. Firstly induces the expression of vascular endothelial growth factor (VEGF) mRNA (135). This induction is direct and not linked to the inhibition of cell proliferation since both cells stimulated in cell cycle progression by and cells respond similarly. inhibited by Like fibroblasts and epithelial cancer cells (135), osteoblastic cultures respond to by up-regulation of VEGF mRNA (136). Interestingly, VEGF has been

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linked to the induction of fenestrated endothelia with a high permeability (137). Similar results have been reported for (131). could therefore exert at least some of its effects on the induction of blood vessels by stimulating paracrine VEGF induction. Smad4 is an essential component of and BMP signaling both the pathways, and is frequently mutated in pancreatic carcinomas as mentioned earlier (9). Re-expression of Smad4 in pancreatic tumor cell lines does not, surprisingly, restore sensitivity, but does inhibit tumor growth. Analysis of differentially expressed genes in such pancreatic carcinomas re-expressing Smad4 led to the identification of Thrombospondin as a Smad4-induced gene, and VEGF as being negatively regulated by Smad4. The authors speculate that the reduced vascular density, observed in tumors induced by Smad4 re-expressing tumor cells, comes as a result of the failure of those tumor cells to induce an angiogenic switch (138). The opposite regulation of both target genes is observed in other cell types (135). It would be in response to interesting to know if re-expression of Smad4 in pancreatic tumor cells restores signaling via the canonical ALK5 signaling pathway or if it mediates BMP receptor or ALK1-induced responses. Expression of a dominant negative receptor in keratinocytes during chemically induced skin cancer was found to be accompanied by highly elevated (139). levels of endogenous Correspondingly VEGF, expression was found to be increased and Thrombospondin expression to be decreased, resulting in increased vascularization of the tumors. Interestingly, even if the tumors were, histologically, rather well differentiated,

Chapter 9 they gave rise to metastasis with an increased frequency (139). is Finally, expression of statistically associated with the expression of vascular endothelial growth factor and with in a poor prognosis in gastric carcinomas (140). induces the migration of endothelial cells, vessel sprouting and angiogenesis in vitro and in vivo. It will be of major interest if the signal is required in vivo at the level of the endothelial compartment or if, as suggested from targeted deletions in mice, the target cells are a mesenchymal stroma cells or smooth muscle cells. 4.2. modulates the immune response All isotypes exhibit a strong immunosuppressive effect. In fact was isolated as a T-cell suppressing factor secreted by malignant glioblastoma cells (141, 142). Further analysis revealed that influences the immune response toward cancer by inhibition of various target cells, such as cytotoxic Tlymphocytes (CTL) and natural killer (NK-) cells (143-146). inhibits Tcell proliferation in response to proliferative cytokines such as IL-1 but does not affect the response to IL-2 (147). In contrast, the IL-2 dependent generation of lymphokine activated killer cells (LAK) is competitively inhibited by (148). Here, inhibits proliferation as well as differentiation of LAK precursor cells, thus reducing the immune response toward tumors at several steps. Interestingly, LAK precursor cells secrete in response to IL-2 and express receptors, thus activating an inhibitory autocrine loop (148). It is important to note that suppresses the production of inflammatory

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cytokines such as Interferon Tumor necrosis factor and thereby interfering with the propagation and differentiation of effector cells. Correspondingly, inhibition of LAK cell development by is most effective in the initial stages of the process; cells which are already activated are only minimally inhibited (149). The inhibitory effect of on NK-cell activity has been shown to result from a dramatic decrease in the cytolytic activity of peripheral blood lymphocytes (PBL) or purified large granular lymphocytes (LGL). Interestingly, the cytolysis was diminished in response to Interferon gamma but not in response to Interleukin2 (IL-2) (150). Recently it has also been shown that antagonizes the inhibitory effects of activation of Jak1 and Stat1 induces the inhibitory Smad7 by direct binding to sequences in the Smad7 promoter (151, 152). Up-regulation of Smad7 leads to inhibition of the receptor-induced phosphorylation of Smad3 . has similar effects on B-cells. inhibits IL-2 dependent B-cell proliferation and factor-dependent immunoglobulin secretion(153). inhibits, for example, the transcription of the immunoglobulin Kapppa which is normally induced by bacterial LPS or Interferon This repression does not to the kappa affect the binding of promoter, indicating that does not antagonize Interferon by inhibiting NFactivation (154). Recently it has been shown that Interferon antagonizes signaling by activation of mediated transcription of the inhibitory Smad7 (59). Smad7 binds and inhibits receptor-induced Smad phosphorylation. Thus does antagonize signaling. In contrast it

199 was found that Smad3 and can jointly induce other promoters such as the junB promoter (58). would thereby induce certain aspects of signaling, might repress certain ILinduced, promoters by attracting or stabilizing transcriptional repressors, like Ski, SnoN or Evi1 to the transcription site, but co-activate other genes where the transcriptional repressor is replaced, for example, by the Histone acetylase. suppresses the T-cell and Bcell-mediated immune defense against act tumors. Interestingly, however, as a chemotactic factor on monocytes, neutrophils and eosinophils ((155-157). activates monocytes, induces them to express of interleukin 1 and interleukin 6 and functions as a chemo-attractant for monocytes (157). Interleukin 4 even in induces the expression of monocytes. This monocyte-derived has been shown to inhibit lymphocyte proliferation in cocultivation experiments and presumably during inflammatory processes and tumorigenesis in vivo (158). Monocytes, macrophages and granulocytes play, however, a crucial role in tumor development by providing metallo-proteases like MMP9 to the growing tumor. In a tumor progression model using transgenic animals expressing HPV 16 transforming proteins in the skin, MMP9 expression in bone morrow derived inflammatory cells is essential for tumor progression (159, 160). Thus regulates the immune response by several mechanisms. The specific anti-tumor response mediated by B-Lymphocytes and T-Lymphocytederived NK and LAK cells is abrogated in response to For granulocytes and macrophages, however, acts as a

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chemo-attractant, inducing the proteolytic modification of the stroma ECM and facilitating tumor invasion and metastasis. 4.3. induces osteolytic bone metastasis Several tumors types such as breast cancer often metastasize to the bone (161). Bone metastases frequently induce bone remodeling, bone demineralization and even hypercalcaemia due to the increased calcium released from the destructed bones. Several lines of evidence have associated the metastasisinduced bone destruction to the secretion of Parathyroid hormone related peptide (PTHRP) in the metastasis (162, 163) and with an increased infiltration by osteoclasts surrounding the cancer metastasis (164) plays an important role in bone formation (165). Expression of in osteoblasts of transgenic animals lead to osteoporosis in mice (166). In these mice the remodeling of trabecular bone is increased and the demineralization of trabecular bone is decreased. Experiments where of signaling was inhibited by overexpression of dominant negative in osteoblasts reveal receptors for the opposite phenotype, namely an increase in trabecular bone and increased mineralisation of the resulting bone (167). Interestingly, however, the osteoporosis is accompanied by an increased terminal differentiation of osteoprogenitor cells into osteoblasts (168). The increased bone resorption in transgenic animals is therefore due most likely to a directly or indirectly induced increase of osteoclast activity. Similar experiments targeting receptors to the dominant negative joint areas lead to the induction of chondrocyte differentiation and resulted in an osteoarthritis-like phenotype (169).

Chapter 9 Those evidences taken together it appears that modifies the activity and differentiation of both the osteoblast and osteoclast compartment, leading as a result to an increased demineralization and resorption of the bone. has been shown to increase Parathyroid hormone related peptide (PTHrP) at the level of mRNA transcription and protein secretion in tumors (170, 171). PTHrP expression is clinically associated with bone metastasis and hypercalcaemic episodes of patients with breast cancer (162). Moreover, when mice injected with human breast cancer cell lines metastasizing to the bone were treated with neutralizing antibodies for PTHrP, the amount of osteolytic bone destruction was significantly decreased (172). Recent experiments bring these two findings together and suggest that induced PTHrP levels play a critical role in the ability of breast cancers to form osteolytic metastases (173, 174). Expression of dominant negative receptors for in human breast cancer lines decreased the amount of osteolysis in mice and increased the survival time. These effects could be overcome by simultaneous overexpression of PTHrP in the same cells. These results suggest that for the destruction of bone matrix by breast tumor metastasis, PTHrP induction by is essential (174). The induced up-regulation of PTHrP seems to be regulated by Smad-induced transcription, since overexpression of a dominant negative Smad2 molecule releases PTHrP from control (173). Interestingly, induces IL-11 in osteoblastic cells, presumably leading to an increased resorption of bone matrix family (175). Other members of the such as various BMP isotypes are also found to be overexpressed in human tumors, suggesting that the development

9.

signaling and cancer metastasis

of osteoblastic bone metastasis might be directly influenced by alteration in signaling (176, 177). 4.4. induces cell migration and invasion The migration of tumor cells on and through extracellular matrices is intimately associated with their aggressiveness and metastasis formation in vivo. has been reported to regulate a number of processes associated with cell migration and invasion. In most cases, induces a more migratory phenotype of the target cell. Stimulation or inhibition of the migration of cells in vitro and in vivo can be modulated by a through peptide growth factor like several direct and indirect mechanisms. Firstly, the migratory machinery, namely the dynamics of the actin cytoskeleton, can be directly altered. Secondly, the adhesive properties of the target cell to the extracellular matrix (ECM) can be influenced. Thirdly, the factor can alter the expression of extracellular matrix components and substrates in the migrating or neighboring cell. In addition, the matrix can be indirectly altered by changing the proteolytic activity of the migrating cells. Finally, factors like family can under members of the certain circumstances, induce a change in the differentiation of the cell type, a process which in this review will be referred to as cell fate change. A change from an epithelial to mesenchymal cell type (epithelial-mesenchymal transition, EMT) changes the overall perception of the cell-cell adhesion and cell-substrate adhesion. induces migration of a variety of cells. Endothelial cells are induced to migrate and form tubular structures in response to (see in

201 angiogenesis). acts as a chemotactic factor on monocytes, neutrophils and eosinophils ((155-157) and see in immune modulation). During embryonic development, family members are involved in various morphogenetic movements. induces migration of primordial germ cells (178). The receptor has an essential function in axon guidance in the nervous system of the worm, Caenorhabditis elegans (179). Finally, the homologue in flies, dpp, is essential during cell migration in the process of dorsal closure (180). In genetic organisms it was clearly established that the concentration of family members at the signal receiving cell surface is crucial for the interpretation of the signal (181, 182). Different signal intensities can indeed induce opposing effects on the same target cells. It is therefore conceivable that the sometimes puzzling differences found in in vitro experiments with are explained by different signaling intensities. Keratinocyte migration is significantly stimulated by (183). As in many other experiments, the stimulation of migration is accompanied by the production of extracellular matrix components such as fibronectin in untransformed keratinocytes, allowing no definitive answer as to the mechanism of migration induction. Similarly, migration of primary tracheal epithelial cells is stimulated by when cells are plated on a supporting matrix (184). The migratory phenotype of epithelial cells in response to involves increased cellular actin dynamics and the upregulation of matrix components such as fibronectin. This migration induction involves a rearrangement of the actin cytoskeleton. Recently in has been shown

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that the rearrangement of the Actincytoskeleton is mediated by the canonical receptor ALK5 and correlates with activation of Smad2/3 (185). The actin reorganization associated with cell migration requires also signaling via the PISkinase pathway (186). It remains unclear which molecular mechanism is the in the actin retarget for organization. Keratinocyte migration in response to involves a quite complex modulation in the adhesiveness to and extracellular matrix substrates. Activin A induce the phosphorylation of focal adhesion components Paxillin and p130(cas), which are commonly associated with migration induction (187). Zambruno and colleagues showed that in keratinocytes the fibronectin receptor consisting of integrin alpha-5 beta 1, the vitronectin receptor alpha v-beta 5, and the collagen receptor alpha 2-beta 1, are strongly upregulated in response to The multifunctional integrin alpha 3 beta 1 heterodimer was found to be downregulated by Moreover, induces the de novo expression and surface exposure of the alpha v-beta 6 fibronectin receptor (188, 189). These changes in the adhesion molecule repertoire of primary keratinocytes are functionally related to the migration of skin keratinocytes over granulation tissue after skin wounding. Simultaneously, granulation tissue does provide a rich source for platelet derived in the wound. Inflammatory cells such as macrophages can produce significant Interestingly, this amounts of macrophage-derived can stimulate invasive properties of hepatoma cells in a transwell in vitro invasion assay (190). This observation might, however, be similarly important for in vivo tumor cell

Chapter 9 invasion, since growing tumors usually attract high amounts of infiltrating macrophages. Alternatively, myofibroblasts of the colon stroma might regulate the migration of the colonic epithelium in a paracrine manner by secreting (191). Many tumor cells, however, secrete significant amounts of in an autocrine manner and are therefore not necessarily dependent on stromal cells for supply (see below). Some of the migration induction observed in response to might also be caused by the increased synthesis of extracellular matrix components such as fibronectin or collagen type I (123, 192). In addition to the secretion of certain upregulates the matrix components, expression of the 92kD and the 72kD type IV collagenases. This up-regulation is epithelial cell-specific and is not observed in fibroblasts (193). The expression of both the Plasminogen activator inhibitor I (PAI-I) and Urokinase-like Plasminogen activator is elevated during endothelial tube formation (194). Taken thus together, regulates cell migration and local invasiveness at various levels. It is presumably the combination of several effectors which a potent inducer of cell make migration and invasion in vitro and in vivo. 5. MOUSE MODELS MODIFYING SIGNALING Analysis of transgenic or knockout mice in which members of the family or their signaling mediators have been modified greatly increased our in understanding of the role of development, tumorigenesis and tumor progression. Firstly, -/- mice have been developed by several labs (195, 196). A certain percentage of these mice

9.

signaling and cancer metastasis

develop normally to birth. They suffer however a massive wasting syndrome caused by a multifocal inflammatory disease, mainly due to lymphocyte infiltration in the lung and heart. Subsequently the massive, and apparently uncontrolled, auto-aggressive immune disorder leads to tissue necrosis and death of the animals. To inhibit the lymphocytemediated immune disorder, immunosuppressive drugs were applied to the -/- mice, or the deletion was genetically combined with mice harboring the severe combined immunodeficiency mutation (SCID-mice). In the -/- / SCID mice, the autoimmune disorder was completely blocked and the animals developed normally into adulthood, indicating the specificity of the -/- phenotype to lymphocytes (197). Loss of also causes a defect in embryonic development due to a failure of the proper development of the extraembryonic mesoderm. -/mice show a defect in yolk sac hematopoiesis, or in vasculogenesis, or both (132, 133, 198). This early embryonic lethality is highly dependent on the genetic background of individual mouse strains (199). receptor type II deficient animals show in all animals the more severe phenotype of the early embryonic lethality observed in -/mice. As mentioned before, mice deficient for the activin receptor-like kinase 1 (ALK1) phenocopy certain aspects of -/- animals (26, 134). Oh and colleagues have shown that binds ALK1 and inhibits certain aspects of signaling transmitted by (26). A more detailed analysis of the epithelia in -/- mice suggested there were disturbances in epithelial organ

203 differentiation similar to the phenotypes in Thrombospondin1-/- mice (200). /- mice display only a mild hyperplasia in the stomach epithelium associated with some inflammatory gastritis. Surprisingly, however they do not show an increased risk of malignancies (201). This might be explained by rescue of the deficiency by other family members such as and Indeed the targeted disruption of genes of show a partially and overlapping phenotypes to the knockouts. In addition, deficient mice show a variety of developmental defects such cardiac, lung, craniofacial, limb, spinal column, eye, inner ear and urogenital malformations (202). Expression of dominant negative receptor type II mutants of has been targeted to various epithelial tissues. When the expression of was localized to the basal layer of the skin, cell cycle progression of basal cells was dramatically enhanced and the in vitro responses of keratinocytes to was abolished (203). When similar transgenic mice were subjected to a chemical carcinogenesis protocol, the numbers of papillomas and carcinomas which formed were increased consistent with the idea of as a tumor suppressor (204). Recently, Go and levels in colleagues analyzed the chemically induced skin cancers expressing dominant negative receptors. Expression of endogenous and VEGF was found to be highly elevated in the well differentiated tumors. These tumors were highly vascularized and despite the low progression grade of the primary tumor, frequently gave rise to metastases (139). It would be interestingly to know if the expression of the dominant negative receptor did suppress

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signaling in the metastasizing tumor cells. Accordingly, overexpression of in the skin dramatically reduced the number of papillomas formed. However, under these experimental conditions the tumor progression and conversion from a benign papilloma to a squamous carcinoma stage were significantly increased (205). Moreover, most of the squamous carcinomas progressed to spindle cell carcinomas, the latter representing a highly invasive tumor type with a strongly increased risk for metastasis. might therefore have a bi-phasic function in carcinogenesis, inhibiting early tumor growth but stimulating tumor progression, especially in later stages. apparently triggers EMT converting rather epithelialdifferentiated, squamous carcinomas, to spindle cell tumors expressing mostly mesenchymal markers. The Smad proteins or transcriptional mediators of have been mutated in mice. Mutation of Smad4 abrogates gastrulation and leads to early embryonic death (206, 207). Interestingly, this phenotype was not due to a cell autonomous defect of the developing mesoderm, but due to a failure of the extra-embryonic tissue, the visceral endoderm, to support the developing embryo. When the extra-embryonic defect was rescued, gastrulation progressed normally, but the embryo showed severe defects in anterior structures (207). Further analysis of cells from Smad4 deficient cells revealed that Smad4 is required for the induction of some, but not all, responses (208). As mentioned before, Smad4 / Dpc4 is mutated in human pancreatic cancer and colon cancer. Smad4 heterozygous mice develop gastric and duodenal polyposis. Even if the remaining copy of Smad4 is

