Molecular Mechanisms in Gastrointestinal Cancer

C O M PA N Y

R.G. LANDES

MEDICAL INTELLIGENCE UNIT

12

B. Mark Evers EVERS

Molecular Mechanisms in Gastrointestinal Cancer

MIU

12

Molecular Mechanisms in Gastrointestinal Cancer R.G. LANDES COM PA N Y

MEDICAL INTELLIGENCE UNIT 12

Molecular Mechanisms in Gastrointestinal Cancer B. Mark Evers, M.D. The University of Texas Medical Branch at Galveston Department of Surgery Galveston, Texas, U.S.A.

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

MEDICAL INTELLIGENCE UNIT Molecular Mechanisms in Gastrointestinal Cancer R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright ©1999 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-590-0

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Molecular mechanisms in gastrointestinal cancer / edited by B. Mark Evers. p. cm. -- (Medical intelligence unit) ISBN 1-57059-590-9 (alk. paper) 1. Gastrointestinal system--Cancer--Molecular aspects. I. Evers, B. Mark, 1957-. II. Series. RC280.D5M635 1999 616.99'43307--dc21 98-43469 CIP

MEDICAL INTELLIGENCE UNIT 12 PUBLISHER’S NOTE

Molecular Mechanisms in Gastrointestinal Cancer

Landes Bioscience produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental. The authors of our books are acknowledged leaders in their fields. Topics are unique; almost without exception, no similar books exist on these topics. Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience. Most of our books are within Branch 90 to 120atdays of receipt of The University ofpublished Texas Medical Galveston the manuscript. WeDepartment would like to thank our readers for their of Surgery continuing interest Galveston, and welcomeTexas, any comments U.S.A. or suggestions they may have for future books.

B. Mark Evers, M.D.

Michelle Wamsley Production Manager R.G. Landes Company

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

CONTENTS 1. Growth Factors, Hormones and Receptors in GI Cancers ..................... 1 B. Mark Evers and Courtney M. Townsend, Jr. Growth Factors ........................................................................................ 1 GI Hormones and Receptors .................................................................. 8 Future Perspectives and Therapeutic Implications ............................. 12 2. Signaling Pathways in GI Cancers .......................................................... 21 Richard A. Ehlers Protein Kinase A .................................................................................... 21 Protein Kinase C .................................................................................... 25 Protein Tyrosine Kinases ...................................................................... 28 Mitogen-Activated Protein Kinases ...................................................... 30 Conclusions ........................................................................................... 32 3. The Role of the COX/Prostaglandin Pathway in GI Cancers ............... 37 Richard T. Ethridge The COX/Prostaglandin Pathway and Colorectal Cancers ................. 38 Clinical Trials Using COX Inhibitors ................................................... 43 Conclusions and Future Perspectives ................................................... 43 4. Cell Cycle and Apoptosis Regulation in GI Cancers ........................................................................................... 49 Harry T. Papaconstantinou and Tien C. Ko Cell Cycle ............................................................................................... 49 Cell Cycle and the Induction of Cancer ............................................... 52 Apoptosis ............................................................................................... 58 Apoptosis and Cancer Induction .......................................................... 61 Metastatic Disease ................................................................................. 67 Cancer Treatment Modalities ............................................................... 68 Conclusions ........................................................................................... 71 5. Oncogenes and Tumor Suppressor Genes in GI Cancer ...................... 79 Mimi Kim and B. Mark Evers Colorectal Cancer .................................................................................. 80 Pancreatic Cancer .................................................................................. 85 Gastric Cancer ....................................................................................... 88 Conclusions ........................................................................................... 89 6. Angiogenesis and GI Cancer ................................................................... 95 David A. Litvak Development of the Angiogenic Phenotype in Tumors ...................... 95 Mediators of Angiogenesis .................................................................... 99 Genetic Alterations and Angiogenesis ................................................ 100 Angiogenesis and Cancer Metastasis .................................................. 101 Angiogenesis as a Prognostic Indicator for Cancers .......................... 103 Novel Angiogenesis-Based Chemotherapeutic Agents ...................... 103 Conclusions ......................................................................................... 105

EDITORS B. Mark Evers, M.D. The University of Texas Medical Branch at Galveston Department of Surgery Galveston, Texas, U.S.A. Chapters 1 and 5

CONTRIBUTORS Richard A. Ehlers, M.D. The University of Texas Medical Branch at Galveston Department of Surgery Galveston, Texas, U.S.A. Chapter 2

David A. Litvak, M.D. The University of California Davis-East Bay Department of Surgery Oakland, California, U.S.A. Chapter 6

Richard T. Ethridge, B.A. The University of Texas Medical Branch at Galveston Department of Surgery Galveston, Texas, U.S.A. Chapter 3

Harry T. Papaconstantinou, M.D. The University of Texas Medical Branch at Galveston Department of Surgery Galveston, Texas, U.S.A. Chapter 4

Mimi Kim, B.A. The University of Texas Medical Branch at Galveston Department of Surgery Galveston, Texas, U.S.A. Chapter 5

Courtney M. Townsend, Jr., M.D. The University of Texas Medical Branch at Galveston Department of Surgery Galveston, Texas, U.S.A. Chapter 1

Tien C. Ko, M.D. The University of Texas Medical Branch at Galveston Department of Surgery Galveston, Texas, U.S.A. Chapter 4

PREFACE Cancers of the gastrointestinal tract are a leading cause of cancer-related deaths. Carcinoma of the pancreas, the stomach, and colorectum account for more than 150,000 new cases of cancer and are responsible for more than 75,000 deaths per year. If localized, these cancers can be effectively treated by surgical resection with excellent results. Unfortunately, many of these cancers have already spread to surrounding lymph nodes or distant organs, thus precluding surgical resection for cure and mandating adjuvant therapy with radiation and/ or chemotherapeutic agents which have only been minimally successful. Novel agents are required in the adjuvant treatment of these cancers if we are to realize an improvement in overall survival. The design of novel agents requires an in-depth understanding of the mechanisms regulating tumor development, progression and subsequent metastasis. With the advent of molecular techniques, a revolution of sorts has taken place in the field of cancer biology and genetics. Although other factors such as diet, environment, and social considerations may contribute in some way to tumor progression, this book specifically focuses on the molecular mechanisms of gastrointestinal neoplasia. The development of cancer is a multifactorial process with a number of cellular mechanisms contributing to cancer progression, growth and subsequent spread. For example, growth factors and gut hormones, acting through specific cell surface receptors, can affect the growth of certain gastrointestinal cancers. Signaling pathways that are important include the protein kinase A, protein kinase C, protein tyrosine kinase, and mitogen-activated protein kinase pathways which transduce the mitogenic signal from the cell surface to the nucleus. We will discuss the potential role of the arachidonic acid/prostaglandin pathway in certain gastrointestinal cancers, particularly colorectal neoplasias. Proteins that regulate the cell cycle as well as those that regulate the process of cell death can also contribute to the alteration of normal cellular growth and result in marked proliferation characteristic of neoplasias. Oncogenes and tumor suppressor genes likewise contribute to this scenario and are crucial to our understanding of the development and progression of these cancers. Lastly, we will discuss an increasingly important role that angiogenesis may play in not only growth but also metastasis of cancers. Elucidating the molecular mechanisms underlying gastrointestinal carcinogenesis is crucial to not only our understanding of these cancers, but also the development of potential novel therapies that target these pathways. Each chapter discusses the potential therapeutic implications and future prospects for treatment based on our increasing knowledge of the molecular pathways contributing to gastrointestinal cancer. The last several years have been particularly exciting with new discoveries made on almost a daily basis. As we approach the next millennium, the ability to treat gastrointestinal cancers appears to be closer to our realization than ever before. In the future, the precise dissection of these molecular pathways and the ultimate novel therapy that can be devised based upon this knowledge may be as important as the sharp dissection of the surgical scalpel in improving overall prognosis and hopefully leading to an eventual cure of these deadly diseases.

CHAPTER 1

Growth Factors, Hormones and Receptors in GI Cancers B. Mark Evers and Courtney M. Townsend, Jr.

O

ver the last two decades great strides have been made in understanding the role that growth factors and hormones play in regulation of growth of gastrointestinal (GI) cancers (most notably gastric, pancreatic and colorectal adenocarcinomas). By definition, any agent that regulates growth can be considered a growth factor; however, these growth-stimulating agents are usually divided into growth factors, which are produced by both normal and neoplastic cells and are thought to act locally to control cancer cell proliferation, and hormones, which are thought to act at a distance.1-3 The local effects of the growth factors, such as the transforming growth factor (TGF) family, epidermal growth factor (EGF), plateletderived growth factor (PDGF), insulin-like growth factor (IGF), and fibroblast growth factor (FGF), can occur by one of two mechanisms. For example, autocrine stimulation occurs when tumor cells produce and release growth factors which then modulate their own growth via a receptor-mediated event. In addition, tumor cells may also secrete growth factors which alter growth of surrounding cells in a paracrine fashion. Various gut hormones can regulate growth of GI cancers usually through an endocrine effect although, on occasion, an autocrine or paracrine mechanism has been postulated for the proliferative effects of these trophic peptides. This review has been divided into the growth factors and specific hormones that affect GI cancer proliferation. The focus of this review will be on the effects of these growthstimulating agents on gastric, pancreatic and colorectal adenocarcinomas since these are the most common GI malignancies and those in which the most information is available on the potential mechanisms contributing to their proliferation.

Growth Factors The ability of cancer cells to proliferate autonomously may be attributed to the production of autocrine or paracrine growth factors which are released locally to stimulate their own growth or the growth of nearby cells. An important point to remember is that many of these growth factors are produced by a variety of normal cells and contribute to the normal proliferative process; however, in cancer cells, there has been an obvious loss of regulatory functions that allows the malignant cell to proliferate without restraints. These autocrine agents normally act through their cell surface receptor to stimulate growth; however, autonomous proliferation may also occur independent of the growth factor through the constitutional activation of the growth factor receptor.3 Well-characterized examples of constitutional activation include the EGF receptor (EGFr) and the EGFr family member, c-erbB2 (also known as HER-2/neu); the overexpression of EGFr and HER-2/neu have both been implicated in the proliferation and prognosis of a number of GI cancers. An important Molecular Mechanisms in Gastrointestinal Cancer, edited by B. Mark Evers. ©1999 R.G. Landes Company.

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Molecular Mechanisms in Gastrointestinal Cancer

clinical consideration is the fact that these growth factor receptors may act as therapeutic targets in which treatment may be directed to either slow down or stop proliferation.

Gastric Cancer Although its incidence has been decreasing in the United States, gastric cancer is a significant cause of GI cancer deaths in a number of endemic countries. Gastric cancer has often spread to surrounding lymph nodes or distantly at the time of initial patient presentation, thus precluding a curative resection. Gastric cancers may produce an array of growth factors which include TGF-α, TGF-β, EGF, PDGF and IGF.4-10 The ligands for a number of these growth factors have also been identified and include EGFr, HER-2/neu, the hepatocyte growth factor receptor encoded by c-met and a heparin-binding growth factor-like receptor encoded by K-sam.8-12 These growth factors not only stimulate proliferation of the cell but can also affect the invasiveness and metastatic rate of these cancers. Gastric cancers, as well as the normal gastric mucosa, express EGFr, a tyrosine kinase receptor, and produce its various ligands (i.e., TGF-α and EGF).4-7 The immunoneutralization of TGF-α or EGF inhibits the proliferation of gastric cancer cell lines in vitro, suggesting that these agents can act in an autocrine fashion through the EGFr.7-13 Production of human EGF-like immunoreactivity by five human gastric cancer cell lines (MKN-1, MKN-28, MKN-45, MKN-74 and KATO-3) have been described.14,15 The degree of gene amplification and the concentration of EGFr appear to correlate directly with the growth of the gastric cancer A431 transplanted into athymic mice.16,17 The presence of both EGF and EGFr has been assessed in various gastric cancers in vivo. No EGF immunoreactivity was found in early cancers, but EGF-positive tumor cells were detected in 38 of 130 (29%) of advanced cancers.18 EGFr was found in 34% of advanced cancers but in only one early cancer; 17 advanced cancers had synchronous expression of EGF and EGFr. These studies suggest that patients with EGFr-positive gastric cancers have a poorer prognosis than those with EGFr-negative cancers. Moreover, gastric cancers which express EGF and the EGFr simultaneously have been shown to have a greater degree of local invasion and lymph node metastasis, and patients with gastric cancers that coexpress EGF and EGFr exhibited a much poorer prognosis than those without EGF and EGFr.18,19 The coexpression of EGF and EGFr in gastric cancers strongly suggests the possibility that the proliferation of these cancers may occur, in part, by an autocrine mechanism. Several studies have assessed the potential efficacy of anti-EGFr monoclonal antibodies to inhibit EGFr-positive gastric cancer and have shown promising results in tumor xenografts in nude mice.20,21 The clinical utility of these antibodies have yet to be established in extensive clinical trials. The EGFr-like receptor kinase, HER-2/neu, is overexpressed in approximately 10% to 30% of gastric cancers.22,23 This overexpression has been associated with a poorer prognosis in certain cases;24 however, this is a controversial point since the examination of archival samples in one study demonstrated that the overexpression of HER-2/neu actually correlated with an apparent improved prognosis.23 In marked contrast, studies of breast cancers have clearly shown that HER-2/neu amplification portends a worse prognosis.25 Despite the apparent controversy regarding prognosis, experimental studies have shown that a combination of monoclonal antibodies to the ligand-binding domain of HER-2/neu inhibits the proliferation of gastric cancer xenografts that overexpress this receptor kinase.26 These studies are encouraging and indicate that HER-2/neu may provide a chemotherapeutic target in the treatment of certain gastric cancers. Future studies will need to delineate the mechanism for this inhibition, as well as the number of gastric cancers which may be affected by this treatment.

Growth Factors, Hormones and Receptors in GI Cancers

3

Other tyrosine kinase receptors present in certain gastric cancers include the hepatocyte growth factor receptor encoded by the c-met protooncogene and the K-sam gene which encodes a receptor tyrosine kinase that appears to be a member of the heparin-binding growth factor receptor family.3,11,12 The overexpression of both of these receptors has been noted in poorly differentiated gastric cancers (e.g., scirrhous carcinomas);27,28 however, the significance of these receptors in gastric cancers has yet to be entirely defined. Future studies are required to analyze additional gastric cancers and clearly determine the incidence of overexpression of these receptors. These genetic alterations must also be characterized in a systematic fashion to ascertain the mechanisms underlying this overexpression. A growth factor that is assuming apparent importance in a number of GI cancers is IGF (both IGF-I and IGF-II). In vitro studies have identified the expression of IGF-II mRNA in a human gastric cancer cell line; this cell line releases IGF-II into the medium and possesses both type I and type II IGF receptors.29 Monoclonal antibodies that neutralize either IGF-I or IGF-II can inhibit the autonomous proliferation of these cells, suggesting that IGF-II may be an autocrine factor for certain gastric cancers. Another growth factor that may play a role in the growth of gastric cancer is the peptide, bombesin (BBS), a tetradecapeptide originally isolated from the skin of the frog Bombina bombina, or its mammalian equivalent, gastrin-releasing peptide (GRP).30 The mitogenic effect of BBS was first reported in human small cell lung cancers which express the GRP receptor and secrete GRP, thus initially suggesting an autocrine mechanism for this peptide growth factor in various tumors.31 Recently, we have identified a human gastric cancer cell line, SIIA, which possesses GRP receptors and is stimulated by the administration of BBS.32 In response to BBS, SIIA cells release intracellular calcium and increase the expression of various AP-1 related proteins which have been shown to play a role in the proliferation of a number of cells.33 This effect can be blocked by specific receptor antagonists and, with the increasing number of specific and potent GRP receptor antagonists, these findings offer potential therapies for gastric cancer. In addition, we have identified expression of GRP receptor in another in vitro gastric cancer cell line (MKN-45) and in three of five human gastric cancer xenografts.33 Consistent with these findings, Preston et al34 demonstrated that 13 of 23 gastric cancers expressed high affinity GRP receptors. The presence of the GRP receptor in gastric cancers appears to play a functional role in the growth of these tumors as noted by Qin et al35 who demonstrated that the GRP antagonist, RC-3095, blocks the growth of the human gastric cancer, Hs746T, both in vitro and when placed as xenografts in nude mice.

Pancreatic Cancer Carcinoma of the pancreas is the fifth leading cause of cancer deaths in industrialized countries.36,37 Prognosis remains poor with a mean survival time after diagnosis of about four to six months.36 Pancreatic cancers produce a number of growth factors, including TGF-α, TGF-β, EGF, FGF and PDGF.3,38-40 Of these, TGF-α and EGF have been noted in a number of pancreatic cancers and have been the growth factors best characterized. These substances may be important for pancreatic cancer growth by acting through the EGFr. Support for this hypothesis has been provided by findings that the growth of certain EGFr-positive pancreatic cancers is stimulated by exogenous TGF-α or EGF and that EGFr-blocking antibodies can inhibit proliferation.41,42 EGF has been shown to increase somewhat the number of PANC-1 cells after six days in culture in which the serum concentration was 0.1%.43 Pancreatic cancer cell lines (ASPC-I, T3M4, PANC-1, COLO 357 and MIA PaCa-2) produce TGF-α and express TGF-α mRNA; however, the amount of TGF-α protein found in the supernatant of these cells did not correlate with the amount of TGF-α mRNA.42 A number of pancreatic cancers demonstrate concomitant overexpression

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Molecular Mechanisms in Gastrointestinal Cancer

of the EGFr and EGF and/or TGF-α, suggesting an autocrine or paracrine stimulatory effect. Furthermore, patients with pancreatic cancers that simultaneously overexpress this EGFr and EGF and/or TGF-α die earlier than those patients who do not overexpress these products.44 Similar to gastric cancers, approximately 20% to 40% of pancreatic cancers demonstrate increased HER-2/neu immunoreactivity; however, in contrast to the EGFr, overexpression of HER-2/neu was associated with better tumor differentiation.45 This overexpression was not associated with advanced tumor stage or shorter postoperative survival. Other candidate growth factors that are found in pancreatic cancers and have been postulated to affect tumor growth include IGF-I, interleukin-3, granulocyte-macrophage colony stimulating factor and pancreastatin.38-40 The FGFs, of which acidic and basic FGF are the prototypes, comprise another growth factor family that may play a role in the growth of pancreatic cancers.38,46,47 Both acidic and basic FGF are overexpressed in a number of pancreatic cancers; however, increased basic FGF, but not acidic FGF, correlated with a decreased survival of patients following tumor resection.47 Keratinocyte growth factor (KGF), which is often expressed in fibroblasts and other mesenchymal cells, may affect pancreatic cancer growth in a paracrine fashion.48,49 Thus, KGF may participate with other growth factors in the pancreatic cancer growth through a number of local effects. The TGF-β gene family, which includes the three homologs TGF-β1, TGF-β2 and TGF-β3, may affect pancreatic cancer growth by promoting angiogenesis, increasing the desmoplastic response of surrounding tissues and acting as immunosuppressive agents.50 All three of the TGF-β homologs are overexpressed in a number of pancreatic cancers.51 Moreover, this overexpression is associated with more aggressive tumor growth and a significantly reduced postoperative survival time. The inhibitory peptide, somatostatin, and its various analogs have been evaluated as antiproliferative agents in experimental pancreatic cancer as human pancreatic cancers are reported to have high-affinity binding sites for somatostatin. Multiple mechanisms of action have been proposed for the antiproliferative effects of somatostatin and its analogs on GI cancers.52 These potential mechanisms include a ‘direct’ effect acting through specific high-affinity biding sites or via ‘indirect’ effects which may include the inhibition of angiogenesis and/or the suppression of growth hormones or IGF secretion. Redding and Schally53 reported significant reductions in tumor weight and volume in Wistar-Lewis rats bearing the pancreatic acinar tumor, DNCP-322. Upp et al54 in our laboratory demonstrated the inhibition of two human pancreatic adenocarcinoma (SKI and CAV) xenografts in nude mice by the administration of the long-acting somatostatin analog, SMS201-995 (Fig. 1.1). Furthermore, the long-acting analog RC-160 has been shown to inhibit proliferation of the human pancreatic cancer MIA PaCa-2 possibly by activating dephosphorylation of the EGFr.55,56 Human trials using SMS201-995 at conventional doses (less than 600 (µg/d) have not demonstrated the inhibition of pancreatic tumor progression;57 however, studies using higher dosages or in combination with other therapeutic agents may prove beneficial, as may newer and long-acting somatostatin analogs.

Colorectal Cancer Colorectal cancer is the second leading cause of cancer deaths in North America and Western Europe.37 A role of autocrine growth regulatory peptides in the growth of colorectal cancer has been suggested by a number of studies. Endogenous growth factors, which have been identified in a variety of these cancers, include TGF-α, TGF-β, PDGF, IGF-I and BBS-like peptides.3,58,59

Growth Factors, Hormones and Receptors in GI Cancers

5

Fig.1.1. Percentage of increase in tumor area during treatment in SKI and CAV treatment groups (dotted bars) and control groups (solid bars). Treatment with SMS 201-995 (100 µg/kg) three times daily was begun 21 days after transplantation (10 tumors, * = p < 0.05). Reprinted with permission from Upp JR Jr, Olson D, Poston GJ; et al, Am J Surg 1988; 155:29 ©1988 Excerpta Medica, Inc.

The coexpression of TGF-α and EGFr have been noted in a number of freshly resected colorectal cancers as well as established cell lines.60-62 Other EGF-related peptides that are often overexpressed in colorectal cancers and may play a role in their growth include amphiregulin, a glycosylated heparin-binding protein, and CRIPTO, a protein that shares a cysteine-rich motif in common with other members of the EGF family. In fact, a majority of colorectal cancers coexpress multiple growth factors of the EGF family.59,63 The ability of immunoneutralizing antibodies to TGF-α and EGFr to inhibit the proliferation of these cancers appears to depend on the presence of high affinity EGFr subtypes.64,65 In addition to the inhibition of the growth of various colorectal cancers using EGFr-blocking antibodies, another novel strategy to block protein function is to prevent the translation of messenger RNA into protein using antisense technology.66 Several studies have demonstrated that antisense oligonucleotides directed against EGF-related growth factors can inhibit the in vitro growth of human colon cancer cell lines (e.g., Caco-2, GEO and CBS).67-72 Moreover, it has been reported that a supra-additive effect occurs when combinations of the antisense oligonucleotides directed against EGF-related growth factors and the EGFr-blocking

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Molecular Mechanisms in Gastrointestinal Cancer

Fig. 1.2. Weight, protein and DNA content of xenografted MIA Paca-2 tumor, jejunum and ileum in tumor-bearing nude mice (* = p < 0.05 vs control; † = p < 0.05 vs NT alone). NT: neurotensin, SR: SR48692. Reprinted with permission from Iwase K, Evers BM, Hellmich MR et al; Cancer 1997; 79:1787 ©1997 John Wiley & Sons, Inc.

antibody are used.68 TGF-β and its receptor are expressed in a subset of well-differentiated colon cancer cell lines.3 Exogenous TGF-β inhibits proliferation and appears to promote differentiation; therefore, TGF-β may serve as an autocrine inhibitory factor for various colon cancers. Colorectal cancers also express other receptor tyrosine kinases to a varying degree, including HER-2/neu and the hepatocyte growth factor receptor;3 the presence of these receptors and their effects on colon cancer growth have yet to be clearly elucidated. Other growth factors that may play a role in the regulation of colorectal cancer growth include IGF-I, IGF-II and platelet-derived growth factors.73-77 IGF-II immunoreactivity has been reported in a greater number of colorectal tumors than in surrounding normal mucosa, and its mRNA is overexpressed in approximately 30% of colorectal cancers.73-75 Colon cancer cell lines also exhibit IGF-I and IGF-II binding sites and show proliferative responses to both of these agents. Similar to TGF-α and EGF, response to these peptides is

Growth Factors, Hormones and Receptors in GI Cancers

7

Fig. 1.3. Comparison of survival of MC-26 tumor-bearing mice after treatment with normal saline solution, pentagastrin treatment for 7 days, or pentagastrin treatment for 14 days. Reprinted with permission from Winsett OE, Townsend CM Jr, Glass EJ et al; Surgery 1988; 99:302 ©1988 C.V. Mosby Company.

Fig. 1.4. Survival of MC-26 tumor-bearing mice after treatment with (solid line) normal saline solution, (long-dash line) proglumide beginning on the day of tumor inoculation, or (short dash line) proglumide beginning 7 days after tumor inoculation. Reprinted with permission from Beauchamp RD, Townsend CM Jr, Singh P, et at; Ann Surg 1985; 202:303 ©1985 Lippincott-Raven.

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Molecular Mechanisms in Gastrointestinal Cancer

Fig. 1.5. Enprostil significantly inhibited MC-26 mouse colon cancer growth from day 15 through day 25. (* p <0.05 vs control). Reprinted with permission from Townsend CM Jr, Singh P, Thompson JC; Gastroenterol Clin North Am 1989; 18:777 ©1989 W.B. Saunders Company.

dependent on a functional receptor. Other potential growth factors include basic FGF in which receptors have been noted in certain colorectal cancer cell lines, and PDGF which has also been found to be produced by colon cancer cells. The overall significance of these growthpromoting agents has yet to be clearly shown. A number of experimental studies have examined the effects of somatostatin on the growth of colon cancers. Milhoan et al78 from our laboratory demonstrated that the long-acting somatostatin analog, MK-678, significantly inhibited growth of two human colon cancer xenografts (RIP and DRUM) in nude mice. In addition, Smith and Solomon79 analyzed three human colon cancers, CXI, X56 and HT-29. Somatostatin had no effect on the growth of X56, but significantly reduced the volume and DNA content of CXI. We speculate that this effect could potentially have been from inhibition of gastrin release or the modulation of gastrin receptors. This hypothesis was further supported by demonstration that the administration of SMS201-995 blocked the gastrin-induced stimulation of the MC-26 colon cancer cell line.80 Gastrin receptors were found to be downregulated in MC-26 tumors following treatment with somatostatin. Consistent with our findings, Smith and Solomon79 demonstrated that somatostatin inhibited the gastrin-mediated proliferation of HT-29 cells. These findings, therefore, suggest that somatostatin may block the growth of certain colon cancers.

GI Hormones and Receptors Hormonal control of the growth of certain malignancies was firmly established in 1896 by the landmark studies of the Scottish surgeon, Beatson,81 who successfully treated a patient with breast cancer by bilateral oophorectomy. Since that time, a number of cancers have been shown to possess receptors for hormones and their growth can be stimulated by these

Growth Factors, Hormones and Receptors in GI Cancers

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Table 1.1 Early follow-up of patients with colon cancer highly positive or poorly positive for gastrin receptors

Gastrin receptors >10 fmol/mg protein Alive Recurrence or dead Gastrin receptors <10 fmol/mg protein Alive Recurrence or dead

A

DUKES’ STAGE B C

D

0 0

11 0

6 0

2 0

1 0

9 0

14 5*

8 8

Length of follow-up ranges from 40 to 972 days with a mean follow-up of 292 days. The presence of >10 fmol of gastrin receptor/mg of protein equals highly positive tumors. The presence of <10 fmol/mg of protein equals poorly positive for gastrin receptors. * Four patients dead and one alive with recurrent cancer. Reprinted with permission from Townsend CM Jr et al, Gastroenterol Clin N Am, 1989; 18:777. ©1989 W.B. Saunders.

agents. Similar to breast and prostate cancers which are the best-described of the hormonally responsive cancers, certain GI cancers possess peptide receptors, are hormone-responsive and growth can be inhibited by peptide deprivation, the antagonism of peptide action or the administration of inhibitory hormones. Thus, the growth of these GI cancers may be regulated by peptides localized to the gut and pancreas. In a similar fashion, it has been shown that gut hormones stimulate the growth of normal gut mucosa and pancreas. Johnson82 demonstrated that gastrin, the hormone produced by G-cells of the gastric antrum, was trophic for the mucosa of the stomach, duodenum and colon. Patients with the Zollinger-Ellison syndrome have excessive amounts of tumor-elaborated gastrin and have massive proliferation of gastric mucosa. Solomon et al83 demonstrated that cerulein (a cholecystokinin analog) and secretin stimulate growth of the normal pancreas. Therefore, the fact that GI cancers possess receptors and are responsive to these various hormonal agents is supported by the fact that they also appear to play a role in normal gut and pancreatic growth.