Chapter 9 lost in hyperplastic epithelium, these polyps apparently do not progress to is a mouse carcinomas (209). model for familial adenomatous polyposis (FAP). Elegant experiments using compound heterozygous mice for both Smad4 and showed a dramatic increase of polyp size and number in the compound heterozygote animals as compared to each of the parental strains. Moreover, the polyps showed increased proliferation and submucosal invasion (210). Smad2 deficient mice display many early embryonic defects including a failure to induce proper mesodermal and endodermal structures (206, 211, 212). So far, an increase in tumor formation in the heterozygous mice has not been reported. In contrast Smad3 deficient mice display no obvious defects during embryogenesis and are viable (213-216). The adult animals show a range of pathological phenotypes. The most severe, reported by one group, being invasive metastasizing adenocarcinomas of the colon (213), a phenotype not observed by other groups. Smad3 -/- tumors metastasize via the lymph drainage and have a relatively differentiated, mucous-secreting phenotype, even in the metastases formed. This comes as a surprise, since Smad3 mutations are not found in human tumors (118, 121, 217). In general Smad3-/- mice have an impaired immune response, an accelerated re-epithelialization of skin wounds and a reduced sensitivity of monocytes and splenocytes to (214-216). Since enhances the migration of keratinocytes during wound healing, the finding that deletion of Smad3 leads to the opposite phenotype offers several possible explanations. The simplest possibility is that Smad2 mediates the migratory effects on keratinocytes. Alternatively, other

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pathways involved in signaling could be responsible for migratory response. Thirdly the primary effect of the deletion of Smad3, accelerated wound healing could involve other cells, normally inhibited by such as inflammatory and stroma mesenchymal cells. Indeed direct effects of Smad3 deletion have been most prominently shown in monocytes, splenocytes and embryonic fibroblasts (214-216). Thus, it seems mutations of Smad4 or Smad3 enhance or induce tumor development in mouse models but only partially recapitulate the findings in human tumors. Our ability to analyze signaling pathway mutations in the in mouse tumor models has generally been hampered due to essential functions of most of its effectors during development of the organism. Conditional knock out strategies and conditional expression of mutant proteins will hopefully further clarify the role of in tumor metastasis. 6. EPITHELIAL-MESENCHYMAL TRANSITION IN TUMORS During tumor progression epithelial tumor cells subsequently lose many characteristics of differentiation . Tumor cells lose, for example, epithelial specific polarity and expression of epithelial adhesion molecules, lose or alter expression of epithelial intermediate filaments, and lose the ability to terminally differentiate. Pathologists have long described the histologically apparent loss of epithelial tissue differentiation as dedifferentiation. Poor differentiation of a tumor is associated with a higher risk for metastasis. Concomitantly many tumors acquire expression of mesenchymal proteins, such as the mesenchymal intermediate filament Vimentin, certain adhesion molecules for the extracellular

205 matrix, and proteases normally limited to mesenchymal cells, in the process which was described earlier as epithelialmesenchymal transition (EMT) (218, 219). Most epithelial derived tumors undergo, at later stages, a partial EMT as indicated by the co-expression of cytoskeletal proteins of both epithelial and mesenchymal cell lineages. Indeed most human tumor cell lines and many transformed epithelial cell lines express Vimentin, a feature not shared by normal cells in their parental organs. It has been proposed that this up-regulation of mesenchymal features increases the ability of tumor cells to attach to, and migrate through, the extracellular matrix component of the mesenchymal stroma. Many extracellular growth factors and signaling pathways have been shown to be capable of inducing EMT in tumor cells, depending on the experimental system or the tumor type. In the following pages, I summarize the dominant role family members play in EMT in tumors as well as during mesoderm induction, the equivalent to EMT which occurs during embryonic development. 6.1 Epithelial-mesenchymal transition and mesoderm induction The loss of epithelial cell differentiation or cell fate markers and the gain of mesenchymal characteristic observed in late stage tumor cells resembles in many aspects the process in the early mammalian embryo when the first mesodermal cells arise from the epithelial epiblast, later giving rise to the mesenchyme of the organism. Since this step in the embryonic development is closely mirrored in critical events during tumor progression such as invasion and metastasis, the next paragraph summarizes the role of family members in mesoderm induction.

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A group of ectodermal cells at the posterior side of the embryo loses its characteristic epithelial tissue architecture. These cells, presumptively the first mesodermal cells, delaminate from the epithelial ectoderm and form the so-called primitive streak, a structure of loosely associated migratory cells, which during streak formation undergo a cell fate and morphology change. This process is called mesoderm induction and takes place in the mouse embryo after day 6.5 d.p.c. (days post coitum). Even if the three dimensional structure of the early mammalian embryo differs quite significantly from those of other vertebrates such as the frog Xenopous laevis, and genetic organisms such as the fruitfly Drosophila melanogaster, the underlying molecular mechanisms are surprisingly conserved. In frog embryos two poles are distinguished, the animal pole giving rise to future ectoderm and the vegetal pole forming the presumptive endoderm, with the poles being separated by the mesodermal layer. This clear spatial separation has researchers allowed early on to search for signals inducing mesodermal cell fates in isolated ectodermal poles. These mesoderm inducing factors or MIF’s do upregulate mesenchymal marker genes such as goosecoid or Smooth Muscle Actin when injected in or added to the isolated ectodermal half of the embryo. It was noted very early on that FGF or and activin family members such as function as mesoderm inducing factors in these assays (220-223). Later analysis of the expression patterns of various family members excluded Activin or as candidate factors for inducing mesoderm in vivo. Moreover, neither have been found to be Activin nor essential for mesoderm induction in mice (196, 224). The subfamily of Nodal

Chapter 9 and Nodal related genes seem, however, to be both sufficient and essential to induce mesodermal cell fates in animal cups and for formation of the primitive streak in mice (225, 226). It seems likely therefore that Nodals induce the ectodermal to mesodermal cell fate decision during embryogenesis. Like Activins, Nodals activate the Activin type II Receptors, ActRIIA and ActRIIB and Activin type I Receptors ActRIB (13, 227). Consistently mouse embryos deficient in ActRIB or embryos with combined deficiencies for both Activin type II receptors ActRIIA and ActRIIB lack the primitive streak and mesodermal structures (228, 229). These results clearly establish that signals derived from the Activin receptor are sufficient and necessary for mesoderm induction. Nodal or nodal-related factors seem to induce receptor activation in the embryo. Activin and Nodals bind specifically to the Activin receptor complex and do not activate other members of the receptor family. Both Activin and receptors share, however, the same transcriptional machinery. Both Activin and activate Smad2 and Smad3 by receptor-mediated phosphorylation and utilize Smad4 to transduce the signal to the nucleus (13). Interestingly, mouse embryos deficient for Smad2 fail to form the primitive streak and embryonic mesoderm (206, 211,212). However, other family members seem to contribute essential components to the activation of mesoderm inducing processes in vivo. Embryos deficient for either BMPRIA / ALK3 or BMPRII are not capable of forming mesodermal tissues (230, 231). Similarly, embryos deficient in BMP4 do not form mesoderm efficiently (232). In these embryos, the primary ectodermal structure, the epiblast, forms but shows

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signaling and cancer metastasis

distinct differentiation defects. Embryonic

ectodermal cell proliferation is reduced

before morphological abnormalities are

prominent. Notably, BMP-induced signals

have also been reported to be essential for

ventral mesodermal cell fates in isolated

ectodermal tissue from Xenopous animal

caps (233-235).

6.2. induces epithelial-

mesenchymal transition in tumor cells

Several extracellular growth factors

and the activation of a number of

signaling molecules have been associated

with EMT. In the following I review our

knowledge of the induction of EMT in

tumor cells, particularly in response to

Activation of fos-ER, a fusion protein

of c-fos to the hormone binding domain of

the human estrogen receptor, by hormone

addition allows for a rapid conditional

induction of a transcriptional response.

Moreover, upon hormone withdrawal

transcriptional activity ceases

immediately. Activation of such a

conditional fos transcription factor in

differentiated, polarized, epithelial cells

induces the loss of epithelial polarization,

when they are activated for several hours

(53). Interestingly, activation of the same

fos-ER molecule for two days is sufficient

to induce the complete loss of epithelial

differentiation and the gain of

mesenchymal features. These strikingly

different effects which result from

differences or gradations of signal

strength and duration mirror the different

effects which developmentally active

ligands have, depending upon their

concentration at the target cell(236).

Interestingly, once the cells undergo

EMT, the mesenchymal cell fate is

stabilized, even if the initiating fos-ER is

inactivated(53). This definitive cell fate

change mirrors EMT during mesoderm

207 induction. Fos-ER-induced EMT, interestingly, involves the nuclear localization and transcriptional activation of (237). Since Smad2/3 interact with c-fos (48), it will be very interesting to elucidate the role of mediated signaling on fos-ER-induced EMT. Activation of a conditional c-junER in the same experimental setting induces loss of epithelial polarity, but is not, however, sufficient for EMT (238). Purified and basic fibroblast growth factor (bFGF) cooperate when added to Xenopous animal caps to induce mesenchymal cell fates in those ectodermal cells (222). Both factors are clearly not identical to the gastrulation inducing factors in the early embryo. However, the signals activated by FGF seem to be sufficient to trigger and EMT in a manner similar to the endogenous signals. The signaling components activated by FGF and are therefore likely to be identical with the endogenous signals inducing mesoderm in the embryo. According to this notion FGF has been found to trigger a cell shape change in a bladder carcinoma cell model (NBT-II ) (239, 240). Here, as probably in most examples of tumor progression, the epithelial-mesenchymal transition is not complete. Even after FGF stimulation the tumor cell remains clearly epithelial, as judged by the cytokeratin-containing cytoskeleton. Similarly, E-cadherin, an epithelial adhesion molecule, is still expressed after the transition. This partial cell fate transition of the NBT-II bladder carcinoma has been observed in response to a variety of extracellular factors, such as FGFs, Scatter Factor / Hepatocyte growth factor (HGF) and ligands of the epidermal growth factor receptor (EGFR) (240). These findings indicate that the high specificity observed during

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embryonic development is presumably lost in tumor progression and that activation of a similar receptor tyrosine kinase can substitute for the embryonically required FGF receptor. It is, however, unclear if in this system the cooperation of FGF and is no longer required or if, alternatively, the present is produced by the tumor cell or derived from the fetal calf serum in the media. on the other hand has been found to induce several mesenchymal characteristics in an untransformed mouse mammary epithelial cell line (241). Here, the gain of mesenchymal characteristics involves re-organization of the actin cytoskeleton and up-regulation of mesenchymal genes like fibronectin. It has been further shown that this process receptor ALK-5 and is involves the accompanied by phosphorylation of Smad proteins (185). This untransformed cell line does not, however, undergo a complete cell fate change; the cytoskeleton remains cytokeratindominated and the observed cell shape change could result from the induced migratory response. Moreover it remains unclear if the increased plasticity observed indeed increases the tumorigenic and invasive capacity of tumor cells. When Ras transformed mouse mammary epithelial cells are exposed to they have been reported to undergo epithelial-mesenchymal transition (46). In this scenario induction of the epithelial polarized, but Ha-v-as transformed cells with switches them to a mesenchymal phenotype which includes up-regulation of various mesenchymal marker genes, such as Collagen I, Fibronectin and Vimentin intermediate filaments. During this transition, the tumor cells gain resistance to the cell

Chapter 9 cycle inhibitory activity of Moreover, cells now in a spindle shaped mesenchymal status express, similar to the majority of human tumor cells, high amounts of In this model system, is shown to serve as an autocrine factor stabilizing the tumor cells in a spindle shaped mesenchymal cell fate. Once the autocrine activation is disrupted, the tumor cells revert to the epithelial polarized phenotype (46). The phenotypic changes are fully reversible and dependent on the presence of extracellular and signaling. Cells in the epithelial fate are non-invasive in vitro. In contrast, the same cells switched to the mesenchymal fate are aggressively invasive. In vivo, during tumor growth, initial doses of host stroma-derived induce the ras transformed epithelial cells to acquire an epithelial-mesenchymal cell expression fate transition, enabling in the tumor cells. Disruption of signaling during tumor growth inhibits tumor growth and inhibits local invasive tumor cell spreading in vivo, and in invasion assays in vitro (109). Similar results have been obtained with human tumor cells of different tissue origin. Exogenous and autocrine allowed most tumor cell lines to be invasive. The cells lost this capacity when signaling was disrupted. Interestingly, due to DNA mismatch repair defects (MIS), human colon carcinoma cells from HNPCC patients (see above) harbor a mutation in and therefore express non functional receptors. Yet, when compared to other colon tumors, MIS+ colon tumors show a comparatively indolent clinical outcome and a relative good prognosis associated with a low frequency of metastasis formation (101).

9.

signaling and cancer metastasis

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Mouse colon cancer models for metastasis expressing functional receptors, such as CT26, cells have been used to analyse a relationship between the formation of distant metastases and signaling was signaling (109). genetically inhibited in these invasive colon cancer cells by expression of a kinase-inactivated receptor. Surprisingly, it turned out that locally invasive growth of tumors was correlated with activity of transcription. Various clones expressing increasing levels of the dominant interfering showed decreasing reporter responsiveness of a construct. The responsiveness of the reporter construct correlated with the ability of these tumor cells to grow as tumors upon subcutaneous injection. Clones in which the transcription was completely abolished did not form tumors in nude mice. Most interestingly, however, in contrast to the parental cell line which metastasized to the lung from the primary subcutaneous injection site, all cells expressing lost this dominant interfering capability. Furthermore, using in vivo cell tracking experiments it became clear that signaling was necessary for both tumor and metastasis formation at the single cell level (109). When co-injected with non-modified tumor cells, cells signaling were found to mutant for be not capable of contributing to tumors or metastases formed by their parental cells. The tumor environment under these experimental conditions was stimulated by the tumor forming parental cells, though providing potential paracrine stimulation of angiogenesis and tumor stroma by Cells expressing the dominant interfering TbRII are therefore inhibited

Chapter 9 due to the defect in signaling in the tumor cell. Similar experiments using activated and dominant negative forms of Smad2 indicated that Smad2 mediates Similar to its EMT in response to function during gastrulation, Smad2, induces a cell fate transition in transformed tumor cells. Furthermore, dependent on its concentration and the coexpression of dominant oncogenes Smad2 induces migration and EMT. Moreover, only high level of Smad2 stimulation allow tumor cells to metastasize (242). During embryonic development, Smad2 is presumably activated in response to Nodal family members to induce mesoderm induction (227). In contrast, receptors are both sufficient and necessary to induce the similar process during tumor progression.

7. CONCLUSION During tumor development, tumor progression, and metastasis, signaling has pleiotropic effects on the tumor cell and the tumor stroma. induces growth inhibition of early tumor stages and infiltrating immune cells. Later, in most cases, metastasizing tumor cells are resistant to the growth inhibition and actually secrete by themselves. But for a few exceptions, this does not result from resistance to mutations in the receptor complex or its signaling mediators. The secreted now exerts various functions associated with tumor cell invasion and metastasis (see Figure 2). inhibits the immune response to growing tumors and metastasis, it stimulates tumor angiogenesis by several different mechanisms and acts as a chemoattractant inducing the migration of macrophages, stromal cells and the tumor

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signaling and cancer metastasis

cell itself. Importantly, however, receptor activation and activation of Smad2 induces signaling events in the transformed cell similar to embryonic cell fate determinations. receptor signaling induces an epithelialmesenchymal transition of epithelial tumor cells. Once having acquired expression of mesenchymal surface proteins and cytoskeletal intermediate filaments, tumor cells become locally highly invasive. Their ability to invade blood vessels and penetrate the

211 endothelium at the site of the distant metastasis is highly dependent on these cells having an active signaling pathway. Increase in signaling must therefore be regarded as one of the major routes for tumor metastasis. Acknowledgements I am indebted to my colleagues Margaret McKinnon and Byron Hann for discussion and many thoughtful comments on the manuscript.

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Chapter 10 VEGF-C/VEGFRS AND CANCER METASTASIS

1

Yutaka Yonemura 1, Yoshio Endou2, Takuma Sasaki2, Kazuo Sugiyama3, Tetumouri Yamashima5, Taina Partaneri1 , Kari Alitalo4

Second Department of Surgery, School of Medicine, Kanazawa Unversity, Takara-Machi 13-1, Kanazawa 920 2 Experimental Therapeutics Cancer Research Institute, Kanazawa University, Takara-Machi 13-1, Kanazawa 920 3 Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuoo-Ku, Tokyo 104 4 Molecular/Cancer Biology Laboratory, Haatman Institute, University of Helsinki, PL 21, 00014 Helsinki, Finland 5 Department of Neurosurgery, School of Medicine, Kanazawa Unversity, TakaraMachi 13-1, Kanazawa 920

Key words: VEGF, VEGF-C, VEGF-R, gastric cancer, KDR, lymphangiogenesis. Abstract:

Vascular endothelial growth factor C (VEGF-C) is the only factor known causing lymphangiogenesis. We report herein the review of recent experimental studies on VEGF family and their receptors and the molecular mechanisms of the lymphangiogenesis in cancer. According to our study, VEGF-C is a potent stimulator in not only the angiogenesis but also the lymphangiogenesis on the chick chorioallantoic membrane. In the clinical specimens from gastric cancer, there is an intimate relationship between the VEGF receptor-3/VEGF-C tissue status and lymphagiogenesis. RT-PCR and immunohistological examinations demonstrated that VEGF-C was mainly produced from cancer cells and that VEGFR-3 expression was restricted in the endothelial cells of lymphatic vessels. VEGF-C and VEGFR-3 mRNA expression were positively correlated in primary gastric cancers and the number of VEGFR-3 positive lymphatic vessels in VEGF-C mRNA positive tumour was significantly larger than that in VEGF-C negative tumours. The number of such vessels in tumour stroma was closely related to the grade of lymphatic invasion of gastric cancer. Accordingly, we conclude that VEGF-C may induce the lymphatic neogenesis in the stroma of primary gastric cancer. In these circumstances, cancer cells can easily intravasate into the lymphatic vessels, because of the increase of the contact point of cancer cells with lymphatic vessels.