Gastric Cancer Gastrin has well-described trophic effects for the mucosa of the normal gastric fundus82 and also may play a role in the growth of certain gastric cancers. Studies have demonstrated that human gastric cancer cell lines possess low as well as high affinity receptors for gastrin.84-86 The growth of these cells can be stimulated by pharmacologic and, in certain instances, physiologic concentrations of gastrin or gastrin analogs both in vivo and in vitro. 1,87-91 Gastrin receptor antagonists block these trophic effects, suggesting mediation through specific gastrin receptors.87,92 Further support of this hypothesis is provided by the findings of studies performed in our laboratory93 as well as studies by Watson et al,94 which demonstrate that enprostil, a synthetic prostaglandin analog that lowers serum gastrin levels in fasted and fed humans and mice, inhibits the growth of gastrin receptor-positive human gastric cancers xenografted into athymic nude mice. Interestingly, enprostil has not been shown to affect the growth of gastrin receptornegative gastric cancers (for example, the CLEES tumor).93 Taken together, these findings

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provide evidence that endogenous gastrin may contribute to proliferation of certain gastric cancers that possess gastrin receptors. Studies on the effects of gastrin on carcinogen-induced stomach cancers have provided conflicting results. Some investigators have noted that gastrin increases the incidence of carcinogen-induced gastric cancer in rats,95 whereas others have found that gastrin actually inhibited the incidence of stomach cancers induced by carcinogens.96 Therefore, based on these results, some investigators believe that the trophic effect of gastrin enhances the effects of various carcinogens, while others postulate that gastrin allows healing of carcinogen-induced injury and thus prevents tumor development.96,97 The timing for administration of gastrin appears critical and a major determinant of enhanced tumor development in a number of these studies. Gastrin given during exposure to MNNG increased gastric cancer development, but gastrin administration later did not enhance the development of gastric cancer. Therefore, the discrepancies in the literature may be explained by differences in experimental design. The long-term administration of high doses of omeprazole, which produces an endogenous hypergastrinemia, results in hyperplasia of enterochromaffin-like cells and gastric carcinoid tumors in rats;98 the development of carcinoids can be prevented by antrectomy.99 In fact, three patients have been reported in whom antrectomy caused disappearance of gastric carcinoids in the remaining stomach.100,101

Pancreatic Cancer Cholecystokinin (CCK) stimulates the growth of the normal pancreas as well as a number of CCK receptor-positive pancreatic cancers.1,83 We have identified CCK and secretin receptors in hamster and human pancreatic cancer cell lines and have shown that the presence of CCK receptors can predict the responsiveness of various human pancreatic cancers to CCK.93,102,103 The CCK analog, cerulein, significantly stimulates the growth of the pancreatic cancer cell line, SKI, which possesses CCK receptors.104 However, CAV, a CCK receptornegative tumor, is not affected by cerulein. Townsend et al105 demonstrated that growth of the hamster pancreatic cancer, H2T, was stimulated by cerulein; this trophic effect was augmented by secretin. Pancreatic tumors in rats can be induced by stimulating endogenous CCK release using trypsin inhibitors, biliary diversion or bile salt binding drugs.106-108 This effect can be blocked by CCK receptor antagonists. Although it appears clear that CCK receptor-positive pancreatic cancers respond to exogenous CCK administration by increased growth, the effect of increasing endogenous CCK on carcinogenesis of the normal pancreas in combination with various chemicals has been somewhat controversial. For example, Johnson et al109 reported that the octapeptide fragment of CCK (CCK-8) inhibited development of nitrosamine-induced pancreatic cancer in hamsters. In contrast, Satake et al110 found that weekly administration of cerulein enhanced the carcinogenic effect of N-nitrosobis(2-hydroxypropyl)amine (BHP) in hamsters. Howatson and Carter111 found that both CCK and secretin enhanced pancreatic carcinogenesis induced by N-nitrosobis(2-oxypropyl)amine (BOP) in hamsters. Therefore, it appears that agents that increase endogenous CCK and stimulate hyperplasia of the normal pancreas may also stimulate carcinogenesis of the pancreas. Future studies are required to further examine the timing of administration as well as the effects of hormonal antagonists. Another gut peptide that may play a role in pancreatic cancer growth is neurotensin (NT), a gut peptide localized primarily to the small bowel.112 NT stimulates the growth of small bowel as well as colonic mucosa,113,114 and Feurle et al115 have shown that NT also stimulates the growth of the normal pancreas. We have shown that human pancreatic cancer cell line, MIA PaCa-2, possesses NT receptors and mobilizes intracellular calcium in response to NT administration.116 NT stimulates the growth of MIA PaCa-2 cells both in vitro and in vivo. In addition, we have recently shown that the NT receptor antagonist,

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Fig. 1.6. Survival rates of MC-26 tumor-bearing mice treated with saline (control), NT 300 µg/kg or NT 600 µg/kg and tumor-free mice treated with NT 600 µg/kg) n = 20 mice/group). Reprinted with permission from Yoshinaga K, Evers BM, Izukura M et al; Surg Oncol 1992; 1:127, ©1992 Elseiver Science Ltd.

SR46892, inhibits intracellular calcium immobilization, IP3 turnover, and MIA PaCa-2 cell growth induced by NT in a dose-dependent fashion.117 In in vivo experiments, NT significantly increased the size, weight and DNA and protein content of xenografted MIA PaCa-2 tumors as well as the normal jejunum and ileum; SR46892 inhibited the effect of NT (Fig. 1.2).117 Recent findings by Reubi et al118 have shown that approximately 75% of pancreatic adenocarcinomas express NT receptors. Therefore, it is speculated that NT receptors may serve as a therapeutic tool to potentially direct radiotherapy or specific analogs directly to the pancreatic cancer.

Colorectal Cancer Gastrin has a trophic effect on normal colonic mucosa as well as certain colorectal cancer cell lines.82,83,119 Gastrin stimulates the growth of the mouse colon cancer, MC-26, which possesses high affinity gastrin receptors both in vitro and in vivo (Fig. 1.3).120-122 Administration of the gastrin receptor antagonist, proglumide, inhibits the growth of this cancer and prolongs survival of tumor-bearing mice (Fig. 1.4).120 Furthermore, we have shown that gastrin stimulates the growth of two human colon cancer cell lines, LoVo and HT-29, but inhibits the growth of a third colon cancer cell line, HCT-116.123 This differential effect suggests different gastrin receptor subtypes or alternate signal transduction pathways. We have studied the effect of suppressing endogenous gastrin with enprostil on MC-26 tumor growth and found that, similar to our results in gastrin receptor-positive gastric adenocarcinomas, enprostil significantly inhibited MC-26 tumor growth in mice (Fig. 1.5).93 Endogenous hypergastrinemia produced by antral exclusion performed after

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carcinogen treatment was begun did not alter tumor incidence, but increased DNA synthesis in carcinogen-induced colon tumors.124 In contrast, Oscarson et al125 reported that endogenous hypergastrinemia (induced by fundusectomy at the onset of carcinogen treatment) did not potentiate DMH-induced colon carcinogenesis. Upp et al126 have analyzed 67 primary colorectal cancers for gastrin receptors and found a spectrum of receptor expression, which suggests a correlation between tumor stage and the presence of gastrin receptors. Some cancers had no detectable receptors. Some had high affinity (Kd = <1.0 nmol) receptors while others had low affinity (Kd = > 1.0 nmol) receptors. Twenty patients (30%) had cancers with gastrin receptor contents > 10 fmol/mg protein. A significantly greater percentage (52%) of patients with early cancers (Dukes’ A and B) had gastrin receptor contents of >10 fmol/mg protein as compared to patients with advanced (Dukes’ C and D) cancer (20%) (Table 1.1). Early follow-up of these patients showed that no patient with a gastrin receptor content of >10 fmol/mg protein had developed recurrence or died, regardless of Dukes’ stage. In contrast, 5 of 19 Dukes’ C patients and 8 of 16 Dukes’ D patients with gastrin contents of <10 fmol/mg protein had developed recurrence or died. A longer follow-up period is required before any prognostic significance can be made from these studies. The actions of gastrin on gastrin receptor-positive colorectal cancers have been well characterized, but the effect of gastrin on colorectal cancer growth in humans is still uncertain. Studies have added to this confusion by demonstrating increased circulating levels of gastrin in some patients with colorectal cancer127-129 but little or no change in others.130-133 Kameyama et al134 reported that the incidence of liver metastasis in colorectal cancer was significantly higher in patients with elevated serum gastrin. In addition to the expression of gastrin receptors, some colorectal cancer cell lines also express gastrin mRNA and release gastrin-like immunoreactivity, suggesting a possible autocrine role for gastrin in these cancers.91 In addition to gastrin, NT has been found to stimulate the growth of normal colonic mucosa and likewise stimulates the growth of the human colon cancer cell line, LoVo (Fig. 1.6). 135 We have shown that approximately 25% of colon cancers analyzed express NT messenger RNA.136 NT appears to promote carcinogenesis in the colon of rats137 and, in a recent study by Rovere et al,138 all 13 human colon cancer cell lines displayed low to moderate levels of pro-NT/neuromedin N protein expression. Only six of these 13 tumors, however, processed the precursor to produce NT and larger precursor fragments ending with the NT or neuromedin N sequence. The receptor for NT has been identified in a number of human colon cancer cell lines.139 Recently Ehlers et al140 from our laboratory has shown that the addition of NT to the human colon cancer cell line, KM20, which expresses high levels of the NT receptor, results in calcium mobilization as well as activation of the MAP kinase (MAPK) pathway. These findings suggest that the proliferative effect of NT may be through the MAPK stimulatory pathway and that, like gastrin, NT may be an autocrine growth factor in certain colon cancers.

Future Perspectives and Therapeutic Implications The trophic effects of growth factors as well as GI peptides may have important implications for the management of patients with gastric, pancreatic and colorectal cancers. By assaying tumors for the presence of these growth factors and receptors and studying the effects of these agents on the tumors that possess receptors and those that do not, we should be able to more definitively elucidate the role these factors play in regulating the growth of GI cancers. More importantly, we must define the precise mechanisms by which these agents act. When defined, the mechanisms become targets so that it may be possible to develop therapeutic strategies for patients with these cancers based upon receptor blockade or the alteration of endogenous hormone levels.141 Signal transduction pathways as therapeutic

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targets for these tumors should be explored with the use of antisense gene therapy techniques. In addition, tyrosine kinase inhibitors have been developed that inhibit a variety of receptor and nonreceptor tyrosine kinases and have been shown to inhibit proliferation of a variety of GI cancer cell lines both in vitro and in vivo.3 Among these are erbstatin, herbimycin-A, and a family of inhibitors called tyrphostins. Erbstatin has been shown to inhibit EGF and serum-stimulated proliferation of gastric carcinoma cell lines, whereas herbimycin-A has been shown to inhibit proliferation of several colon carcinoma cell lines. Somatostatin and the long-acting somatostatin analogs also hold promise as adjuvant therapy for various cancers;53 however, to date the clinical trials using the somatostatin analog, SMS201-995, to inhibit tumor growth have been disappointing. A further understanding of the regulatory mechanisms involved in cancer cell growth will allow for the development of noncytotoxic modalities of therapy for GI cancers. Certainly no one agent alone will be effective in treating patients with GI cancers, but it is hoped that by using multiple agents that block different receptors or signal transduction pathways we can increase the therapeutic efficacy and decrease toxicity.

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102. Townsend CM Jr, Singh P, Thompson JC. Gastrointestinal hormones and gastrointestinal and pancreatic carcinomas. Gastroenterology 1986; 91:1002-1006. 103. Singh P, Townsend CM Jr, Poston GJ et al. Specific binding of cholecystokinin, estradiol and somatostatin to human pancreatic cancer xenografts. J Steroid Biochem Mol Biol 1991; 39:759-767. 104. Upp JR Jr, Singh P, Townsend CM Jr et al. Predicting response to endocrine therapy in human pancreatic cancer with cholecystokinin receptors. Gastroenterology 1987: 92:167 (Abstract). 105. Townsend CM Jr, Franklin RB, Watson LC et al. Stimulation of pancreatic cancer growth by caerulein and secretin. Surg Forum 1981; 32:228-229. 106. Lamers CB, Jansen JB, Woutersen RA. Cholecystokinin and gastrointestinal cancer. J Steroid Biochem Mol Biol 1990; 37:1069-1072. 107. Beardshall K, Frost G, Morarji Y et al. Saturation of fat and cholecystokinin release: implications for pancreatic carcinogenesis. Lancet 1989; 2:1008-1010. 108. Miazza BM, Widgren S, Chayvialle JA et al. Exocrine pancreatic nodules after longterm pancreaticobiliary diversion in rats. An effect of raised CCK plasma concentrations. Gut 1987; 28:269-73. 109. Johnson FE, LaRegina MC, Martin SA et al. Cholecystokinin inhibits pancreatic and hepatic carcinogenesis. Cancer Detect Prev 1983; 6:389-402. 110. Satake K, Mukai R, Kato Y et al. Effects of cerulein on the normal pancreas and on experimental pancreatic carcinoma in the Syrian golden hamster. Pancreas 1986; 1:246-253. 111. Howatson AG, Carter DC. Pancreatic carcinogenesis-enhancement by cholecystokinin in the hamster-nitrosamine model. Br J Cancer 1985; 51:107-114. 112. Reinecke M. Neurotensin. Immunohistochemical localization in central and peripheral nervous system and in endocrine cells and its functional role as neurotransmitter and endocrine hormone. Prog Histochem Cytochem 1985; 16:1-172. 113. Chung DH, Evers BM, Shimoda I et al. Effect of neurotensin on gut mucosal growth in rats with jejunal and ileal Thiry-Vella fistulas. Gastroenterology 1992; 103:1254-1259. 114. Evers BM, Izukura M, Chung DH et al. Neurotensin stimulates growth of colonic mucosa in young and aged rats. Gastroenterology 1992; 103:86-91. 115. Feurle GE, Muller B, Rix E. Neurotensin induces hyperplasia of the pancreas and growth of the gastric antrum in rats. Gut 1987; 28 Suppl:19-23. 116. Ishizuka J, Townsend CM Jr, Thompson JC. Neurotensin regulates growth of human pancreatic cancer. Ann Surg 1993; 217:439-446. 117. Iwase K, Evers BM, Hellmich MR et al. Inhibition of neurotensin-induced pancreatic carcinoma growth by a nonpeptide neurotensin receptor antagonist, SR48692. Cancer 1997; 79:1787-793. 118. Reubi JC, Waser B, Friess H et al. Neurotensin receptors: a new marker for human ductal pancreatic adenocarcinoma. Gut 1998; 42:546-550. 119.Townsend CM Jr, Singh P, Thompson JC. Hormonal effects on gastrointestinal cancer growth. In: J Morisset, TE Solomon (Eds), Growth of the Gastrointestinal Tract: Gastrointestinal Hormones and Growth Factors. Florida: CRC Press, 1991:207-221. 120. Beauchamp RD, Townsend CM Jr, Singh P et al. Proglumide, a gastrin receptor antagonist, inhibits growth of colon cancer and enhances survival in mice. Ann Surg 1985; 202:303-309. 121. Winsett OE, Townsend CM Jr, Glass EJ et al. Gastrin stimulates growth of colon cancer. Surgery 1986; 99:302-307. 122. Singh P, Le S, Beauchamp RD et al. Inhibition of pentagastrin-stimulated upregulation of gastrin receptors and growth of mouse colon tumor in vivo by proglumide, a gastrin receptor antagonist. Cancer Res 1987; 47:5000-5004. 123. Ishizuka J, Townsend CM Jr, Bold RJ et al. Effects of gastrin on 3',5'-cyclic adenosine monophosphate, intracellular calcium, and phosphatidylinositol hydrolysis in human colon cancer cells. Cancer Res 1994; 54:2129-2135. 124. McGregor DB, Jones RD, Karlin DA et al. Trophic effects of gastrin on colorectal neoplasms in the rat. Ann Surg 1982; 195:219-223.

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125. Oscarson JE, Veen HF, Ross JS et al. Dimethylhydrazine-induced colonic neoplasia: dissociation from endogenous gastrin levels. Surgery 1982; 91:525-530. 126. Upp JR Jr, Singh P, Townsend CM Jr et al. The clinical significance of gastrin receptors in human colon cancers. Gastroenterology 1988; 94:A472 (Abstract). 127. Smith JP, Wood JG, Solomon TE. Elevated gastrin levels in patients with colon cancer or adenomatous polyps. Dig Dis Sci 1989; 34:171-174. 128. Wong K, Beardshall K, Waters CM et al. Postprandial hypergastrinaemia in patients with colorectal cancer. Gut 1991; 32:1352-1354. 129. Seitz JF, Giovannini M, Gauthier A. Elevated gastrin levels in patients with colorectal cancer. J Clin Gastroenterol 1989; 11:362. 130. Suzuki H, Matsumoto K, Terashima H. Serum levels of gastrin in patients with colorectal neoplasia. Dis Colon Rectum 1988; 31:716-717. 131. Yapp R, Modlin IM, Kumar RR et al. Gastrin and colorectal cancer. Evidence against an association. Dig Dis Sci 1992; 37:481-484. 132. Kikendall JW, Glass AR, Sobin LH et al. Serum gastrin is not higher in subjects with colonic neoplasia. Am J Gastroenterol 1992; 87:1394-1397. 133. Penman ID, el-Omar E, Ardill JE et al. Plasma gastrin concentrations are normal in patients with colorectal neoplasia and unaltered following tumor resection. Gastroenterology 1994; 106:1263-1270. 134. Kameyama M, Fukuda I, Imaoka S et al. Level of serum gastrin as a predictor of liver metastasis from colorectal cancer. Dis Colon Rectum 1993; 36:497-500. 135. Yoshinaga K, Evers BM, Izukura M et al. Neurotensin stimulates growth of colon cancer. Surg Oncol 1992; 1:127-134. 136. Evers BM, Zhou Z, Dohlen V et al. Fetal and neoplastic expression of the neurotensin gene in the human colon. Ann Surg 1996; 223:464-471. 137. Tatsuta M, Iishi H, Baba M et al. Enhancement by neurotensin of experimental carcinogenesis induced in rat colon by azoxymethane. Br J Cancer 1990; 62:368-371. 138. Rovere C, Barbero P, Maoret JJ et al. Pro-neurotensin/neuromedin N expression and processing in human colon cancer cell lines. Biochem Biophys Res Commun 1998; 246:155-159. 139. Maoret JJ, Pospai D, Rouyer-Fessard C et al. Neurotensin receptor and its mRNA are expressed in many human colon cancer cell lines but not in normal colonic epithelium: binding studies and RT-PCR experiments. Biochem Biophys Res Commun 1994; 203:465-471. 140. Ehlers RA II, Bonnor RM, Wang X et al. Signal transduction mechanisms in neurotensin-mediated cellular regulation. Surgery 1998; 124:239-247. 141. Bold RJ, Ishizuka J, Townsend CM Jr. Progress toward hormonal therapy of gastrointestinal cancer. Ann Surg 1996; 223:4-11.

CHAPTER 2

Signaling Pathways in GI Cancers Richard A. Ehlers

C

ancer is classically described as a disease of cell proliferation; cells interact with external factors that bind to membrane receptors to transmit signals to the cell. Cells receive signals to proliferate or to stop proliferating and those signals are transmitted to the nucleus through an ever increasing network of signal transduction pathways. Efforts are currently underway to decipher this network; however, studies are limited by the redundant nature of most signaling mechanisms. This appears to be a defense mechanism for the cells, so that they can adapt to genetic challenges such as loss of, or overexpression of, a particular enzyme. Most investigators now believe that the development and progression of cancer involves the sequential mutation of multiple genes.1 Mutations, when they occur in signaling pathways, confer a more rigid structure to the signaling network and decrease the control over cell growth.2 In fact, many genetic mutations identified in cancers involve these pathways. Scientists are now focusing on understanding and modifying components of these signaling pathways as a means to treat or even prevent cancer. In normal cells, extracellular factors transmit signals through the extracellular membrane by several mechanisms. Growth factors can bind to tyrosine kinase receptors (e.g., PDGF) which in turn phosphorylate and activate a signaling pathway such as the Src kinase pathway. Other types of receptors discussed previously in this book activate second messengers including cAMP, 1,2-diacylglycerol (DAG), Ca2+ and G-proteins. These second messengers then activate downstream pathways such as protein kinase A, protein kinase C and the Ras-MAPK pathway. In cancer cells, as opposed to normal cells, these pathways can be activated by second messengers in response to gut hormones or can be activated independently of extracellular factors by genetic alterations.3 Although there are many such pathways we will discuss four that are commonly affected in gastrointestinal (GI) cancers: Protein kinase A (PKA), Protein kinase C (PKC), Protein Tyrosine Kinase (PTK), and Mitogen-Activated Protein Kinase (MAPK) (Fig. 2.1).

Protein Kinase A One of the best characterized signal transduction pathways, both in vitro and in vivo is that of the cyclic AMP-dependent protein kinase, also known as Protein kinase A (PKA). The second messenger cAMP was discovered by Sutherland in 1957 to be a mediator of hormonal signals.4 Present in all eukaryotic tissues and cells, cAMP exerts its effects via the cAMP-dependent protein kinase.5-7 In the absence of cAMP, PKA is inactive. PKA has a unique structure among protein kinases in that it is composed of two catalytic, or C, subunits and two regulatory, or R, subunits (Fig. 2.2). Cyclic AMP binds to the regulatory subunit inducing a conformational change in PKA structure resulting in release, and thereby dysinhibition, of the two catalytic subunits. These free subunits go on to catalyze Molecular Mechanisms in Gastrointestinal Cancer, edited by B. Mark Evers. ©1999 R.G. Landes Company.

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Fig. 2.1. Signal transduction pathways implicated in GI cancers.

phosphorylation reactions, resulting in a wide variety of cellular functions including gene transcription and cell proliferation or cell growth arrest and differentiation, as well as ion efflux.8 PKA exists in two distinct isoforms, type I (PKAI) and type II (PKAII), which are distinguished by isoforms of the R subunit, RI and RII, respectively.9 Additionally, there are two isoforms of each R subunit (RIα, RIβ, RIIα, RIIβ) as well as three known isoforms of the C subunit (α, β, and γ)10 (Table 2.1). Although the C subunit isoforms show considerable homology and little, if any, difference in action and substrate specificity, there are significant differences in the R isoenzymes. Specifically, the RII group contains an autophosphorylation site within the amino-terminus, whereas RI contains a pseudophosphorylation site at this location thus preventing this important event.11 From a functional standpoint, PKAI/RI and PKAII/RII appear to have quite different functions within cells. PKAII expression mediates cell growth arrest and differentiation. PKAI, on the other hand, has been implicated in cellular proliferation and transformation in a wide variety of tissues.12,13 In particular, several GI malignancies have been studied and PKA specifically evaluated. Using a rat model for stomach cancer induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), total PKA activity was shown to be elevated in stomach tumors.14 Moreover, this study showed that the proliferative effect of the hormone gastrin on these tumors involved the PKAI isoenzyme. More recently, Hahm et al15 has shown that histamine-mediated proliferation of human gastric cancer cell lines is associated with increased PKA activity; both proliferation and PKA activity were significantly reversed by the H2-receptor antagonist cimetidine.

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Fig. 2.2. Model of the PKA holoenzyme. The regulatory subunit (R) dimerizes at the NH2 terminal domain and the substrate site (RRXS) is one point of R/C interaction. The sites A and B toward the carboxyl terminal region represent the two cAMP-binding sites on each R subunit. The catalytic subunit (C) is shown interacting with both the substrate sites and other regions of the R subunit. (Reprinted with permission from McKnight GS, Clegg CH, Uhler MD et al. Recent Prog Horm Res 1998; 44:307-335 ©1998 Academic Press).

PKA may also play a role in pancreatic cancer proliferation. Lieberman et al16 showed that the agent forskolin (FSK), which increases cAMP levels, inhibited epidermal growth factor (EGF)-mediated proliferation in vitro as well as EGF-mediated activation of other signal transduction pathways, namely the MAPK pathway.16 In contradistinction to these findings, recent work by Tortora et al17 demonstrated that PKAI, specifically through the RIα subunit, induces MAPK stimulation in response to EGF treatment of epithelial cells. This difference may simply be a reflection of differential PKA expression in the cell systems used in these studies. The earlier study, however, did not characterize which isoform of PKA was induced or the PKAII/PKAI ratio before and after FSK treatment. The relative of amounts of each PKA (or the PKAII:PKAI ratio) has been shown to be an important factor in determining the effect of PKA within cells, and normalizing this ratio can restore normal growth to cells. If PKAII were increased relative to PKAI by FSK treatment in these pancreatic cells, they would be expected to stop proliferating as they did. Treatment with the site-selective cAMP analog 8-chloro-cAMP (8-Cl-cAMP) produced the same effects on pancreatic cancer cells as FSK.16 This compound has been shown to selectively downregulate PKAI and upregulate PKAII.18 One could speculate that FSK induction of cAMP in this particular pancreatic cancer cell line may restore a more normal PKA expression pattern. However, this point was not examined in this study and selective PKA effects in response to FSK have not been previously shown. For now, the exact role of PKA in pancreatic cancer remains unclear, but further delineation of the effects of PKA isoenzymes are needed. In contrast to pancreatic and stomach cancer, PKA expression in colon cancer has been well studied and has resulted in some important and exciting recent therapeutic findings. PKAI has been clearly implicated in colon cancer growth in a number of human cell lines including HT-29, LS-174T, and GEO.19 Moreover, a study in human surgical colon cancer

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Table 2.1 Subunit types Subunit Type I regulatory RIα

RIβ Type II regulatory RIIα RIIβ

Catalytic Cα Cβ

Description

Constitutively expressed in most tissues examined including brain Corresponds to the published amino acid sequence of RI from bovine skeletal muscle mRNA expressed in brain Induced in developing spermatids at the round spermatid stage Constitutively expressed in all tissues examined including brain Corresponds to the 54-kDa RII Expressed in brain, adrenal, adipose Induced in granulosa cells in response to FSH and estrogen Induced in Friend erythroleukemic cells during differentiation Corresponds to the 51-kDa form Constitutively expressed in all tissues examined including brain Major site of mRNA expression is brain with much smaller but detectable amounts in other tissues

Reprinted with permission from McKnight GS, Clegg CH, Uhler MD et al. Recent Prog Horm Res 1998; 44:307-335 ©1998 Academic Press.

specimens showed PKAI was expressed at higher levels than PKAII in cancer tissue, comprising 80% of the total PKA activity. Adjacent and distant normal mucosa from the same patients showed much less PKAI.20 Selective regulation of PKA isoenzymes has become possible now with the advent of site-selective cAMP analogs. In particular, RIα can be downregulated and RIIβ can be upregulated resulting in a restoration of PKAII/PKAI balance.21 8-Cl-cAMP is the most effective of this class of compounds and shows no toxic effects on cells.21 Treatment of human colon cancer cell lines with 8-Cl-cAMP resulted in decreased RI levels, increased RII levels, and RII nuclear translocation where its normal actions are performed.22 More importantly, 8-Cl-cAMP inhibited growth of the colon cancer cells prompting clinical trials with the drug which are currently ongoing. The importance of the site-selective analogs goes beyond their own role in the treatment of GI cancers in that they have introduced the concept of selective isoenzyme regulation. This concept has now been extended to the use of antisense (AS) oligonucleotides to control the expression of PKA isoenzymes. These compounds bind to target mRNA, such as the RIα subunit of PKA, thereby inhibiting expression into proteins. Recently, this system was studied using a single subcutaneous injection of AS-RIα in nude mice bearing the human colon carcinoma LS-174T.23 With AS therapy, RIα expression was acutely reduced. Furthermore, PKAI activity was suppressed and PKAII was induced, resulting in a PKA ratio similar to control animals. These changes in PKA expression and activity resulted in an inhibition of tumor growth without toxic side effects. The effects of AS on tumor growth persisted even after RIα suppression had ceased, suggesting the possibility that, once restored, PKA balance restores a normal growth pattern and stops further tumor progression. Similar results have been found in multidrug-resistant (MDR) colon cancers treated with

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Fig. 2.3. PKC gene structure and tissue expression. The conserved C1 motif is a cysteine-rich, zinc-binding domain that binds the activators DAG and phosphatidylserine (PS). C2 adds calcium sensitivity to the activation process and also binds PS. C3 and C4 are highly conserved domains providing the ATP binding site and core catalytic domain, respectively. The βI and βIIPKC isozymes are splicing variants differing by 50 residues in the C-terminal. The novel (n) and atypical (a) isozymes are described as lacking the C2 common domain present in the classical (c) isozymes. Predominant tissue expression (heterogeneity within a tissue is common): Br, brain; Ht, heart; Kd, kidney; Lu, lung; Ly, lymphoid; Mu, muscle; Sk, skin; Ts, testes; Ub, ubiquitous. (Reprinted with permission from Mochly-Rosen D, Kauvar LM. Adv Pharmacol 1998; 44:91 ©1998 Academic Press).

AS.24 These clinically frustrating tumors are known to have high levels of RIα. Treatment with AS lowered RIα, induced RIIβ, and inhibited tumor growth.24 In fact, these studies demonstrated that AS treatment in MDR tumor cells induced apoptosis. Although promising, AS therapy alone is not a cure for colon cancer, or any other cancer. However, it may play an important role eventually in multimodal therapy of colon cancer. A recent study demonstrated a synergistic effect between AS therapy and a number of cytotoxic chemotherapeutic agents, particularly paclitaxel, without increasing the toxicity of these drugs.25 As in previous AS studies, combination therapy blocked tumor growth and induced apoptosis in tumor cells as measured by flow cytometry. Combination therapy not only reduced tumor size, but also resulted in a survival advantage in mice implanted with colon cancers.25 The precise role for agents such as site-selective analogs and AS remains to be defined. Indeed, clinical use of AS in patients is probably many years away. Nonetheless, these agents will certainly aid in studying the exact role for the important signaling pathway in GI cancers.

Protein Kinase C Protein kinase C (PKC) was originally identified over 20 years ago as a calciumactivated, phospholipid-dependent kinase.26 Over the ensuing years, molecular cloning and biochemical studies have continued to more precisely characterize PKC as a family of serine/ threonine-specific protein kinases composed of, at last count, 12 different isoforms composing a total of three different subclasses.27 Based on activation requirements, the subclasses are as follows: (1) conventional, or classical PKCs: α, βI, βII γ are dependent on calcium and diacylglycerol (DAG) and are activated by phorbol esters; (2) novel PKCs: δ, ε, η, θ are calcium independent, but can be activated by DAG and phorbol esters; (3) atypical PKCs: ζ, λ, ι, µ are calcium and DAG independent, not activated by phorbol esters, but are

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Fig. 2.4. Schematic representation of PKC. (Reprinted with permission from Hofmann J. FASEB J 1997; 11:649-669 ©1998 Academic Press.)

activated by other phospholipids such as ceramide and retain some sequence homology with the other PKCs27-30 (Figs. 2.3, 2.4). Since the original discovery of PKC, an enormous body of scientific work has been accomplished, better defining the structure, function, regulation, and interactions of this important enzyme family. This work, however, has been complicated considerably by the number, varied distribution, and tissue-to-tissue functional variations of these isoenzymes. A complete review of all of this work is clearly beyond the scope of this discussion, but interested readers are referred to a number of excellent recent

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reviews of PKC.28,29,31-34 Our focus will be on recent work examining the location, regulation and modification of PKC isoenzymes in colon cancer. While some PKC isoforms are ubiquitous in their tissue distribution, others are more restricted. Isoforms also vary in their subcellular distribution, ostensibly reflecting their activation status. Indeed, not all isoforms have been found within the colon. Animal studies have identified the presence of PKC α, βII, δ, ε, and ζ in normal colonocytes in rats.35,36 These same isoforms, with the exception of PKC ε, have been implicated in the normal growth cessation and/or differentiation of enterocytes and colonocytes in the rat.37,38 In human colon samples, PKC α, β, δ, ε, and ζ were detected as well as PKC η.39 Within both rats and humans, PKC α, β, and ζ appear to be associated more with the cytosolic portion of the cell implying that, while present, they are not completely activated. In contrast, PKC δ, ε, and, in humans, η, are normally associated with the particulate, or membrane, fraction and are activated in a normal state.36,39 In light of the fact that PKC plays a significant role in normal colonic growth and development, it is not surprising that these normal expression patterns are significantly altered in colon cancer. A number of studies have shown a decrease in total PKC activity in rat models of colon cancer.40,41 More careful investigations of the individual isoenzymes reveals that α, δ, and ζ are decreased, βII is increased, and ε is unchanged.36,42 Moreover, these changes in PKC are accompanied by an increase in DAG mass. Although PKC ε was unchanged in this model, its possible role has been suggested by transfection studies in which PKC is overexpressed in colonic cells in vitro. Transfection of PKC ε into poorly metastatic colon cancer cells resulted in marked morphologic changes, anchorage independence and also caused tumors with 100% incidence in nude mice whereas controls produced no tumors.43 PKC alterations have been noted in both human adenomas and carcinomas. As in rat models, total PKC activity is decreased in tumors compared to surrounding normal adjacent and distant mucosa and normal mucosa from control patients.39,44,45 This decrease was also noted in precancerous adenomas indicating that it occurs early in the carcinogenic process.44,45 However, analysis of the pattern of isoenzymes and their subcellular distributions is more complex. Of the three cited studies examining PKC activity in human colon samples, one did not characterize isoenzyme expression at all.45 It did, however, show a relative increase in the membrane-associated/total PKC activity ratio despite lower overall PKC activity, suggesting but not proving a relative increase in δ, ε, η and a relative decrease in α, β, ζ.45 The other studies provide conflicting results. Levy et al44 showed a significant decrease in both total PKC activity and PKC β mRNA expression. In contrast to these findings, but consistent with rat models, Davidson et al39 demonstrated a decreased total PKC activity, but an increase in PKCβII expression at the protein level. Adding further to this confusion is the finding that overexpression of PKC βI in HT-29 human colon cancer cells inhibits growth and tumorigenicity when implanted into nude mice.46 Whether these differences are due to post-transcriptional modifications or reflect that individual isoenzyme expression by itself is unimportant remains to be elucidated. However, recent evidence of the importance of selective PKC isoenzymes is accumulating. PKC α appears to play an important role in cellular adhesion by moderating fibronectin, laminin and CEA expression.47 PKC α expression is also important in p21WAF1 expression and thus in cellular differentiation.48 Moreover, PKC β expression activates other kinase pathways and provides a link to other signal transduction mechanisms.49,50 Recently, Davidson et al51 has noted a correlation between colon cancer incidence and the ratio of PKC βII:PKC ζ. Even more importantly, they have characterized this relationship using colon cells excreted in feces and RT-PCR to screen for PKC isoenzyme expression. They demonstrated that this technique is possible in humans and correlates well with results

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obtained from colon scrapings.39 This novel, noninvasive technique may become an important method of screening patients in the future. The importance of PKC in colon cancer involves not only its possible role as a biomarker for early detection, but also its possible regulation in the prevention of cancer. It has long been postulated that diet plays a significant role in colon cancer. General consensus, on the basis of epidemiological data, is that high dietary fat and low fiber content increases the incidence of colon cancer.52,53 There are several theories explaining this effect that involve the PKC pathway. High fat content in the diet increases exposure of the colon to bile acids which are known to selectively modulate various PKC isoenzymes both in animal models and humans.36,54 However, the influence of bile acids on colon cancer in general is quite controversial. Studies in humans have shown conflicting data on the incidence of colon cancer after cholecystectomy, a condition producing increased bile acid exposure. Regardless of the effect of bile acids, high dietary fat has been shown to directly alter the levels of intracellular lipids that act as second messengers in the PKC pathway, namely DAG and ceramide.55 Chronic high dietary fat is thought to increase these second messengers leading to persistent activation followed by downregulation of PKC isoenzymes. This effect can be suppressed by dietary fish oil, a suspected cancer-preventing agent.56 Fish oil, probably via the Ras pathway, abrogates the increased levels of DAG thus removing the initial PKC stimulus.56 In addition to dietary modifications, some drugs are capable of acting on the PKC pathway. PKC inhibitors such as staurosporine and safingol, which have been effectively used as combination therapy with mitomycin C in gastric cancer in vitro,57 may eventually play a role in cancer treatment. In summary, PKC appears to play a role in the pathogenesis of colon cancer and dietary factors likely contribute to the observed PKC alterations. Defining the exact role of the PKC isoenzymes in colon carcinogenesis remains an important goal and is being actively pursued. The development of selective isoenzyme inhibitors and even isoenzyme knockout animal models will aid immensely in this work.

Protein Tyrosine Kinases Protein tyrosine kinases (PTKs), as the name indicates, differ from other kinases in that they catalyze the phosphorylation of tyrosine residues as opposed to serine/threonine residues. There are two main classes of PTKs: the transmembrane receptor PTKs, which will be discussed elsewhere, and the nonreceptor PTKs, of which the most studied and well characterized is the Src family.58 Similar to other intracellular kinase signaling mechanisms, the Src family is composed of numerous related kinases, now totaling nine, that share significant sequence homology and regulate a wide variety of cellular responses to extracellular signals.59,60 While some members of the Src family are ubiquitously expressed, others show tissue selective expression and function. For this discussion, we will refer primarily to the prototype tyrosine kinase, Src. An extensive review of all PTKs, even one of the Src family alone, is well beyond the scope of this section. We will address the salient data pertaining to the role of Src in gastrointestinal cancers, particularly colon cancer. Src was first identified in its mutated form, the v-src oncogene, in the Rous sarcoma virus. The normal cellular homolog of v-src, c-src, was later recognized and found to be present in normal, that is noncancerous, cells.61 Both v-src and c-Src encode membraneassociated proteins with tyrosine-specific protein kinase activity. The distinguishing functional feature between these two proteins is their intrinsic kinase activity. Whereas normal c-Src activity is tightly controlled, v-Src and other oncogenic forms of c-Src have much higher constitutive levels of kinase activity.60 Overexpression and activation of c-Src has been demonstrated as an early event in colon cancer both in vitro and in vivo.62,63 Moreover, elevated c-Src activity has been noted in known premalignant conditions such

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as adenomas and even ulcerative colitis, particularly when it involves tissue dysplasia, suggesting a possible role for this enzyme in carcinogenesis.64 These previous studies used similar techniques measuring in vitro kinase activity from colon cancer specimens. With the recent development of improved monoclonal antibodies for Src, more precise localization of c-Src in the colon is now possible using immunohistochemical techniques. Two recent studies, one in the United States and the other in Japan, have examined human adenocarcinomas, premalignant adenomas, and normal colon mucosa with some surprising results. In both studies active c-Src was present at high levels in adenomatous tissue and showed a significant difference when compared to normal mucosa. However, only a small number of adenocarcinomas in these two studies strongly expressed c-Src (15% and 25%, respectively). Furthermore, only a minority (32%) of metastatic lesions expressed c-Src in the one study that examined metastasis.65,66 These important findings corroborate, on an in situ level, that c-Src expression and activation is an early event in the carcinogenesis process in the colon. Recent studies have examined c-Src activity in other gastrointestinal malignancies, such as pancreas.67 Both primary pancreatic cell cultures and human pancreatic cancer cell lines showed a significant increase in c-Src activity. Furthermore, using immunohistochemical studies, Src was overexpressed in all 13 human pancreatic cancer surgical specimens examined. Src was either not detected, or faintly detected in pancreatic specimens from 2 normal patients.67 These data indicate, for the first time, that Src may play a role in pancreatic carcinogenesis. Although studies now implicate Src in the carcinogenesis of both colon and pancreas, one wonders what the mechanism is for increased Src activity. No studies have demonstrated convincingly that Src activity correlates with Src expression alone. In fact, the aforementioned studies found no correlation between activity and expression. Therefore, it is likely that some post-transcriptional mechanism is involved. Regulation of Src activity remains incompletely understood. However, the enzyme Csk (C-terminal Src kinase) is a known negative modulator of the entire Src family.68,69 By phosphorylating Src at the Tyr-527 position, Csk downregulates Src activity. One could speculate that alterations in Csk levels or activity may control c-Src activation in colon or pancreatic cancer. Unfortunately, Src activity did not show a correlation with Csk expression in pancreatic cancer.67 Csk activity was not assessed, nor has Csk expression or activity been studied in human colon cancer specimens. Other allosteric c-Src modulators may be involved as well such as the polyoma virus middle-sized tumor antigen (MTAg). Although lacking any kinase activity of its own, MTAg binds c-Src and blocks the Tyr-527 site from Csk-mediated phosphorylation, effectively disinhibiting c-Src.70 Indeed, one group has shown that MTAg transfection into a premalignant colon cell line induces tumorigenicity in nude mice.71 Other mechanisms of Src regulation may include intracellular phosphatases or alternate signal transduction pathways such as Ras.60 Nonetheless, much work remains to be done clarifying the regulation and dysregulation of c-Src activity in colon and pancreatic cancer. Despite uncertainty over its regulation, c-Src function is becoming somewhat clearer. Src most certainly plays a role in cell proliferation in GI cancer. It is known to be associated with transmembrane protein kinase receptor c-Met, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF).72-74 Moreover, Src inactivation with the PTK inhibitor herbimycin A resulted in growth inhibition in both colon and pancreatic cells.67,75 The exact mechanism by which Src mediates proliferation, however, remains unknown. Src has numerous substrates exerting a wide variety of functions.60 One of the more intriguing of these is the focal adhesion kinase (FAK). A tyrosine kinase itself, FAK is important in cell motility and adherence and has been suggested to play a role in the metastatic ability of several cancers, including colon cancer.76,77 FAK is associated with, and its phosphorylation

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regulated by c-Src.78 It has been postulated that Src, through its interaction with FAK, may influence the metastatic ability of colon cancer.72,79 However, it is unlikely that FAK is an essential factor in metastasis as overexpression of FAK in human colon carcinoma cell lines does not increase the rate of metastasis when implanted into nude mice.80 Nonetheless, c-Src may use FAK to mediate other important signaling pathways such as the Ras/MAPK pathway.72

Mitogen-Activated Protein Kinases Of the signal transduction pathways involved in GI cancers, the Mitogen-Activated Protein Kinases (or MAPKs) are appropriately discussed last. This ubiquitous family of enzymes is composed of three main members: the extracellular-signal regulated kinases (ERK1 and ERK2), the c-Jun N-terminal kinases, which are also called stress-activated protein kinases (JNK/SAPK), and the p38 kinase.81,82 The ERKs are the best characterized of the three and are intimately associated with the process of cell growth and proliferation. Activated ERKs translocate to the nucleus and phosphorylate transcription factors such as Elk-1 inducing transcription of AP-1 proteins. In fact, the constitutive activation of MAPK itself is sufficient for tumorigenesis.83 The ERK cascade is vital in the transmission of mitogenic signals from the cell surface to the nucleus and appears to represent a mechanism for the convergence of many other signaling pathways (Fig. 2.5).81,84-86 The JNK/SAPK pathway was originally characterized in response to cellular stress, but may be involved with cell proliferation as well by phosphorylating c-Jun, inducing c-Jun dimerization, and AP-1 activation.87 Some of the most important and potent mitogenic signals within the GI tract are gut hormones acting through their cell surface receptors, primarily the G-protein coupled class of receptors (GPCRs). Under physiologic conditions, the GPCRs have been shown to activate the MAPK pathway in response to ligand binding.81,85 An increasing number of studies are now examining the role of the MAPK pathway in hormone-mediated cancer proliferation. Not unexpectedly, ERK is stimulated in pancreatic cancer cells in response to the hormones gastrin (via the CCK-B receptor), cholecystokinin, and bombesin.88,89 In our laboratory, we have shown that neurotensin (NT) induces its proliferative effects on colon cancer cell lines expressing the NT receptor by stimulating the ERK cascade.90 Furthermore, we have shown that NT stimulates the JNK pathway in the human pancreatic cancer cell line MIA Paca-2 (unpublished data). JNK cascade activation has also been demonstrated in isolated rat pancreatic acini in response to treatment with CCK and, to a lesser extent, with bombesin.91 These data together are not entirely surprising given that these hormones are known mitogens. The MAPK cascade has been studied in a rat model of 1,2-dimethylhydrazine-(DMH) induced colon carcinogenesis with interesting results.92 Licato et al92 compared ERK and JNK activity in colon cancers from DMH-treated rats and normal colon from the same animals and normal controls. Both ERK and JNK activity were increased in the tumors compared to the normal colon of control animals. Furthermore, there was no difference between ERK activity in normal colon from DMH animals compared to the normal controls.92 The increase in ERK and JNK activity also resulted in AP-1 activation. Together, these studies suggest that both ERK and JNK are important in neoplasia and not simply increased proliferation.92 These findings contrast with several studies in human GI cancers. In a series examining human gastric adenocarcinomas, ERK activity was actually decreased in the membrane fraction of the tumors and unchanged within the cytosol.93 ERK protein expression was also decreased. Similar results were found in human colon adenocarcinoma taken from surgical specimens: membrane-associated MAPK was decreased and cytosolic MAPK was

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Fig. 2.5. Activation of the ERK cascade and its effects. The MAPKKKs of the Raf subfamily are activated by processes that may involve Ras, GTP, c-Src PTKs, PKC and/or 14-3-3 proteins. Activated Raf phosphorylates and activates the MAPKKs, MEK1 and MEK2. This in turn leads to phosphorylation and activation of the MAPKs, ERK1 and ERK2. ERK1 and ERK2 phosphorylate a number of enzymes/proteins involved in the regulation of a diverse range of cellular processes. (Reprinted with permission from Sugden PH, Clerk A. Cell Signal 1997; 9:337-351 ©1997 Elsevier Science Inc.)

unchanged.94 Although MAPKs are not typically thought to be membrane-associated, the identification of decreased activity along with no change in cytosolic MAPK in human cancers seems curious. Several possibilities may explain these results. Given the small number of tumors examined, sampling error from the tumor could have occurred (i.e., the tumor collected could have been necrotic). Patient-to-patient variability may also provide an explanation for these findings. Finally, there may be some real, but as yet unknown, difference between rat tumors, which showed increased MAPK activity, and human tumors which have shown decreased activity. A more recent study sought to characterize MAPK activity in untreated human pancreatic cancer cell lines. They found that, in cell lines expressing a mutant form of K-Ras, MAPK activity was significantly decreased when compared to nonmutant K-Ras cell lines.95 No studies have yet been published investigating JNK activity in human cancers from patients. The finding of decreased ERK activity in mutant Ras cells is an interesting one. The MAPK pathway is believed to begin with G-protein activation of Ras by GTP binding. Ras activation leads to Raf recruitment to the membrane.96 Once activated, Raf, in turn, activates the MAPK/ERK kinase (or MEK) which then activates MAPK itself.86,97 Many GI cancers have been shown to have a high rate of expression of mutant Ras, which is permanently GTP-bound and active.98 According to the model, increased Ras activity in GI cancer would lead to increased Raf, MEK, and MAPK activity. The actual data in human cancers, there-

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fore, present some important information about the MAPK pathway. In addition to investigating MAPK activity in pancreatic cancer, Berger et al95 assessed the upstream activator of MAPK, Raf-1. They demonstrated that Raf-1 expression did not correlate with MAPK activity suggesting some alternate pathway for signal transduction through the MAPK cascade. In fact, MEK and MAPK have been shown to be activated in a Ras-independent manner, in addition to the known Ras-dependent pathway.99 Moreover, MAPK, when activated by some alternate route, is capable of downregulating Raf providing feedback inhibition for itself.100 It is possible, therefore, that MAPK is activated during the process of tumorigenesis but acts upstream to inhibit its own activation in later stages of cancer. This might explain the decreased MAPK activity noted in human cancers. However, this has not yet been proven in GI cancers and may be due to another more complex mechanism entirely. The use of Raf-specific inhibitors is underway and will help to clarify this system.101 We are clearly in the early stages of understanding the role of the MAPK pathway in GI cancers. As with other signaling pathways, MAPKs act on a number of substrates, some of which are signal transduction proteins themselves. Cyclic AMP, PKC, PTK (both receptor and nonreceptor) can all modulate the MAPK pathway thus adding to the complexity of this system.

Conclusions Although much work has been done to examine the role of signaling pathways in GI cancer, we remain at a very early stage in our knowledge of these mechanisms. New classes of agents which inhibit various members of these signaling pathways are being developed which are cytostatic, as opposed to more traditional cytotoxic chemotherapeutic agents. These agents may play an important role in future adjuvant combination therapy of GI cancer, preventing progression of metastatic disease or even inhibiting local disease until tumor cells die by attrition.

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32. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular response. FASEB J 1995; 9:484-486. 33. Mochly-Rogen D. Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 1995; 268:247-251. 34. Hug H, Sarre TF. Protein kinase C isoenzymes: Divergence in signal transduction? Biochem J 1993; 291:329-343. 35. Bissonnette M, Wali RK, Hatrmann SC et al. 1,25-Dihydroxyvitamin D 3 and 12-0-Terradecanoyl phorbol 13-Acetate cause differential activation of Ca2+-dependent and Ca2+-independent isoforms of protein kinase C in rat colonocytes. J Clin Invest 1995; 95:2215-2221. 36. Wali RK, Frawley BP, Hartmann S et al. Mechanism of action of chemoprotective ursodeoxycholate in the azoxymethane model of rat colonic carcinogenesis: Potential roles of protein kinase c-α, βII and ζ. Cancer Res 1995; 55:5257-5264. 37. Saxon ML, Zhao X, Black JD. Activation of protein kinase C isozymes is associated with postmitotic events in intestinal epithelial cells in situ. J Cell Biol 1994; 126:747-763. 38. Jiang YH, Aukema HM, Davidson LA et al. Localization of protein kinase C isozymes in rat colon. Cell Growth Diff 1995; 6:1381-1386. 39. Davidson LA, Jiang YH, Derr JN et al. Protein kinase C isoforms in human and rat colonic mucosa. Arch Biochem Biophy 1994; 312:547-553. 40. Wali RK, Baum CL, Bolt MJG et al. Down regulation of protein kinase C activity in 1,2-dimenthylhydrazine-induced rat colonic tumors. Biochim Biophys Acta 1991; 1092:119-123. 41. Baum CL, Wali RK, Sitrin MD et al. 1,2-dimenthylhydrazine-induced alterations in protein kinase C activity in the rat preneoplastic colon. Cancer Res 1990; 50:3915-3920. 42. Craven PA, DeRubertis FR. Alterations in protein kinase C in 1,2-Dimethylhydrazine induced colonic carcinogenesis. Cancer Res 1992; 52:2216-2221. 43. Perletti GP, Folini M, Lin HC et al. Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells. Oncogene 1996; 12:847-854. 44. Levy MF, Pocsidio J, Guillem JG et al. Decreased levels of protein kinase C enzyme activity and protein kinase C mRNA in primary colon tumors. Dis Colon Rectum 1993; 36:913-921. 45. Kopp R, Noelke B, Sauter G et al. Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas. Cancer Res 1991; 51:205-210. 46. Choi PM, Wang KMT, Weinstein IB. Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression. Mol Cell Biol 1990; 10:4650-4657. 47. Chakrabarty S, Rajagopal S, Moskal TL. Protein kinase Cα controls the adhesion but not the antiproliferative response of human colon carcinoma cells to transforming growth factor βI: Identification of two distinct branches of postprotein kinase Cα adhesion signal pathway. Lab Invest 1998; 78:413-421. 48. Abraham C, Scaglione-Sewell B, Skarosi SF et al. Protein kinase Cα modulates growth and differentiation in Caco-2 cells. Gastroenterology 1998; 114:503-509. 49. Sauma S, Yan Z, Ohno S et al. Protein kinase βI and protein kinase βII activate p57 mitogen-activated protein kinase and block differentiation in colon carcinoma cells. Cell Growth Differ 1996;7:587-594. 50. Sauma S, Friedman E. Increased expression of protein kinase Cβ activates ERK3. J Biol Chem 1996; 271:11422-11426. 51. Davidson LA, Aymond CM, Jiang YH et al. Noninvasive detection of fecal protein kinase C βII and ζ messenger RNA: putative biomarkers for colon cancer. Carcinogensis 1998; 19:253-257. 52. Potter JD, Slattery ML, Bostick RM et al. Colon cancer: A review of the epidemiology. Epidemiol Rev 1993; 15:499-545. 53. Reddy BS. Dietary fat, calories, and fiber in colon cancer. Prev Med 1993; 22:738-749. 54. Pongracz J, Clark P, Neoptolemos JP et al. Expression of protein kinase C isoenzymes in colorectal cancer tissue and their differential activation by different bile acids. Int J Cancer 1995; 61:35-39.

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55. Jiang YH, Lupton JR, Chang WCL et al. Dietary fat and fiber differentially alter intracellular second messengers during tumor development in rat colon. Carcinogenesis 1996; 17:1227-1233. 56. Chapkin RS, Jiang YH, Davidson LA et al. Modulation of intracellular second messengers by dietary fat during colon tumor development. Adv Exp Med Biol 1997; 422:85-96. 57. Schwartz GK, Haimovitz-Friedman A, Dhupar SK et al. Potentiation of apoptosis by treatment with the protein kinase C-specific inhibitor safingol in mitomycin C-treated gastric cancer cells. J Natl Cancer Inst 1995; 87:1394-1399. 58. Boardman LA, Karnes WE. Src family of tyrosine kinases: A role of c-Yes in colon carcinogenesis. Gastroenterology 1995; 108:291-294. 59. Cooper JA, Howell B. The when and how of Src regulation. Cell 1993; 73:1051-1054. 60. Brown MT, Cooper JA. Regulation, substrates and functions of Src. Biochim Biophys Acta 1996; 1287:121-149. 61. Jove R, Hanafusa H. Cell transformation by the viral Src oncogene. Ann Rev Cell Biol 1987;3:31-56. 62. Budde RJA, Ke S, Levin VA. Activity of pp60c–src in 60 different cell lines derived from human tumors. Cancer Biochem Biophys 1994; 14:171-175. 63. Cartwright CA, Meisler AI, Eckhart W. Activation of the pp60c–src protein kinase is an early event in colonic carcinogenesis. Proc Natl Acad Sci USA 1990; 87:558-562. 64. Cartwright CA, Coad CA, Egbert BM. Elevated c-Src tyrosine kinase activity in premalignant epithelia of ulcerative colitis. J Clin Invest 1994; 93:509-515. 65. Iranvani S, Mao W, Fu L et al. Elevated c-Src protein expression is an early event in colonic neoplasia. Lab Invest 1998; 78:365-371. 66. Sakai T, Kawakatsu H, Fujita M et al. An epitope localized in c-Src negative regulatory domain is a potential marker in early stage of colonic neoplasm. Lab Invest 1998; 87:219-225. 67. Lutz MP, Eber IBS, Flossmann-Kast BBM et al. Overexpression and activation of the tyrosine kinase Src in human pancreatic carcinoma. Biochem Biophys Res Commun 1998; 243:503-508. 68. Okada M, Nada S, Yamanashi Y et al. CSK: A protein-tyrosine kinase involved in regulation of Src family kinases. J Biol Chem 1991; 266:24249-24252. 69. Sabe H, Okada M, Nakagawa H et al. Activation of c-Src in cells bearing v-Crk and its suppression of CSK. Mol Cell Biol 1992; 12:4706-4713. 70. Perez L, Paasinen A, Schnierle B et al. Mitosis-specific phosphorylation of polyomavirus middle-sized tumor antigen and its role during cell transformation. Proc Natl Acad Sci USA 1990; 90:8113-8117. 71. Empereur S, Djelloui S, DiGioia Y et al. Progression of familial adenomatous polyposis (FAP) colonic cells after transfer of the src or polyoma middle T oncogenes: cooperation between src and HGF/Met in invasion. Br J Cancer 1997; 75:241-250. 72. Parsons JT, Parsons SJ. Src family protein tyrosine kinases: Cooperating with growth factor and adhesion signaling pathways. Curr Opin Cell Biol 1997; 9:187-192. 73. Hiscox SE, Hallett MB, Puntis MCA et al. Expression of the MGF/SF receptor, c-met, and its ligand in human colorectal cancers. Cancer Invest 1997; 15:513-521. 74. Mao W, Irby R, Coppola D et al. Activation of c-Src by receptor tyrosine kinases in human colon cancer cells with high metastatic potential. Oncogene 1997; 15:3083-3090. 75. Garcia R, Parikh NU, Saya H et al. Effect of herbimycin A on growth and pp60c-src activity in human colon tumor cell lines. Oncogene 1991; 6:1983-1989. 76. Richardson A, Parsons JT. A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK. Nature 1996; 380:538-540. 77. Owens LV, LiHui X, Craven RJ et al. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 1995; 55:2752-2755. 78. Schaller MD, Hildebrand JD, Shannon JD et al. Autophosphorylation of the focal adhesion kinase, pp125 FAK , directs SH2-dependent binding of pp60 src . Mol Cell Biol 1994; 14:1680-1688.