223 W.G. Jiang et al. (eds.), Growth Factors and their Receptors in Cancer Metastasis, 223–239. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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1. ANGIOGENESIS AND LYMPHANGIOGENESIS Vascular endothelial cells are derived from precursor cells known as hemangioblasts. During embryogenesis, the precursor cells are first detected in the AGM region (aorto-gonad-mesonephros) during embryonic day E8 to E9 (1), and have great roles in the angiogenesis of not only embryo but also adult. Hemangioblasts differentiate into endothelial cells and hematopoietic progenitor cells, and the differentiation depends on the molecules expressed on the hemangioblasts. Vasculoendothelial growth factor (VEGF) is the most important cytokine of the differentiation from hemangioblasts to endothelial cells (2), and CD34 and angiopoietin (Ang) receptor (Tie) are expressed on the endothelial cells (3). In contrast, α4integrin is specifically detected from hematopoietic cells, and Flk-1 (fetal liver kinase-1, KDR/VEGFR-2) is expressed in the hematopoietic cells and endothelial cells (4). Actually, flk-1 deficient mouse died at day E9 due to the disturbance of development of both endothelial cells and hematopoietic cells. During development, the expression of the specific receptors and their ligands for the proliferation of endothelial cells is switched on sequentially at approximately 24 hours intervals. Usually, endothelial cells express more than one receptor and react to several stimuli at the same time. As a result, vascularization in the embryo is completed by the complex interaction of paracrine or autocrine loops, consisting of growth factors and their receptors. In the adult, all the receptors and growth factors related to angiogenesis are downregulated, but are upregulated during some physiological and pathological processes (5). Recently, Asahara et al have proved that human peripheral blood contains

Chapter 10 endothelial cell (EC) progenitors or angioblasts, which can in vitro differentiate into endothelial cells (6). In adult tissue, these EC progenitor cells are speculated to be incorporated into sites of active angiogenesis, like inflammation or cancer, and to become a new vascular network. In spite of the enthusiastic studies for angiogenesis and vasculogenesis, the research on the mechanisms of the formation of lymphatics has lagged. At the beginning of this century, two theories concerning the origin of the lymphatic system have been postulated. Sabin proposed a theory of venous origin with centrifugal spread (7), and Huntington (8) and Kampmeier (9) postulated a theory of mesenchymal origin with centripetal spread. Sabin reported that lymphatic vessels sprout from large central veins called anterior cardinal veins in the neck and inferior vena cava during the embryonic period (Figure-1), and these premordial lymph sacs spread gradually into the periphery of the human body by repeat branching and proliferation of endothelial cells. However, precise mechanisms of the proliferation of lymphatic vessels remains unclear. The reasons were the difficulty for the establishment of primary culture of lymphatic endothelial cells, and the lack of the histological determination method to confirm the vessels as the lymphatic vessels, because there had been no reliable molecular marker for lymphatic endothelial cells. Recently, however, Alitalo et al clarified the role of VEGF-C as the lymphangiogenesis factor (10). In addition, they found that VEGF-C is a ligand for VEGFR-2 and VEGFR-3 (10). We herein will present the recent results about the mechanisms of the angiogenesis and lymphangiogenesis in cancer.

10. VEGF and VEGFR in cancer metastasis 2. VEGF-C In 1983, VEGF was discovered as a vascular permeability factor (VPF) from media of tumour cell lines by Senger et al (11), and then, Ferrara et al named the molecule as vascular endothelial growth factor (VEGF), which can induce mitogenic activity selectively for endothelial cells (12). Since the identification of VEGF, four VEGF related genes have been characterized, including the placenta growth factor (PIGF), VEGF-B, VEGF-C and VEGF-D.

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In 1996, VEGF-C was identified as a ligand for VEGFR-2 (KDR) and VEGFR3 (Flt-4) by Joukov et al (13). VEGF-C gene has 7 exons (14), and the core growth factor domain is encoded by exon 3 and 4, a feature conserved in all the members of the VEGF family (15). VEGF-C mRNA transcription is upregulated by PDGF, EGF, and IL-1 (16), but not by hypoxia which induce VEGF mRNA transcription.

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During mouse embryogenesis, VEGFC mRNA detected by in situ hybridization technique is first found in cephalic mesenchyme at day E8.5 and at E12.5 the signal increases around metanephros and jugular area, where primitive lymphatic vessels sprout from venous-sak like structures (17) (Figure-1). In addition, mRNA expression of VEGFR-3, which is a receptor of VEGF-C was detected on the developing venous sacs. These findings strongly suggest the existence of paracrine loop between VEGF-C and VEGFR-3 (17), and the paracrine may have a role in the lymphangionenesis of embryo. In adult tissue, VEGF-C mRNA expression decreased below the detection level except in lung, heart, liver and kidney (18). However, VEGF-C mRNA expression is upregulated in the pathological condition, like inflammation and cancer (19,20,21). VEGF-C is translated from mRNA as a 61 kDa prepropeptide with signal peptide (13). Then, the stepwise proteolytic processing of VEGF-C generated several forms with increased activity against VEGFR-3. Finally, the homodimer of the fully processed 21 kDa VEGF-C could activate VEGFR-2 and VEGFR-3. Mature VEGF-C (21 kDa) increased vascular permeability, as well as the migration and proliferation of endothelial cells. Transgenic mice expressing VEGF-C under a basal epidermal keratin promotor developed a hyperplastic lymphatic vessel network in the skin (10). Oh et al reported that recombinant mature VEGF-C induced a selective growth of lymphatic vessels in 13 day chick chorioallantoic membrane (23). Proteolytically processed VEGF-C was also capable of stimulating VEGFR-2 and had weak angiogenic activity (13). We studied the angiogenic and lymphangiogenic ability by VEGF, VEGF-C and mutant VEGF-C on

Chapter 10 differentiated avian chorioallantoic membrane (CAM). Mutant VEGF-C in was replaced by a Ser which residue bound and activated VEGFR-3 but neither bound VEGFR-2 nor activated its autophosphorylation or downstream signaling to the ERK/MAPK pathway (32). Accordingly, mutant VEGF-C may provide a valuable tool for the analysis of VEGF-C effects mediated via VEGFR-3 (Fig 2). Figure 3 shows the angiogenic activity on the third day after implantatation of a methylcellulose disk impregnated with VEGF (l-5µg/egg), VEGF-C (5µg/egg) and mutant VEGF-C (5µg/egg). Neovascularization was prominent in VEGF and VEGF-C- treated eggs, and the angiogenic effect increased in a dose dependent manner (Fig 3). However, the neovascularization was not found in the mutant VEGF-C-treated CAM. Angiogenic index of VEGF was three-times greater than that of VEGF-C, but mutant VEGF-C did not show any angiogenic activity (Fig 4). Histological examination demonstrated many microblood vessels in VEGF and VEGFC treated CAM. In contrast, neogenesis of microlymphatic vessels was found in the CAM treated with VEGF-C and mutant VEGF-C. In ultrathin sections of mutant VEGF-C treated CAM, immature lymphatic vessels with thin endothelial lining, numerous lamellinodia, and anchoring filaments were detected (Fig 5). As shown in Figure-2, VEGF family have the dual function by the activation of two different receptors. VEGF-C activates both VEGFR-2 and VEGFR-3, but the receptor binding affinity of recombinant VEGF-C for VEGFR-3 is approximately threefold higher than for VEGFR-2. Accordingly, VEGF-C is angiogenic and lymphangiogenic growth factor, and mutant VEGF-C has lymphoangiogenic activity.

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3. RECEPTOER FOR VEGF-C (VEGFR-2 AND VEGFR-3) VEGFR-2 was cloned by Millauer et al (23), and identified as the tyrosin kinase receptor to bind VEGF and VEGF-C (13). VEGFR-2 mRNA is detected in a high level from E7.0 in the embryonic mesoderm which is later forming the but the expression decreased in low levels in the adult tissue where angiogenesis has ceased (25). VEGFR-2 was not expressed after systemic hypoxia in mice (26), but Moriyama et al reported an increased expression of VEGFR-2 in the stroma of human oral cancer (27). VEGFR-3 was isolated from a human placental cDNA library, and differs from the other two VEGFR-1 and VEGFR-2 (Figure-1). The cleavage of precursor protein (190/195 kDa) occurs in the extracellular domain and results in two polypeptide chains (75+120/125 kDa), and these two polypeptides bind with disulfide bonds (28) (Figure-1). During the early embryogenesis in mouse, VEGFR-3 mRNA was found in the angioblasts (29). Since VEGFR-3 knock out mice showed disorganization of endothelial cells and severely retardation of the growth of major vessels, VEGFR-3 is speculated to have a role in the early development of the cardiovascular system before the formation of lymph system30). During the later embryogenesis, VEGFR3 was detected in the lymphatic endothelium on the lymph sacs derived from anterior cardinal vein (Figure-1) (30). VEGFR-3 also has a big role in the lymphangiogenesis of tumours (20,21,27). 4. VEGF-C/VEGFRS EXPRESSION IN CANCER The adult vasculature is quiescent but become activated to form new capillaries during tumourigenesis. Tumours in the

Chapter 10 prevascular phase is less than a few cubic millimeters, and can not grow up to larger than several millimeter in diameter without angiogenesis (33). Angiogenesis and lymphangiogenesis are also essential for tumour metastasis, because tumours with vascular or lymphatic neogenesis in their stroma could be predisposed to metastatic spread via blood vessels and lymphatics. Until now, some clinical reports have indicated a significant correlation between the incidence of metastasis and angiogenesis, and it has been clear that tumour angiogenesis is associated with a worse prognosis (34,35). Among many kinds of angiogenic factors, VEGF and its receptors are believed to be mainly responsible for the mechanisms of tumour angiogenesis. Vessel density in tumour stroma correlates with the intensity of VEGF expression and metastasis (36,37) In lung cancer, Ohta et al reported that VEGF and VEGFR-1 are expressed in autocrine and/or paracrine manner, and that the significant correlation between VEGF mRNA expression and the survival period was found (39). VEGF gene consists of 8 exons, of which exon 6 and 7 are alternatively used in the generation of the longer isoforms (39). All the isoforms (VEGF121, VEGF 165, VEGF 189, VEGF 206) contain the exon 1-5 encoded N-terminal domain which is responsible for interaction with VEGFR-1 and VEGFR-2. VEGF 121 and VEGF 165, which were translated from short transcript, are efficiently secreted and promote mitogenesis of endothelial cells. On the other hand, the longer forms (VEGF 189 and VEGF206) are associated with vascular permeability (40). In lung cancer, VEGF121 mRNA expression level showed a significant correlation with survival of patients who underwent curative resection (41).

10. VEGF and VEGFR in cancer metastasis Although many reports about VEGF and cancer have been reported, there are few articles describing the relationship between VEGF-C/VEGFRs and cancer metastasis. Generally, VEGF-C mRNA is not expressed in adult normal tissue except in lung, heart, liver and kidney, but is upregulated in cancer tissue (18). Salven et al reported that VEGF-C mRNA was expressed from melanoma, squamous cell carcinoma, and sarcoma (19). In human malignant mesothelioma, VEGF-C mRNA expression was positively associated with lymphatic vessel density in tumour stroma and showed significant relation with VEGFR-3 mRNA expression (21). We studied the expression of VEGFC, VEGFR-2, and VEGFR-3 in gastric cancer. In eight gastric cancer cell lines, VEGF-C mRNA was expressed in four cell lines (TMK-1, NKPS, AZ521, and MKN-45), but was not expressed in KATOIII, NKPS, NUGC-3, and MKN28. In contrast, VEGFR-3 mRNA was detected only in one (NKPS) cell line, and VEGFR-2 mRNA was expressed in MKN-28, NUGC-3, AZ521, and KKLS. In gastric cancer cell lines, autocrine or paracrine mechanism via VEGF-C and VEGFR-3 could not be found. In the clinical gastric cancer, overexpression of VEGF-C mRNA was found in gastric cancer, but was not detected in normal mucosa (Figure-6). In contrast, there was no significant difference in the VEGFR-3 expression between normal gastric mucosa and primary tumour. However, expression of VEGF-C mRNA and VEGFR-3 mRNA in primary tumours had a significant positive relationship (table 1). Immnohistochemical analysis demonstrated that the expression of VEGF-C was detected in cancer cell cytoplasm (Figure-7), and the VEGFR-3

233 immunoreactivity was exclusively detected in the endothelial cells of lymphatic vessels in normal mucosa and in the stroma of primary tumours (Figure8). VEGFR-3 immunoreaction was found only in one of 85 primary gastric cancers, and the tumour was poorly differentiated adenocarcinoma of early gastric cancer. The number of VEGFR-3 positive lymphatic vessels in the microscopic field of x400 magnification in VEGF-C positive tumours was significantly larger than that in negative tumours (table 2) These results suggest that VEGF-C could be an important factor regulating the mutual paracrine relationship between tumour cells and endothelial cells in lymphatic vessels. Lymphangiogenesis may be induced by VEGF-C, produced by gastric cancer. In our study, there was a significant relation between VEGF-C protein overexpression and lymph node metastasis. Distant lymph node stations were more frequently involved in VEGF-C-positive tumours than in VEGF-C-negative gastric cancer (20). In oral squamous cell cancer, VEGFR-3 expression significantly correlated with lymph node metastasis (28). These observations strongly indicate that the VEGF-C/VEGFR-3 paracrine system promotes lymphangiogenesis in the stroma of primary tumour. In these circumstances, cancer cells easily invade into the lymphatic vessels, because of the increased opportunities in the contact of cancer cells and lymphatic vessels. Furthermore, mutual connection of the endothelial cells in the immature lymphatic vessels are not so tight. Accordingly, cancer cells intravasate into lymphatic vessel through the gaps of the lymphatic endothelial cells, even if these cancer cells have low production of matrix-digesting enzymes and motility factors.

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10. VEGF and VEGFR in cancer metastasis More interestingly, patients with VEGF-C positive rumour had a significantly poorer prognosis than did those in low VEGF-C expression group (20). In addition, Cox proportional hazard model indicated that immunohistological tissue status of VEGF-C in gastric cancer emerged as an independent prognostic parameter (20). Accordingly, VEGF-C tissue status may be a powerful indicator for poor prognosis in patients with gastric cancer.

237 From the clinical point of view, patients with VEGF-C positive tumour should be treated with adjuvant chemotherapy, and should be carefully managed by periodical examination using computed tomography and serum tumour markers even after curative resection. The preoperative determination of VEGFC/VEGFR-3 tissue status by RT-PCR using endoscopic biopsied materials may be useful in deciding the extent of surgical lymph node dissection.

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Chapter 11 HGF-c-MET RECEPTOR PATHWAY IN TUMOR INVASION-METASTASIS AND POTENTIAL CANCER TREATMENT WITH NK4

Kunio Matsumoto and Toshikazu Nakamura Division of Biochemistry, Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan

Key words: Angiogenesis inhibitor, c-Met, HGF, NK4, tumor-stromal interaction

Abstract:

Hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, has mitogenic, motogenic, anti-apoptotic, and angiogenic activities. In tumor tissues, HGF potently enhances dissociation and invasion of a wide variety of tumor cells, thereby facilitating tumor metastasis. Aberrant expression of the c-Met receptor in cancer tissues, up-regulation of the HGF gene expression in tumor cells and/or host stroma, and mutational activation of c-Met receptor tyrosine kinase are particularly associated with the malignant progression of tumors. Likewise, in a variety of carcinomas (tumors of epithelial origin), HGF functions as a stromal-derived mediator in tumor-stromal interaction which confers malignant behavior in carcinoma cells. Therefore, blockage of HGF-c-Met receptor coupling or c-Met receptormediated signals has potential value for treatment of cancer patients. NK4, originally prepared as a competitive antagonist for HGF, is an internal fragment of HGF and it contains N-terminal hairpin and four kringle domains. NK4 binds to but does not activate the c-Met receptor, thereby competitively antagonizing biological activities of HGF. Unexpectedly, NK4 was subsequently shown to be an angiogenesis inhibitor and this angioinhibitory activity is independent of its action as an HGF-antagonist. Importantly, NK4, HGF-antagonist/angiogenesis inhibitor, inhibits tumor invasion, growth, angiogenesis, and metastasis of tumors in laboratory animals. Invasion and subsequent establishment of metastasis are devastating events in patients with cancer, but many past approaches did not address what is perhaps a most important issue in cancer treatment, i.e., invasion and metastasis. The therapeutic strategy of NK4 is based on suppression of intrinsic characteristics of malignant tumors. The possibility that NK4 can be an effective therapeutic for cancer patients warrants ongoing attention.

241 W.G. Jiang et al. (eds.), Growth Factors and their Receptors in Cancer Metastasis, 241–276.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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1. INTRODUCTION Tumor metastasis is the most important and determinant factor affecting survival of cancer patients. The dissociation and invasion of tumor cells and their adhesive interaction towards other cells and the extracellular matrix (ECM) play a definitive role in tumor metastasis. Although mutational alterations that occur in oncogenes and tumor suppressor genes are of genetical background for tumorigenic transformation of cells, metastatic behavior of tumor cells is largely regulated by extracellularly acting growth factors and cytokines. Among these factors, hepatocyte growth factor (HGF), a ligand for c-met protooncogene product, potently affects metastatic behavior in a wide variety of cells (1-3). In normal tissues, HGF plays roles in dynamic construction and reconstruction of tissue architectures during organogenesis and organ regeneration. In tumor tissues, however, tumor cells utilize the biological actions of HGF for their dissociative, invasive and metastatic behavior. Definitively, abrogation of the HGF-c-Met receptor coupling or c-Met receptor-mediated signalling events presents one strategy toward prevention of tumor metastasis. NK4, a four kringles-containing fragment of HGF, was isolated as a competitive antagonist. Subsequent studies showed that NK4 is bifunctional: it is HGF-antagonist and an angiogenesis inhibitor. Bifunctional properties of NK4 suppressed tumor invasion, angiogenesis and metastasis in experimental metastatic tumors in mice. Application of NK4 has the potential for treatment of cancer patients, by inhibiting malignant behavior of tumor cells, rather than directly killing tumor cells as is the case for most of cytotoxic anti-tumor drugs. This overview focuses on tumor invasion and metastasis

Chapter 11 regulated by the HGF-c-Met system and putative cancer treatment with NK4. 2. BACKGROUND HGF was first identified (4, 5) and purified as a mitogenic polypeptide for fully differentiated hepatocytes (6-8). HGF is a heterodimeric molecule composed of a 69kDa and 34 kDa (6-8). Molecular cloning of HGF in 1989 revealed that the contains the N-terminal hairpin domain and subsequent four kringle domains while the contains a serine protease-like domain (Figure 1) (9, 10). HGF has a 38% amino acid sequence homology to plasminogen. Plasminogen is composed of a five kringle-containing A-chain and Bchain of the serine protease catalytic subunit, whereas HGF has no serine protease activity because of amino acid substitution in the catalytic center. In 1985, Stoker et al. identified a fibroblast-derived epithelial cell motility factor, termed scatter factor (11). Scatter factor was purified in 1989-1990 (12, 13) and subsequent characterization unexpectedly revealed that scatter factor is identical to HGF (14-16). Similarly, bioactive polypeptides purified using different bioassay systems revealed identity of the polypeptide to HGF, including fibroblast-derived epithelial cell growth factor (17), tumor cytotoxic factor (18), and fibroblast-derived epithelial morphogen which induces unique branching tubulogenesis in epithelial cells (19). Thus, HGF has multiple biological effects on a wide variety of cells, including mitogenic, motogenic, morphogenic, and anti-apoptotic activities (Figure 1). The receptor for HGF was shown to be a c-met protooncogene product in 1991 (20, 21). The c-Met/HGF receptor is

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composed of a 50 kDa and 145 (22). The is exposed kDa extracellularly, while the is a transmembrane subunit containing an intracellular tyrosine kinase domain (Figure 1). Binding of HGF to the c-Met receptor induces tyrosine phosphorylation in the tyrosine kinase domain, the result being enhanced tyrosine kinase activity and the subsequent phosphorylation of Cterminally clustered tyrosine residues (2325). Phosphorylated C-terminal tyrosine residues recruit intracellular signaling molecules containing the src homology (SH) domain, including Gab-1, phospholipase Ras-GTPase activating protein (Ras-GAP), phosphatidylinositol-4, 5-bisphosphate 3kinase (PI-3 kinase), c-Src, and Grb-2 (Figure 1). Although intracellular signaling pathways leading to specific or preferential activation of each biological response (i.e., mitogenic, motogenic, morphogenic, or anti-apoptotic) driven by HGF-c-Met receptor coupling are not fully understood, preferential activation of signaling molecules and induction of gene expression responsible for activating specific cellular responses are evident. Phosphotyrosine-dependent recruitment of the Grb-2/SOS complex activates Ras and subsequent phosphorylation events, including extracellular signal regulated kinase (ERK). Activation of the Ras-ERK pathway is required for cellular proliferation (23). On the other hand, association and tyrosine phosphorylation of Gab-1, a docking protein that couples the c-Met receptor with multiple signaling proteins such as PI-3 kinase, Shp2, and Crk-2, plays a definite role in HGFinduced morphogenesis and cellular movement (26, 27). The expression of Gab-1 in MDCK renal epithelial cells induces cell movement and branching

Chapter 11 tubulogenesis (26, 28). Association of Gab-1 with Shp-2 protein tyrosine phosphatase but not PI-3 kinase and Crk-2 is essential to induce epithelial branching tubulogenesis (27, 29). Instead, activation of PI-3 kinase, and the Rho family GTPases (Rho, Rac, Cdc42), and Ras play critical roles in HGF-induced cell movement (27, 30-35). Rho GTPases regulate actin-related cytoskeletal organization and contractive force for cell movement and concomitant cell shape rearrangement. In terms of anti-apoptosis, activation of PI-3 kinase and downstream Akt (protein kinase B) (36), and induction of Bcl-2/Bcl-xL are likely pathways responsible for protection of cells from apoptosis by HGF (37, 38). Knockout of HGF or the c-Met receptor gene in mice defined the essential role of HGF-Met system in mammalian development (39-41). Mice with a disrupted HGF or c-Met receptor gene were embryonic lethal due to impaired organogenesis of the placenta and liver. HGF is also involved in development of embryonic tissues, including the kidney, lung, mammary gland, teeth, and skeletal muscle (42). In mature tissues, HGF has an organotrophic role in regeneration and protection of various tissues, including the liver, lung, kidney, stomach, nervous tissue, pancreas and heart (43-47). Likewise, HGF has angiogenic activity for vascular endothelial cells (48-51). Administration of HGF to laboratory animals has therapeutic effects on various models of diseases (45,47). 3. HGF IN INVASIVE AND METASTATIC CANCER BEHAVIOR Invasion of tumor cells is regulated by distinct cellular functions, including cellcell adhesion, cell-matrix association, proteolytic breakdown of the extracellular matrix, and cellular locomotion. As HGF

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affects these processes, and HGF exhibits profound effects on the invasive behavior of a wide variety of tumor cells. Figure 2 shows typical examples of the tumor cell invasion stimulated by HGF. HGF potently stimulates invasion of tumor cells through basement membrane components (Figure 2A) and in collagen gel matrix (Figure 2B). Enhancement of cell motility and the invasive behavior by HGF has been noted in a wide variety of tumor cells (Table 1). Cell-cell adhesiveness is regulated by distinct types of cell adhesion molecules, while the adhesive potential is often decreased or lost in various types of malignant tumors as the result of genetic and/or epigenetic events (52). Among various cell adhesion molecules, epithelial cadherin- (E-cadherin-) mediated cell-cell adhesion is widely considered to contribute to the invasive characteristics of variety of tumor cells of epithelial origin (52, 53). E-cadherin binds to and catenin, and the complex associates with which links cadherin complexes to the actin cytoskeleton (53). HGF stimulates dissociation of cell-cell adhesion through tyrosine phosphorylation of catenins and by regulating expression and distribution of the cadherin-based adhesive machinery. HGF induces tyrosine phosphorylation of and (54-57). HGF reduces the amount of complex in human colorectal cancer (58), while HGF decreases expression of E- and P-cadherin in gastric cancer cells (59). Moreover, the c-Met receptor associates with E-cadherin (60) and localizes at the basolateral plasma membrane domain of polarized cells (61), thereby suggesting that rapid and direct tyrosine phosphorylation of the E-cadherin/catenin complex by the

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activated c-Met receptor. Likewise, HGF reduces expression of proteins involved in tight junctional complex, including zonula occludin-1 and barmotin/7H6 (62-64). The reduction in tight junctional complex proteins may reduce junctional communication between cells, thereby facilitating cellular migration. Cellular migration is regulated by multiple extracellular and intracellular processes organized by a complex signaling cascade. While the entire story for molecular mechanisms involved in cellular migration has yet to be defined, regulation of cell-substratum interaction and concomitant change in cell shape and cytoskeletal rearrangement are major components related to cell movement. Focal adhesions, adhesive complexes for anchoring of cells to extracellular matrix components, are newly formed in frontier regions of migrating cells, whereas they are dissociated in terminal regions. Coupling with active turnover (association and dissociation) of focal adhesion complexes, rearrangement and retraction of cytoskeletal networks occur in migrating cells. Cells adhere to the extracellular matrix (ECM) components via integrin family receptors and integrins transduce signals within cells for ECMdependent cell shape arrangement and cell movement. In adherent cells, HGF induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin (6569), presumably through activation of Src upon HGF stimulation. Expression of kinase-negative FAK in cells and antibody against α2 integrin inhibited HGFinduced migration of cells (69). Thus integrin-dependent cell-ECM binding and intracellular tyrosine phosphorylation of FAK are required for cell migration. Additionally, HGF induces tyrosine phosphorylation of ezrin (70, 71). Ezrin is involved in cross-linking between plasma

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membrane and actin filaments and is localized at microvilli, ruffling membrane, and cadherin- and integrin-based adherens junctions. Tyrosine phosphorylation of ezrin may be involved in the rapid induction of HGF-induced membrane ruffling, the formation of focal adhesion togethar with FAK, and rearrangement of actin stress fibers to ruffling membranes and focal adhesions. Small GIP-binding proteins (Rho, Rac, Cdc42, etc) play crucial roles in the regulation of cell shape and movement through remodeling of cytoskeletal networks and their interaction with plasma membranes and intracellular organellas. Cdc42, Rac, and Rho respectively affect the actin cytoskeleton to produce filopodia, lamellipodia, and stress fibers and focal adhesions. HGF activates Rho, Rac, Cdc42 (30, 32, 34, 35). Although involvement of Rho in HGF-induced cell spreading and migration is somewhat controversial when different assay systems are used, HGF-induced lamellipodia formation and cell spreading require activation of PI-3 kinase and these events are inhibited by dominant-negative Cdc42 or Rac (32, 35). HGF-induced activation of Rac but not Cdc42 is dependent on PI-3 kinase (35). Likewise, the microinjection of dominant-negative Ras into cells inhibited HGF-induced cell spreading and scattering (32). Extracellular proteolysis of extracellular matrix proteins plays a critical role in tumor invasion and metastasis. Among various types of proteases, the urokinase type plasminogen activator (uPA)-dependent proteolytic network and matrix metalloproteinases (MMPs) affect invasiveness of a wide variety of tumour cells. uPA anchors to cell surfaces through the uPA receptor, the cell surface-anchored uPA activates plasmionogen to plasmin, and plasmin

Chapter 11 subsequently activates some types of MMPs. HGF induces uPA gene expression and strongly stimulates uPA production in many types of normal and tumour cells, including MDCK renal epithelial cells (72), gallbladder carcinoma cells (73, 74), pancreatic carcinoma (75), human squamous carcinoma (76), and mammary carcinoma (77). Similarly, establishment of an autocrine activation loop of HGF-Met in leiomyosarcoma cells resulted in marked activation of uPA and the uPA receptor system and concomitant acquisition in invasiveness and metastatic potential of the cells (78). In addition to uPA induction, HGF stimulates production of some MMPs, membrane-type MMP-1, collagenase-1, and stromelysin in distinct types of cells. HGF stimulates collagenase-1, MMP-3 (stromelysin-1) and MMP-9 in keratinocytes (79, 80), and MMP-2 and MMP-9 in gallbladder carcinoma cells (73, 74). In human colon cancer cells, HGF stimulates MMP-1, MMP-2, MMP-9 production (81). Rosenthal et al. found that HGF stimulates expression of uPA, uPA receptor, MMP9, membrane type MMT-1, collagenase-1, and stromelysin-1 in a human squamous carcinoma cell line, and HGF triggers invasion of the cells through type I collagen (76). They also found that inhibitors for MMPs, but not for the uPAplasmin system, inhibited HGF-induced invasion of cancer cells. Figure 3A collectively describes HGF-induced signalling events involved in invasive behavior of tumor cells. Angiogenesis, the formation of new blood vessels from preexisting blood vessels, is a critical process involved in embryonic development, tissue regeneration, and pathological conditions such as tumorigenesis. Angiogenesis is critical for tumor growth (82, 83), and

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increased angiogenesis coincides with increased tumor cell entry into the blood circulation and thus facilitates metastasis (84). Vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been particularly strengthened to be involved in tumor angiogenesis, whilst HGF has potent angiogenic activity in vivo as well as in vitro (48-51). Potent angiogenic activity of HGF over VEGF has been demonstrated in vivo (49). Although a role for HGF in tumor angiogenesis has yet to be extensively investigated, there are reports of involvement of HGF in tumor angiogenesis. When the HGF gene was stably expressed in human breast cancer cells and in glioma cells, these tumor cells exhibited more extensive tumor angiogenesis and enhanced growth in nude mice (85, 86). Expression of HGF and the Met receptor are overexpressed and these expressions are associated with an increased microvessel density in malignant pleural mesothelioma (87). Moreover, HGF enhances expression of VEGF mRNA in cultures of human glioma cells and in vivo when administrated into tissues (49, 88). These results indicate that HGF is one of the growth factors affecting tumor angiogenesis. In addition to angiogenesis, HGF stimulates attachment of tumor cells to endothelial cells by increasing CD44 expression in endothelial cells, a molecule which may play a critical role in tumorendothelial interactions and establishment of metastasis (89). Likewise, endothelial integrin cell-derived HGF enhances expression in human hepatoma cells, thereby facilitating adhesion of the tumor cells to endothelial cells (90). Together with its potent action to enhance tumor cell motility/invasion, HGF is likely to confer metastatic potential in many types of tumor cells, via

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enhancement of tumor angiogenesis, as well as tumor invasion. Figure 3B schematically describes participation of HGF in early steps leading to cancer metastasis. HGF stimulates 1) dissociation of cancer cells at the primary site, 2) invasion through the basement membrane and host stroma, via enhancing cell-matrix interactions, protease networks for breakdown of the extracellular matrix, and motogenic responses, 3) angiogenesis in tumor tissues, and 4) interaction with blood vessels. 4. c-MET ACTIVATION THROUGH DISTINCT PATHWAYS 4.1. Gain-of-function mutation in human cancer Hereditary papillary renal carcinoma is an inherited renal cancer characterized by a predisposition to develop multiple, bilateral papillary renal tumors. Genetical analysis of papillary renal carcinoma in patients indicated that missense mutations in the c-met gene are the causative genetical disorder in inherited and some sporadic papillary renal carcinomas (91, 92). All mutations that occur in eight distinct positions in the c-met gene are missense and are located in the tyrosine kinase domain of the c-Met receptor (Figure 4A). Jeffers et al. found that NIH3T3 cells transfected with the c-met cDNA with corresponding mutations were tumorigenic in nude mice and that the cMet receptor with these mutations had enhanced tyrosine kinase activity (93). Therefore, c-Met receptor mutations originally identified in patients with hereditary and some sporadic papillary renal carcinoma are likely to be the gainof-function mutation and thus may play a determinant role in papillary renal carcinoma. The possibility that similar

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gain-of-function mutations in the c-met gene participate in development of other neoplastic diseases remains to be addressed. 4.2. Overexpression of the c-Met receptor Over-expression of growth factor receptors were noted in a variety of tumors and some growth factor receptors were identified as oncogenes overexpressed in various tumor cells. Overexpression of the c-Met receptor was noted in a wide variety of tumor cells and tissues, including carcinoma, lymphomas, and soft tissue tumors (Table 2). Although two distinct mechanisms by which the cMet receptor is over-expressed in cancers can be considered, i.e., transcriptional activation or amplification of the c-met gene, the mechanism involved in the overexpression of the c-met gene has yet to be determined. In case of colorectal cancers, over-expression of the c-Met receptor was related to transcriptional activation in 90% of the primary tumors, whereas 8 among 9 cases of metastases of colorectal cancers accompanied amplification of the c-met gene (94). Amplification of the c-met gene may occur more frequently in late stage metastatic tumors than in primary tumors, presumably due to chromosomal instability in advanced cancers. It should be emphasized that the level of c-Met receptor expression tends to correlate with the progression of tumors in many types of tumors; a higher expression of the cMet receptor is seen in the late stage or in metastatic tumors. The c-Met receptor may be activated in a ligand-independent manner in some tumors wherein c-Met expression is extremely high, through increased susceptibility to ligandindependent receptor dimerization (Figure 4A). Alternatively, an over-expressed cMet receptor in other tumor tissues may

Chapter 11 be activated by HGF produced by tumor cells themselves or by host stromal cells, the result being acquisition of malignant behavior in many types of tumor cells (see below). 4.3. c-Met receptor activation through an autocrine loop Ligand-dependent constitutive activation of receptor tyrosine kinases through establishment of an autocrine loop in growth factors and their receptors associates with tumorigenic transformation of cells. Since the c-Met receptor is predominantly expressed in epithelial cells but not in mesenchymal or stromal cells, while HGF is predominantly expressed in mesenchymal or stromal cells but not in epithelial cells, two distinct types of gene transfer experiments established the autocrine loop of the HGFc-Met receptor: Expression of HGF in cMet-positive (but HGF-negative) epithelial cells, or expression of c-Met receptor in HGF-positive (but c-Metnegative) mesenchymal/stromal cells. Stable expression of the c-Met receptor gene in NIH-3T3 fibroblasts conferred tumorigenic and invasive characteristics in nude mice (95-97). Similarly, expression of the c-Met receptor gene in human leiomyosarcoma cells and mouse 127 cells resulted in establishment of the HGF-c-Met autocrine loop and concomitant progression to invasive and metastatic cancers in nude mice (78, 98). On the other hand, transfection of the HGF gene in hepatic epithelial cells induced tumorigenic transformation (99) or conferred invasive and metastatic behavior (100). Thus, autocrine activation of the c-Met receptor participates in tumorigenic, invasive and metastatic behavior in cancer cells. Consistent with the results in gene transfer experiments in cells, transgenic

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overexpression of the HGF gene in various tissues from early embryonic development in mice was found to be associated with aberrant development and tumorigenesis in several tissues and cells, including melanocytes, hepatocytes and mammary gland epithelial cells (101-103). Melanoma in HGF-transgenic mice was metastatic. However, it is noteworthy that transgenic expression of HGF gene in a tissue-specific manner in mice was not associated with tumorigenesis. Transgenic mice expressing HGF under an albumin promoter had a greatly increased potential for liver regeneration, but hepatocellular carcinoma did not occur (104). Keratinocyte-specific expression of HGF affected melanocyte development, leading to melanocytosis but tumors did not occur, even from the skin (105). Likewise, transgenic mice expressing HGF in pancreatic cells did not develop tumors, and mice were resistant to the diabetic effects of streptozotocin (106). It is noteworthy that hepatocarcinogenesis caused by transgenic overexpression of myc or the transforming growth gene in the liver was suppressed in double transgenic mice of HGF and myc genes or HGF and genes (107, 108). Moreover, hepatocarcinogenesis promoted by phenobarbital was also strongly inhibited by the transgenic expression of HGF in the liver (107). While the mechanism by which transgenic expression of the HGF gene suppressed hepatocarcinogenesis has yet to be defined, the inhibition of hepatocarcinogenesis may be related to growth inhibitory effects of HGF on hepatocellular carcinoma cells (109, 110). On the other hand, abundant expression of HGF in various tissues from early developmental stages in transgenic mice might affect differentiation, maturation, or