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79. Brunton VG, Ozanne BW, Paraskeva C et al. A role for epidermal growth factor receptor, c-src, and focal adhesion kinase in an in vitro model for the progression of colon cancer. Oncogene 1997; 14:293-293. 80. Irby R, Mao W, Coppola D et al. Overexpression of normal c-Src in poorly metastatic human colon cancer cell enhances primary tumor growth but not metastatic potential. Cell Growth Differ 1997; 8:1287-1295. 81. Sugden PH, Clerk A. Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors. Cell Signal 1997; 9:337-351. 82. Woodgett JR, Arruch J, Kyriakis J. The stress activated protein kinase pathway. Cancer Surv 1997; 27:127-138. 83. Mansour SJ, Matten WT, Hermann AS et al. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 1994; 265:966-970. 84. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 1997; 9;180-186. 85. Gutkind JS. The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 1998; 273:1839-1842. 86. Hill CS, Triesman R. Transcriptional regulation of extracellular signals: Mechanisms and specificity. Cell 1995; 80:199-211. 87. Minden A, Karin M. Regulation and function of the JNK subgroup of MAP kinases. Biochim Biophys Acta 1997; 1333:F85-F104. 88. Todisco A, Takeudi Y, Urumov A et al. Molecular mechanisms for the growth factor action of gastrin. Am J Physiol 1997; 273:G891-G898. 89. Duan R-D, Williams JA. Cholecystokinin rapidly activates mitogen-activated protein kinase in rat pancreatic acini. Am J Physiol 1994; 267:G401-408. 90. Ehlers RA, Bonnor RM, Wang XF et al. Signal transduction mechanisms in neurotensinmediated cellular regulation. Surgery 1998; 124:239-247. 91. Dabrowski A, Grady T, Logsdon CD et al. Jun kinases are rapidly activated by cholecystokinin in rat pancreas both in vitro and in vivo. J Biol Chem 1996; 271:5686-5690. 92. Licato LL, Keku TO, Worzelmann JI et al. In vivo activation of mitogen-activated protein kinases in rat intestinal neoplasia. Gatroenterology 1997; 113:1589-1598. 93. Atten MJ, Attar BM, Helian O. Decreased MAP kinase activity in human gastric adenocarcinoma. Biochem Biophys Res Commun 1995; 212:1001-1006. 94. Attar BM, Atten MJ, Holian O. MAPK activity is downregulated in human colon adenocarcinoma: correlation with PKC activity. Anticancer Res 1976; 16:395-400. 95. Berger DH, Jardines LA, Chang H et al. Activation of Raf-1 in human pancreatic adenocarcinoma. J Surg Res 1997; 69:199-204. 96. Stokoe D, Macdonald SG, Cadwallader K et al. Activation of Raf as a result of recruitment to the plasma membrane. Science 1994; 264:1463-1467. 97. Howe LR, Leavers SJ, Gomez N et al. Activation of the MAP kinase pathway by the protein kinase raf. Cell 1992; 71:335-342. 98. Bos JL, Fearon ER, Hamilton SR et al. Prevalence of ras gene mutations in human colorectal cancers. Nature 1987; 327:293-297. 99. Xu S, Khoo S, Dang A et al. Differential regulation of mitogen-activated protein/ERK kinase (MEK)1 and MEK2 and activation by a Ras-independent mechanism. Mol Endocrinol 1997; 11:1618-1625. 100. Williams NG, Paradis H, Agarwal S et al. Raf-1 and p21v–Ras cooperate in the activation of mitogen-activated protein kinase. Proc Natl Acad Sci USA 1993; 90:5772-5776. 101. Naumann V, Eisenmann-Tappe I, Rapp UR. The role of Raf kinases in development and growth of tumors. Recent Results Cancer Res 1997; 143:237-244.

CHAPTER 3

The Role of the COX/Prostaglandin Pathway in GI Cancers Richard T. Ethridge

A

rachidonic acid (5,8,11,14-eicosatetraenoic acid) normally resides in the plasma membrane where it is esterified to phospholipids. Upon its liberation by a variety of phospholipases, arachidonic acid becomes the substrate for further enzymatic modification.1-3 Once arachidonic acid is released from the membrane, it may be utilized by one of three different enzymatic pathways. These include the lipoxygenase, cytochrome P450, or cyclooxygenase (COX) enzymatic pathways. Of these three pathways, the one that is currently being implicated as having a role in tumorigenesis and carcinogenesis is the cyclooxygenase pathway (Fig. 3.1).4 Two isoforms of the COX enzyme have been identified. Both isoforms have cyclooxygenase and peroxidase activities, which convert arachidonic acid to prostaglandin G2 (PGG2) by inserting two oxygen molecules and then reducing PGG2 to PGH2 (Fig. 3.2). This unstable metabolite can then be converted to a variety of prostaglandins depending on the enzymes present in a particular cell type. Cyclooxygenase-1 (COX-1) or prostaglandin synthase-1 (Ptgs-1) was first purified from bovine vesicular glands in 1976 and is expressed at constitutive levels in most tissues and is not inducible with treatment of growth factors and other stimuli.5 Its genomic DNA has been characterized in numerous sources including human, bovine, and mouse tissues.6-8 The COX-1 gene is composed of 11 exons and 10 introns which span 22.5 kb, however, this large gene only yields a 2.7 kb mRNA. The COX-1 protein encoded by this mRNA is 565-residues and is 65 kDa in size. In humans, this locus is located on chromosome 9q32-q33.3.9 Recently, the localization of COX-1 activity has been demonstrated to be primarily in the endoplasmic reticulum.10 Cyclooxygenase-2 (COX-2) or prostaglandin synthase-2 (Ptgs-2) was more recently cloned by two groups working separately.11,12 COX-2 is not normally expressed in most tissues, however, it is highly inducible upon stimulation by mitogens, lipopolysaccharide, growth factors, and cytokines.2 COX-2 is composed of 10 exons and 9 introns. In general, the genomic structures of COX-1 and COX-2 have been conserved with the exception that exon 2 is absent in COX-2 and the sizes of the introns are reduced (Fig. 3.3). The gene product for COX-2 is 4.5 kb and the protein translated is approximately 70 kDa in size. Using fluorescence in situ hybridization, COX-2 has been localized to chromosome 1q25.2-q25.3 in humans.13 COX-2 activity is localized in both the endoplasmic reticulum and the perinuclear envelope.10 The COX-1 and COX-2 genes and proteins exhibit approximately 60% and 75% homology, respectively; however, there are several important differences between these genes. For example, the COX-2 gene contains a TATA box and regulatory sites for Molecular Mechanisms in Gastrointestinal Cancer, edited by B. Mark Evers. ©1999 R.G. Landes Company.

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Fig. 3.1. Schematic diagram of arachidonic acid metabolism via the cyclooxygenase pathway.

glucocorticoids and cytokines while the COX-1 gene does not.2 There are also differences in the primary amino acid sequence; the amino terminus is truncated in the COX-2 protein and the carboxy terminus has an 18-residue insert that is absent from COX-1. However, the peroxidase and cyclooxygenase regions are conserved between the two proteins.14 The clinical relevance of these enzymes is that they are the targets of nonsteroidal antiinflammatory drugs (NSAIDs), such as aspirin. Aspirin acetylates a serine in the active site in these two enzymes. With COX-1, this acetylation completely inhibits its ability to utilize arachidonic acid, however, with COX-2, its oxygenase activity persists allowing the conversion of arachidonic acid to 15 (R) HETE. This difference is thought to be due to the slight differences in the protein sequence of their active sites.

The COX/Prostaglandin Pathway and Colorectal Cancers Early reports demonstrated elevated levels of prostaglandins in colorectal cancer compared to normal mucosa15-17 and more recently, a more comprehensive study showed a significant increase in PGE2 in 21 surgically excised colorectal cancers.18 Stemming from this observed increase in prostaglandin synthesis in colorectal cancer, recent studies have analyzed the levels of the COX enzymes in colorectal cancer. Eberhart et al19 measured COX-1 and COX-2 mRNA levels in adenocarcinomas and the paired normal mucosa of 14 surgical patients with cancers classified as Dukes’ Stage B to D. Low to undetectable levels of COX-2 mRNA were observed in the normal mucosa. However, COX-2 expression was apparent in 12 of the 14 carcinomas (86%). This increase in COX-2 varied from a 2- to 50-fold elevation compared to normal mucosa and the degree of upregulation did not appear to correlate

The Role of the COX/Prostaglandin Pathway in GI Cancers

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Fig. 3.2. Cyclooxygenase and peroxidase activities of PGH synthesis in the metabolism of arachidonic acid.

with the stage or the differentiation state of the tumor. There were no differences in the level of COX-1 expression in the colon cancer compared with normal mucosa. This increased expression of COX-2 in colon cancers has been confirmed by other groups and, in addition, these studies have demonstrated a concomitant increase in COX-2 protein levels. Therefore, these studies demonstrate that COX-2, and not COX-1, may contribute to colon carcinogenesis.

COX-2 and Carcinogenesis Dubois et al20 has shown that transforming growth factor-α (TGF-α), which is associated with the progression of transformed properties in human colon cancer cells,21 results in the induction of COX-2 expression and increased levels of eicosanoids in a nontransformed rat intestinal epithelial (RIE-1) cell line. This increase in COX-2 is associated with an increase of the immediate-early genes, such as c-myc and junB, which have been shown to play a direct role in growth regulation.22,23 As a logical extension of these experiments, Tsujii and DuBois24 transfected the RIE-1 cells with a COX-2 cDNA to achieve overexpression of COX-2 and then assessed the ability of these cells to adhere to extracellular matrix. RIE cells overexpressing COX-2 demonstrated a 6-fold increase in their adherence to Matrigel and laminin-coated plates which suggests that the cellular adhesion pathway is altered with COX-2 overexpression. In addition, RIE cells overexpressing COX-2 showed an increased resistance to apoptosis, an increased expression of the anti-apoptotic protein Bcl-2, and a decrease in the TGFβ2 receptor and E-cadherin levels, which have been implicated in colorectal carcinogenesis and tumor invasiveness.25-29 These data suggest that increased levels of COX-2 play a crucial

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Fig. 3.3. Human cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) genomic structure. (Reprinted with permission from: Williams CS, DuBois RN, Am J. Physiol. 1996; 270:G393-G400. ©The American Physiological Society).

role in carcinogenesis by affecting phenotypic changes that are associated with tumorigenesis in a nontransformed cell line.

COX-2 and Metastatic Potential The increased mortality associated with colorectal cancer is due to its ability to spread beyond the large intestine prior to diagnosis. The ability of a cancer to spread is dependent upon its ability to release itself from its basement membrane. Caco-2 cells, a human colon cancer cell line, transfected with COX-2 demonstrated increased levels of metalloproteinase-2 (MMP-2) which is involved in extracellular matrix degrading activity.30 Using an invasion chamber analysis, these cells showed a 6-fold increase in the invasion of the matrix compared with the nontransfected Caco-2 cells. Treatment of Caco-2 cells overexpressing COX-2 with sulindac inhibited the increased PGE2 production and invasiveness in a dosedependent fashion suggesting that increased expression of COX-2 results in increased metastatic potential by increasing the expression of MMP-2. While this in vitro study demonstrated an apparent correlation of COX-2 expression and a more invasive phenotype, studies using surgically excised specimens have not shown a similar correlation. Therefore, COX-2 expression may only be one of the factors that determine invasiveness of a tumor.

COX-2 and Angiogenesis Several recent studies have demonstrated the importance of angiogenesis (i.e., the formation of new vascular supply) for the growth of tumors.31,32 The increase in vascular supply is thought to be necessary for a tumor to grow beyond 2-3 mm in size. A recent report by Seed et al33 demonstrated that the topical administration of a nonselective NSAID (diclofenac) resulted in the decreased growth of colon-26 cells, which produce high levels of PGE2, after implantation into nude mice. This observed inhibition of growth was thought to be due to the reduction of angiogenesis. To further determine whether the prostaglandin pathway was important in the induction of angiogenesis, Tsujii et al34 studied cells that expressed high levels of COX-2. They used two cell types: Caco-2 cells that are stably transfected with COX-2, and HCA-7, a human colon cancer, which constitutively expresses COX-2. 35 The Caco-2 cell line that overexpresses COX-2 demonstrated an 8-fold increase in migration compared to control cells. This increased migration was reduced with treatment using a COX-2 selective inhibitor, NS-398. Both of these COX-2 expressing cells were shown to have increased levels of angiogenic factors such as VEGF, bFGF, bFGF-binding protein, TGF-β, PDGF-B, endothelin-1, and iNOS.

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Fig. 3.4. Multivariate relative risk for fatal colon cancer in men and women who use aspirin occasionally, HS, or 16+ times per month compared to persons who use no aspirin. (Reprinted with permission from Thun MJ, Namboodiri MM, Heath CW et al, Cancer Res., 1993; 53:1322-1327. ©Plenum Publishing Corporation).

Prostaglandin E2 and Colorectal Cancer PGE2 has long been known as a major modulator of the acute inflammatory response, however, several recent reports have implicated PGE2 in carcinogenesis. For instance, it has been shown that PGE2 is increased in both surgically excised colon cancer specimens18 and cultured colon cancer cell lines.35 A report using the HCA-cell line, demonstrated that treatment with exogenous PGE2 resulted in a 4-to 5-fold increase in Bcl-2 production but did not result in increases in Bcl-x or Bax.36 This study also showed that treatment with a specific COX-2 inhibitor, SC-58125, increased the rate of apoptosis in these cells. These data indicate that the mechanism of COX-2 inhibition of apoptosis may be through modulation of Bcl-2 levels. The carcinogenic role of PGE2 may not be due entirely to the inhibition of apoptosis since PGE2 is known to be proangiogenic.37 PGE2 is also immunosuppressive, which may aid in the progression of cancer.38 Another interesting aspect of increased PGE2 production is that it can induce COX-2 expression through an autocrine mechanism, therefore, cancer cells may modulate their own expression of COX-2 furthering their malignant potential.39

In Vivo Studies Linking the COX/Prostaglandin Pathway to Colorectal Cancer Several groups have tested the effectiveness of NSAIDs in the prevention of adenomas in the Min/+ mouse model which was developed by treating C57BL/G1 mice with ethylnitrosurea and selecting the transmission of germline mutations resulting in the predisposition to develop multiple adenomas throughout the intestine.40 One NSAID, piroxicam, has been shown to produce a dose-dependent effect on tumor multiplicity41.

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Fig. 3.5. Schematic of colon cancer progression and the potential interventional site of COX-2 selective inhibitors.

Sulindac, another NSAID, has been observed to suppress tumor formation in these animals and increases apoptosis.42,43 A recent report demonstrated increased COX-2 levels in the adenomas of the Min/+ mice compared to normal mucosa.44 In addition, the adenomas of the Min/+ mice have been examined for expression of the TGFβ2 receptor.45 TGFβ2 inhibits growth of epithelial cells and its receptor appears not to be expressed in human colon cancer cell lines. 26 Immunohistochemistry was used to demonstrate that TGFβ2 immunoreactivity was not detected in the adenomas examined. A decrease in TGFβ2 was also observed in the RIE-1 cells transfected with COX-2,24 suggesting that in vivo as well as in vitro, the increase of COX-2 results in an apparent decrease of the TGFβ2 receptor, thus contributing to the progression of adenomas. The APC∆716 knockout mouse model, developed by Oshima et al,46 results in the development of numerous polyps in the intestinal tract as early as three weeks of age secondary to a truncation mutation in the APC gene. Due to the loss of the full-length wild type APC allele in the proliferative zone cells, these microadenomas originate from single crypts by forming abnormal outpockets into the lacteal side of the neighboring villi. Oshima et al47 demonstrated that COX-2 expression is necessary for the formation of polyps by crossbreeding the APC∆716 knockout mice with the COX-2 knockout mice to generate doublemutant mice that carried the APC∆716(+/-) Ptgs(+/-) and APC∆716(+/-) Ptgs(-/-) mutations. The APC ∆716(+/-) Ptgs(+/+) littermates were used as the positive controls. The APC ∆716(+/-) Ptgs(+/-) mice developed 66% fewer polyps compared to the controls and the APC ∆716(+/-) Ptgs(-/-) mice developed 86% fewer polyps when compared with the APC∆716(+/-)Ptgs(+/+) littermates. The APC∆716(+/-) Ptgs(-/-) mice showed significantly smaller polyps and no polyp was larger than 2 mm in diameter. This indicates that the expression of COX-2 increases polyp number and size in a gene dosage-dependent manner in the APC∆716 knockout mice. These APC∆716 knockout mice were also treated with a COX-2 selective inhibitor which demonstrated a 62% decrease in polyp number, however, animals treated with the nonspecific COX inhibitor, sulindac, showed no significant decrease in polyp numbers. The effects of NSAIDs have also been evaluated in the azoxymethane (AOM) model of colon cancer. Treating rats with AOM produces colonic lesions that later develop into carcinomas.4 Reddy et al48 demonstrated that aspirin reduced the size, multiplicity, and incidence of colon tumors in this model. Similar effects were noted with sulindac, piroxicam, ibuprofen, and SC-58125, a COX-2 selective inhibitor.49-51 Furthermore, the use of SC-58125 reduced aberrant crypt formation by 50%. Prostaglandin levels were higher in the colon tumors than in the normal mucosa; these levels were markedly suppressed with sulindac treatment. The levels of COX have also been evaluated in the colon tumors of AOM-treated rats. It was

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noted that COX-2, but not COX-1, mRNA and protein expression was increased in the colonic tumors.

Clinical Trials Using COX Inhibitors Epidemiological Studies Colorectal cancer is a major cause of death in Western countries, ranking as the second leading cause of death from cancer in the United States. Increasing evidence, both in vivo and in vitro, points to the COX/prostaglandin pathway as playing a major role in colon carcinogenesis. For example, NSAIDs have been shown to inhibit tumor growth in a wide variety of experimental models of cancer, including tumors of the colon,52-55 tongue,56 esophagus,57,58 and pancreas,59 however, the majority of the recent studies has been restricted to the colon. The first observation of the chemoprotective effects of NSAID administration came with Waddell and Loughry’s60 finding that treatment with indomethacin and sulindac resulted in the disappearance of rectal polyps in a patient with Gardner’s syndrome.60 More recently, large epidemiological studies have been performed which demonstrate a lower risk of colorectal cancer in persons regularly taking NSAIDs. One such study demonstrated a 40% lower risk of colon cancer in those individuals who took an aspirin on 16 or more occasions during a month’s time compared to those individuals who took no aspirin.53 This study was particularly noteworthy due to the large size of the study and the fact that risk decreased with both the frequency and the duration of aspirin use (Fig. 3.4).

NSAID Use in Patients with FAP Familial adenomatous polyposis (FAP) is an autosomal dominant disorder characterized by formation of many colorectal adenomas at an early age and is associated with an increased risk of developing colorectal cancer. This disease is due to a genetic mutation in the adenomatous polyposis coli (APC) gene, which is located on chromosome 5.61,62 In fact, up to 50% of all spontaneous colorectal cancers appear to have mutations in the APC gene.63,64 The first report of FAP patients having a reduction in polyp size and number with NSAID use was in 1983.60 There have been several more recent reports on NSAID use and reduction of polyps in FAP patients. One such study was by Labayle et al65 which analyzed patients with FAP who were given either 100 mg of sulindac three times a day or placebo for two separate 4-month periods. Patients treated with sulindac demonstrated a significant decrease and often total regression of rectal polyps. In another study by Giardiello et al,66 22 patients with FAP were randomized to receive either sulindac 150 mg twice a day for nine months or placebo. The individuals treated with sulindac demonstrated a 44% decrease in polyp number compared to baseline levels and a 35% decrease in the size of polyps suggesting a correlation of NSAID usage and a reduction in both the size and number of polyps seen in FAP patients.

Conclusions and Future Perspectives Since the characterization of the COX-2 gene in 1991, there has been a considerable amount of evidence indicating that COX-2 has a role in carcinogenesis. With the current data, it seems apparent that COX-2 not only plays a crucial role in polyp formation, but is intimately involved in later phenotypic characteristics of cancer, such as metastatic potential and angiogenesis. Since COX-2 expression has been implicated as a crucial step in the formation of adenocarcinomas, it has been proposed that COX-2 may serve as a target for novel treatment regimens.

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The sequence of events leading to colon cancer, as understood today, is shown in Figure 3.5. The APC gene has been proposed to act as the ‘gatekeeper’, ensuring that all division is balanced by cell death.67 It appears that the APC gene is necessary, but not sufficient for the formation of adenomas,68 therefore, there must be external factors present in the gut or in the diet that contribute to tumor progression of colorectal cancer. Once this checkpoint is passed, other mutations such as Ras or p53 can occur which lead to later stage carcinomas. It is believed that the influence of COX-2 expression influence is between the APC gene mutation and later mutations, therefore, the inhibition of COX-2 potentially could be an effective site of adenocarcinoma prevention. Traditional NSAIDs, such as aspirin, inhibit both COX-1 and COX-2 and with chronic use can result in gastroduodenal bleeding and damage the kidneys. These side effects have lead to an increased effort to develop selective inhibitors of COX-2. To date, there have been approximately a dozen COX-2 selective inhibitors developed, each with differing specificities to the COX enzymes.69,70 The early limitations of these COX-2 selective inhibitors is that, unlike aspirin which transfers an acetyl side group to the active site of the COX enzymes, the first wave of COX-2 selective inhibitors are not permanent, therefore, have only temporary effects. A recent report by Kalgutkar et al,71 designed and described a COX-2 selective inhibitor which retained aspirin’s acetyl side group, thus resulting in a permanent inactivation of COX-2. This type of strategy may lead to effective inhibition of the deleterious effects of COX-2 activity while having no effect on COX-1 activity, thus allowing for chronic usage without the toxicity observed with traditional NSAIDs.

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36. Sheng H, Shao J, Morrow JD et al. Modulation of apoptosis and bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 1998; 58:362-366. 37. Form DM, Auerbach R. PGE2 and angiogenesis. Proc Soc Exp Biol Med 1983; 172:214-218. 38. Appleton I, Thomlinson A, Mitchell JA et al. Distribution of cyclooxygenase isoforms in murine chronic glanulomatous inflammation. Implications for future anti-inflammatory therapy. J Pathol 1995; 176:413-420. 39. Tjandrawinata RR, Hughes-Fulford M. Up-regulation of cyclooxygenase-2 by product-prostaglandin E2. In: Honn KV et al., eds. Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation, and Radiation Injury 3. New York:Plenum Press, 1997:163-170. 40. Moser AR, Pitot HC, Dove WF. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 1990; 247:322-325. 41. Jacoby RF, Marshall DJ, Newton MA et al. Chemoprevention of spontaneous intestinal adenomas in the Apc Min mouse model by the nonsteroidal anti-inflammatory drug piroxicam. Cancer Res 1996; 56:710-714. 42. Boolbol SK, Dannenburg AJ, Chadburn A et al. Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res 1996; 56:2556-2560. 43. Beazer-Barclay Y, Levy DB, Moser AR et al. Sulindac suppresses tumorigenesis in Min mouse. Carcinogenesis 1996; 17:1757-1760. 44. Williams CS, Luongo C, Radhika A et al. Elevated cyclooxygenase-2 levels in Min mouse adenomas. Gastroenterology 1996; 111:1134-1140. 45. Zhang T, Nanney LB, Peeler MO et al. Decreased transforming growth factor beta type II receptor expression in intestinal adenomas from Min/+mice is associated with increased cyclin D1 and cyclin-dependent kinase 4 expression. Cancer Res 1997; 57:1638-1643. 46. Oshima M, Oshima H, Kitagawa K et al. Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene. Proc Natl Acad Sci USA 1995; 92:4482-4486. 47. Oshima M, Dinchuk JE, Kargman SL et al. Suppression of intestinal polyposis in Apc∆716 knockout mice by inhibition of cyclooxygenase-2 (COX-2). Cell 1996; 87:803-809. 48. Reddy BS, Rao CV, Rivenson A et al. Inhibitory effect of aspirin on azoxymethaneinduced colon carcinogenesis in F344 rats. Carcinogenesis 1993; 14:1493-1497. 49. Rao CV, Rivenson A, Simi B et al. Chemoprevention of colon carcinogenesis by sulindac, a nonsteroidal anti-inflammatory agent. Cancer Res 1995; 55:1464-1472. 50. Reddy BS, Maruyama H, Kelloff G. Dose-related inhibition of colon carcinogenesis by dietary piroxicam, a nonsteroidal antiinflammatory drug during different stages of rat colon tumor development. Cancer Res 1987; 47:5340-5346. 51. Reddy BS, Rao CV, Seibert K. Evaluation of cyclooxygenase-2 inhibitor for potential chemopreventive properties in colon carcinogenesis. Cancer Res 1996; 56:4566-4569. 52. Thun MJ, Namboodiri MM, Heath CW et al. Aspirin use and reduced risk of fatal colon cancer. New Engl J Med 1991; 325:1593-1596. 53. Thun MJ, Namboodiri MM, Calle EE et al. Aspirin use and risk of fatal cancer. Cancer Res 1993; 53:1322-1327. 54. Smalley WE, DuBois RN. Colorectal cancer and nonsteroidal anti-inflammatory drugs. In: August JT et al, eds. Advancements in Pharmacology, Vol. 39. New York:Academic Press, 1997:1-21. 55. Thun MJ. NSAID use and decreased risk of gastrointestinal cancers. Gastroenterol Clin North Am 1996; 25:333-348. 56. Tanaka T, Nishikawa A, Mori Y et al. Inhibitory effects of nonsteroidal anti-inflammatory drugs, piroxicam and indomethacin on 4-nitroquinoline 1-oxide induced tongue carcinogenesis in male ACI/N rats. Cancer Lett 1989; 48:177-182. 57. Rubio CA. Antitumoral oral activity of indomethacin on experimental esophageal tumors. J Natl Cancer Inst 1984; 72:705-707. 58. Rubio CA. Further studies on the therapeutic effect of indomethacin on esophageal tumors. Cancer 1986; 58:1029-1031.

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59. Takahashi M, Furukawa F, Toyoda K et al. Effects of various prostaglandin synthesis inhibitors on pancreatic carcinogenesis in hamsters after initiation with N-nitrosobis (z-oxopropyl) amine. Carcinogenesis 1990; 11:393-395. 60. Waddel WR, Loughry RW. Sulindac for polyposis of the colon. J Surg Oncol 1983; 24:83-87. 61. Kinzler KW, Nilbert MC, Su LK et al. Identification of FAP locus genes from chromosome 5q21. Science 1991; 253:661-665. 62. Nishisho I, Nakamura Y, Miyoshi Y et al. Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients. Science 1991; 253:665-669. 63. Miyaki MM, Konishi M, Kikuchi-Yanoshita R et al. Characteristics of somatic mutation of the adenomatous polyposis coli gene in colorectal tumors. Cancer Res 1994; 54:3011-3020. 64. Powell SM, Zilz N, Beazer-Barday Y et al. APC mutations occur early during colorectal tumorigenesis. Nature 1992; 359:235-237. 65. Labayle D, Fischer D, Vielh P et al. Sulindac causes regression of rectal polyps in familial adenomatous polyposis. Gastroenterology 1991; 101:635-639. 66. Giardiello FM, Hamilton SR, Krush AJ et al. Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis. N Engl J Med 1993; 328:1313-1316. 67. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996; 87:159-170. 68. Gould KA, Dove WF. Action of Min and Mom1 on neoplasia in ectopic intestinal grafts. Cell Growth Differ 1996; 7:1361-1368. 69. Peunisi E. Building a better aspirin. Science 1998; 280:1191-1192. 70. Masferrer JL, Isakson PC, Seibert K. Cyclooxygenase-2 inhibitors. A new class of antiinflammatory agents that spare the gastrointestinal tract. Gastroenterol Clin North Am 1996; 25:363-372. 71. Kalgutkar AS, Crews BC, Rowlinson SW et al. Aspirin-like molecules that covalently inactivate cyclooxygenase-2. Science 1998; 280:1268-1270.

CHAPTER 4

Cell Cycle and Apoptosis Regulation in GI Cancers Harry T. Papaconstantinou and Tien C. Ko

I

n normal tissues, organ size remains relatively constant, and is a consequence of the homeostatic processes that maintain normal cell number. This tissue homeostasis is achieved through a balance between cell proliferation, growth arrest and cell death (Fig. 4.1A). Cell proliferation and growth arrest is controlled by a family of regulatory proteins that signal the progression or arrest of the cell cycle. Cell elimination, or cell death, can be further classified into two distinct processes, apoptosis and necrosis. The gastrointestinal (GI) tract is composed of a variety of organs each with distinct characteristics of homeostatic balance. The normal small bowel and colonic epithelium possess a dynamic nature of mucosal turnover that is a result of rapid stem cell proliferation in the crypts to counterbalance the rapid cell elimination in the villus. This is in contrast to normal solid organs such as the liver and pancreas, which exhibit a more static nature of cell turnover where once normal size and development have been achieved, rates of cell division and cell elimination decrease. The loss of cell cycle control or the loss of apoptotic induction would result in increased cell proliferation and cell number, which are the basic principles of tumorigenesis. Abnormalities in cell cycle regulation that lead to uncontrolled proliferation include factors that increase activation or decrease inhibition of certain cell cycle checkpoint controls (Fig. 4.1B). Conversely, a decrease in pro-apoptotic regulators or an increase in antiapoptotic regulators would promote cell survival and increase cell number (Fig. 4.1C). These abnormalities have a profound influence on tissue homeostasis, and have been implicated in cancer formation. In this chapter we present a review of the molecular mechanisms that regulate the mammalian cell cycle and apoptosis, and discuss the current research implicating the loss of regulation of these two processes in the induction, progression and establishment of malignant characteristics of GI cancers. Furthermore, we will explore how current therapeutic manipulations of the cell cycle and apoptosis are used to treat GI cancers, and discuss future developments for clinical application.

Cell Cycle Over the past several decades, research into the eukaryotic cell cycle and cellular growth control has expanded our understanding of the molecular mechanisms regulating cell proliferation. The cell cycle is an organized sequence of complex biological interactions that mediate cell growth, DNA synthesis, chromosome condensation, nuclear envelope breakdown, segregation of chromosomes, and cytokinesis resulting in two identical daughter cells. These events occur during four discrete phases—G1 (postmitotic presynthetic), S-phase (DNA replication), G2 (postsynthetic premitotic), and M (mitotic) (Fig. 4.2A). The Molecular Mechanisms in Gastrointestinal Cancer, edited by B. Mark Evers. ©1999 R.G. Landes Company.

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Fig. 4.1. The induction of tumor formation through alterations in cell cycle and apoptosis regulation. (A) Normal tissue homeostasis is achieved through a balance between cell proliferation and apoptosis. (B) Alterations of cell cycle regulation leading to increased activation or decreased inhibition, results in uncontrolled proliferation. (C) A decrease in pro-apoptotic and an increase in anti-apoptotic regulators results in increased cell survival and cell number. These events are implicated in the induction of tumor formation.

progression of the mammalian cell cycle through these specific phases is governed by the sequential activation and inactivation of a highly conserved family of regulatory proteins, cyclin-dependent kinases (Cdks).1,2 The activation of Cdks requires binding to regulatory proteins called cyclins (Fig. 4.2B).2-4 The active cyclin/Cdk complex is involved in the phosphorylation of other cell cycle regulatory proteins. These posttranslational modifications play a key role in the control of cellular proliferation. Currently, there are five distinct groups of human cyclins, A through E, which are grouped according to sequence similarity.3,5 The homology between cyclins is confined to a 100-150-amino acid region, or ‘cyclin box’, which is conserved among all species, and is believed to be the site of interaction with Cdks for the formation of a functional complex.3 Each cyclin exhibits a cell cycle-phase specific pattern of fluctuation (Fig. 4.2C).3,5 Increased expression during specific stages of the cell cycle allows for discrete interactions with their respective constitutively expressed Cdks for activation of factors that regulate cell cycle progression.2,3,5 Conversely, a family of Cdk inhibitory proteins (CKIs) regulates cell cycle arrest at specific checkpoints by complexing with and inactivating the Cdks or the Cdk/cyclin active complex (Fig. 4.2A).2,6 Therefore, the molecular machinery responsible for the progression and cessation of the cell cycle are regulatory units comprising cyclins, Cdks, and CKIs. Thus, these phase-specific interactions of molecular subunits of the cell cycle are the basic overall mechanisms that regulate cellular proliferation. The mammalian cell cycle is regulated at two major checkpoints, the G1/S and the G2/M transitions (Fig. 4.2A).7 The progression from early to mid G1 phase is dependent

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Fig. 4.2. Cell cycle Regulation. (A) Cell cycle progression through 4 specific phases (G1, S, G2 and M) is controlled by protein complexes composed of cyclins and cyclin-dependent kinases (Cdks). These protein-protein interactions function to regulate progression through the cell cycle by phosphorylating downstream regulatory subunits such as retinoblastoma (pRb) protein. In the hypohosphorylated form pRb sequesters transcription factors such as E2F. Upon phosphorylation, pRb releases E2F transcription factors, which are responsible for the transcription of cellular genes involved in cell cycle progression through the G1/S transition. Cell cycle arrest is controlled at various phases by the CIP/KIP and INK family of cyclin-dependent kinase inhibitors. (B) Cdk activation is achieved through protein complex formation with the cyclin regulatory proteins. (C) Cell cycle phase-specific expression of cyclins is responsible for the careful orchestration of cyclin-Cdk complex activity for cell cycle progression. Adapted from Cordon Cardo C, Am J Pathol.1995;147:547, ©American Society for Investigative Pathology, with permission.

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upon an increased expression of D-type cyclins (D1, D2 or D3) and their interaction with Cdk4 and/or Cdk6 for kinase activation.2,7,8 The transition between G1/S requires the sequential activation of Cdk2 by its regulatory cyclins E and A.2,7-9 Finally, progression through the G2/M transition is dependent upon the activation of Cdk1 (cdc2) by complexing with its regulatory protein, cyclin B.2,3,7 Therefore, these interactions clearly show how the careful orchestration of cyclin expression regulates Cdk activation and the normal progression through the cell cycle. Similar specific protein-protein interactions are required for the inhibition of cell cycle progression. This negative regulation is mediated by two families of Cdk inhibitory proteins, CIP/KIP and INK (Fig. 4.2A). The CIP/KIP family binds to cyclin/Cdk complexes and inhibits Cdk-associated kinase activity.2,6,7,10 This family of proteins, which includes p21CIP1, p27KIP1 and p57KIP2, share a conserved amino-terminal domain responsible for these protein-protein interactions.11-13 Each CIP/KIP protein can inhibit all known Cdks, and when overexpressed can cause cell cycle arrest at the G1/S or G2/M transition checkpoints (Fig. 4.3A). The INK proteins (p15INK4B, p16INK4A, p18INK4C, and p19INK4D) selectively bind and inhibit Cdk4 and Cdk6 (Fig. 4.2A), and are expressed in a tissue-specific pattern.2,6,7,10 Increased expression of these proteins inhibits cell cycle progression through the G1 phase. The retinoblastoma tumor suppressor protein (pRb) belongs to the family of Rb pocket proteins, which includes pRb, p107, and p130. These proteins regulate transcription factors, and are key targets for G1 Cdk activity (Fig. 4.2A).7,14 While the pRB pool level does not change during progression of the cell cycle, the phosphorylation state of pRB is cell cycledependent and is a target for the enzymatic activity of cyclin/Cdk complexes.2,7,15 In the hypophosphorylated form, pRB sequesters cell cycle regulatory transcription factors and DNA binding proteins such as E2F (Fig. 4.2A).16 E2F protein family members are believed to be responsible for traversing the G1/S regulation point in the cell cycle.17 In this series of protein modifications and interactions, the sequential phosphorylation of pRb by cyclin D-dependent kinases occurs in early to mid G1, then cyclin E/Cdk2 during late G1 to release E2F/DP transcription factors, which are essential for gene activation associated with S phase entry.16 The phosphorylated form of pRB is maintained up to the G2/M transition point, where dephosphorylation events cause the sequestration of E2F and other transcription factors prior to mitosis. Our present understanding of cell cycle progression has made it apparent that its control is accomplished at several levels of regulation that include transcription, translation and posttranslational processes. However, major underlying regulatory processes that facilitate the functions of the various subunits that activate and suppress the cell cycle are interconnected through protein modification by phosphorylation, dephosphorylation and protein-protein interactions. Thus, perturbations of these processes at any regulatory step that alters their interactions can have severe effects on regulation of cell proliferation, and may be factors in the loss of cell cycle control, and cancer cell formation.

Cell Cycle and the Induction of Cancer Classically, neoplastic diseases have been defined as proliferative disorders characterized by unregulated cell growth and proliferation.18 The multitude of regulatory proteins and their specificity of action upon distinct phases of the cell cycle make their genes highly plausible candidates as targets for mutagenic agents that induce neoplastic disease. Each step in the cell cycle is characterized by the activation or inactivation of various proteins and protein complexes. Thus, mutation events that result in loss of function or abrogation of gene expression of CKIs, cyclins, and pRB could result in unregulated cellular proliferation and cancer formation.

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Fig. 4.3. Schematic model for the action of p53 and MTS1 (p16) in cell cycle control. (A) the mutational inactivation of p53 would result in loss of p21 transcription and activation resulting in loss of normal control in cell cycle progression checkpoints. (B) The mutational inactivation or loss of homology of MTS1 gene would result in p16 protein inactivation. Conceptually, this event would result in continued cyclin D-Cdk4 activity with maintenance of pRb phosphorylation and E2F activation resulting in loss of G1/S cell cycle checkpoint. These mutational events are thought to play a major role in GI cancer formation with inappropriate entry of cells into the phases of DNA synthesis and cell division. Reproduced with permission, Surgical Clinics of North America 1995; 75:862, ©W.B. Saunders Company.

The research on tumorigenesis has led to the discovery of proto-oncogenes, which are homologs of normal growth regulatory genes. Proto-oncogenes are activated to oncogenes resulting in uncontrolled cell proliferation, and are thought to be one factor contributing to tumor progression. The activation events include tumor-specific clonal DNA abnormalities such as gene amplification, gene rearrangement, or point mutation, which result in deregulation of proto-oncogene expression and/or the alteration of mRNA which results in altered protein structure. Certain human cyclin genes have been associated with various clonal genetic aberrations associated with neoplastic disease, which suggests their involvement in tumorigenesis. Cyclins compose a family of proteins responsible for the regulation of Cdk activity and phase-specific cell cycle progression, and have been targets of mutagenic processes. These events lead to abnormal levels of cyclins, which are required for cell cycle progression, and result in uncontrollable proliferation.5,19 Cyclin D is one of the most frequent cyclin abnormalities found in cancers, and is a good model to describe the cyclin role in tumorigenesis.19 The cyclin D1 gene has been localized to the long arm of chromosome 11, at the q13 band. This location has been associated with the t(11;14) translocations which are involved in hemoproliferative malignancies.19-21 Therefore, the 11q13 locus was termed BCL1 because of its proto-oncogene characteristics and involvement in tumor-associated translocation events.22 The cyclin D1 gene is located 110 kb downstream from the BCL1 breakpoint, indicating that cyclin D1 is a target gene for this translocation event.22 This oncogenic event is the result of uncontrollable activation of the cyclin D1 gene and subsequent

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overexpression of mRNA and normal protein, indicating these mutations are in the regulatory regions of the gene. Cyclin D1 regulates the G1 phase of cell cycle control, and therefore, overexpression may prevent cells from exiting the cell cycle for differentiation.23 The most frequent abnormality of cyclin D1 is DNA amplification.19 The amplification factors or driver of the 11q13 amplicon gene are believed to be related to CCND1 genes, and include fibroblast growth factor 3 (FGF3)-int2 and FGF4-hst1 signaling pathways.24 These genes were first identified in gastric cancer, but have subsequently been found in a variety of human malignancies.19 Gene amplification has been shown to be a major factor in cyclin D1 overexpression in head and neck tumors and esophageal cancers.25,26 Although translocation amplification is an attractive mechanism for cyclin D1 overexpression, there is evidence that other factors may be involved according to tissue type. In many tumors the 11q13 translocation amplicon is a rare event or has never been detected despite the aberrant accumulation of cyclin D1 in over 40% of the cases.27 Thus, overexpression may be a consequence of mutations that regulate mRNA turnover. Whether amplification, or post-transcriptional/translational modifications occur, these events result in the stable overexpression of cyclin D1 and the loss of cell cycle control with the acquisition of tumor cell characteristics. These findings strongly implicate the level of expression of the cyclin D1 gene and its products as major factors in the induction of some cancers through cell cycle aberrations. The counterpart to the oncogene is the tumor suppressor gene, which normally confers negative control on cellular proliferation. Negative control of cell cycle progression plays an important role in embryonic tissue development, differentiation, senescence, and cell death. Therefore, the role of CKI proteins on the progression of the cell cycle is an attractive target for the study of regulatory processes in tumorigenesis. In many cases, cell cycle arrest results from DNA damaging agents, many of which are potent carcinogens. Failure to arrest proliferation would result in propagation of highly unstable genomes, which is an early event and primary characteristic of cancer cells. The protein p21Waf1 is one of several negative regulatory proteins involved in the inactivation of Cdks and cyclin/Cdk complexes,2,6,11 which are the regulatory kinase components that target pRB for phosphorylation. Although the incidence of p21 gene mutations appears to be a rare event,5,28 the mechanism for its inactivation in most tumors could be the result of changes in its protein pool levels. This is based on the observation that mutations of p53, which is a transactivating factor that regulates the transcription of p21, are prevalent events in human cancer, and could have a negative effect on p21 expression (Fig. 4.3A).29 Interestingly, the induction of p21 has also been shown to occur in a p53-independent pathway.30 The role of this pathway in human cancer production is still subject to experimental study. The CKI proteins p15, p16, and p18 can complex with both Cdk4 and Cdk6, inhibiting their catalytic regulation of pRB phosphorylation (Fig. 4.3B).6,19,29 Therefore, loss of p15, p16, or p18 function through mutation events would cause an increase in G1 cyclin/Cdk activity that would lead to phosphorylation and inactivation of pRB with uncontrolled cell cycle progression and cell proliferation. Both p15 and p16 genes are located on the short arm of chromosome 9 (9p21), while the p18 gene is located on chromosome 1p32. These genes are located in regions involved in translocations and mutational deletions in various human cancers,5,29 which suggests a causal role of these inhibitory proteins in cancer induction. The cell cycle regulatory protein pRB is a prototype tumor suppressor, which functions to regulate DNA binding proteins and transcription factors important for progression through the cell cycle. In the hypophosphorylated state, pRB molecules can interact with viral proteins such as SV40 large T antigen, the adenovirus E1A protein, and the papillomavirus E7 protein inactivating pRB function, which renders the cell immortal.5 The deregulation of cell proliferation by inactivated pRB is compensated by p53 function, another tumor suppressor gene.31 Therefore, the loss of pRB and p53 function would result

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in a continuous high level of E2F activity resulting in unregulated cell proliferation and tumor formation.31,32 Furthermore, since the tumor virus proteins that inactivate pRB can also inactivate p53, these proteins appear to be components of a common pathway for tumorigenesis in certain tissue neoplasms. Although the mechanisms of these tumor suppressor genes have been elucidated using cells in culture, these findings are supported by experiments utilizing RB and/or p53 knockout murine models, which clearly demonstrate their biological effects. Although homozygous RB mutations result in death in utero,33,34 p53-homozygous and p53- or pRB-heterozygous mice which are viable, develop a high incidence of a variety of tumors. The additive effect of p53 and RB mutations has been investigated, and reveals an increased tumor burden and metastasis.35

Colorectal Cancer The expression of cyclin D1 protein was examined in normal colonic mucosa and primary and metastatic lesions.36 Normal tissue and 56% of primary tumors revealed weak expression of cyclin D1 gene products, whereas 23% and 21% of primary carcinomas expressed moderate to strong overexpression of cyclin D1, respectively. Moreover, the metastatic lesions examined exhibited immunohistochemical staining characteristics similar to those of the primary lesion. Furthermore, in cell lines overexpressing cyclin D1, the antibody-mediated knockout of activity demonstrated a positive regulatory role of the cyclin D1 protein whose function was required for progression through the G1 phase of the cell cycle.36 These data suggest that cyclin D1 is overexpressed in a subset of colorectal tumors, but it is not clear whether this is a result of abnormalities of gene amplification. Although this study does not directly address the oncogenic role of the cyclin D1 gene product in the induction of colorectal cancers, the knowledge of cyclin D gene amplification and overexpression in other tumors supports a mechanism in colorectal cancer induction.19 Many mechanisms of cyclin D overexpression have been described. There is also documentation that there is an abundance of tumor types that lack any significant frequency of 11q13 amplification but also exhibit cyclin D1 overexpression. These data imply that the expression of this cell-cycle regulatory proto-oncogene which plays a significant role in human oncogenesis has multiple levels of regulation. Since cyclin D is involved in the inactivation of the growth-restraining function of wild type pRB through the cyclin D-kinase associated catalytic unit, this has been proposed as one step in the multistep process of colorectal tumorigenesis.37 Interestingly, some colorectal carcinomas exhibit concurrent increased expression of cyclin E and Cdk2 genes,38 which may act through pRB regulation to induce uncontrolled proliferation in a similar manner to that of cyclin D1. Tumor suppressor genes exert a negative effect on cell cycle control and proliferation, which includes CKI proteins. Although prevalent in many cancers, mutations in p16 and other INK family Cdk inhibitors have not been found in colon cancer.39 However, loss of p16 transcription by de novo methylation has been found in >92% of colon cancer cell lines.40 The development of null mutation-gene knockout technology has provided rodent transgenic models and a novel approach for the investigation in tumorigenesis. The induction of human colon cancer is achieved through a progression of mutations that encompass the progression of change from normal mucosa to adenomas and then carcinomas. Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary condition linked to the inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene, which results in numerous adenomatous polyps in the colon that progress to colorectal carcinoma at a high rate.41,42 The murine APC knockout model of multiple intestinal neoplasm, (Min)/+, is a good model to study the molecular mechanisms associated with the progression of adenomatous disease to colon cancer, because its progression is similar to that seen

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in the human FAP disease.43 Recent investigation using this in vivo model system has revealed that cyclin D1 and Cdk4 exhibit concurrent overexpression in the intestinal adenomas which was accompanied by an increase in cell proliferation indicated by 5-bromo-2'-deoxyuridine incorporation.23 These investigators compared their rodent model observations with human tissue and found a similar cyclin D1 and Cdk4 overexpression in the majority of intestinal adenomas from FAP patients. These results indicate that overexpression of G1 cell cycle proteins is an early event in the progression of premalignant disease to cancers of the colon. Further studies using this model have shown an inverse correlation of TGF-βII receptor expression to cyclin D1 and Cdk4 expression at the transcriptional and translational levels.44 Since TGF-β functions to inhibit proliferation through the induction of various CK1 protein expressions, the loss of this signal transduction pathway may be one of the mechanisms involved in the upregulation of these positive G1 cell regulators. This event subsequently leads to the failure of adenomatous cells to exit the cell cycle 23 as should normally occur during the process of intestinal cell differentiation. Recently, other Cdks have been associated with colorectal adenomas and carcinomas. In a study of 50 colonic adenomas, and 15 adenomas with focal carcinoma, overexpression of Cdk2 and cdc2 (Cdk1) were found in 28% and 87%, respectively.45 Although loss or deletion of RB gene has been demonstrated in several human malignancies,46,47 this event is rare in carcinomas of the colon and rectum. However, since pRB is inactivated through phosphorylation,16,48 the mechanism of action in rendering these cells into a state of unregulated proliferation lies in the regulatory role of these overexpressed Cdks in the phosphorylation and inactivation of pRB.49

Cancers of the Esophagus and Stomach Several genetic abnormalities involving cell cycle regulators have been implicated in the induction and progression of esophageal squamous cell carcinoma, and includes amplification of the chromosome 11q13 region associated with the hst-1, int-2 and cyclin D genes.19 Esophageal cancers exhibit an amplification of cyclin D expression in 34% of combined series investigations.19 Studies have concluded that amplification of cyclin D1 in esophageal cancer closely correlated to tumor staging, depth of tumor invasion, distant metastasis, and indicates a reduced overall survival.19,50 Therefore, overexpression of cyclin D gene is a good biological marker of high malignancy for esophageal carcinoma. As indicated in colorectal carcinomas, the role of cyclin D in regulation of G1 phase progression makes it likely that its overexpression could lead to uncontrollable cell growth and proliferation of these tumor cells. Transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) are autocrine growth factors synthesized and released by esophageal cancer cells.51 Since cyclin D acts as a growth factor sensor to promote cell proliferation, it has been hypothesized that overexpression of EGF/TGF-α by tumor cells could lead to cyclin D amplification in esophageal tumors. Mutations in the multiple tumor suppressor (MTS1) gene, which encodes the Cdk4 inhibitor p16, are observed in approximately 50% of esophageal squamous cell cancer.52 In a review of the literature, p16 was mutated in about 30% of esophageal cancers.19 In addition to p16, it has been shown that p15 has both point mutations and homozygous deletions in primary esophageal tumors and tumor-derived cell lines.53 The inhibitory role of p15 and p16 in G1 cyclin/Cdk complexes may be another pathway involved in the overexpression of cyclin D, and inactivation of pRB resulting in accelerated cell proliferation (Fig. 4.3B) seen in esophageal cancers. Finally, loss of heterozygosity of various tumor suppressor genes has been found in esophageal cancers and includes the p53, RB, and MTS1 genes.50,53 Point mutations in p53

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have been found in primary squamous cell carcinoma of the esophagus54 as well as Barrett’s associated adenocarcinoma and adjacent dysplasia or metaplasia.55 Series studies have shown a high incidence of p53 mutations in both squamous and adenocarcinoma of the esophagus.50,53 Similar p53 mutations have been found in premalignant and early cancers of the esophagus, which lead to the conclusion that p53 mutation is an early event in esophageal cancer induction.50,53,55 There also appears to be a unique mutation profile of p53 in esophageal cancer that consists primarily of nonsense mutations.55 However, p53 mutation was found to confer no prognostic significance on patient survival in esophageal cancer.56 Other mutations include the loss of homology of RB gene on chromosome 13q, and the MTS1 or p16 gene on chromosome 9p.52,55 Aberrant transcripts of RB and DNA amplification of cyclin D have been noted in esophageal cancer.50,53 The role of pRB is to sequester and release transcriptional factors for DNA replication and progression through G1/S cell cycle transition. Cyclin D regulates its Cdk4 partner to phosphorylate pRB which releases these transcription factors. The CKI protein, p16, blocks the cyclin D/Cdk4 complex to prevent pRB phosphorylation and cell cycle progression (Fig. 4.3B). Therefore, one can see how the mutations of pRB and p16, as well as the amplification of cyclin D, are involved in promoting uncontrolled cellular proliferation in esophageal cancers. Gastric cancers express a broad spectrum of growth factors that act as autocrine and paracrine growth regulators. Overexpression of EGF/TGF-α and EGF receptors contributes to the biologic malignancy of gastric cancers. TGF-β is often overexpressed in poorly differentiated adenocarcinoma and scirrhous carcinomas.57 Growth factors regulate cell proliferation through their signaling pathway interactions with cell cycle regulators. As in esophageal cancers, increased EGF/TNF-α and EGF receptors are linked to an increase in cyclin D expression. Aberrant CKI expression has been implicated in gastric cancers, and involves p21. Activation of wild type p53 by genetic damage is known to induce p21 expression at the transcriptional level, whereas mutant p53 loses this ability (Fig. 4.3A). It has been observed that gastric cancer cell lines expressing the mutant p53 gene product are associated with low to undetectable levels of p21 mRNA.50,57,58 Since p21 is known to be an inhibitor of G1-Cdk/cyclin complexes, it is not surprising that increased levels of cyclin E is frequently associated with gastric cancers.50 These findings suggest that mutated p53 is not capable of inducing p21 expression, leading to the subsequent overexpression of Cdk2 and G1 cyclins, which are responsible for the deregulated growth characteristics of gastric cancer cells with mutant p53. Gastric cancers with cyclin E amplification and overexpression are associated with a more aggressive characteristic with a higher incidence of lymph node metastasis.59 Allele loss and mutation of the p53 gene is detected in >60% of gastric cancers regardless of histological type.57,58 A good correlation has been found between the nature of p53 gene mutation and histological atypia of gastric adenomas. Nonsense mutations or frame shift mutations that affect the structure of the gene product results in a high-grade atypia, whereas silent mutations have a low-grade atypia.60

Pancreatic Cancer As with other tumors, the induction of pancreatic cancer is facilitated through the activation of proto-oncogenes and the inactivation of tumor suppressor genes. In pancreatic cancer, there is an increased incidence of mutations in the tumor suppressor genes p53 and MTS1.29,61 As described above, p53 is a nuclear phosphoprotein, which binds to specific DNA sequences to activate gene transcription and induce cell cycle arrest or apoptosis. Mutations resulting in changes of the p53 amino acid sequence can prevent its binding to these DNA regulatory sites.29 Sequence analysis has found that 50 to 70% of pancreatic

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carcinomas had mutations of the p53 gene. One of the target genes for p53 activated transcription is the CKI protein p21. This negative regulator of cell cycle progression complexes with G1 phase-specific cyclin/Cdk complexes to inhibit phosphorylation of pRB, which is required for initiating DNA transcription and cell cycle progression. Mutated p53 exhibits a loss in p21 transcription ability (Fig. 4.3A), and would therefore result in the maintenance of cyclin/Cdk activity in the G1 phase with subsequent loss of pRB regulatory function of DNA transcription factors. Pancreatic cancers were the first type of cancer found to have high frequency of p16 inactivation.62 MTS1/p16 tumor suppressor gene was found to be mutated in 38% of pancreatic carcinomas with loss of the wild type allele, and homozygously deleted in another 40% of pancreatic carcinomas.61 Furthermore, methylation associated with the silencing of p16 gene expression can be found in the majority of the remaining cases.63 The loss of inhibition of G1 cyclin/Cdk activity, and thus pRB regulation, through mutation or loss of p16 function (Fig. 4.3B) is one of the possible mechanisms involved in the uncontrolled proliferation seen in pancreatic cancers. Recent evidence suggests that loss of p16 protein expression in pancreatic cancer is associated with advanced clinical stage and decreased survival rates.64 Growth factors regulate cell cycle progression by increasing cyclin/Cdk activity. Recent studies have shown that cyclin D1 mRNA and protein levels are overexpressed in pancreatic cancer tissues and cell lines when compared to normal pancreatic tissue.65 Furthermore, cyclin D1 overexpression was found to be associated with decreased survival rates (6.5 vs 15.5). This observation, coupled to the increased susceptibility of pancreatic cancers to growth factor regulation, is a proposed mechanism for loss of cell cycle control and tumorigenesis.3