Chapter 11 epithelial renewal, the result being in tumor development in various tissues. Tumorigenesis and invasive and metastatic progression in cells through the experimental establishment of the HGF-cMet autocrine loop suggest the presence of the HGF-c-Met autocrine loop in naturally developed cancer cells. Autocrine activation of the c-Met receptor was noted in various types of tumor cell lines, including human myeloma (111, 112), human osteosarcoma (113), human glioma and glioblastoma (114-117), human small cell lung carcinoma (118), rat bladder carcinoma (119), rat cholangiocarcinoma (120), and mouse mammary carcinoma (121). In these cancer cells, autocrine activation of the cMet receptor affects tumor growth and confers anti-apoptotic, invasive and metastatic characteristics. In addition to autocrine activation of the c-Met receptor noted in various tumor cell lines, coexpression of HGF and the c-Met receptor has been detected in a variety of tumors, and the incidence of co-expression and their expression pattern varies depending on tumor types. Table 3 summarizes the co-expression of HGF and c-Met receptor in tumors, as detected by histological analysis (for more details, see reviews: 2, 3). Particularly, it is noteworthy that coexpression of HGF and c-Met is more frequently observed in sarcomas, gliomas, and myeloma/lymphoma, than in carcinomas. Since mesenchymal/stromal cells are a predominant source of HGF in various normal tissues, these findings mean that activation of the c-met gene in mesenchymal/stromal cells may be associated in tumorigenesis or malignant progression in sarcomas, gliomas, and myeloma/lymphoma. In contrast, since most normal epithelial cells are c-Metpositive but HGF-negative, activation of HGF gene expression seems to be

11.HGF-c-MET receptor pathway and NK4 associated with tumorigenesis or malignant progression in some carcinomas, whereas the source of HGF in most carcinomas seems to be host stromal cells (see below). 4.4. HGF in tumor-stromal interaction Host stromal influences on epithelial neoplasia, tumor angiogenesis and malignant behavior of carcinomas have been noted in various types of cancers, including prostate, stomach, skin, oral cavity, mammary gland, and colon cancers (122-125). In vivo growth of various carcinoma cells was accelerated by a broad spectrum of fibroblasts, and both in vitro and in vivo growth and invasiveness of carcinoma cells was enhanced by the co-existence of stromal fibroblasts (65, 77, 126-137). The host stromal-derived factor(s) is one key molecule that enhances invasive and metastatic potential in cancer cells. Figure 5 shows in vitro invasion of four distinct types of human carcinoma cells through the Matrigel basement membrane components, in the absence or presence of stromal fibroblasts (134). Although basal invasive potentials of these carcinoma cells somewhat varied in each cell type, invasion of these tumor cells was potently enhanced by cocultivation with stromal fibroblasts. The result clearly indicated that invasiveness of tumor cells is stimulated by fibroblastderived soluble factor(s). Importantly, an antibody against HGF almost completely abrogated invasion of the cancer cells in the co-culture system. Similar results were noted in various types of cancer cells, including oral squamous cell carcinoma cells (65, 135), mammary carcinoma (77), gallbladder carcinoma (130, 131), esophageal cancer (133), and prostate cancer (136). These results indicate that invasion of various tumors depends on a

257 fibroblast-derived factor and that HGF is likely to be a predominant factor responsible for cancer cell invasion mediated by tumor-stromal interaction. Consistent with a lack of autonomous potential to aggressively invade into the scaffold of ECM in many of carcinoma cells, most carcinoma cells do not secrete HGF. In over 70 distinct carcinoma cells, only a few types of tumor cell lines secrete HGF (our unpublished data). However, several reports noted that many types of tumor cells secrete a variety of HGF-inducers, through which HGF production in stromal fibroblasts is upregulated (77, 131, 132, 134-136, 138, 139). These tumor-derived HGF-inducers were identified to be interleukin-1 (IL-1), bFGF, platelet-derived growth factor and prostaglandin E2. (PDGF), These observations suggest a mutual interaction between tumor cells and stromal fibroblasts, as mediated by HGF and the HGF-inducer loop: tumor cells secrete HGF-inducers for stromal cells, while stromal cells secrete HGF which may stimulate tumor invasion, angiogenesis, and metastasis (Figure 4C). The mutual interaction between tumor cells and stromal fibroblasts, as mediated by HGF and the HGF-inducer loop was demonstrated in several types of tumor cells, using a co-culture method (77, 131, 132-134, 136). 5. EXPERIMENTAL CANCER TREATMENT WITH NK4, AN HGFANTAGONIST AND ANGIOGENESIS INHIBITOR Understanding molecular and cellular mechanisms of tumor invasion, angiogenesis, and metastasis should lead to establishment of new therapeutics for treatment of cancer patients. Close involvement of c-Met receptor activation in acquisition of malignant characteristics

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in a wide variety of tumors suggests therapeutic strategies for prevention of tumor invasion, angiogenesis, and metastasis, through blockage of c-Met receptor-mediated signaling. For this purpose, a specific antagonistic molecule was prepared. Recent studies demonstrated that blockage of c-Met receptor-mediated signaling by the HGFantagonist does inhibit tumor invasion and metastasis. 5.1. Structure and function of NK4 Studies on the structure-function relationship in the HGF molecule revealed a functional domain for receptor binding and for subsequent biological activities. Small molecules consisting of the Nterminal hairpin plus the first and second kringle domains (designated NK2) and the N-terminal hairpin and the first kringle domains (designated NK1) are in naturally biosynthesized variant forms of HGF (140, 141). Both NK1 and NK2 can bind the c-Met receptor, thus NK1 serves a minimum set of domains responsible for binding to c-Met receptor. Interestingly, NK1 and NK2 exhibit antagonistic activity on HGF-induced mitogenesis (140, 142), while they function as agonists in terms of cell motility activity (118, 141, 143). Silvagno et al. showed that NK3, a larger variant consisting of the N-terminal hairpin and subsequent three kringle domains induces angiogenic responses in endothelial cells (144). Therefore, NK1, NK2, and NK3, all variants of the containing binding domains to the c-Met receptor retain agonistic activity, not fully but in part, and approaches to obtain a complete antagonist for HGF were unsuccessful. On the other hand, Date et al. first prepared the HGF-antagonist (NK4) which competitively inhibits HGF-c-Met receptor coupling from elastase-digested

Chapter 11 human HGF. NK4 is composed of Nterminal 447 amino acids of the of HGF, thus contains the N-terminal hairpin domain and subsequent four kringle domains (Figure 6A) (145). NK4 can bind to the c-Met receptor, however, NK4 does not activate c-Met receptor and is devoid of c-Met-mediated agonistic biological activities. Thus, NK4 competitively inhibits the binding of HGF to the c-Met receptor and occupies the cMet without inducing tyrosine phosphorylation, thereby antagonizing biological activities of HGF (Figure 6A). NK4 has the receptor binding domains (i.e., NK1 or NK2), whereas it has no agonistic activities after binding to c-Met receptor. One possible explanation is that the fourth kringle domain inhibits receptor dimerization required for tyrosine phosphorylation of the c-Met receptor. Figure 6B and 6C respectively demonstrate antagonistic activities of NK4 on HGF-induced c-Met receptor tyrosine phosphorylation and in vitro invasion of human tumor cells. NK4 has a 10-fold lower affinity for the c-Met receptor than does HGF and almost completely inhibits the biological activities of HGF at a 300-1000-fold higher concentration than does HGF (145, 146). Likewise, Hiscox et al. and Parr et al. reported that NK4 inhibits motility and invasion of breast cancer and colon cancer cells, respectively (147, 148). 5.2. NK4 is an angiogenesis inhibitor Since vascular endothelial cells express the c-Met receptor and HGF has angiogenic activity (48-51), it was predicted that NK4 might inhibit angiogenic responses induced by HGF. However, recent studies showed that four kringle domains (K1-4), as well as NK4, have anti-angiogenic activity to inhibit the

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11.HGF-c-MET receptor pathway and NK4 angiogenic responses induced by bFGF, VEGF, and HGF (149, 150). Figure 7 shows inhibitory effects of NK4 on proliferation of human microvascular endothelial cells in culture. HGF stimulates endothelial cell proliferation, whereas NK4 inhibits HGFstimulated endothelial proliferation. Importantly, NK4 inhibits endothelial proliferation stimulated by bFGF and VEGF (Figure 7A). Similar inhibitory effects of NK4 were noted in bFGF- and VEGF-induced endothelial cell migration in various types of endothelial cells, and inhibitory effects of NK4 on bFGFinduced cell proliferation and migration was not evident using non-endothelial cells (149). Moreover, NK4 inhibits bFGF-induced in vivo angiogenesis in the rabbit cornea (Figure 7B) and chick allantoic membrane assay. When a pellet containing bFGF was implanted under the rabbit cornea, bFGF induced extensive angiogenesis (Figure 7B). The coexistence of NK4 with bFGF in the pellet potently inhibited the bFGF-induced angiogenesis. These results strongly suggest that NK4 has anti-angiogenic activity and that the angioinhibitory effect of NK4 may be independent on its original activity as an HGF-antagonist. Subsequent studies showed that deletion of the N-terminal hairpin domain (an essential domain responsible for c-Met receptor binding in NK4 and HGF) in NK4 led to a selective loss of HGFantagonist activity, yet the remaining four-kringle variant of HGF (K1-4) retains angioinhibitory effect (Figure 7C) (150). Taken together, NK4 is bifunctional as it is an HGF-antagonist and an angiogenesis inhibitor, and domains responsible for angiogenesis inhibition reside in four kringles. In this context, it is noteworthy that kringle domains in some kringlecontaining molecules have angioinhibitory

267 activity, including angiostatin (151), the fifth kringle domain of plasminogen (152), and the kringle-2 of prothrombin (153). Particularly, angiostatin and NK4 or K1-4 have structural similarities and four kringle domains (1st to 4th) in both plasminogen and HGF have angioinhibitory actions (Figure 7C). It should be emphasized, however, that angiostatin has no apparent activity to antagonize HGF. Moser et al. reported that binding of angiostatin to ATPsynthase on the plasma membranes is likely to be involved in angiostatininduced endothelial inhibition (154). Although mechanisms by which NK4 and K1-4 inhibit angiogenic responses in endothelial cells has remained an open question, NK4 and K1-4 may exert angiostatic signals through putative binding molecules present on endothelial cells. 5.3. Experimental cancer treatment by NK4 The bifunctional property of NK4 as an HGF-antagonist and angiogenesis inhibitor means that NK4-treatment of patients with cancer may be one approach to suppress the malignant behavior of cancer. Recent studies on cancer treatment with NK4 in experimental models showed the unique therapeutic potential of NK4 (73, 149). The therapeutic effects of NK4 were examined in case of human invasive gallbladder carcinoma (73) and murine metastatic carcinoma, Lewis lung carcinoma and mammary carcinoma cells (149). Figure 8 shows inhibitory effects of NK4 on in vitro invasion (Figure 8A) and in vivo therapeutic effects of NK4 on tumor growth, angiogenesis, and metastasis in Lewis lung carcinoma (Figure 8B-E). In vitro, HGF and NK4 had no direct effect on proliferation of the

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cells yet HGF potently stimulated the invasion of Lewis lung carcinoma cells, whereas NK4 blocked the invasion of the tumor cells induced by HGF (Figure 8A). When Lewis lung carcinoma cells were subcutaneously inoculated into nude mice, these tumor cells exhibited malignant characteristics, including extensive tumor angiogenesis and metastasis. The mice were then infused with NK4 for 14 days from the 4th day after tumor implantation (Figure 8B-E). NK4 did not directly inhibit tumor cell proliferation in vitro, but continuous infusion of NK4 inhibited growth of these tumors (Figure 8B). The volume of NK4treated Lewis lung carcinoma was inhibited to 30.1% on the 28th day, as compared with findings in control tumors. The number of blood vessels in tumors of NK4-treated mice decreased to 55.8% of that of control tumors, and some tumor vessels in NK4-treated mice were disrupted and had a short diameter (Figure 8C). In addition, NK4-treatment led to a 2.25-fold increase in the number of apoptotic cells, without changing the number of proliferating tumor cells (Figure 8D). Thus, NK4 seems to suppress primary tumor growth mainly by inhibiting tumor angiogenesis. By the 28th day, Lewis lung carcinoma cells metastasized to the lung. The number of lung metastases was decreased to 9.46% of control mice by NK4-treatment (Figure 8E). Suppression of metastasis of Lewis lung carcinoma may be achieved by bifunctionality of NK4 as an HGFantagonist and angiogenesis inhibitor. In addition to Lewis lung carcinoma, similar inhibitory effects of NK4 on in vivo tumor growth, angiogenesis, and metastasis were obtained with human gallbladder carcinoma and murine mammary carcinoma (73, 149). Furthermore, NK4-treatment strongly

Chapter 11 inhibited disseminative metastasis of human pancreatic cancer cells orthotopically implanted into nude mice and prolonged survival time of these mice, even when NK4-treatment was initiated from a relatively advanced stage (our unpublished data). All these results indicate that NK4 inhibits tumor invasion, metastasis and angiogenesis, thus leading to suppression of malignant behavior of tumor cells. 6. CONCLUDING REMARKS Aberrant expression of the c-Met receptor in cancer tissues, up-regulation of HGF gene expression in tumor cells and/or host stroma, and mutational activation of c-Met receptor tyrosine kinase are associated with carcinogenesis and particularly with the malignant progression of tumors. Since HGF has potent activity to enhance the breakdown of ECM and cell movement/invasion, the HGF-c-Met system in a wide variety of tumor cells is operative in acquisition of invasive and metastatic potentials. On the other hand, HGF has biological activities involved in dynamic tissue remodeling during embryogenesis and tissue regeneration. Breakdown of ECM and concomitant cellular migration, mitogenesis, and morphogenesis driven by the HGF-c-Met system make way for construction and reconstruction of tissues through epithelial-mesenchymal (or stromal) or other cell-cell interactions for organogenesis and organ regeneration. Functions of the HGF-c-Met system in embryonic, wounded and cancer tissues may confer similarity in gene expression profiles for these tissues. The simile that “cancer is never-healing wound” seems pertinent here. Undoubtedly, the final objective of cancer research is to cure affected patients with cancer. The many past approaches

11.HGF-c-MET receptor pathway and NK4

used to treat cancer patients (i.e., chemotherapy, radiotherapy, and surgery) have often led to unsatisfactory outcomes. Since both chemo and radiotherapies are based on the concept to directly kill cancer cells, these therapies have severe side effects that result in reduction in quality of life and/or in decrease in immune responses. Likewise, the emerge of drug-resistant cancer cells restricts long-term usage of chemical anti-cancer drugs. The surgical removal of cancer after early detection of a cancer can be associated with disease-free survival of patients in some malignant cancers, yet surgical treatment often results in incomplete removal, allowing invasion and subsequent micro-metastasis of cancer cells. Most past approaches did not address perhaps a most important issue in cancer treatment, i.e., invasion and

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metastasis. On the basis of findings that aberrant utilization of HGF-c-Met system is closely involved in tumor invasion and metastasis, abrogation and inhibition of HGF-c-Met coupling and signal transduction from c-Met may prove to be therapeutic strategies to prevent cancer metastasis. NK4, the HGFantagonist/angiogenesis inhibitor can inhibit tumor invasion, growth, angiogenesis, and metastasis of tumors in laboratory animals. The possibility that NK4 and other inhibitors of HGF-c-Met signaling can be therapeutic for cancer patients warrants ongoing further investigations. Acknowledgment: We are grateful to M. Ohara for helpful comments.