Apoptosis Cell elimination occurs by two distinct well-characterized biological processes, necrosis or apoptosis. Necrosis is a passive, ATP-independent form of cell death that requires an acute nonphysiologic injury (i.e., ischemia, mechanical injury, and toxins) that results in destruction of the cytoplasmic and organellar membranes with subsequent cellular swelling and lysis.66,67 The lysis of necrotic cells releases cytoplasmic and organelle contents into the extracellular milieu resulting in inflammation with surrounding tissue necrosis and destruction. In contrast, apoptosis is a highly regulated energy requiring form of cell death that is genetically programmed.66,67 Apoptotic cells undergo the following sequence of morphological and biochemical events:67-70 (1) In the early phase of apoptosis, cells exhibit a shrunken cytoplasm and detach from neighboring cells. One of the earliest biochemical features of apoptotic cells is the externalization of phosphatidyl serine residues on the plasma membrane. It has been proposed that these signaling intermediates may be involved in alerting surrounding cells of the occurrence of apoptosis. (2) Middle events include chromatin condensation with resultant crescent shaped nuclei and subsequent nuclear fragmentation. During this phase endonuclease activation results in the fragmentation of DNA into 180-200-base pair (bp) internucleosomal sized fragments. (3) Late in apoptosis the cells begin to fragment into discrete plasma membrane-bound vesicles termed ‘apoptotic bodies’ which are then phagocytized by neighboring cells and macrophages without inducing an inflammatory response. Apoptosis has been implicated in various physiologic functions including the remodeling of tissues during development, removal of senescent cells and cells with genetic damage beyond repair, and the maintenance of tissue homeostasis.68,69 In this section we will define the current knowledge of the molecular machinery involved in the induction of apoptosis. The molecular interactions involved in apoptosis can be divided into 3 parts (Fig. 4.4): (1) signaling of apoptosis by a stimulus; (2) regulation by pro- and anti-apoptotic factors;

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Fig. 4.4. The apoptotic pathway of cell death. The molecular mechanisms involved in apoptosis are divided into 3 parts: (1) stimuli of the apoptotic pathway include DNA damage through ionizing radiation or chemotherapeutic agents (p53 activation), activation of death receptors such as Fas and TNF-α, free radical formation, or loss of growth factor signaling. (2) The progression of these stimuli to the central execution pathway are either positively or negatively regulated by expression of the Bcl-2 family of proteins. (3) The execution phase of apoptosis involves the activation of a family of evolutionarily conserved proteases called caspases. Caspase activation targets various nuclear and cytoplasmic proteins for activation or destruction leading to the morphologic and biochemical characteristics of apoptosis.

and (3) the execution machinery.67,70 These molecular events result in the morphologic and biochemical characteristics of the apoptotic cell. Many stimuli activate the process of apoptosis (Fig.4.4). These include DNA damage through ionizing radiation, growth factor and nutritional deprivation, activation of certain death receptors (e.g., Fas receptor [FasR] and tumor necrosis factor receptor [TNF-R1]), metabolic or cell cycle perturbations, oxidative stress, and many chemotherapeutic agents.68,72 Signal sensors proximal in the apoptotic pathway recognize these stimuli, and include cell surface receptors requiring ligand binding, or intracellular sensors detecting the loss of an advantageous environment for survival or irreparable damage.67,68 The nerve growth factor/tumor necrosis factor (NGF/TNF) receptor family is the typical example of membrane receptor signal sensors, and includes the FasR and TNF-R1 receptor.73 FasR is a 45 kDa protein expressed at the surface of activated T cells, hepatocytes and enterocytes,

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and can be found expressed in tissues including the liver, heart, lung, kidney, and small intestine.68,74 Extensive studies with the T cell model have revealed the downstream events of receptor activation. The binding of a death-promoting ligand to the receptor triggers the death signal resulting in a conformational change in the intracellular region of the receptor. This protein structure change allows binding of cytoplasmic adapter proteins. These receptor-adapter protein complexes such as the Fas activated death domain (FADD)75 catalyze the activation of downstream proteases involved in the execution phase of apoptosis.67,68,76 Intracellular signal sensors include the p53 tumor suppressor gene. The identification of DNA damage activates p53 functional activity, and results in G1 phase cell cycle arrest to allow DNA repair; however, irreparable damage commits the cell to death by apoptosis.77,78 This differential function may be a result of intracellular expression levels of p53.79 Finally, the lack of certain survival factors results in decreased cytoplasmic signals from cell surface receptors such as IL-2 receptors on activated T cells.67,80 This loss of exogenous survival signals results in the activation of the endogenous death program.67 Similar results have been seen with serum withdrawal or growth factor receptor blockade, both of which induce apoptosis.81 Regardless of the many different signals and signal sensors involved in the activation of apoptosis, each of these pathways converge to activate a common central execution process, the caspase cascade.67,76,82,83 Caspases, or cysteine aspartate proteases, are highly conserved proteins first recognized as the ced-3 gene product from the nematode Caenorhabditis elegans.82,83 The sequence of Ced-3 exhibits homology to the mammalian interleukin-1β (IL-1β) converting enzyme (ICE) which is now known as caspase 1.84 To date there are 10 known mammalian caspases,67,82,83 each of which are intimately involved in the conserved biochemical pathway that mediates apoptotic cell death. These proteolytic enzymes are synthesized as inactive proenzymes requiring cleavage for activation. Each activated caspase has specific functions, which may overlap with other caspases. This overlap in function shows the evolutionary significance of apoptosis. The protein substrates cleaved by activated caspases play a functional role in the morphologic and biochemical features seen in apoptotic cells. As indicated in Figure 4.4, activated caspases results in the destruction of cytoskeletal and structural proteins (α-fodrin and actin), nuclear structural components (NuMA and lamins), and cell adhesion factors (FAK). They induce cell cycle arrest through Rb cleavage, cytoplasmic release of p53 by cleavage of the regulatory double minute 2 (MDM2) protein with subsequent nuclear translocation, and PKC δ activation. DNA repair enzymes such as poly (ADP-ribose) polymerase and the 140-kDa component of DNA replication complex C are inactivated by caspase proteolysis. Finally, DNA fragmentation is induced by the activation and nuclear translocation of a 45 kDa cytoplasmic protein called DNA fragmentation factor (DFF). Although there is no known caspase involved in the redistribution of phosphatidyl serine residues on the plasma membrane, caspase inhibitors have been shown to block this event. Overall, the net effect of caspase activation is to halt cell cycle progression, disable homeostatic and repair mechanisms, initiate the detachment of the cell from its surrounding tissue structures, disassemble structural components, and mark the dying cell for engulfment by surrounding cells and macrophages.83 The process of apoptosis is regulated by the expression of certain intracellular proteins belonging to the Bcl-2 family of genes (Fig. 4.4). Bcl-2 is a potent inhibitor of apoptosis, and is predominantly expressed in cholangiocytes, colonic epithelial cells, and pancreatic duct cells.85 The precise mechanism of apoptotic inhibition by Bcl-2 is not known, but this protein is found on organelle membranes86 and may function as an antioxidant, protease inhibitor, or gatekeeper preventing the apoptotic machinery from entering a target organelle.67,87,88 Other proteins in this family include Bcl-xL, Bcl-xs, Bax, Bak, and Bad. Bcl-xL is another inhibitor of apoptosis. Bcl-xs, Bax, Bak, and Bad function as pro-apoptotic

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regulators by dimerizing with Bcl-2 and Bcl-xL inhibiting their function.87-89 Furthermore, it has been shown that the pro-apoptotic protein Bax exhibits channel-forming activity in lipid membranes, which is blocked by Bcl-2.90 Increasing evidence has suggested that the balance or ratio of these pro- and anti-apoptotic proteins is important for signaling the cell to commit to or inhibit apoptosis. The complex molecular machinery of apoptosis involving signal and activation, promotion or inhibition regulation, then execution, is a carefully choreographed process. Perturbations of this process at any of these three phases can result in loss of the apoptotic cell elimination pathway. Since apoptosis is a key regulator of cell number and, therefore, tissue homeostasis (Fig. 4.1), it is easy to see how dysregulation of apoptosis can result in cancer induction.

Apoptosis and Cancer Induction With the evolving role of apoptosis in tissue homeostasis and removal of genetically damaged cells, it has been postulated that the loss of its regulation can be an important factor in the induction and progression of cancers. Mutations or dysregulation of the apoptotic machinery has been implicated in GI cancers and includes the tumor suppressor gene p53, the anti-apoptotic protein Bcl-2, and the pro-apoptotic protein Bax. The loss of the apoptotic pathway results in increased survival of cells with genetic mutations resulting in the increased probability of further mutations or oncogenic progression with subsequent tumor formation (Fig. 4.5). In this section we will discuss the current theories and evidence involved in the implication of these factors in cancers of the GI tract. The tumor suppressor gene p53 is involved in surveying the genome for DNA damage, and functions as a regulator of the cell cycle and an inducer of apoptosis.78 Once DNA damage is detected, p53 expression is upregulated at the translational level and is translocated to the nucleus where it binds to cis-activating DNA sequences that regulate transcription of various DNA damage response genes.78,91 These DNA damage response genes encode the following proteins: (1) p21Waf1, a CKI which regulates G1 cell cycle arrest; (2) Bax, a pro-apoptotic regulator protein; (3) GADD45, a DNA damage-induced protein of unknown function; and (4) MDM2, a cellular protein that binds to and regulates p53. The p53 induction of p21 expression results in G1 cell cycle arrest (Fig. 4.3) which allows the cell to either repair the damaged DNA, or commit to cell death by apoptosis. The significance of this event is that it protects the cell with DNA damage from entry into S-phase, which can result in the daughter cells having unstable chromosomal structure allowing possible recombination events, or stable mutations with resulting malignant transformations. The complex regulatory role of p53 in the differential response to cell cycle arrest and DNA repair versus the induction of apoptosis has been thought to be cell type specific. However, others have shown that p53 response levels to DNA damage may be implicated in this differential function.79,92 Low to moderate p53 expression results in cell cycle arrest with subsequent DNA repair, while a high level of p53 response results in cell progression to apoptosis. Collectively, the role of p53 is an essential process functioning to prevent mutant cell propagation through regulation of DNA repair and repair enzymes, cellular growth arrest, and inducing apoptosis following genotoxic stress. Our current knowledge of p53 function suggests that the loss of p53 activity or expression through mutation would result in decreased apoptosis with a subsequent increased population of cells bearing DNA damage (Fig. 4.5). Although it has been shown that p53 mutations have no bearing on subsequent mutation, it does promote the propagation of mutant cells by increasing cell survival.93 The p53 gene is the most commonly mutated gene in human malignancy. Despite traditional thinking that p53 mutations occur late in tumorigenesis, there is increasing evidence that mutations of p53 in the neoplastic process

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Fig. 4.5. The role of the apoptotic pathway in the induction and progression of cancer. The normal function of wild type p53 tumor suppressor gene after cell exposure to apoptotic stimuli or DNA damage is the induction of apoptotic cell death. One of the mechanisms involved in p53-mediated apoptosis is increased expression of the pro-apoptotic protein, Bax. Mutations in p53, or increased expression of negative regulators of apoptosis (i.e., Bcl-2, Bcl-xL or Bfl-1) leads to prolonged cell survival. This prolonged longevity increases cell exposure to mutagens with increased risk for further oncogenic lesions. The culmination of these events leads to cancer cell transformation and malignant progression. Reproduced with permission Pan H et al. Apoptosis and cancer mechanisms. In: Kastan MB (ed). Cancer Surveys. Checkpoint Controls and Cancer 1997; 29:314.©Cold Spring Harbor Laboratory Press; Plainview, NY.

of colon cancer may be an early event.94,95 On the basis of crystallographic data and biochemical studies, the p53 amino acid residues most frequently mutated in human cancers alter or destroy p53 sequence-specific DNA binding function.96,97 Therefore, the transcriptional regulation of DNA repair genes, cell cycle regulators, and pro-apoptotic regulators may be the mechanism by which p53 mutations influence tumorigenesis. One of the theories that explain the prevalence of p53 mutation in cancers lies in its role in the induction of apoptosis following hypoxia. In tissue regions exhibiting low oxygen tension, such as the center of poorly vascularized solid tumors, cells bearing wild type p53 are more readily removed from the malignant tissue so that the majority of cells would be those with the mutated gene.98 This process may be one of the selective advantages of p53 mutations in the progression of malignant disease. Other regulators of the apoptotic cell death pathway implicated in tumor induction are the Bcl-2 family of genes. Bcl-2 is a negative regulator of apoptosis. The bcl-2 gene was first isolated from human follicular B cell lymphoma, where chromosomal t(14:18) translocation moved the gene into juxtaposition with the immunoglobulin heavy chain transcription enhancer resulting in increased Bcl-2 mRNA synthesis and protein expression. The cancer resulting from this mutation was a consequence of extended B cell lifespan due to the Bcl-2-mediated suppression of apoptosis which resulted in predisposition to B cell lymphoma.87,88 Increased expression of other anti-apoptotic regulators such as Bcl-xL and Bfl-1, and decreased expression or mutation of pro-apoptotic genes (e.g., Bax and Bak), would lead to decreased apoptosis with increased cell survival. Therefore, alterations in apoptotic regulators can increase cell survival and cell number resulting in cancer formation.

Cancers of the Small Bowel and Colon The incidence of cancer in the colon is about 100 times more prevalent than cancer of the small bowel. Traditionally it has been thought that this was secondary to the rapid transit time of the small bowel, decreasing exposure to carcinogens and intraluminal toxins. As

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described above, the tissue homeostasis of both the small bowel and colonic epithelium are very dynamic where the proliferating stem cell compartment located in the crypts rapidly replaces cells loosed from the mucosal surface or villus. Recent evidence has shown that apoptosis plays a significant role in regulating the crypt stem cell units. Furthermore, the differences in expression of apoptotic mediators and location of apoptosis in the proliferative unit of the small bowel and colon may provide new insight into the differences between cancer induction in these organs. In the normal small intestine, there is a persistent low frequency (<1% of crypt cell number) of apoptosis found in the stem cell zone, and has been termed spontaneous apoptosis.69,99 It is believed that apoptosis in this region functions to regulate stem cell number. Tight control of the stem cell population within the crypts helps maintain villus size through steady migration of cells from the crypt to villus axis. In the colon, spontaneous apoptosis in the proliferating zone is a rare event.69 The location of these apoptotic events are scattered throughout the crypt and are rarely located in the stem cell unit. Therefore, spontaneous apoptosis in the colon is unlikely to regulate stem cell number. Potten et al69 proposed that the positional differences in apoptotic incidence between the small bowel and colon has direct implications on cancer incidence. They argue that the small bowel, capable of removing excess cells from the proliferating crypt unit, can regulate the output of progenitor cells to maintain constant cell number in the villus. Without apoptosis as a regulating mechanism, the colon is unable to remove excess stem cells, which can result in an increased cell number in the proliferating unit resulting in a hyperplastic crypt. These hyperplastic crypts result in an increased cell number exposed to, and therefore susceptible to, carcinogens and mutagens. In addition to the positional differences of apoptosis between the small and large bowel, Merrit et al100,101 found important regional differences in the expression of p53 and Bcl-2 family proteins which directly relate to tumor incidence in these regions of the GI tract. In the small bowel, the anti-apoptotic protein Bcl-2 was not expressed in the small intestinal epithelial cells, whereas the pro-apoptotic protein Bax was present.85,101 This natural organspecific distribution of apoptotic protein regulators would localize apoptosis to specific small bowel enterocytes, which would explain the relatively low incidence of small bowel adenocarcinomas despite their rapid proliferative rate. In contrast, Bcl-2 is expressed in the base of the colonic crypts,101 suggesting that Bcl-2 expression predispose the colonic epithelium to the formation of adenomas by prolonging the survival of stem cells and increasing the time of exposure to intraluminal carcinogens. It has become increasingly evident that genes that regulate apoptosis are mutant in colon cancers, and that the transformation of the colorectal epithelium to carcinoma is associated with a progressive inhibition of apoptosis.102 Fearon and Vogelstein103 reported that colorectal tumorigenesis was a result of mutational activation of oncogenes coupled to the mutational inactivation of tumor suppressor genes. Furthermore, formation of malignant tumors required the accumulation of multiple mutational events.104 In adenocarcinomas of the colon, p53 mutations and decreased Bcl-2 expression are frequent, and result in the loss of cell ability to regulate apoptosis (Fig. 4.5).105,106 The importance of p53-mediated apoptotic cell death in the induction and progression of colon cancer has been shown by the introduction of wild type p53 to colon cancer cell lines with known p53 mutations. These wild type p53 transfection experiments resulted in inhibition of cell growth and the induction of apoptosis.107,108 These data suggest that functional loss of p53 activity increases cancer cell survival and propagation of mutational events through the loss of the apoptotic pathway. The tumor suppressor gene p53 is located on chromosome 17p, which regulates cell cycle progression and the induction of apoptosis as a result of DNA damage. In colon cancer, loss of heterozygosity on chromosome 17p is associated with a worse prognosis and

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advanced metastatic disease.109 The bcl-2 gene is located on chromosome 18q, and is a negative regulator of apoptosis. It has been shown that the progression from normal mucosa to carcinoma is associated with a dramatic reduction in apoptosis102 through differential expression of the pro-apoptotic gene bcl-2 and mutation of p53.105 Immunohistochemical analysis of tissues for Bcl-2 protein revealed an increased expression in adenomas, and a most striking expression in colorectal carcinomas.105,110 Furthermore, there is an inverse relationship of Bcl-2 to p53 expression in adenomas, with coexpression in adenocarcinomas. 105 The recent discovery of Bfl-1, an anti-apoptotic protein, has led to the observation that there is an increased expression through the development of colonic adenomas and carcinomas.111 These data provide evidence that increased cell survival through increased expression of anti-apoptotic regulators (Bcl-2 and Bfl-1) is an early event, and p53 mutation is a late event in the induction and progression of adenocarcinoma of the colon. Others have shown a loss in the expression of Bcl-2 once the tumor has progressed to a carcinoma.112 This is partly because 69% of colorectal cancers are associated with a loss of chromosome 18q, which is the locus of the bcl-2 gene.113 The loss of Bcl-2 expression may be from the increased expression of p53, since both wild type and mutant p53 downregulate bcl-2 by binding to a transcriptional silencer element within the bcl-2 promoter.114 Clinically, the relevance of Bcl-2 expression in certain subsets of colon cancer is a favorable clinical outcome.105 This seems counter-intuitive because of the association of Bcl-2 with cell survival, but whether or not this is a mutated protein is not known. Furthermore, it has been postulated that the loss of Bcl-2 expression may be just a marker of acquisition of a more potent survival factor.115 Finally, immunohistochemistry staining of FasR and Fas ligand (FasL) expression has been studied in colorectal adenomas and carcinomas.116,117 FasL is a triggering agent of FasR-mediated apoptosis within the immune system.68 Expression of FasR is also seen in the normal colonic epithelium,118 and is also responsible for the induction of apoptosis in these cells. Moller et al118 showed that FasR expression is decreased or lost in 40 to 50% of colorectal cancers examined, and 100% of colon cancers with established metastasis. Recent evidence suggests that the decreased expression of FasR in colon cancer is not a result of Fas gene disruption, but a loss of Fas gene transcription through epigenetic gene silencing.116 These data support the findings of decreased apoptosis in the induction and progression of colorectal tumors through the loss of an apoptosis-inducing receptor. Furthermore, others have shown that there is an increased FasL expression in colon cancer, which induces apoptosis in activated T cells in culture.117,119 Collectively, these data suggest a FasL counterattack model for immune escape and metastatic establishment of colorectal cancers (Fig. 4.6).

Cancers of the Esophagus and Stomach Gastric carcinomas have also been associated with dysregulation of apoptosis. Increased Bcl-2 expression is a frequent occurrence in malignant and premalignant conditions of the stomach such as chronic atrophic gastritis, gastric epithelial dysplasia and gastric cancer.120 There is accumulating evidence to suggest different genetic pathways for well differentiated and poorly differentiated gastric cancers.57,58 Kasagi et al121 measured apoptosis in various gastric cancer tissues using the TUNEL assay. Their results showed a decrease in apoptotic index between well differentiated and poorly differentiated tumors (10.9% vs 4.0%). Furthermore, the inhibition of apoptosis through Bcl-2 expression appears to be specifically associated with the promotion of poorly differentiated gastric adenocarcinomas. Studies have shown that the loss of heterozygosity of the bcl-2 gene is a common event in well differentiated adenocarcinoma, whereas overexpression of bcl-2 gene is observed in poorly differentiated adenocarcinoma.122 Bcl-2 expression is also increased in gastric adenocarcinomas of the intestinal morphotype, but have no correlation to patient survival.120 Finally,

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Fig. 4.6. Hypothetical model of FasL-expressing human colonic adenocarcinomas in tumor escape from immune cell surveillance and liver colonization. FasR induces FasL-mediated apoptosis. FasR is normally expressed in immunocytes, hepatocytes, and epithelial cells of the gut. Colon cancer formation is associated with decreased expression or functional loss of FasR with increase in FasL. Increased FasL expression results in immune cell apoptosis and loss of normal tumor cell surveillance and elimination. FasL expression is also thought to play a role in liver metastatic establishment through the induction of hepatocyte apoptosis. Reproduced with permission, Shiraki K et al. Proc Natl Acad Sci USA 1997; 94:6424. ©The National Academy of Sciences.

Bcl-2 expression has been shown to improve cancer cell survival and enhance peritoneal dissemination with a 4-fold tumor weight increase in an in vivo model.123 Therefore, increased Bcl-2 expression appears to be an early event in gastric carcinogenesis, and a factor for tumor aggressiveness and peritoneal dissemination. Transforming growth factor-beta 1 (TGF-β1) can induce apoptosis through a receptormediated event. Ito et al124 and co-workers demonstrated reduced levels of the TGF-β receptor in poorly differentiated tumors. These data provide further mechanistic implications for the differences in the apoptotic index between well and poorly differentiated gastric adenocarcinomas. The tumor suppressor gene p53 has also been implicated in the induction and progression of gastric cancers. Allele loss and mutations of the p53 gene are detected in >60% of

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gastric cancers regardless of histological type.58 However, p53 abnormalities are found in 30% of gastric adenomas, and are a good correlation to histological atypia.125 It is known that activation of wild type p53 induces bax expression to accelerate apoptosis.78,91 The Bcl-2 and Bax proteins can form heterodimers which regulate the ability of Bcl-2 protein to block apoptosis. Decreased pro-apoptotic regulatory proteins (Bax and Bak) have been found in 25 to 70% of gastric cancer cases when compared to normal tissues.126 However, the expression of Bax does not correlate to gastric tumor histology.127 These data suggest that p53 mutations alter pro-apoptotic protein expression, which may be involved in the increased survival and chemoresistance of gastric cancer cells in vitro and in vivo. The induction of esophageal cancers has also been associated with the activation of proto-oncogenes and mutations in tumor suppressor genes. Among them, p53 was the first target tumor suppressor gene shown to undergo frequent point mutation in esophageal cancer tissues and cell lines.128 Similar findings were found in Barrett’s esophagus.129 These data were supported by other studies showing the high prevalence of p53 mutations in squamous cell and adenocarcinoma of the esophagus.130 In all, p53 mutations are found in approximately 50-80% of esophageal cancers.131,132 The mutation of p53 and accumulation of nuclear p53 protein occur during Barrett’s metaplasia progression both before and after cancer diagnosis.133,134 The accumulation of p53 in Barrett’s esophagus has been associated with an increased potential (56%) for malignant progression.135 These data suggest that p53 mutation is an early event in the development of squamous and adenocarcinoma of the esophagus,129,130 and may be of diagnostic significance in identifying the early progression of Barrett’s esophagus to adenocarcinoma. As in other cancers of the GI tract, the Bcl-2 gene family of apoptotic regulators is altered in a subset of esophageal cancers. The bcl-2 proto-oncogene is a known inhibitor of apoptosis. Immunohistochemical staining studies have examined Bcl-2 protein expression in both normal and cancer tissues of the esophagus. Immunoreactivity to Bcl-2 was found in 27% of invasive squamous cell carcinomas, and was more frequently expressed in poorly differentiated cell types.136 However, Bcl-2 expression showed no correlation to tumor size, depth of invasion, nodal status, or overall survival. The expression of Bcl-xL, another negative regulator of apoptosis, has been examined in the progression of normal to squamous cell carcinoma in the esophageal epithelium. In this study, Bcl-xL protein expression was decreased in malignant lesions of the esophagus, and correlated with decreased tumor differentiation and decreased patient survival.137 However, there was an inverse correlation of Bcl-xL to Bcl-2 expression. Bax expression, a pro-apoptotic regulatory protein, was also examined in normal and cancer tissues of the esophagus. Cytoplasmic staining for Bax protein was found uniformly in all layers of the normal esophageal squamous epithelium, with a gradual loss of expression in a fraction of preneoplastic and neoplastic lesions.136 This may be a result of loss of transcriptional regulation of the Bax gene through p53 mutations. However, as with Bcl-2, Bax expression showed no correlation to tumor size, depth of invasion, nodal status, or overall survival. These data indicate that the collective alterations in the apoptotic regulatory machinery may play a role in the development of esophageal cancers, but other factors determine tumor aggressiveness and metastatic spread. As described above, FasR is a cell surface receptor that mediates the induction of apoptosis by binding with FasL. FasR and FasL have been shown to be expressed in the normal epithelium of the esophagus in a positional-dependent manner.138 Squamous cell esophageal cancer cell lines were shown to possess FasR and FasL through reverse transcriptase-PCR analysis. Functional FasL was determined through the induction of apoptosis in Jurkat T leukemia cells, which are sensitive to FasL. Furthermore, through immunohistochemical staining, this study showed that FasL expression was found in >50% of tumor cells in 95% of invasive esophageal squamous cell carcinomas. In contrast, 80% of tumors

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showed no FasR expression. Therefore, similar to that seen in cancers of the colon, upregulation of FasL and downregulation of FasR expression are early events frequently associated with the evolution of esophageal squamous cell carcinomas.