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Chapter 11 islet of transgenic mice increases beta cell proliferation, enhanced islet mass, and induces mild hypoglycemia. J Biol Chem 2000; 275: 1226-1232. 107.Santoni-Rugiu E, Preisegger KH, Kiss A, Audolfsson T, Shiota G, Schmidt EV, Thorgeirsson SS. Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model. Proc Natl Acad Sci USA 1996; 93: 9577-9582. 108. Shiota G, Kawasaki H, Nakamura T, Schmidt EV. Characterization of double transgenic mice expressing hepatocyte growth factor and transforming growth Res Commun Mol Pathol Pharmacol 1995; 90: 17-24. 109.Shiota G, Rhoads DB, Wang TC, Nakamura T, Schmidt EV. Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells. Proc Natl Acad Sci USA 1992; 89: 373-377. 110.Tajima H, Matsumoto K, Nakamura T. Hepatocyte growth factor has potent antiproliferative activity in various tumor cell lines. FEBS Lett 1991; 291: 229-232. 111.Borset M, Lien E, Espevik T, Helseth E, Waage A, Sundan A. Concomitant expression of hepatocyte growth factor/scatter factor and the receptor c-MET in human myeloma cell lines. J Biol Chem 1996; 271: 24655-24661. 112. Borset M, Hjorth-Hansen H, Seidel C, Sundan A, Waage A. Hepatocyte growth factor and its receptor c-met in multiple myeloma. Blood 1996; 88: 3998-4004. 113.Ferracini R, Di Renzo MF, Scotlandi K, Baldini N, Olivero M, Lollini P, Cremona O, Campanacci M, Comoglio PM. The Met/HGF receptor is over-expressed in human osteosarcomas and is activated by either a paracrine or an autocrine circuit. Oncogene. 1995; 10: 739-749. 114.Moriyama T, Kataoka H, Tsubouchi H, Koono M. Concomitant expression of hepatocyte growth factor (HGF), HGF activator and c-met genes in human glioma cells in vitro. FEBS Lett. 1995; 372: 78-82. 115. Shiota G, Kawasaki H, Nakamura T. Coexpression of hepatocyte growth factor and its receptor (c-met oncogene) in HGL4 glioblastoma cells. Oncology 1996; 53:511-516. 116. Koochekpour S, Jeffers M, Rulong S, Taylor G, Klineberg E, Hudson EA, Resau JH, Vande Woude GF. Met and hepatocyte growth factor/scatter factor expression in human gliomas. Cancer Res 1997; 57: 5391-5398. 117. Abounader R, Ranganathan S, Lal B, Fielding K, Book A, Dietz H, Burger P, Laterra J. Reversion of human glioblastoma malignancy by U1 small nuclear RNA/ribozyme targeting of scatter factor/hepatocyte growth factor and c-met

11. HGF-c-MET receptor pathway and NK4 expression. J Natl Cancer Inst. 1999; 91: 15481556. 118. Itakura Y, Yamamoto T, Matsumoto K, Nakamura T. Autocrine stimulation of motility in SBC-5 human lung carcinoma cells by a twokringle variant of HGF. Cancer Lett 1994; 83: 235-243. 119.Tamatani T, Hattori K, Iyer A, Tamatani K, Oyasu R. Hepatocyte growth factor is an invasion/migration factor of rat urothelial carcinoma cells in vitro. Carcinogenesis. 1999; 20: 957-962. 120. Lai GH, Radaeva S, Nakamura T, Sirica AE. Unique epithelial cell production of hepatocyte growth factor/scatter factor by putative precancerous intestinal metaplasias and associated "intestinal-type" biliary cancer chemically induced in rat liver. Hepatology. 2000; 31: 1257-1265. 121. Rahimi N, Tremblay E, McAdam L, Park M, Schwall R, Elliott B. Identification of a hepatocyte growth factor autocrine loop in a murine mammary carcinoma. Cell Growth Differ 1996; 7: 263-270. 122. Sakakura T. New aspects of stroma-parenchyma relations in mammary gland differentiation. Int Rev Cytol 1991; 125: 165-202. 123. Van den Hoff A. Stromal involvement in malignant growth. Adv Cancer Res 1988; 50: 159-196. 124.Wernert N. The multiple roles of tumour stroma. Virchows Arch 1997; 430: 433-443. 125. Sokoloff MH, Chung LW. Targeting angiogenic pathways involving tumor-stromal interaction to treat advanced human prostate cancer. Cancer Metastasis Rev 1999; 17: 307-315. 126. Schor SL, Schor AM, Winn B, Rushton G. The use of three dimensional gels for study of tumour cell invasion in vitro. Int J Cancer 1982; 29: 5762. 127. Picard O, Rolland Y, Poupon MF. Fibroblastdependent tumorigenicity of cells in nude mice: implication for implantation of metastases. Cancer Res 1986; 46: 3290-3294. 128. Grey AM, Schor AM, Rushton G, Elias I, Schor SL. Purification of the migration stimulating factor produced by fetal and breast cancer patient fibroblasts. Proc Natl Acad Sci USA 1989; 86: 2438-2442. 129. Camps JL, Chang S, Hsu TC, Freeman MR, Hong S, Zhau HE, von Eschenbach AC, Chung LWK. Fibroblast-mediated acceleration of human epithelial tumour growth in vivo. Proc Natl Acad Sci USA 1990; 85: 75-79. 130. Shimura H, Date K, Matsumoto K, Nakamura T, Tanaka M. The induction of invasive growth in a gallbladder cancer cell line by hepatocyte growth

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Hepatocyte growth factor (HGF)/NK1 is a

naturally occurring HGF/scatter factor variant

with partial agonist/antagonist activity. J Biol

Chem 1996; 271: 13110-13115.

142. Lokker NA, Godowski PJ. Generation and characterization of a competitive antagonist of human hepatocyte growth factor, HGF/NK1. J Biol Chem 1993; 268: 17145-17150. 143.Hartmann G, Naldini L, Weidner KM, Sachs M, Vigna E, Comoglio PM, Birchmeier W. A functional domain in the heavy chain of scatter factor/hepatocyte growth factor binds the c-Met receptor and induces cell dissociation but not mitogenesis. Proc Natl Acad Sci USA 1992; 89: 11574-11578. 144. Silvagno F, Follenzi A, Arese M, Prat M, Giraudo E, Gaudino G, Camussi G, Comoglio PM, Bussolino F. In vivo activation of met tyrosine kinase by heterodimeric hepatocyte growth factor molecule promotes angiogenesis. Arterioscler Thromb Vasc Biol 1995; 15: 18571865. 145.Date K, Matsumoto K, Shimura H, Tanaka M, Nakamura T. HGF/NK4 is a specific antagonist for pleiotropic actions of hepatocyte growth factor. FEBS Lett 1997; 420: 1-6. 146.Matsumoto K, Kataoka H, Date K, Nakamura T. and Cooperative interaction between chains of hepatocyte growth factor on c-Met

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147. Hiscox S, Parr C, Nakamura T, Matsumoto K, Mansel RE, Jiang WG. Inhibition of HGF/SFinduced breast cancer cell motility and invasion by the HGF/SF variant, NK4. Breast Cancer Res Treat 2000; 1627: 1-10.

Chapter 11 148. Parr C, Hiscox S, Nakamura T, Matsumoto K, Jiang WG. NK4, a new HGF/SF variant, is an antagonist to the influence of HGF/SF on the motility and invasion of colon cancer cells. Int. J. Cancer 2000; 85: 563-570. 149. Kuba K, Matsumoto K, Date K, Shimura H, Tanaka M, Nakamura T. HGF/NK4, a fourkringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice. Cancer Res 2000, 60: 6737-6743. 150. Kuba K, Matsumoto K, Ohnishi K, Shiratsuchi T, Tanaka M, Nakamura T. Kringle 1-4 of hepatocyte growth factor inhibits proliferation and migration of human microvascular endothelial cells. Biochem Biophys Res Commun 2000, 279: 846-852. 151.O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79: 315-328. 152. Cao Y, Chen A, An SSA, Ji RW, Davidson D, Llinas M. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem. 1997; 272: 22924-22928. 153. Lee TH, Rhim T, Kim SS. Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells. J Biol Chem 1998; 273: 28805-28812. 154. Moser TL, Stack MS, Asplin I, Enghild JJ, Hojrup P, Everitt L, Hubchak S, Schnaper HW, Pizzo SV. Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci U S A. 1999; 96: 2811-2816

Chapter 12 GROWTH FACTOR RECEPTORS AND CELL ADHESION COMPLEXES IN CYTOSKELETAL ASSEMBLY/ANCHORAGE Gaynor Davies1, Malcolm D. Mason2 and Wen G. Jiang1 1

Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, and Department of Medicine, Section of Clinical Oncology, Velindre Hospital, Cardiff, UK

Key words: Paxillin, FAK, adherens junctions, cadherin, catenin, focal adhesions and tyrosine phosphorylation Abstract:

Cell adhesions mediate major sites for the facilitation of cytoskeletal anchor protein assembly and organisation within cells, forming tightly regulated membrane domains. These membrane domain anchor proteins, play key roles in providing secure scaffolding/linkage attachment for junction assembly structure, and for the construction of different adhesions into ordered junctional complexes. Adhesion complexes provide pivotal sites for the localisation of a number of proteins implicated in the signal transduction pathway. There is continual interchange and interplay between these junctional complexes within this pathway which regulates the fate of cells by controlling a number of biological and biochemical processes such as, gene transcription, cell proliferation, progression and cell signalling. This review summarises some of the recent observations reported to date, on two main types of cell adhesions (focal adhesions and adherens junctions) and their involvement with growth factor receptors, extracellular matrix proteins and anchor proteins within the signal transduction pathway.

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1. THE ASSEMBLY OF CYTOSKELETAL ANCHOR PROTEINS IN CELL ADHESION AND THEIR INVOLVEMENT IN SIGNAL TRANSDUCTION 1.1. Cytoskeletal anchor protein interactions and cell adhesion complexes There are numerous distinctive extracellular ligands which provide an adhesive surface for cell attachment. These include various components of the extracellular matrix (ECM) and basement membranes together with specialised transmembrane glycoproteins called cell adhesion receptors which mediate cell adhesions. Interplay between extracellular ligands and their associated receptors initiates the formation of two main groups of cell adhesions, termed cell-matrix adhesions and cell-cell adhesive junctions respectively. Intercellular adhesion processes are mediated by four major groups of junctional plaques; adherens junctions (1), gap junctions (2), tight junctions (3) and desmosomes (4-5). These cell adhesions are responsible for facilitating cell-cell adhesion complexes by binding to the cytoskeleton via linkage to anchorage proteins. Attachment to the extracellular matrix is mediated by two specialised types of cell adhesions called hemidesmosomes (6) and focal adhesions (7) respectively. As mentioned previously this review will focus on adherens junctions (cadherins) and focal adhesions (integrins), since both cell adhesions are linked to actin filaments (F-actin) and have in common a number of anchorage proteins for mediated linkage to the actin cytoskeleton (vinculin, catenins, α-actinin, tensin, VASP and zyxin). In addition, cadherins and integrins are two-way signalling receptors which play a vital role in cell adhesion.

Chapter 12 Focal adhesions or focal contacts are mediated by integrin transmembrane receptors which allow cells to attach to the ECM (7-8). Integrins belong to a family of heterodimers which are formed by the non-covalent interactions of their and chains (9). Integrins have short, nonenzymatic cytoplasmic domains (10), enabling them to interact with the F-actin filament via their association with cytoskeletal linker proteins (7, 11-12). Currently, there have been about 8 different chains identified which bind to at least 15 different chains, resulting in 21 possible permutations of individual receptors (10). Focal adhesions are the pivotal anchorage points of cells and are formed by clusters of integrins binding to both the external ECM proteins like fibronectin, as well as to internal cytoplasmic linker proteins such as talin and (7, 1112). Furthermore, vinculin a polypeptide enriched in focal adhesions forms a complex with talin, paxillin and actin, providing stability to the entire structure (7, 11). These actin associated proteins (reviewed in 12) have also been shown to form a focal adhesion complex with the cytoplasmic domain of integrin after their isolation from round cells bound to fibronectin, or to magnetic microbeads coated with the RGD peptide (13-14). The association between these dynamic cytoskeletal structures thus provides a solid scaffold for attachment to the ECM, which is reinforced by connection to an elaborate actin network formed by the actin-binding protein filamin (15). When a focal contact has formed and it matures, the actin filaments elongate to form prominent structures called stress fibres and their organisation within focal adhesions is regulated by phosphorylation of several cytoskeletal proteins mediated

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by integrins (12). In addition, the formation of actin stress fibres are induced by a member of the Rho family of small GTPases which also aid in the regulation of the actin cytoskeleton (16). Stress fibres have been implicated to control the deposition of oriented extracellular matrix, via junctional complexes involving vinculin and α5ß1 integrins; thus promoting the formation of oriented collagen and fibronectin, at and between the cell surface respectively (17). Apart from mediating cell-matrix adhesion, focal contacts have also been implicated as communication ports between the inside and outside of the cell (18). The formation of focal contacts via integrin-mediated cell adhesion, initiates a cascade of tyrosine phosphorylation events in the absence of soluble factors which can in turn, directly lead to the activation of numerous downstream targets such as mitogen-activated protein kinase (MAP kinase) (19-21). It has been postulated that integrins help regulate the MAP kinase pathway through their association with caveolin-1, a transmembrane protein and via the recruitment of an adapter protein called Shc (22). Therefore, signalling by integrin-mediated adhesion is a complex process which can occur at the receptor level, or as previously stated, it can target further downstream via a signalling pathway (23). Furthermore, it appears that focal adhesions can even direct signals back to the extracellular matrix, a process referred to as inside-out signalling (18). Such signalling events contribute to trigger a multitude of cellular events such as differentiation, cell activation and motility (24). Adherens junctions are characterised by the specific interactions they form with the contractile microfilament system (11, 25). They are mediated by classical

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cadherins (P-, N- and E-cadherins) which

function as transmembrane receptors (26)

and interact with a group of cytoplasmic

proteins termed catenins and catenins; or in some cases p120cas); mediating tightly regulated calcium dependent cell-cell adhesion complexes in the N-terminal portion of the extracellular domain (25, 27-30). Classical cadherins were originally classified according to their shared structural and functional similarities. Typically, classical cadherins are composed of five tandem repeats in the N-terminal extracellular domain, and a single transmembrane segment containing a carboxy terminal in the intracellular domain (31-32). The assembly of adherence junctions is particularly important to the biology of epithelial cellcell contact with neighbouring cells, and to the subsequent organisation of tissue formation. This is mediated by the joining of adherence junctions to dense bundles of actin filaments which thus stabilise the whole epithelial architecture. or (plakoglobin) interact more directly with the carboxy-terminal cytoplasmic domain of classical cadherins, while provides mechanical linkage to the actin-containing cytoskeleton; via its association with the N-terminal regions of catenins or plakoglobin) forming a structure termed an adherence junction (12, 26, 33). Furthermore, connection of this membrane-associated complex to the actin cytoskeleton via -catenin is initiated either directly through both its amino and carboxy termini, or indirectly by association with another actin binding protein termed (34-35). catenin is homologous to the actin-binding protein vinculin, a cytoskeletal component critical for the assembly of focal contacts via its association with both and

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talin (36). and plakoglobin share about 65% sequence homology due to the presence of multiple copies of the Drosophila protein armadillo located within their central binding regions (33, 36-37). These armadillo repeats are composed of a 42 amino acid motif which are necessary for binding to the conserved cytoplasmic domain of cadherins (36). In addition to their roles in cell-cell adhesion, and plakoglobin are components of the Wnt signalling pathway but plakoglobin has been shown to have different transactivation capacities compared to thus leading to distinct signalling properties which have been reviewed elsewhere (33, 37-38). Other actin associated cytoskeletal proteins present in the assembly of both adherence junctions and integrin-mediated adhesion include; the ERM (ezrin, radixin and moesin) family proteins (39), tensin and zyxin (11, 40). The ERM family members are responsible for cross-linking actin filaments through their carboxy terminal domain to plasma membranes via their amino terminal domain and a binding partner such as CD44 to form microvilli (39, 41). The activation of ERM proteins as cross-linking components between the actin filament, and the plasma membrane in cytoskeletal organisation, is regulated by downstream signalling transduction involving the small GTPase Rho (39,42). Zyxin is another membrane linker molecule which regulates actin filament assembly by interacting with the actin binding region of while VASP (vasodilator-stimulated phosphoprotein) provides mechanical linkage to the cytoskeleton through attachment to Factin filaments (12, 43). The binding of zyxin to Ena/VASP family members in vitro, also appears to be an important factor in the re-organisation of the actin cytoskeleton during its spreading (44).

Chapter 12 DNA sequence analysis has shown that the amino terminal domain of zyxin is rich in proline and composed of three tandemly arrayed LIM regions (Lin-II, Islet I and Mer 3); which have previously been identified in proteins that play key roles in the regulation of transcriptional and cellular differentiation processes (45). Tensin is a cytoskeletal anchorage protein whose key role is to maintain mechanical tension by connecting F-actin to the plasma membrane, via interactions with other actin associated proteins in order to initiate both focal and cell-cell adhesions respectively (46). Tensin has binding sites for a number of cytoskeletal components which include vinculin, paxillin, phosphatidylinositol-3-kinase (12, 47). (PI-3K), FAK, Src and In addition, tensin also has sequence homologies to several other cytoskeletal proteins including the vinculin binding site of the cell-cell adhesion component (46, 48). Recently, it has been reported that over-expression of tensin results in the activation of the c-Jun amino-terminal kinase (JNK), and p38 signalling pathways respectively; through downstream signalling events via SEK (a kinase involved in the JNK pathway), or through independent activities from members belonging to the small family of G proteins such as Rac and Cdc42 (49). 1.2. The role of cell-cell and cell-matrix junctions in signal transduction During the last few years there have been major advances made in understanding the exact role of cell adhesion receptors, and this has been demonstrated by the central roles they play in signalling pathways, by operating as transducing units. Both and plakoglobin are mainly found at sites of cell-cell contact, linking cadherin

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molecules to F-actin filaments through anchorage by the cytoskeletal protein catenin (25). Furthermore, the transcriptional activator has also been implicated in the negative regulation of cadherin/catenin complexes by controlling the conformation of (50). Evidence to support this comes from induced detachment from the cadherin/catenin complex after phosphorylation by either the epidermal growth factor (EGF) receptor, or by the motogen hepatocyte growth factor/scatter factor (HGF/SF) (51-53). is a central player in signal transduction whose level is elevated in response to signalling from the morphogenic Wnt pathway (54-55). A schematic representation of signalling in the Wnt pathway is summarised in Figure 1. In the absence of Wnt signalling levels are downregulated by phosphorylation from a (glycogen synthase component of the Axin complex composed of APC (adenomatous polyposis coli) and conductin (or Axin). (55-58). The binding of to the Axin complex aids in regulating both its stability and subcellular distribution within the cell (59-60). Furthermore, phosphorylation of cytoplasmic by results in its degradation by the ubiquitin-proteasome pathway, thus levels by keeping an regulating equilibrium between complexed and uncomplexed pools (23). When the cell surface receptor Frizzled is stimulated by the binding of Wnt proteins, it activates the cytoplasmic protein Dishevelled which in turn inhibits the down-regulation of by (38). The inhibition of results in the accumulation of

Chapter 12 within the cytoplasm, where it then goes on to form a complex with the high mobility group (HMG)-domain proteins, Lef/Tcf (Lymphocyte enhancing factor/Tcell factor) family of transcription factors (59). The translocation of this complex into the nucleus activates transription of Wnt target genes through association with a co-activator of Lef termed ALY which aids in the modulation of gene expression (61). The identification of the Wnt target genes c-myc, fra-1, c-jun and cyclin D1 (62-64), has revealed the location of Tcf binding sites within their promoter regions which are believed to act as transcriptional repressors rather than activators (38). Direct evidence for this comes from the phosphorylation of Tcf by the binding of a NEMO-like kinase (NLK), which inhibits the binding affinity of the complex to DNA (65). signalling is also inhibited by transcriptional repression of the Lef/Tcf complex by the binding of corepressor factors TLE/Groucho (66-67). Other nuclear binding proteins which have recently been identified, include Pontin52 and Duplin (38, 68). Pontin52 functions by providing bridging between complexes and transcriptional machinery (68); while Duplin negatively regulates the function of within the nucleus, inhibiting the activation of signalling by competing for Tcf binding regions in catenin (38). Signal transduction events involving cell-matrix interactions become activated upon tyrosine phosphorylation from intracellular signalling pathways which help facilitate cell adhesion, cytoskeletal organisation, cell proliferation and cell differentiation (69-71). Tyrosine phosphorylation events triggered by

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integrin attachment to ECM proteins have been reported for epidermal carcinoma cells, where cross-linking of the integrin results in transient tyrosine phosphorylation of the plasma membranespanning glycoprotein pp130 (72). Additional evidence to support the role of tyrosine kinases in integrin signalling comes from induced cell spreading on ECM proteins (fibronectin, collagen type IV or laminin) via integrin clustering, which in turn increases the tyrosine phosphorylation of a nonreceptor protein tyrosine kinase (PTK) found to co-localize with cytoskeletal proteins talin and paxillin at points of focal contact (69, 71,73-74). The binding regions for talin and paxillin attachment to focal adhesion kinase (FAK or are positioned on the focal adhesion targeting sequence (FAT), which also mediates the subcellular localisation of FAK (75-77). Furthermore, the binding of talin to FAK may also aid in the activation of tyrosine phosphorylation through integrin mediated signalling pathways, via its dependence on the integrity of the actin-cytoskeleton through multiple intracellular signalling pathways which converge to FAK activation and its autophosphorylation (69). Autophosphorylation of FAK creates a binding region for the Src homolgy 2 (SH2) domain of Src kinase (78-79), which in turn, phosphorylates downstream signalling components such as pl30 cas , tensin and paxillin at focal adhesions (71, 80-81). Signalling pathways initiated by the association of Src, FAK, paxillin and after FAK autophosphorylation, have also been implicated in the regulation of cell spreading and mobility upon integrin engagement with ligands via the recruitment of adapter proteins (12, 82).