Pancreatic Cancer Pancreatic adenocarcinoma is a disease with extremely poor prognosis, and is the fifth leading cause of cancer death. Insights into the molecular aberrancies that result in pancreatic cancer formation have evolved through the study of apoptosis-related genes, specifically p53 and bcl-2. Mutations in the p53 gene are frequent in pancreatic adenocarcinoma, occurring in 50 to 70% of the tumors.139-141 Investigation of premalignant hyperplastic lesions and carcinoma in situ of the pancreas revealed the presence of p53 mutations in 35% of cases.142 These data suggest that mutations of the p53 gene may represent early genetic transformation in the induction of pancreatic cancer, and may help identify precursor lesions with malignant predisposition. The prognostic significance of p53 mutations in this disease is somewhat unclear. One study reports that p53 mutations are an independent prognostic factor and conferred no significant differences in overall survival rates in pancreatic cancers.143 However, when evaluated in surgical patients undergoing resection or palliation, p53 mutations were associated with decreased survival (6.2 months vs. 15.0 months). Furthermore, in patients undergoing curative resection, the median survival of those with p53 mutations was 12.8 months vs. 38.6 months in those patients without p53 mutations.144 Therefore, it does appear that p53 mutations have a prognostic significance in pancreatic cancers, but further studies are required to confirm these findings. A prevalent mechanism in the increased survival of cancer cells and the induction of malignant disease is through the increased expression of Bcl-2, a negative regulator of apoptosis. Normal pancreatic ductal cells exhibit no Bcl-2 staining.145 Recent immunohistochemical staining experiments have revealed increased Bcl-2 expression in 53% of pancreatic adenocarcinomas studied. Bcl-2 expression was not related to tumor grade, DNA ploidy or S-phase fraction, but did predict a favorable outcome when compared to Bcl-2 negative tumors.146 These data indicate that pancreatic cancers with increased Bcl-2 expression behave less aggressively; however, the mechanism remains unknown. Overall, the prognostic value of Bcl-2 expression is unclear, since other tumors show no prognostic significance with Bcl-2 expression.147,148

Metastatic Disease With the evolving field of apoptosis and cancer induction, the question arises as to whether deregulation of apoptosis is associated with tumor metastasis. The role of apoptosis in removal of excess cells is important in preventing hyperplasia and the expansion of cells beyond normal organ functional and structural boundaries. It has been postulated that the removal of cells from their native confines within an organ results in the loss of growth factors and other survival factors that would lead to cell death by apoptosis.149 The establishment of metastatic disease requires the cancer cell to exit its normal microenvironment, which may be associated with the loss of essential survival signals. Early work before the discovery of apoptosis showed that most cells leaving the primary tumor through the circulation die before they can establish metastatic deposits.150 This may be the result of altered survival signals, or the loss of cell-cell attachment, both of which have been shown to induce apoptosis. However, cellular alterations that promote cell survival such as p53 mutations, Bcl-2 overexpression, and alterations of the Fas- and TNF-mediated death pathway may have direct implications in the establishment of metastatic disease. The role of apoptosis suppression and the incidence of metastatic disease were examined using a murine melanoma cell line exhibiting Bcl-2 overexpression. Compared to the

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parental cell line, Bcl-2 expression was associated with a significantly increased rate of pulmonary metastasis.151 Similar findings have also been found in prostate cancer.152 Although these findings seem logical, Bcl-2 expression and the occurrence of metastatic disease does not hold true for all cancers. It has been reported that metastatic potential in human colon cancer is associated with an increased sensitivity to apoptosis through increased pro-apoptotic protein expression (i.e., Bak).149 Also, pancreatic cancers exhibiting increased expression of Bcl-2 are associated with a lower probability of metastatic disease and longer survival. These data suggest that Bcl-2 expression may be one of the factors in the establishment of metastatic disease in certain tumor tissue types. FasL and its receptor FasR induce the activation of apoptotic machinery in many cell types.68 Abnormal FasL expression has been found in various human cancers, including colon cancer cells.116,117 Recently, FasL expression has been implicated in the establishment of liver metastasis in human colonic adenocarcinomas.119 This conclusion was made through the observation that FasL, which has low expression in normal colonic tissue, exhibits an increased expression in colon cancers with liver metastasis (Fig. 4.6). Since FasL is responsible for Fas-mediated apoptosis in receptor-expressing cells (i.e., activated T lymphocytes and hepatocytes), these data suggest that the evolvement of FasL expression may be an adaptive process gained by cancer cells to evade immune destruction and facilitate colonization during the establishment of metastatic disease (Fig. 4.6). Although curative resection of select tumors can achieve a significant disease-free interval, many patients develop metastatic disease. This is probably a result of microscopic metastasis established prior to resection, which remains dormant for a significant period of time.153 The kinetics of microscopic to macroscopic metastasis progression was examined in a murine model of lung metastasis, which showed that dormancy was associated with a significantly higher rate of apoptosis compared to growing lesions.154 Later, it was found that angiogenesis was the likely factor in these differences.155 These data suggest mechanisms by which apoptosis plays a possible factor in the establishment and progression of metastatic disease. The role of apoptosis in metastasis is probably tissue-type specific, and may vary depending on regional involvement. Further studies are needed to characterize this role, but could have implications on treatment of metastatic disease.

Cancer Treatment Modalities Radiation and chemotherapy are two modalities used to treat GI cancers and improve survival rates. Anticancer drugs are targeted to a multitude of intracellular components disrupting normal cellular homeostasis and/or inducing cellular damage. Despite this diversity of intracellular targets, cytotoxic anticancer drugs converge to a common response, cell cycle arrest and the induction of apoptosis.71 Furthermore, it is evident that the same oncogenes and mutations in tumor suppressor genes that are implicated in the induction of cancers can also explain, in part, the resistance of certain tumors to these conventional treatment modalities. In this section we will discuss the role of the cell cycle and apoptotic machinery in the cytotoxic effects of and resistance to chemotherapeutic agents and radiation therapy. The use of chemotherapy in the treatment of GI cancers is to target presumed micrometastasis and established macrometastasis, which are beyond the scope of surgical resection, to improve overall survival rates. Many chemotherapeutic agents with diverse mechanisms of action have been shown to induce apoptosis in a variety of GI tumors and cancer cell lines. Despite this diversity of chemotherapeutic action, the cytotoxic process which follows have been broken down into four stages which lead to the induction of apoptosis.149 In the first stage, the chemotherapeutic agent disrupts cellular homeostasis

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through a specific interaction with an intracellular target (i.e., RNA, DNA, or microtubules) resulting in its dysfunction. The second stage involves the recognition by the cell of homeostatic disruption (i.e., p53 response to DNA damage). In the third stage, the cell determines the severity of the insult, and makes the decision whether to repair the injury or proceed to apoptotic cell death. Finally, stage four is the induction of the apoptotic machinery leading to the morphological and biochemical features of this process. Although many chemotherapeutic agents are used to treat GI malignancies, the specific mode of action of each drug in relation to the induction of apoptosis is not well understood.71 The observation that antitumor drugs with disparate modes of action converge to induce cell death by apoptosis suggests that it is not the drug-induced lesion that causes apoptosis, but subsequent events such as disruption of growth.71 Indeed many of the current chemotherapeutic agents have a profound impact on cell cycle progression through cross-linking nucleic acids (alkylating agents), interfering with DNA and RNA synthesis (antimetabolites), and inhibiting mitosis through the binding of microtubules (vinca alkyloids). Each of these drugs has been shown to induce apoptotic cell death.71,156 In our laboratory, we have studied the effects of olomoucine and roscovitine, novel compounds designed to inhibit Cdks, on four human gastric cancer cell lines157 and five pancreatic cancer cell lines (unpublished data). We found that these compounds completely block Cdk2 and cdc2 activities and inhibited cellular proliferation with cell cycle arrest at the G2/M transition. Furthermore, roscovitine resulted in increased apoptosis by biochemical and morphological criteria in each of the pancreatic cancers studied. These data suggest that disruption of cell cycle events can act as a trigger to initiate the apoptotic cascade. Furthermore, these data show how ‘designer’ drugs aimed at the cell cycle machinery can be effective in inhibiting cancer cell proliferation and inducing cell death. Radiation and chemotherapy are treatments used to decrease tumor burden. The primary impact of radiation on the cell is a result of DNA damage, which activates p53 for G1 cell cycle arrest to allow for DNA repair, or the induction of apoptosis. It is known that chemotherapeutic agents induce p53-dependent cell cycle arrest, and it has been suggested that anticancer agents induce p53-mediated apoptosis.71 Although this may explain, in part, the resistance seen in some cancers with mutated p53 gene, it provides no insight as to why such treatment modalities are used to successfully treat tumors. Recently, a hypothesis has emerged that implicates intact cell cycle checkpoints involving cell cycle arrest as critical mediators of the response to chemotherapy and radiation. The p21 gene is transcriptionally activated by p53, and is responsible for the G1 cell cycle arrest following DNA damage. It has been shown that cancer cells with a defective p21 response as a result of deletion or p53 mutation underwent apoptosis as a result of DNA damage or chemotherapeutic drug treatment.158 However, cancer cells with an intact p21 checkpoint, which is also found in normal human cells, entered a stable cell cycle arrest. Furthermore, it was demonstrated that loss of the p21 checkpoint resulted in the continuation of DNA synthesis without mitosis, resulting in polyploidy and apoptosis. Similar results were obtained in a recent in vivo study using xenografts which were established from isogenic colon cancer cell lines differing only in their p21 checkpoint status.159 This uncoupling of mitosis and S phase after DNA damage suggests a mechanism to account for the specificity of commonly used chemotherapeutic agents and ionizing radiation, and supports the investigation of novel compounds that induce checkpoint-dependent cell cycle arrest as anticancer drugs. Resistance of cancer cells to the effects of chemotherapy and radiation treatment seems to have a common central theme, reduced sensitivity to the induction of apoptosis.149 Many human cancers express a mutated p53 tumor suppressor gene. Wild type p53 is a potent inducer of apoptosis after DNA damage. It has been shown that loss of p53 function results in drug and radiation resistance to apoptosis.160 Furthermore, increased expression of

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Table 4.1 Challenges to gene therapy I. Technical obstacles A. Delivery 1. Improvements in anatomic physical delivery systems for effective gene transfer to solid tumors 2. High-efficiency tumor-specific transduction producing a direct or indirect biologic effect against all tumor cells 3. Design of safe tumor-restricted replication effective vectors 4. Development of novel vectors capable of carrying large complex gene constructs 5. Cost-effective large scale production of vectors B. Effector gene strategies 1. Development of tumor-specific strategies (e.g., tumor specific receptor mediated uptake, tumor-specific promoters) 2. Requirement for a range of effective antitumor gene constructs tailored to tumors of a particular molecular genetic profile (e.g., p53-deficient, DNA mismatch repair-deficient) 3. Inducible systems to control transgene expression II. Potential or proven risks A. Toxicity 1. Vector related (e.g., viremia, inflammation) 2. Vector DNA recombination, leading to production and release of replication-competent virus 3. Large volumes of cationic lipids 4. Deleterious effects of suicide gene on normal tissues (e.g., liver) 5. Prodrug toxicity (not a major problem with current drugs) 6. Transgene toxicity B. Induction of neoplastic transformation 1. Insertional mutagenesis 2. Unknown effects of p53 overexpression in nontumor cells C. Induced autoimmunity against ‘self’ antigens after successful tumor killing/immune modulation Reproduced with permission (Zwacka RM, Dunlop MG, Hematology/Oncology Clinics of North America 1998; 12:599.

anti-apoptotic regulators (i.e., Bcl-2 and Bcl-xL), or decreased expression of pro-apoptotic regulators (i.e., Bax) correlate with resistance to radiation and chemotherapy in a variety of human cancers.161 It is evident that certain genetic mutations that confer cell survival advantages in tumorigenesis are also responsible for the resistance to treatment. Apoptosisrelated genes and their mutations seem to provide a prognostic marker and predictor to chemoresponsiveness in many cancers. As described in this review, aberrancies in cell cycle regulation and the apoptotic pathway are key events in the induction and progression of cancers. The abundance of research in these fields has provided information on the genetic mutations involved in cancer formation. Although cancer generally arises as a result of multiple genomic perturbations, the detailed knowledge of these events has opened new opportunities to develop novel therapeutic modalities, such as gene therapy. Gene therapy involves the introduction of genetic material into target tissue to achieve therapeutic benefit.162 One of the major strategies utilized in cancer gene therapy is the re-establishment of wild type tumor suppressor gene function.163,164 Of particular interest is the p53 tumor suppressor gene because of its

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frequent incidence of mutation in many GI cancers. As described above, wild type p53 is necessary for cell cycle arrest and the induction of apoptosis after genotoxic damage. Loss of this functional role results in uncontrolled proliferation and the progression of genetic mutations to daughter cells, and has also conferred resistance to standard chemotherapy. Therefore, the re-establishment of wild type p53 gene to p53 mutant tumor cells could restore normal regulation of the proliferative and apoptotic signaling pathways. In vitro studies using colon cancer cell lines with known p53 mutations showed that transfection of wild type p53 resulted in inhibition of cell growth and the induction of apoptosis.107,108 Furthermore, replacement of p53 function increases tumor radiation sensitivity and apoptotic response.162,164 In vivo studies using colon cancer xenografts in nude mice showed that the combination of gene therapy and chemotherapy was greater than single agent treatment or controls.164 These data indicate that genetic manipulations, such as wild type p53 gene therapy, can be an effective treatment modality or adjuvant therapy for malignant disease and chemoresistant cancers. Currently, gene therapy protocols are progressing from preclinical to Phase I clinical trials.164 Despite the advances made, there still remains many potential risks and challenges to the successful application of gene therapy for cancer treatment (Table 4.1). Major challenges lay in the specific targeting of these genes to tumor cells and/or the determination of long-term effects of gene transfer to noncancerous cells. Recent developments in gene therapy involve the establishment of a mutant adenovirus that replicates only in p53-deficient cells.165 This virus will provide specific tumor cell targeting for specific gene therapy. The scientific field of gene therapy is continually evolving, and is an exciting novel approach to the treatment of cancer.

Conclusions We have presented the major current concepts in the induction of cancer through perturbations of cell cycle control and the apoptotic pathway. The molecular mechanisms regulating these processes require careful regulation through various protein-protein interactions. Alterations in these protein regulators through mutation, increased expression, and inactivation can lead to uncontrolled proliferation and increased cell survival, which are basic to the transformation to malignant disease. Furthermore, these same processes involved in cancer formation can have a profound impact on treatment response and the establishment of metastatic disease. The knowledge of malignant transformation has provided new concepts in the re-establishment of cell cycle control and the apoptotic pathway through genetic manipulations. Future research may give clues for the early diagnosis of premalignant disease, and the early recognition of genetic factors predisposing GI tissues to malignant transformation.

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113. Jen J, Kim H, Piantadosis S et al. Allelic loss of chromosome 18q and prognosis in colorectal cancer. N Engl J Med 1994; 331:213-221. 114. Miyashita T, Harigai M, Hanada M et al. Identification of a p53-dependent negative response element in the bcl-2 gene. Cancer Res 1994; 54:3131-3135. 115. Watson AJ, Merritt AJ, Jones LS et al. Evidence of reciprocity of bcl-2 and p53 expression in human colorectal adenomas and carcinomas. Br J Cancer 1996; 73:889-895. 116. Butler LM, Hewett PJ, Butler WJ et al. Downregulation of Fas gene expression in colon cancer is not a result of allelic loss or gene rearrangement. Br J Cancer 1998; 77:1454-1459. 117. OíConnell J, OíSullivan GC, Collins JK et al. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med 1996; 184:1075-1082. 118. Moller P, Koretz K, Leithauser F et al. Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium. Int J Cancer 1994; 57:371-377. 119. Shiraki K, Tsuji N, Shioda T et al. Expression of Fas ligand in liver metastases of human colonic adenocarinomas. Proc Natl Acad Sci USA 1997; 94:6420-6425. 120. Lauwers GY, Scott GV, Hendricks J. Immunohistochemical evidence of aberrant bcl-2 protein expression in gastric epithelial dysplasia. Cancer 1994; 73:2900-2904. 121. Kasagi N, Gomyo Y, Shirai H et al. Apoptotic cell death in human gastric carcinoma: analysis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. Jpn J Cancer Res 1994; 85:939-945. 122. Ayhan A, Yasiu W, Yokozaki H et al. Loss of heterozygosity at the bcl-2 gene locus and expression of bcl-2in human gastric and colorectal carcinomas. Jpn J Cancer Res 1994; 85:584-591. 123. Yawata A, Adachi M, Okuda H et al. Prolonged cell survival enhances peritoneal dissemination of gastric cancer cells. Oncogene 1998; 16:2681-2686. 124. Ito M, Yasui W, Kyo E et al. Growth inhibition of transforming growth factor beta on human gastric carcinoma cells: receptor and postreceptor signaling. Cancer Res 1992; 52:295-300. 125. Tohdo H, Yokozaki H, Haruma K et al. p53 gene mutations in gastric adenomas. Virchows Arch B Cell Pathol Include Mol Pathol 1993; 63:191-195. 126. Krajewska M, Fenoglio-Preiser CM, Krajewski S et al. Immunohistochemical analysis of Bcl-2 family proteins in adenocarcinomas of the stomach. Am J Pathol 1996; 149:1449-1457. 127. Komatsu K, Suzuki S, Ohara S et al. Expression of Bcl-2 and Bax in human gastric cancer tissue. Nippon Rinsho 1996; 54:1929-1934. 128. Hollstein MC, Metcalf RA, Welsh JA et al. Frequent mutation of the p53 gene in human esophageal cancer. Proc Natl Acad Sci USA 1990; 87:9958-9961. 129. Casson AG, Mukhopadhyay T, Cleary KR et al. p53 gene mutations in Barrettís epithelium and esophageal cancer. Cancer Res 1991; 51:4495-4499. 130. Meltzer SJ. The molecular biology of esophageal carcinoma. Recent Results Cancer Res 1996; 142:1-8. 131. Montesano R, Hollstein M, Hainaut P. Molecular etiopathogenesis of esophageal cancers. Ann Ist Super Sanita 1996; 32:73-84. 132. von Brevern MC, Hollstein MC, Cawley HM et al. Circulating anti-p53 antibodies in esophageal cancer patients are found predominantly in individuals with p53 core domain mutations in their tumors. Cancer Res 1996; 56:4917-4921. 133. Casson AG, Manolopoulos B, Troster M et al. Clinical implications of p53 gene mutation in the progression of Barrettís epithelium to invasive esophageal cancer. Am J Surg 1994; 167:52-57. 134. Hardwick RH, Shepherd NA, Moorghen M et al. Adenocarcinoma arising in Barrettís oesophagus: evidence for the participation of p53 dysfunction in the dysplasia/carcinoma sequence. Gut 1994; 35:764-768. 135. Younes M, Ertan A, Lechago LV et al. p53 protein accumulation is a specific marker of malignant potential in Barrettís metaplasia. Dig Dis Sci 1997; 42:697-701. 136. Sarbia M, Bittinger F, Porschen R et al. bcl-2 expression and prognosis in squamous-cell carcinomas of the esophagus. Int J Cancer 1996; 69:324-328.

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164. Zwacka RM, Dunlop MG. Gene therapy for colon cancer. Hematol Oncol Clin North Am 1998; 12:595-615. 165. Bischoff JR, Kirn DH, Williams A et al. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 1996; 274:373-376.

CHAPTER 5

Oncogenes and Tumor Suppressor Genes in GI Cancer Mimi Kim and B. Mark Evers

A

t its most fundamental level, cancer is caused by the accumulation of mutations in a critical combination of genes over time manifested by the disruption and dysregulation of cell machinery controlling growth and proliferation. Ultimately, normal cell growth and proliferation hinges upon the fidelity of message transmission from extracellular stimuli to the nucleus where the blueprints for protein synthesis are stored. When the protein components of signal transduction pathways are altered due to exposure of DNA to mutagens such as UV light, chemicals, or radioactivity, frequently the transmitted signal becomes distorted, amplified, or silenced altogether. Two basic classes of genes can be mutated in cancer: oncogenes, which are mutant versions of normal growth genes called proto-oncogenes that become overexpressed or constitutively active as a result of the mutation and tumor suppressor genes (also known as recessive oncogenes, recessive cancer genes, antioncogenes, and cancer susceptibility genes), which normally provide a counterbalance to the proto-oncogenes by inhibiting cellular growth; cancer may result from loss of expression of their products (Fig. 5.1). While oncogenes produce phenotypic changes in a cell with only one of the two copies mutated, tumor suppressor genes require mutated or lost expression of both copies to exert a tumorigenic effect. Oncogenes and tumor suppressor genes involve multiple levels of signal transduction pathways. Growth factors that are produced by the cell can stimulate the cell in an autocrine fashion and are sometimes overexpressed as a consequence of proto-oncogene mutations. Mutations may occur in genes encoding growth factor receptors such as c-erbB2, disabling the receptor’s regulatory function or increasing quantities of the receptor in the cell. Genes encoding signal transduction proteins such as the Ras family of proteins can also be affected. Finally, nuclear regulatory proteins, which influence transcription of growthrelated genes by binding to DNA, may be mutated to promote cancer. No single lesion independently causes malignancy. Rather, it is the cumulative effect of multiple mutations acquired over time producing a broad range of phenotypic changes that together create a cell which bears the special characteristics of a malignant cell, such as uncontrolled proliferation, loss of intracellular adhesion, ability to invade surrounding tissues, and potential for metastasis and seeding of remote tissues.1 Each mutation brings the cell a step closer to the fully malignant phenotype; the accumulation of mutations confer upon cells a growth advantage due to dysfunctional cell cycle regulation so that these cells and their progeny propagate more efficiently and acquire errors in the genome more quickly, hence accelerating neoplastic progression. For this reason neoplasms usually stem from one Molecular Mechanisms in Gastrointestinal Cancer, edited by B. Mark Evers. ©1999 R.G. Landes Company.

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Fig. 5.1. Normal regulation of cellular proliferation depends upon equilibration between the growth-promoting influence exerted by proto-oncogenes and the growth-inhibiting activity of tumor suppressor genes. Cancer occurs as a consequence of the uncontrolled cellular proliferation that follows disruption of this balance through genetic alterations that activate oncogenes and inactivate tumor suppressor genes.

or two cells more prolific than the rest because of their particular combination of mutations. The majority of tumors contain cells which are very similar genotypically, if not among tumors of the same type, then at least among cells of a single tumor. This fact and the observation that the set of genomic errors in cells of a clone appear to correspond closely with the histological features of a particular neoplasm has formed the basis for the construction of models which attempt to describe the molecular pathogenesis of colorectal, pancreatic, and gastric cancer in correlation with the histological progression from dysplasia and metaplasia to adenoma, carcinoma, and finally to invasive and metastatic cancer (Figs. 5.2, 5.3). This chapter will review the role of the better characterized oncogenes and tumor suppressor genes that have been implicated in the development and progression of colorectal, pancreatic and gastric cancers.

Colorectal Cancer K-ras Mutations in the proto-oncogene c-K-ras (meaning the cellular version of the K-ras oncogene that is expressed in normal cells) occur in approximately 40% of primary colorectal carcinomas and appear to be an early event in colorectal tumorigenesis.2,3 It lies on the short arm of chromosome 12 (12p12) and has a 2.0kb transcript. Its product, a 21kD G-protein found on the inner surface of the plasma membrane, is involved in the transduction of growth-related messages from the cell surface to the nucleus, where ultimately genes controlling cell differentiation and proliferation are activated.4,5 Other members of this family which are not as frequently found in colorectal cancer but bear significant homology to K-ras are N-ras (N for neuroblastoma) and H-ras (for Harvey-ras). While point mutations in codons 12, 13 and 61 of ras genes are found in a multitude of human tumors,6 codon 12 point mutations predominate in colorectal cancer. Point mutations at one of these three codons result in alterations of critical amino acids and the Ras protein loses its inherent ability to convert GTP to GDP. As a result, the protein becomes permanently stuck in its active conformation and continues to transmit its signal to downstream targets even in the absence of upstream stimuli. This can, in turn, lead to overexpression of downstream growthpromoting genes and dysregulated cell replication in cells with this mutation.

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Fig. 5.2. A model for the molecular pathogenesis of colorectal cancer. (Reprinted with permission from Howe JR, Guillem JG, Surg Clin North Am 1997; 77:175-195 ©1997 W.B. Saunders).

Laurent-Puig et al7 observed a decreased 5-year survival among patients with colorectal cancers with 17p allelic loss compared to patients whose cancers without the deletion. However, Kern et al8 found no significant increase in tumor aggressiveness resulting from K-ras activation. One group found a positive correlation between simultaneous mutation in codons 12 and 13 of K-ras and long-term survival in 89 patients with colorectal cancer.9 Thus, although efforts are already under way to develop diagnostic tests involving K-ras detection,10 the evidence accumulated regarding the prognostic value of K-ras mutations, though promising, is unconvincing and further studies are clearly needed before detection of these mutations in patients is of clinical utility.11-14

p53 The tumor suppressor p53 is found at chromosome locus 17p13. Present in approximately 50% of all human cancer, it is the most common genetic aberration in human cancer. p53 encodes a nuclear phosphoprotein of 393 amino acids that is normally expressed only transiently by the cell in the presence of damaged DNA. The p53 protein causes the transcription of genes whose products inhibit cell cycle progression from G1 (resting) to S (synthesis) phase, giving the cell time to repair its DNA before DNA replication and cell division in the presence of damaged genetic material. In this manner, p53 prevents accumulation of genetic lesions in the genome as cells divide from one generation to the next. By mechanisms still unclear, if p53 detects that DNA is irreparably damaged, it can also stimulate the transcription of apoptotic genes that bring about cell self-destruction. Functional p53 exists in the cell as a tetramer; alterations at the interface conjoining the subunits disable the protein. This accounts for the unusual characteristic of certain mutant forms of p53 which require only one mutated allele to abrogate p53 function. It appears that these mutant products do so by forming complexes with the wild type p53 protein with the consequent disruption of normal function secondary to lost DNA binding specificity of the tetramer. This phenomenon in which the product of a single mutant copy of a tumor suppressor gene silences or interferes with the protective function of the normal product is known as a dominant-negative effect. Thus, not only is p53 a tumor suppressor gene, but certain mutant forms have the capacity to behave like oncogenes because of this property.4 Baker et al15 reported that loss of heterozygosity in 17p was observed in 75% of colorectal carcinomas; after point mutation on one allele, loss of the other soon followed so that there was a high incidence of concomitant p53 mutation and chromosomal loss.16 p53 mutation

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Fig. 5.3. A model for the progression of molecular lesions in gastric cancer. It is believed that a unique set of genetic changes is responsible for the phenotypic evolution of each type of human cancer, including gastric cancer. Evidence suggests that the two histological subtypes of gastric adenocarcinoma, diffuse and intestinal gastric cancers, originate from different combinations of molecular lesions. (Reprinted with permission from Tahara E, World J Surg 1995; 19:487 ©Springer-Verlag New York Inc.).

appears to occur as a late event in colorectal cancer progression for both FAP and sporadic cases, probably triggering the conversion from a severely dysplastic adenoma into a carcinoma.17 p53 mutations, particularly deletion of the second copy on chromosome 17p, are associated with poor patient prognosis.8,16 Some p53 mutants have the additional property of increased stability in the cell in comparison to the wild type; consequently, since they are not as easily broken down, the mutant protein tends to accumulate in the cell. In cells containing a p53 mutation, therefore, elevated concentrations of the p53 product can often be detected. This may provide the basis for a clinically useful diagnostic test involving an assay such as immunohisto-chemistry or ELISA. The utility of mutant plasma protein levels for prognostication in colorectal cancer is presently being investigated.18,19

APC Studies of the familial colorectal cancer syndromes have proven instrumental in elucidating the molecular pathogenesis of GI cancer. One of these syndromes, familial

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Fig. 5.4. E-cadherin (A) and APC (B) compete to form complexes with β-catenin, α-catenin, and γ-catenin (plakoglobulin). (A) The cytoplasmic region of E-cadherin binds to β-catenin and γ-catenin which link E-cadherin to β-catenin, a molecule anchored to the cytoskeleton. (B) β-catenin and γ-catenin can instead be bound to APC. The significance of this interchange is not precisely understood, but mutations in E-cadherin and APC have both been shown to interfere with normal cellular adhesion. (Reprinted with permission from Birchmeier W, BioEssays 1995; 17:98, ©John Wiley & Sons, Ltd.).

adenomatous polyposis (FAP), occurs as a consequence of an inherited mutated version of the tumor suppressor gene adenomatous polyposis coli (APC) and subsequent mutation or loss of the remaining normal copy. Although cases of FAP account for but a small proportion of colorectal cancers, according to one study, 60% of sporadic colorectal carcinomas and 63% of adenomas contained somatic mutations in the APC gene,20 indicating that the gene is important in the pathogenesis of sporadic colorectal cancer as well. Mutations of the APC gene appear to be one of the earliest changes in sporadic tumor development, and are possibly the initiating lesion in a great majority of these nonfamilial cases.1,21 APC is located on chromosome 5p21 (that is, the short arm of chromosome 5) and encodes a protein of 2843 residues. The coding region consists of 15 exons; the 15th exon accounts for 77% of the coding region.1 The APC protein is expressed cytoplasmically in a number of tissues and bears little resemblance to any other known protein but contains sequences similar to intermediate filament proteins such as myosin and keratin.22 The vast majority of APC mutations in sporadic and FAP cancers result in truncated proteins and one study found that about half of mutations involve a region spanning less than 10% of the gene.20,23,24 While both wild type and mutant forms are cytoplasmic, mutant proteins are soluble whereas wild type proteins are not.25 Furthermore, it appears that certain mutant versions may interfere with the function of the normal protein in cells heterozygous for the mutation by formation of partially insoluble aggregates in a dominant-negative fashion.4 The APC protein appears to play an integral role in cell-cell adhesion and indirectly in the transcription of genes during development. By sequestering β-catenin, APC prevents it from associating with E-cadherin through α- and γ-catenin (plakoglobulin); E-cadherin has an extracellular domain which protrudes from the cell surface and participates in

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cell-to-cell adhesion by binding to E-cadherins which extend from neighboring cells (Fig. 5.4). APC may also play a role in cytoplasmic microtubule assembly.26 Morin et al27 suggest that APC may influence the balance between cell renewal and death through a role in control of apoptosis.