Chapter 12 is a docking protein which recruits the adapter proteins Crk and Nck (83-85), where the former, is required along with FAK and Src to activate the JNK pathway which regulates the G1 phase of the cell cycle (71). The activation of JNK results in its translocation into the nucleus where it phosphorylates c-Jun, a transcription factor which binds to c-Fos to form a transcription factor complex (AP-1), required for the regulation of genes involved in cell proliferation (71, 86). 2. CROSS-TALK BETWEEN GROWTH FACTOR RECEPTORS AND CELL ADHESION MOLECULES There is considerable cross talk and interplay between cell adhesion molecules and growth factor receptors, which in turn, regulate a number of signalling pathways. Such co-operation can occur at many different levels ranging from EGF receptor signalling within the plasma membraneproximal compartment, to multiple inputs into common pathways such as the FAK, Shc and MAP kinase cascades (54, 71, 87). Much of the evidence to date, for the involvement of growth factor receptors with integrins comes from their localisation within the focal adhesion complex (88) Adapter proteins such as and paxillin which are found to be localised at points of focal contact, become tyrosine phosphorylated upon stimulation with EGF in Rat-1 cells expressing the EGF receptor (89), presumably enabling the recruitment of PTP’s into this adhesion complex (90). A schematic representation of the interplay between growth factor receptors and integrin signalling is shown in Figure 2. The growth factor receptor binding protein 2 (GRB2) initites integrinmediated activation of the MAP kinase

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pathway by binding to phosphorylated FAK, via the creation of an SH2 binding domain by Src kinase (79, 82, 91-92). Another growth factor receptor involved in the regulation of integrin signalling is the platelet derived growth factor (PDGF) receptor. Evidence for this association comes from the phosphorylation of PDGF receptor via its involvement with the integrin, in response to stimulation by PDGF upon integrin engagement onto vitronectin (93-94). Integrin clustering of or its engagememt onto the sub unit ECM proteins has also been reported to result in the initiation of integrin-growth receptor complexes for PDGF in the absence of this factor (95). The motogen HGF/SF has been shown to phosphorylate its proto-oncogene receptor c-Met upon binding, which in turn, stimulates tyrosine phosphorylation of both FAK and paxillin, thus promoting their adhesion to the artificial basement membrane matrigel (96). In addition, HGF/SF upon binding to its c-Met receptor has been shown to result in epithelial cell-cell disengagement, due to tyrosine phosphorylation of the E-cadherin associated protein (52-53). Furthermore, disassembly of this Ecomplex has been shown to result in invasive behaviour in a variety of epithelial derived cells (52, 97). Thus indicating that the interplay and interchange between growth factors and cell adhesion molecules differ in their influence upon adhesive function. 3. PERSPECTIVES Cytoskeletal assembly of actin associated proteins play key roles in the formation of cell adhesion complexes to either neighbouring cells via cadherins, or through attachment to proteins of the ECM via integrins. A fundamental requirement for the assembly of this cytoskeletal

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scaffold is to provide anchorage to the Factin filament, thereby allowing communication via integrated signalling between integrins and growth factor receptors. This signalling regulates a number of biological processes such as growth, differentiation and cell death. The binding of soluble growth factors to their corresponding transmembrane receptors are thought to represent critical sites for the initiation of signal transduction pathways (14). Thereby, unmasking tyrosine kinase activity within the cytoplasmic domain of these receptors and by creating additional binding sites that facilitate the direct association with other intracellular signalling molecules such as (14, 98). Integrins have short cytoplasmic domains exhibiting no endogenous kinase activity, and they rely on integrated signals from soluble growth factors; components of the ECM upon integrin engagement; or after integrin clustering (9). Therefore, integrins themselves act as transducers by activating intracellular signalling pathways upon their attachment to proteins of the ECM. The focal adhesion complex (FAC) provides an insoluble scaffold immobilising growth factor receptors, by bringing them into close proximity within the FAC, which in turn, facilitates crosstalk between multiple signalling pathways that would otherwise function at many sites within the cell (14). The involvement of growth factor receptor signalling on cell-cell adhesion complexes remains unclear at present. However, it has been reported that the EGF receptor can in fact reduce the formation of cell-cell adhesion complexes, by inducing phosphorylation of which results in its subsequent detachment from the cadherin/catenin complex within the cytoskeleton (51). Therefore, we cannot rule out the possibility that

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cadherins may be involved in much more complex signalling pathways than originally thought, which may in fact, parallel those governing integrin mediated signalling events. The involvement of integrin linked kinase (ILK) on translocation into the nucleus is just one example of cadherin/integrin crosstalk (99) which may impinge on growth factor receptor signalling interactions. Certainly

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Index

12(S)-HETE, (12(S)hydroperoxyeicosa176 tetraenoic acid 3T3 fibroblasts, ras-transformed 120, 136, 254

ALK5, see also activin receptor-like kinase 5 197, 198, 202 ALS 110, 123 AMF, autocrine motility factor 176 AML 2,10,12, 192, 222 Anchorage-independent growth, prostate cancer cells 99, 122 Androgen receptor 99, 100 Androgen-induced growth, prostate cancer cells 99 Angiogenesis, 54, 141, 176,224, 226, 232,241, 250 Angiogenic factors 52, 54, 55, 57, 59, 61, 140-143, 196,232 Angiostatin 61, 141 Anoikis 176 Antimetastasis options 185 Antisense oligonucleotide 57, 121, 155 Antisense, 35 AP1 family 191 APC, see adenomatous polyposis 204, 281, 282 coli 204 Aphidicolin, cell cycle synchronisation 38 Apoptosis.. 11, 15, 27-29, 33, 41, 47, 48, 61, 83, 89, 114, 122, 125, 126, 141, 146, 169, 171, 175, 176, 191, 193, 195, 244 168 Artherosclerosis

A

Activation-induced cell death 28 (AICD) 188, 202, 206 Activin Activin Activin receptor-like kinase 1 197, 203 (ALK1) Activin receptor-like kinase 5 197 (ALK5) 206 ActRIB 206 ActRIIA 206 ActRIIB Adenomatous polyposis coli (APC) 204, 281, 282 Adenoviral vector 79, 80, 83, 86, 87 Adherens junctions 277, 278, 286 83, 85, 87 AdmIL-12 94 Adrenocortical cells AICD, see activation-induced cell 28 death AIDS-related Kaposi sarcoma (KS) 173 cells, the role of PDGF 116, 143, 169, 244,283 Akt ALK1, see also activin receptor-like kinase 1 190, 197, 198,203,212

291

292

Index

Articular chondrocytes, see also 67 bone marrow Stromal cells Astrocytes 8, 94 AT-3 cell line 95 ATF-2 192 Autocrine IL-2 pathway 39, 47 Autocrine regulation, IGF-I 13,16, 123,233 Autophosphorylation, growth factor receptor 168,284 Axin 281,282 233 AZ521 gastric cancer cell B

BAEC 135 Barmotin/7H6 245 BB-94 138, 145 BDEC, bone-derived endothelial cells 71,75 BHK cells 136 Biliary adenocarcinomas, role of TGFß . 195 Bladder carcinoma 100, 248, 255 BMP, see also bone morphogenetic proteins .. 188, 190, 198, 200, 207, 212 BMPRIA 206 BMPRII 206 Bone marrow stromal cells .... 18, 67, 76,77 Bone metastasis 67 Bone morphogenetic proteins (BMP) 188 Bone remodelling 68 71 Bone-derived endothelial cells Bovine aortic endothelial cells (BAEC) .135 Breast cancer.... 10, 13,67, 118, 128, 129, 183,248,255,259 BT20 cell line 10,13

C

C6 glioblastoma cells 125 Cadherin 287 Caenorhabditis elegans 188, 201, 211 Calcitonin-gene-related-peptides ..59 Capan-1 cell line 58 Capillary endothelial cells, angiogenesis 101, 140, 177 Cardiotropin-1 1 Catechins 179 Catenins 245, 278, 279, 287, 288, 289 CathepsinD 100,128,140 CBP/p300 193 CCA, see cell cycle arrest (CCA).27, 33,48, 193, 196 CD4 positive T cells 9, 82, 83, 86 CD44 61,175,251,273,280 CD68 positive cells 83 CDS positive T cells 9, 82, 83, 85, 125 Cdc25A 193 Cdc42 244,250,280 CDK, see cyclin-dependent kinase 27,41,44,47 CDK4 193 Cell adhesion molecules 277, 278, 280 Cell cycle arrest (CCA), IL-2 induced..27, 33, 48, 193, 196,214 Cell cycle progression, IL-2 41,47, 179, 193, 194, 197,203 Cervical cancers 59 c-fos 14,102,116,191,192,207 Chemokine ...54, 55, 62, 63, 64, 118, 119, 134, 135, 136, 145 Chemotactic factor, TGFß as 199, 201 Chemotaxis, cancer cells 176 Chicken chorioallantoic membrane (CAM) 119,142,226

Index

CHO cells 136 248 Cholangiocarcinoma Choriocarcinoma11, 15, 58, 64, 173, 183 chronic myelomonocytic leukemia 174 (CMML) Ciliary neurotrophic factor (CNTF).. 2, 16, 23, 24, 68 191, 192, 207, 282 c-jun Clonal expansion, cancer cells 79, 120, 143 c-Met proto-oncogene, HGF/SF receptor 241-245, 251-261, 267, 268, 269, 285 CMML, see chronic 174 myelomonocytic leukemia 123, 125, 156 c-myb 102, 114, 178, 282 C-myc CNTF, see ciliary neurotrophic factor 2, 19, 68 174, 202, 284 Collagen type I 284 Collagen type IV 250 Collagenase-1 202 Collagenases 94 Collagens 16 COLO-16 SCC cell line 282 Conductin Confocal microscopy 45, 47 75 COX-1 75 COX-2 58 CRX2 receptor 121 CSF-1 CTAP-II, connective tissue 54 activating protein-2 () CTL, see also cytotoxic T82, 85, 198 lymphocytes 52 CX3C 51, 52, 54, 55, 61 CXC 55, 58 CXCR1 55, 58 CXCR2 193, 282 Cyclin D

293

Cyclin-dependent kinase (CDK) 27, 38, 41, 44, 47 Cyclins 38, 41, 282 Cyclooxygenase-1 (COX-1) 75 Cyclosporin A 41 Cytokeratin-containing cytoskeleton 207 Cytoskeleton 278, 285 Cytotoxic T-lymphocytes (CTL) 82, 85, 198 Cytotrophoblasts 173 D

Dendritic cells 75, 78, 80, 86 Dermatofibrosarcoma 174 Desmosomes 278, 286 D-factor, see leukemia inhibitory factor 2, 3, 24 DIF, see also leukemia inhibitory factor 2, 3 Differentiation factor (D-factor), see alsoleukemia inhibitory factor2, 8, 17, 78 Differentiation inhibitory activity, see also leukemia inhibitory factor 2 Differentiation-inducing factor (DIF), see also leukemia inhibitory factor 2 Differentiation-retarding factor (DRF), see also leukemia inhibitory factor 2 Dishevelled 282 DNA mismatch repair (MMR) 194, 208 Dpc4, deleted in pancreatic cancer 195, 204, 220 Dpp, TGF homology protein 201, 211 DRF, see leukemia inhibitory factor 3

Index

294

Drosophila melanogaster 188, 206, 211 DU-145 cell line 93, 97, 135, 144, 154 Ductal breast carcinoma 139 Duplin 281,282 E

E-cadherin 175, 207, 245, 248, 249, 279,285 ECM, extracellular matrix 56,133, 134, 138, 139, 141, 145, 187, 196, 200, 201, 242, 245, 248, 249, 257, 268, 278, 283, 284, 285 EGF, see epidermal growth factor 105, 119, 120, 121, 150, 152, 153, 213,225,273,282,284,285 EGFR, see also Epidermal growth factor receptor 29, 61, 118, 119, 207 Egr1 142 Embryogenesis, the role of FGF101,, 131, 197, 204, 206, 224, 226, 232, 268 Embryogenesis, the role of TGFß 101, 131, 197, 204, 206, 224, 226, 232, 268 EMT, see also epithelialmesenchymal transition 187, 201, 204, 205, 207, 210 ENA-78, epithelial neutrophil activating protein-78 54, 55, 63 Endocytosis, IL-2 48 Endogenous IL-2.. 27, 39, 41, 44, 45, 47, 48 Endometrial adenocarcinoma 255 Endometrial cancer 142, 165, 195, 215 Eosinophils 9,17,199,201 Ependymomas 172 Epidermal growth factor (EGF) 29, 61,65, 118, 119, 120, 121,207,

213, 225, 271, 272, 273, 282, 284, 285 Epidermal growth factor receptor (EGFR).29, 61,65, 118, 119, 154, 207 Epithelial-mesenchymal transition (EMT) 187,201,205,207,208, 211 ERM proteins, see also ezrin, radixin, and moesin 280 ER-negative breast cancer cells 117 ER-positive breast cancer cell lines 117, 118, 119, 128, 129 Esophageal cancer 248 Estrogen receptor 13,108,117,152, 193, 207 Estrogen receptor (ER) 13, 117-119, 128-130, 144, 145, 193,207, 213, 222 Evi-1 192, Ewing-sarcoma (EWS) 175 Extracellular matrix (ECM)4, 59, 61, 93,94,99, 100, 131, 133, 137, 141, 146, 187, 201, 202, 205, 214, 216, 219, 242, 244, 245, 250, 251, 277-279 94 Extracellular matrix molecules Extravasation , tumour cells 176, 197 Ezrin 245, 250, 272, 280 F

FAK, focal adhesion kinase133, 134, 136, 146, 245, 249, 250, 272, 277, 280, 283, 284, 285 FAK, seel focal adhesion kinase 133, 134, 136, 146, 245,249,250, 277, 280, 283, 284, 285 Familial adenomatous polyposis (FAP) 204 FAP, see also familial adenomatous polyposis 204 FAS 114

295

Index

FASTI/2 192, 193 Fer kinase 169, 170 FGF receptor93-95, 97, 99, 101, 102, 208 FGF-1 93, 99 FGF-2 105 FGF-3 97 FGF-4 105 FGF-7 93, 94 FGF-8 93-95, 99 FGF-9 16-22, 48, 83, 85, 88,99, 100, 113, 115, 129, 138, 139, 144, 147, 179, 194, 237, 250, 254, 285, 286, FGF-10.22, 48, 139, 144, 147, 179, 194, 237, FGFR-1, see also flg gene 94, 95, 97, 103 FGFR-2 94, 95, 97, 101 FGFR-3 94 FGFR-4 94, 102 Fibroblast-derived epithelial cell growth factor, see also hepatocyte growth factor 242 Fibroblast-derived epithelial morphogen, see also hepatocyte growth factor 242, 270 Fibronectin.201, 202, 208, 278, 279, 284 Fibrosarcoma 164 Fibrotic conditions, the role of PDGF 168 FK506 41 flg gene 94 Flk-1 224 Flow cytometry, cytokine analysis 29,31, 33, 35, 39, 41, 47 Fluorescence intensity (MFI), IL-2R detection 29 Focal adhesion kinase (FAK) 116, 133, 134, 136, 146, 160, 245, 249, 250, 272, 277, 280, 283, 284, 285

Forkhead transcription factors.... 192, 193 Fra-1 182 FYVE domain protein 190 G

G proteins 118, 280 Gab-1 244 Gallbladder cancer 248 Gap junctions 278 GBK-1 gall bladder cancer cell 15 GCP-2, Granulocyte chematactic protein-2 54 Gene therapy 79 Gene therapy, adenoviral..79, 80, 83, 86, 87 Gene therapy, IL-12 83, 85, 86, 87, 88 GH receptor 115 coupled chemokine receptor 118, 119, 135, 137 Gleason score 97, 99 Gli3 192, 222 Glioblastoma 55, 121, 142, 143, 174, 175, 178, 198, 248,256,259, 271 Glioma 248, 255, 259 Glioma-derived angiogenesis inhibitory factor 142 Glucocorticoid receptor (GR) 191, 213 Glycosylphosphatydil inositolanchored 137 GM1 137 gp130 1, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 19, 22, 23, 24, 25, 67, 68, 69, 76 Gp130 5, 6, 9-15, 16, 19, 67, 68, 69, 76 GPCR, see G-protein-coupled receptors 118, 119

296

Index

G-protein-coupled receptors 55, 118, 152, 153 GR, see also Glucocorticoid receptor 19, 20, 63, 76, 77, 191, 192 Granulosa 94 Grb2 116, 170, 171 Growth and differentiation factors (GDFs) 188 54, 55, 58 GTPase activating protein... 169, 244 GTPases 169, 213, 244, 279 H

Haemorrhagic telangiectasia (HHT) 197 HDAC, see also histone deacetylases 191, 193 Heparanase 100, 176 Hepatocellular carcinoma 248, 255, 259 Hepatocyte growth factor/scatter factor (HGF/SF)52, 141, 242, 269, 282 Hepatocyte-stimulating factor III (HSF III), see leukemia inhibitory factor 2 Hepatoma. 11, 15, 24, 142, 143, 153, 202, 251 Hepatoma cells 15, 24, 142, 143, 202, 251 HER-2 82, 89 Herpes Simplex Virus thymidine kinase + ganciclovir 79, 87 HGF/SF, see hepatocyte growth factor/scatter factor HGF-antagonist 241, 242, 260, 261, 267, 268, 269 HGF-c-Met receptor coupling 241, 242, 244, 260 HHT, see also Haemorrhagic telangiectasia 197 Histiocytomas 178

Histone deacetylases (HDAC) 191 hMLHl 194 hMSH2 194 HNPCC, see also non polyposis colon cancer 194, 195 Hoxc-8 192 HPV16v 199 HR gastric carcinoma cell line 18, 20, 35, 41 HRE, hypoxia response element 142, 143 HSF III, see leukemia inhibitory factor 2 HSV-tk+GCV, see Herpes Simplex Virus thymidine kinase + ganciclovir 79, 87 Hypercalcaemia, role of TGF 200, 218 Hypoxia 64, 142, 176 I

ICAM-1, see also intercellular cell adhesion molecule 14 IGF 29, 48,105, 108-123, 126-146 IGF antagonists 117 IGF receptors 108, 111, IGF-1R 108-121, 123, 124, 125, 129, 130, 133-135, 137, 143-147 IGF-2R109-113, 115, 117, 121, 124, 140, 143 IGFBP 108-113, 117, 123, 127-130, 138, 142, 144-147 IGFBP-1 110, 117, 128, 142 IGFBP-2 128, 142, 144 110-113, 117, 123, 128, IGFBP-3 138, 142 IGFBP-4 128 IGF-I 48, 108-119, 123-131, 134145, 147, IGF-II 108- 128, 138, 140, 142, 143, 144,147

297

Index

IL-1, see interleukin-1 1, 7, 9, 10, 12, 15, 18, 20-22, 24, 52, 56, 59, 63, 67-75, 80, 82, 83, 85, 86, 87, 88, 176, 198, 200, 225, 255, 257 IL-2, see interleukin-217, 28-31, 33, 35, 38- 45, 47, 48, 56, 63, 135,

161, 198, 199 IL-2R 28, 35 28, 29, 31, 45 IL-2R IL-2R 27, 35 21, 22, 56, IL-4, see interleukin-4 115, 199 IL-6, see interleukin-6 4, 5, 8, 9, 12, 14, 16, 67, 68, 71, 72, 73, 165, 176 IL-8, see interleukin-8 52, 54, 55, 57-65, 135, 141, 142 IL-8RA 55, 58 IL-10, see interleukin-10 21 IL-11, see interleukin-11 67, 68, 7176, 200 IL-12, see interleukin-12 80, 82, 83, 85, 86, 87, 88 Inositol(l ,4,5) trisphosphate 169 Insulin receptor substrate-1 (IRS-1) 113 Insulin-like growth factors 29, 48, 105, 108-146, 147, Integrins 171, 285 Intercellular cell adhesion molecule (ICAM-1) 14, 89 Interferon 23, 24, 54, 82, 105, 199 Interferon- induced monokine54, 61 9-12, 15, 52, 56, 59, Interleukin-1 67, 68, 71-75, 79-89, 90, 176, 198, 200, 217, 225, 255, 257 Interleukin-2 27-49, 50, 56, 135, 161, 198, 199 Interleukin-4 21, 22, 56, 115 Interleukin-6 20 Interleukin-8 51, 52, 54-63, 135, 141, 142

Interleukin-11.24, 67, 68, 71, 72, 73, 74, 75, 76, 200 Interleukin-12 79, 80, 82-88 Internalisation, exogenous IL2 45 Intracellular calcium, cellular signalling 94, 100 Intravasation, tumour cells .119, 154, 196 Invasion, cancer cells 62, 162, 176, 241, 244, 246, 258 IP-10, interferon inducible protein 54, 61 IRS-1 ..113, 115-118, 122, 123, 129, 137 IRS-2 113, IRS-3 115 IRS-4 115 J

Jak-STAT pathway, LIF 6 JNK 169, 219, 280, 284, 287 JNK, c-Jun N-terminal kinase 169, 219, 280, 284, 287 K

KATOIII gastric cancer cell 233 KDR 223-225 Keratinocyte 201, 202, 256 Keratinocyte growth factor93-95, 99, 101 KGF, see Keratinocyte growth factor Ki-67 44 Kidney development, the role of PDGF 168, 179 Kidney tumour 102 Knock-out mice, LIF 7, 8 L

LAK cells, see also Lymphokine activated killer cells 82, 198, 199

298

Index

Lamellipod extension, in cell 136 motility Laminin 94, 284 Large granular lymphocytes (LGL).

199 LEF, see also Lymphocyte enhancing factor/T-cell factor 281,

282 Leiomyosarcomas Leukaemia inhibitory factor (lif), 17, 19, 21, 23, 24, 25, 68 Leukemia i, 2, 3, 8-10, 12, 16-25, 48, 76, 121, 152, 248, 255 Leukemia inhibitory factor (LIF)1, 3,

16, 18-24

272 MCF-7 breast cancer cell 10, 13, 117-119, 134-140 MCP-1, see monocyte chemoattractant protein 63, 135 MDCK renal epithelial cells 244, 250 Medulloblastomas 24, 172, 182 Melanoma 2, 11, 14, 17, 18, 24-29, 48, 56-59, 71, 88, 121, 136, 139,

141, 173, 177, 233, 249, 255

44 LI, labeling index, Ki-67 LIF, see also leukemia inhibitory 17, 19, 21, 23, 24, 25, 68 factor 2,4-15 LIFR, LIF receptor LNCaP prostate cells 25, 90, 95, 97,

100 Lung cancer ..11, 15, 57, 59, 67, 85, 88, 97, 111, 113, 121, 142, 173, 177, 179, 195, 203, 210, 244, 256, 258, 261, 265-268 Lymphocyte enhancing factor/T-cell factor (Lef/Tcf) 282 Lymphokine activated killer cells 82, 198, 199 Lymphokine activated killer cells (LAK) 198 10, 22, 248, 255 Lymphoma M Macrophages

MAPK 5, 116-118, 133-135, 146, 226, 283 Matrix metalloproteinase (MMP).57, 58, 61-64, 93, 99-101, 105, 111, 137-146, 176, 218, 248, 249, 250,

8, 12, 56, 59, 83, 86,

202 Mad homology domain 1 (MH1) 190 MAEC3, mouse aortic endothelial cell line 71 Mannose-6-phosphate 112 Map kinase 190

Melanoma-derived lipoprotein lipase inhibitor (MLPLI), see leukemia inhibitory factor 2, 14 Membrane ruffling 133, 159 Meningiomas 172, 174 Mesoderm induction, 206, 207, 210 Mesothelioma 121, 172-175, 233, 249, 251, 255, 259 Metastasis 51, 184, 269, 272, 275 Metastatic-type calcifications, bones

9 Methy cyclodextrin 137 MGSA, Melanoma growth stimulatory factor 54, 55, 63 MH1, see also Mad homology domain 1 (MH1) 190, 192 MHC 82, 85, 88 Microsatellite instability (MSI) .. 194 MIG, interferon induced monokine 54, 61 Mitogen-activated protein kinase .. 5, 94, 100, 102, 115-118, 133, 134, 226, 272, 279, 283 Mitotic index 47, 129 MKN-28 gastric cancer cell 233 MKN-45 gastric cancer cell 233

299

Index MLPLI LIF, see leukemia inhibitory factor 3 MLT3 breast cancer 175 MMP-1 58 100-111,138-141,149, 248, 250 MMP-2 58, 64, 100-111, 139, 149, 163, 248, 250 MMP-3 138, 250 MMP-9 57, 101, 138, 139, 144, 149, 162, 218, 250 MMPs 57, 61, 99-111, 137, 138-141, 143-146, 248-250 MMR, see also DNA mismatch repair 194 MMTV 139 Moesin 280 Monocyte chemoattractant protein (MCP)-l 63, 135 Monocytes 2, 3, 28, 54, 59, 67, 80, 199, 201, 204 Motility, cellular 176 Msa, see Multiplication-stimulating activity 108 MSF, motility stimulating factor 176 MSG1 192, 222 MSI, seel also microsatellite instability 194, 195 MT-MMP-1 250 Multiplication-stimulating activity (MSA) 108 Mutation, receptor 194 94, 174, 178, 256 myc Myelosclerosis 9 Myristylation, src family 170 N NAP-1 55 Natural killer (NK-) cells49, 82, 135, 161, 198 Natural killer cells31, 38, 41, 56, 8285, 88, 135, 198 NBT-II cells 100, 207

Neoplastic transformation, the role of 101,120, 174 Neovascularisation 52, 59 neu/HER-2 82 Neuroepithelioma 12 Neutrophils 8, 51-54, 199 NF 191, 199 Nicotine-induced carcinogenesis 175 NIH 3T3 cell line 179, 182, 213, 274 NK cells, see also Natural killer (NK-) cells 31, 38, 41, 54, 56, 82, 83, 85, 88, 181, 198, 199 NK1 260, 275, 276 NK2 260 NK3 260 NK4.. 241, 242, 257, 260- 265, 267, 268 NKPS gastric cancer cell 233 Nodal related genes 206, 210, 221 Non polyposis colon cancer (HNPCC) 194 NUGC-3 gastric cancer cell 233 O OAF, see leukemia inhibitory factor 2 ODF/OPGL 75 Oligodendrogliomas 172 Oncostatin M (OSM) 2, 14, 68 Oropharynx tumours 31 OSM, oncostatin-M 2, 14, 68 3, 5, 13, 67, 68, 71, 75, Osteoclast 200 Osteoclast, LIF 2,13, 23, 71, 200 Osteolysis 14, 71 Osteotropic factors 67 Ovarian cancer 249, 255 P p130(cas)

245 202

300

280-284 193 p21 38, 41, 82, 115, 154 T27, 41, 193 P53 90, 111, 123, 128, 141-145, 176,

193 116, 190, 193 Paclitaxel 58 PAI-1 140 PAI-2 139 Plasminogen activator inhibitor I163, 202 Pancreatic carcinoma 15, 121, 198, 250 Papilloma virus E5 protein 121, 141 Papillomas 203 Paracrine regulation, IGF-I 13, 16, 23, 51, 57-62, 82, 119-129 144147, 167- 179, 198, 202, 224, 226, 232 Parathyroid hormone 200 Parathyroid hormone related peptide (PTHrP) Parathyroid hormone related peptide 200 Paxillin..34, 160, 202, 245, 277-278, 280-285 PC-3 cell line 11, 14, 25, 57, 93, 97, 261 PD-ECGF, platelet-derived endothelial growth factor 141 PDGF, see platelet-derived growth factor 52, 112, 118, 121, 135, 141, 142, 167-179, 225, 257 Pepsinogen C 137 Pertussis toxin 119 PF-4, platelet factor-4 (PF-4) 54 PG cell line 15 73 Phosphatidyl inositides 94 Phosphatidyl inositol-3 kinase 114116, 122, 133, 280, 283

Index

phosphatidylinositol-4, 5bisphosphate 3-kinase (PI-3 kinase) 244,250 Phosphorylation of insulin receptor 115 Phosphotyrosine binding domains, PDGF receptor 116,169 PI-3 kinase, phosphatidylinositol-4, 5-bisphosphate 3-kinase 244, 250 PI-3K 114 Placenta growth factor (PIGF) 225 Plakoglobin 245,279-281 Plasmin 139,250 Plasminogen 202, 242, 269 Plasminogen activator inhibitor I (PAI-I) 202 Platelet aggregation, PDGF 168 Platelet-derived growth factor 20, 52, 112, 118, 121, (PDGF) 135, 142, 160-180, 225, 257, 285 PLC 169, 244 168 Pleckstin homology, PDGF PNT1acell line 99 Prostate cancer 14, 80, 90, 95, 249, 255 Prostate specific antigen (PSA) 80, 129, 138, 158 Protein kinase C 94, 100-104, 116, 169, 176 protein phosphatase 2A 190, 193 PTEN 143, 166 PTH 75 PTHrP, see also Parathyroid hormone related peptide 73, 200 R

R3327 cell line 95 R3327, androgen-responsive cell line 95 Rac 244, 250, 280 Radixin 280 RANTES 64,119,135

Index

301

Rapamycin 41 Ras oncogene 134, 153, 155, 160, 170, 176, 208, 244, 250, 270 Ras-ERK pathway, in HGF-c-MET signalling 244 Ras-GAP 244 RB, see also retinoblastoma gene 48, 58, 104, 183, 193 RC29 renal cancer cells 102 RCC tumour cell line 33 Receptor regulated Smads (RSmads) 188 Receptor tyrosine kinase (RTK) 118 Renal cell carcinomas 14, 101 249, 255 Retinoblastoma gene, RB 193 RGD peptide 176, 278 Rhabdomyosarcoma 121, 249 Rho family ...78, 169, 244, 250, 270, 271, 279 Ribosomal S6-protein, in signaling 190 RM-9 prostate cancer model 83, 86 RM-9 tumour cells 83 RNase protection assays, IL-2R 31 R-Smads, see also receptor regulated Smads 188-192 RUNT transcription factor 192 S

Saos-2 cells 71 SARA, see also Smad anchor for receptor activation 190 SBE, see also Smad Binding Element 190, 193 Scatchard analysis, IL-2R 31 Scatter factor, see also hepatocyte growth factor 242 Schwann cells 172 SCID mice 57, 203 SEK 280

SF, scatter factor, see also hepatocyte growth factor 242 SH2 domains 115,116,123,170, 174, 244, 284 SH2 tyrosine phosphatase 116, 134, 170 SH3 domains 170 Shedding of antigen 85 SHP-2 116, 134, 170 Shp-2 protein tyrosine phosphatase 244 SiHa cervical cancer cells 175 SIP1 192 SK-BR3 cell line 13 Ski / SnoN 192 Smad 1 190, 192 Smad 2188-190, 196- 198, 200, 204207, Smad 3 190-193, 196-199, 204-206 Smad 4 190-195, 198, 204-206 Smad 5 190 Smad 6 190 Smad 7 190, 199 Smad anchor for receptor activation 190 Smad Binding Element (SBE, 5’AGAC-3’), 190 Smad family 188 Smooth muscle94, 100, 131, 179182,196 SnoN 193, 199 SPBP transcription factor 176 Splenocytes 204 SPRE, PDGF-responsive element 176 Squamous carcinoma 8, 31, 63, 97, 103, 204, 250 Squamous carcinoma of the head and neck (SCCHN) 31, 32, 33, 35, 38,44 Src homology 2 (SH2) domains 115, 168-176, 244, 283-285

302

Index

Src kinase 168-171,176, 244- 246, 280 STAT 5, 171 54 Stromal cell-derived factor-1 Stromal cells 56, 127, 139 250 Stromelysin-1 174 Suramin SV40 large T antigen 10, 13, 121, 123-126, 138 Synoviocytes, see also bone marrow stromal cells 67 T

T box transcription factor 82 T10 cells 71, 72, 177 T-47D cell line 13 TAK1, see also activated kinase 190 Tamoxifen 130 TATA elements, LIF gene 4 T-cell growth factor (TCGF), see interleukin-2 28, 49 TCF, see also lymphocyte enhancing factor/T-cell factor 288 28, 49 TCGF, see interleukin-2 Tensin 278, 280, 284 210 activated kinase (TAK1) 190 family receptors 188 188 type I receptor type II receptor (TpRII)v 194 -/- mice 203 2, 68, 117, 256 TGIF 192 TGN, trans-Golgi network 136 Th1 cells 82, 85 Th2 cells 82 101, 141, 198 Thrombospondin Thyroid-stimulating hormone 112, 120, 124 Tight junctions 278 TIMP-1 138

TIMP-2 138, 145 TMK-1 gastric cancer cell 57, 233 TNF 82 Transforming growth factor 90, 105, 158,211 Transforming growth factor 2,68, 117,256 Trophoblast development, LIF 5 TSU cell line 14 Tumor cytotoxic factor, see also hepatocyte growth factor 242 Tumour infiltrating lymphocytes 82 Tumour stroma 187, 194, 196, 197, 210 Tumour-associated hypoglycemia 108, 123, 156 TUNEL assays 33, 265 Tyrphostins 179 see also type II receptor 194, 195, 203, 208, 210 U uPA

95, 139, 250

uPA receptor uPA, see also Urokinase-like Plasminogen activator Urokinase Urological Cancers. Uveal melanoma

250 202 202 93 249

V

Vascular endothelial cells 52, 58, 140, 141, 244, 260 Vascular endothelial growth factor (VEGF) 52, 54, 57, 101, 141-146, 176, 197, 203, 224- 226, 232-237, 251, 263, 267 VASP 278, 280 VDR 192 VEGF, see Vascular endothelial growth factor

Index

VEGF-B 225, 237 VEGF-C 224-226, 232-237 VEGF-D 225 VEGFR-2 224-226, 233, 237 VEGFR-3 223-226, 232-236 v-fms oncogene 120 v-H-ras 120 Vimentin 205, 208 Vinculin 134, 278- 280, 283 Vitronectin 133-140, 202, 285 Vitronectin receptor 134, 138-140, 285 v-K-ras 120 v-mos oncogene 120 v-ras 120 v-sis oncogene 172

303 W WEHI 3BD+cell line 12 Wilms tumour 175 Winged-helix transcription factor 192 Wnt signalling 280-282 Wobbler mouse model, LIF 8 WT1 tumour suppressor genes 123, 156, 175 X Xenopous laevis

188, 206 Z

Zn finger transcription co-repressor 192 Zonula occludin-1 245 Zyxin 278, 280