DCC DCC, short for deleted in colon cancer, is a tumor suppressor gene of 29 exons spanning over a million base pairs on the long arm of chromosome 18 (18q). It encodes a 1447 amino acid transmembrane protein whose extracellular domain resembles the neural cell adhesion molecule (NCAM) family of proteins.28 The exact function of the protein is not fully understood, but it appears that DCC may participate in signaling pathways which control cell proliferation and differentiation. Further supporting this conjecture is the finding that in many mature epithelia DCC expression appears restricted to the proliferative compartment.28,29 A recent study by Fazeli et al30 has raised doubts concerning the role of DCC by suggesting that although DCC appears to play a role in neural development, it is likely not the tumor suppressor from 18q that was thought to promote colorectal carcinoma. However, most of the evidence accumulated supports the notion that DCC participates in the pathogenesis of colorectal cancer.31 Losses of heterozygosity in 18q occur in more than 70% of colorectal cancers, and the altered region includes the DCC locus in over 90% of carcinomas with 18q allelic loss.28 Studies comparing the incidence of DCC allelic loss among adenomas and carcinomas indicate that loss in this region is a relatively late event in the progression of cancer.32 There has been evidence to suggest that tumors with allelic loss at 18q metastasize more readily and behave more aggressively; thus DCC mutations may be of prognostic value.8,33,34

Mismatch Repair (MMR) Genes Hereditary nonpolyposis colorectal carcinoma (HNPCC) is an autosomal dominantly inherited family cancer syndrome which has been instrumental in elucidating the central role of mismatch repair genes in cancer progression in approximately 15% of sporadic cases of colorectal carcinoma.35,36 In about 80% of HNPCC patients, the propensity towards development of colorectal tumors results from an inherited mutation in one or more DNA mismatch repair genes.37 These tumor suppressors are critical in maintaining the stability of the genome as cells multiply; loss of function in these genes leads to replication errors accumulating at a rate of 1,000-fold of normal cells for each cycle, consequently accelerating cancer progression. The mismatch repair genes specific to HNPCC and sporadic cases of colorectal cancer each target specific point mutations in microsatellite repeats, special sequences of DNA which consist of multiple repeats of one or two nucleotides. These mutations occur as deletions and expansions which result from physical damage to the DNA, misincorporation of nucleotides during DNA replication, or mismatched nucleotides that occur during genetic recombination.38 Cells with this type of error are said to be replication-error positive (RER+) and to possess microsatellite instability (MI). The most common genes affected are MSH2 and MLH1, followed by PMS1, PMS2, and MSH6.39 Mutation of different MMR genes appear to influence the phenotype differently, resulting in the variation in clinical characteristics in HNPCC patients. Mutations in MSH2 are more commonly linked with cancer of the renal pelvis, ureter, stomach and ovary than MLH1 mutations, and mutations in MSH6 are more frequently associated with atypical HNPCC involving endometrial and ovarian cancers more than colorectal neoplasms. In addition, the form of mutation appears to be associated with the phenotype. The phenotype of tumors with missense mutations in MMR

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genes appear to be less severe than tumors resulting from frameshift or nonsense mutations, and silencing mutations of MLH1, which produce no protein, are observed to be associated with a far fewer number of extracolonic manifestations of HNPCC when compared with other mutants in which the protein is merely truncated.40,41 One candidate tumor suppressor gene that has been found to be consistently associated with RER+ colorectal neoplasms is the gene encoding the type II tissue growth factorbeta (TGF-β) receptor. Characteristically a frameshift mutation occurs within a string of 10 adenines (A10) from nucleotides 709 to 718 upstream of the coding region of the gene.42 Colorectal neoplasms with this mutation have a definitive growth advantage which is clearly not a byproduct of the generalized instability in these tumors.43 TGF-βRII may play an important role in mediating differentiation and apoptosis of gut epithelial cells. Recent studies suggest that the RER+ phenotype in colorectal cancer has a positiveprognostic significance and that these tumors are less likely to have allelic loss at 5q, 17p, and 18q.36,44 In the future, molecular screening of susceptible individuals for replication errors may be used in conjunction with currently available conventional tests to improve survival through early detection and early selection of the most effective treatment against the cancer.39,45

Pancreatic Cancer K-ras Present in approximately 85% of pancreatic exocrine malignancies as well as in a significant proportion of benign pancreatic tumors, the oncogene K-ras appears to play an important role in initiating transformation in many pancreatic tumors.46,47 In these neoplasms, K-ras mutations occur almost exclusively on codon 12, in contrast to other tumors with mutated ras genes and N-ras and H-ras mutations have been found infrequently. Although a number of studies have been conducted to ascertain whether ethnic differences or different carcinogens account for the variety of nucleotide substitutions observed, this hypothesis remains to be proven.48-50 The ubiquitous presence of K-ras mutations in pancreatic adenocarcinomas has generated a great deal of interest in recent years towards developing methods of using the K-ras gene or its protein product as molecular markers or targets for detection, diagnosis and therapy of pancreatic cancer. One group, upon observing that cells with the activated K-ras oncogene were far more susceptible to the suppression of KRAS/p21 than cells with wild type K-ras,51 suggested that since the cells with the activated K-ras oncogene appeared to depend heavily upon a KRAS/p21-mediated growth signal pathway for their growth, K-ras may potentially be a good target for treatment. In addition, because K-ras mutations are likely early lesions in pancreatic cancer, are few in number, and because they usually affect single amino acids, they may potentially be useful as a gene-based diagnostic test.47 The relatively high incidence of K-ras mutations in pancreatic adenocarcinoma may be helpful in distinguishing these exocrine tumors from endocrine tumors, periampullary tumors, and pancreatitis. Numerous methods employing K-ras detection for diagnostic or prognostic purposes are under development, including fine needle aspiration based cytology used with molecular analysis and tests involving the detection of K-ras in duodenal fluid, peripheral blood, and stool (reviewed in Howe and Conlon 1997).46

p53 p53 mutations occur in approximately 50% of all pancreatic adenocarcinomas and allelic loss is found in up to 80% of tumors.52 Most p53 point mutations are missense mutations within the coding region, particularly in the evolutionarily conserved exons 5

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through 9 which encode the DNA binding domain of the protein responsible for initiating the transcription of growth-control genes downstream of the p53 binding site.46 Mutations in pancreatic cancer correlate with more advanced tumor stage and local lymph node involvement, but not with a higher incidence of distant metastases.53 In contrast, one group found that in a series of 136 primary and 23 metastatic sporadic and familial ductal pancreatic adenocarcinomas, p53 mutations are a frequent early event in pancreatic tumorigenesis not associated with metastasis development, and that there was no significant correlation between p53 mutation and tumor grade, TNM stage, or smoking history.54 p53 overexpression appears to be unique to ductal adenocarcinoma, indicating that it may be a useful tool for differentiating between various diseases of the pancreas.46 However, the sensitivity of tests developed thus far has been low, limiting its usefulness as a tumor marker.47,55,56

p16/MTS1 p16 is a tumor suppressor gene found at chromosome 9p21. It is transcribed from the CDKN2A gene, which also encodes the p19ARK protein; the two products are obtained through alternative splicing of RNA. Like p53, it functions to prohibit cell-cycle progression in the presence of genetic mutation at critical junctions, thus controlling neoplastic growth. It is thought that the p16 product suppresses tumors by inhibiting Cdk/cyclin phosphorylation of the RB/E2F complex. Cdk4 and Cdk6 phosphorylation permits release of E2F, thus stimulating DNA replication. The downstream effect of the normal p16 protein activity is inhibition of transcription of genes that are responsible for growth and differentiation through hyperphosphorylated Rb (Fig. 5.5). p16 is known to act in a regulatory feedback circuit with Cdk4, D-type cyclins, and Rb protein.57 p16 appears to be inactivated through three basic mechanisms: point mutation with loss of heterozygosity within CdkN2A, homozygous deletion, and through methylation of the 5' promoter region.58 As this is a relatively new candidate tumor suppressor, much regarding the precise mechanistic activity of p16 remains to be learned. According to Caldas et al,59 38% of pancreatic carcinomas were found to contain mutations in p16, all with loss of the wild type allele; in addition, the total frequency of mutation and of homozygous deletion of both copies of the p16 gene approaches 80%. There has been evidence to suggest that p16 plays a significant role in exocrine pancreatic carcinogenesis, particularly in ampullary carcinomas.60,61

DCC While a high frequency of loss at chromosome 18q has been reported (88%),61 other studies have found a low expression of DCC in pancreatic cancer.62,63 The significance and role of the DCC mutation in pancreatic cancer is yet unclear and merits further study; however, because the gene is approximately a million base pairs in length, characterization is expected to continue to progress slowly until the Human Genome Project is completed.31

DPC4/Smad4 DPC4 (deleted in pancreatic carcinoma) is a tumor suppressor gene, which, like DCC, is located on chromosome 18p21.1. Its expression is reported to be inactivated in about 50% of all pancreatic adenocarcinomas;64,65 the total combined frequency of mutations and of homozygous deletions approaches 80%. Homozygous deletion occurs as a two step process: first, loss of a larger chromosomal region, followed by loss of a smaller region that targets the tumor suppressor gene.61 DPC4 mediates many of the actions of the serinethreonine kinase receptor for TGF-β; overexpression of DPC4 alone has the potential to

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Fig. 5.5. The CdkN2A gene encodes two products created through alternate splicing, p16 and p19. p16 appears to contribute to the pathogenesis of a significant proportion of pancreatic cancers. By inhibiting the activity of cyclin-dependent kinase complexes Cdk4/cyclinD and Cdk6/ cyclin D, whose function is to inactivate the growth-inhibitory Rb/E2F complex, p16 is able to block cell cycle progression in normal cells. Inactivation of this tumor suppressor gene leads to uncontrolled proliferation. (Reprinted with permission from Clurman BE, Groudine M, New Engl J Med 1998; 338:910-912 ©Massachusetts Medical Society).

induce transactivation of genes that inhibit cell cycle progression as in the presence of TGF-β.66 Structural analysis of the DPC4 protein revealed that the functional form of the protein is a trimer joined through specific protein-protein interactions, which are disrupted in the presence of one of several missense mutations. Shi et al65 indicated that the trimeric assembly of the protein that is disrupted by tumorigenic mutations is important for proper signal transduction. It is suspected that DPC4 is responsible for the familial aggregations of pancreatic carcinoma, but the evidence is thus far inconclusive and further studies are needed.67

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Gastric Cancer p53 By far, p53 appears to be the most common cancer gene altered in gastric neoplasms, regardless of histological classification or staging. Abnormalities of the p53 gene, which occur as missense mutations, frame shifts, and allelic loss,68 are found in at least 60% of gastric cancers and 30% of gastric adenomas.69,70 Lesions in p53 appear to occur early in the adenoma-carcinoma sequence and may play an important role in gastric cancer expansion even from early stages.71 The discrete epidemiological and histological characteristics, combined with the accumulating evidence of different molecular profiles and biological activity, of the two subtypes of gastric cancer as defined by Lauren72 suggests the existence of distinct molecular pathways leading to each type of cancer. Sano et al73 found allelic loss at the p53 chromosomal locus in 67% of well differentiated tumors and 60% of poorly differentiated tumors studied; in another study of 163 patients with gastric cancer, overexpression of p53 was found more frequently in early intestinal tumors than early diffuse tumors, and appeared to participate in the tumor stage progression of diffuse but not intestinal gastric tumors.74 Thus research concerning the role of these lesions in the two histological subtypes has been inconclusive. Gastric carcinomas containing mutations in p53 have been associated with a considerably poorer prognosis in comparison to tumors negative for p53 mutation,75 possibly by enabling tumors to grow vertically into the gastric wall.76 In one recent study by Ichiyoshi et al76 of 196 advanced gastric cancers, tumors overexpressing p53 were associated with an increased number of vessel invasions and more extensive metastasis to lymph nodes; however, p53 was not associated with depth of cancer invasion or tumor stage.

APC Normally, the adenomatous polyposis coli (APC) tumor suppressor gene plays an important role in adhesion between cells, cytoskeletal anchoring, and possibly in cell signaling pathways. Mutation and allelic loss appears to occur early in the development of gastric cancers, especially of the well-differentiated intestinal type;74,77 they may play an important role in the initial development of intestinal gastric cancers. One study identified 5q allelic loss involving the APC locus in 60% of early well-differentiated tumors but found no such abnormalities in poorly differentiated tumors.73 While in familial adenomatous polyposis and sporadic colorectal cancer nonsense mutations predominate, APC alterations in gastric cancers are most commonly missense mutations. The nature of APC and p53 mutations appears to correlate with the histological atypia of gastric cancers and may prove useful in the future as a diagnostic marker.78

DCC Loss of heterozygosity in the DCC tumor suppressor gene occurs in about half of welldifferentiated gastric tumors79 and may promote their development.80 One group suggested that these mutations precede p53 allelic loss in the adenoma-carcinoma sequence in gastric tumors.81 In a recent study of 163 gastric cancers, loss of heterozygosity in DCC occurred primarily in advanced intestinal gastric cancers and less prominently in advanced diffuse or early gastric cancers.74 Inadequate levels of DCC protein are thought to be associated with disrupted control of growth restriction and altered adhesion of mucosal cells,82 ultimately promoting cancer progression.

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c-erbB2 The erbB2 oncogene represents the human homolog of the neu oncogene first isolated in rat neuroblastoma cells; the proto-oncogene c-erbB2 is located on chromosome 17p11 and encodes a 183kD tyrosine kinase which closely resembles the epidermal growth factor receptor (EGFR). In gastric cancer, overexpression of c-erbB2 results primarily from gene amplification—that is, an abnormal increase in the number of copies of a gene in a cell by several times normal. What causes gene amplification is not known, but it is a common abnormality in malignant cells. Overexpression of c-erbB2 was observed in one study to occur mostly in intestinal gastric cancers as well as advanced gastric cancers of both types;74 according to Tahara et al,78 c-erbB2 amplification occurs exclusively in well differentiated gastric cancers. In addition, c-erbB2 overexpression has been found to correlate closely with liver metastasis, invasion, and nodal involvement and a generally more aggressive course in differentiated gastric tumors.74,83,84 One group suggested that the high malignant potential of cancers with amplification of this gene may be directly related to a dramatically increased potential for lymph node metastasis in these tumors.85

c-met The c-met proto-oncogene encodes the hepatocyte growth factor receptor (HGFR), a 65kD tyrosine kinase with autophosphorylation activity. Of proto-oncogene encoding tyrosine kinase receptors, c-met is the most commonly involved in gastric cancer; amplification of c-met is extremely rare in both esophageal and colorectal cancer.78,86 Overexpression of c-met is most common in advanced cancers, particularly in the poorly differentiated scirrhous carcinomas.86 Most gastric cancers overexpress two transcripts of 7.0 kb and 6.0 kb; the latter is expressed preferentially by tumors and correlates with biological malignancy.87 According to one hypothesis, the interaction of c-met amplified in tumor cells and HGF produced by activated stromal cells may promote the development of gastric cancer.88

Conclusions A clearer understanding of the genetic lesions driving cancer progression will be crucial in improving patient prognosis by opening doors for the development of better methods of detection, diagnosis and therapy. Since many gastrointestinal tumors remain clinically silent until advanced stages at which intervention offers little more than palliation, molecular tools permitting early and accurate detection or screening of asymptomatic highrisk individuals could dramatically improve survival rates. Molecular techniques currently available that are specific for protein include immunohistochemistry, immunoprecipitation, ELISA; the polymerase chain reaction (PCR), an assay used for the analysis of DNA and RNA, has played a critical role in cancer research thus far and will continue to be an important tool as molecular diagnosis makes the transition from the bench to the bedside. Current problems of some of the methods include high cost, as in the example of stool analysis for K-ras mutations in colorectal cancer, and the lack of adequate sensitivity of some of these tests. In addition, many of the oncogenes and tumor suppressor genes are shared among tumors, thus limiting their specificity. For many genes, the size or the multitude of possible alterations has proven to be the prohibitive factor. In the case of pancreatic cancer, the prevalence of a limited number of K-ras mutation, most of which are single amino acids changes, occurring in 80-90% of tumors has encouraged exploration into methods focused on detection of K-ras in stool, serum, bile, and pancreatic juice. These tests may ultimately be useful for screening susceptible individuals with an inherited predisposition to cancers or those who are simply at high risk.

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Oncogenes and tumor suppressor genes may also be useful for assessment of patient prognosis and thus may potentially serve as an indicator for the best course of therapy. As discussed previously, certain mutant oncogenes and tumor suppressor genes have been observed to increase the biological aggressiveness and malignant potential of neoplasms. Detection of these lesions in a patient may in the future guide a clinician’s decision to pursue a more aggressive course of therapy from the outset in hopes of improving the prognosis. Additionally, the presence of a particular mutation in a neoplasm may be useful as a predictor of tumor response to certain types of radiation and chemotherapy. For example, the presence of mutations in genes regulating apoptosis such as p53 could conceivably interfere with the success of therapies that depend on induction of apoptosis of cells with damaged DNA due to radiation or drugs. The expanding knowledge of oncogenes and tumor suppressor genes and their functional mechanisms offers hope for the treatment of cancer as well. Avenues presently being explored include chemotherapy targeted at critical mutant gene products, immunotherapy, and gene therapy through use of viral or other vectors to introduce antisense oligonucleotides into oncogene-driven cancer cells and to replace mutated tumor suppressor genes. An obstacle that has been encountered repeatedly in conventional therapy is the problem of selectivity and the identification of targets unique to cancer cells; this issue has also proven difficult to resolve in the molecular treatment modalities currently under investigation. Farnesyl transferase inhibitors show promise; these agents inhibit the posttranslational modification of a number of cellular proteins including Ras but thus far appear to be remarkably specific for mutated ras products. Furthermore, cells transformed by ras mutations appear to be more sensitive to the drug than normal cells. Although much of the practical application is still in its initial stages of development, our growing understanding of the genetic mutations driving neoplastic progression is certain to soon play an instrumental role in reducing mortality through improvements in detection, diagnosis, and therapy.

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36. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993; 260:816-819. 37. Aaltonen LA, Peltomaki P, Mecklin JP et al. Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer Res 1994; 54:1645-1648. 38. Fishel R, Lescoe MK, Rao MR et al. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 1993; 75:1027-1038. 39. Aaltonen LA, Salovaara R, Kristo P et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. N Engl J Med 1998; 338:1481-1487. 40. Lynch HT, Smyrk TC. Identifying hereditary nonpolyposis colorectal cancer. N Engl J Med 1998; 338:1537-1538. 41. Jager AC, Bisgaard ML, Myrhoj T et al. Reduced frequency of extracolonic cancers in hereditary nonpolyposis colorectal cancer families with monoallelic hMLH1 expression. Am J Hum Genet 1997; 61:129-138. 42. Samowitz WS, Slattery ML. Transforming growth factor-beta receptor type 2 mutations and microsatellite instability in sporadic colorectal adenomas and carcinomas. Am J Pathol 1997; 151:33-35. 43. Myeroff LL, Parsons R, Kim SJ et al. A transforming growth factor beta receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability. Cancer Res 1995; 55:5545-5547. 44. Lothe RA, Peltomaki P, Meling GI et al. Genomic instability in colorectal cancer: relationship to clinicopathological variables and family history. Cancer Res 1993; 53:5849-5852. 45. Lukish JR, Muro K, DeNobile J et al. Prognostic significance of DNA replication errors in young patients with colorectal cancer. Ann Surg 1998; 227:51-56. 46. Howe JR, Conlon KC. The molecular genetics of pancreatic cancer. Surg Oncol 1997; 6:1-18. 47. Wilentz RE, Chung CH, Sturm PD et al. K-ras mutations in the duodenal fluid of patients with pancreatic carcinoma. Cancer 1998; 82:96-103. 48. Lemoine NR, Mayall ES, Wyllie FS et al. High frequency of ras oncogene activation in all stages of human thyroid tumorigenesis. Oncogene 1989; 4:159-164. 49. Pellegata NS, Losekoot M, Fodde R et al. Detection of K-ras mutations by denaturing gradient gel electrophoresis (DGGE): a study on pancreatic cancer. Anticancer Res 1992; 12:1731-1735. 50. Scarpa A, Capelli P, Villanueva A et al. Pancreatic cancer in Europe: K-ras gene mutation pattern shows geographical differences. Int J cancer 1994; 57:167-171. 51. Aoki K, Yoshida T, Matsumoto N et al. Suppression of K-ras p21 levels leading to growth inhibition of pancreatic cancer cell lines with K-ras mutation but not those without Ki-ras mutation. Mol Carcinog 1997; 20:251-258. 52. Redston MS, Caldas C, Seymour AB et al. p53 mutations in pancreatic carcinoma and evidence of common homocopolymer tracts in DNA microdeletions. Cancer Res 1994; 54:3025-3033. 53. Yokoyama M, Yamanaka Y, Friess H et al. p53 expression in human pancreatic cancer correlates with enchanced biological aggressiveness. Anticancer Res 1994; 14:2477-2483. 54. Ruggeri BA, Huang L, Berger D et al. Molecular pathology of primary and metastatic ductal pancreatic lesions: analyses of mutations and expression of the p53, mdm-2, and p21/WAF-1 genes in sporadic and familial lesions. Cancer 1997; 79:700-716. 55. Laurent-Puig P, Lubin R, Semhoun-Ducloux S et al. Antibodies against p53 protein in serum of patients with benign or malignant pancreatic and biliary diseases. Gut 1995; 36:455-458. 56. Matsubayashi H, Watanabe H, Nishikura K et al. Determination of pancreatic ductal carcinoma histogenesis by analysis of mucous quality and K-ras mutation. Cancer 1998; 82:651-660. 57. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/Cdk4. Nature 1993; 366:704-707.

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58. Shapiro GI, Rollins BJ. p16INK4A as a human tumor suppressor. Biochim Biophys Acta 1996; 1242:165-169. 59. Caldas C, Hahn SA, da Costa LT et al. Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nature Genet 1994; 8:27-32. 60. Imai Y, Tsurutani N, Oda H et al. p16INK4 gene mutations are relatively frequent in ampullary carcinomas. Jpn J Cancer Res 1997; 88:941-946. 61. Hahn SA, Kern SE. Molecular genetics of exocrine pancreatic neoplasms. Surg Clin North Am 1995; 75:857-869. 62. Hohne MW, Halatsch ME, Kahl GF et al. Frequent loss of expression of the potential tumor suppressor gene DCC in ductal pancreatic adenocarcinoma. Cancer Res 1992; 52:2616-2619. 63. Simon B, Weinel R, Hohne M et al. Frequent alterations of the tumor suppressor genes p53 and DCC in human pancreatic carcinoma. Gastroenterology 1994; 106:1645-1651. 64. Hahn SA, Bartsch D, Schroers A et al. Mutations of the DPC4/Smad4 gene in biliary tract carcinoma. Cancer Res 1998; 58:1124-1126. 65. Shi Y, Hata A, Lo RS. A structural basis for mutational inactivation of the tumor suppressor Smad4. Nature 1997; 388:87-93. 66. Atfi A, Buisine M, Mazars A et al. Induction of apoptosis by DPC4, a transcriptional factor regulated by transforming factor-beta through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling pathway. J Biol Chem 1997; 272:24731-24734. 67. Moskaluk CA, Hruban RH, Schutte M et al. Genomic sequencing of DPC4 in the analysis of familial pancreatic carcinoma. Diagn Molec Pathol 1997; 6:85-90. 68. Imazeki F, Omata M, Nose H et al. p53 gene mutations in gastric and esophageal cancers. Gastroenterology 1992; 103:892-896. 69. Tahara, E. Molecular mechanisms of stomach carcinogenesis. J Cancer Res Clin Oncol 1993; 119:265-272. 70. Tohdo H, Yokozaki H, Haruma K et al. p53 gene mutations in gastric adenomas. Virchows Archiv B Cell Pathol 1993; 63:191-195. 71. Oiwa H, Maehara Y, Ohno S. Growth pattern and p53 overexpression in patients with early gastric cancer. Cancer 1995; 75:1454-1459. 72. Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal type carcinoma. Acta Patholog Microbiol Scand 1965; 64:31.

73. Sano T, Tsujino T, Yoshida K et al. Frequent loss of heterozygosity on chromosomes 1q, 5q, and 17p in human gastric carcinomas. Cancer Res 1991; 51:2926-2931. 74. Wu M-S, Shun C-T, Wang H-P et al. Genetic alterations in gastric cancer: relation to histological subtypes, tumor stage, and Helicobacter pylori infection. Gastroenterology 1997; 112:1457-1465. 75. Poremba C, Yandell DW, Mellin W et al. Adenocarcinoma of the cardia in a young man: detection of a somatic p53 mutation by immunohistochemistry and automated direct sequencing. Pathol Res Pract 1995; 191:1004-1009. 76. Ichiyoshi Y, Oiwa H, Tomisaki S et al. Overexpression of p53 is associated with growth pattern and prognosis in advanced gastric cancer. Hepato-Gastroenterology 1997; 44:546-553. 77. Stemmerman G, Heffelfinger SC, Noffsinger A et al. The molecular biology of esophageal gastric cancer and their precursors: oncogenes, tumor suppressor genes, and growth factors. Hum Pathol 1994; 25:968-981. 78. Tahara E, Semba S, Tahara H. Molecular biological observations in gastric cancer. Semin Oncol 1996; 23:307-315. 79. Tahara E. Carcinogenesis and progression of human gastric cancer. Trans Soc Pathol Jpn 1992; 81:21-49. 80. Uchino S, Noguchi M, Ochiai A et al. p53 mutation in gastric cancer: a genetic model for carcinogenesis is common to gastric and colorectal cancer. Int J Cancer 1993; 54:759-764. 81. Seruca R, David L, Holm R et al. p53 mutations in gastric carcinomas. Br J Cancer 65 1992; 708-710.

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82. Fearon ER, Cho KR, Nigro JM et al. Identification of a chromosome 18p gene that is altered in colorectal cancers. Science 1990; 247:49-56. 83. Mizutani T, Onda M, Tokimaza A et al. Relationship of c-erbB-2 protein expression and gene amplification to invasion and metastasis in human gastric cancer. Cancer 1993; 72:2083-2088. 84. Shun CT, Wu MS, Lin JT et al. Relationship of p53 and c-erbB2 expression to histopathological features, Helicobacter pylori infection and prognosis in gastric cancer. Hepatogastroenterology 1997; 44:604-609. 85. Yonemura Y, Ninomiya I, Yamaguchi A et al. Evaluation of immunoreactivity for each erbB-2 protein as a marker of poor short term prognosis in gastric cancer. Cancer Res 1991; 51:1034-1038. 86. Kuniyasu H, Yasui W, Kitadai Y et al. Frequent amplification of the c-met gene in scirrous type stomach cancer. Biochem Biophys Res Commun 1992; 189:227-232. 87. Kuniyasu H, Yasui W, Yokozaki H et al. Aberrant expression of c-met mRNA in human gastric carcinoma. Int J Cancer 1993; 55:72-75. 88. Tahara E, Kuniyasu H, Yasui W et al. Abnormal expression of growth factors and their receptors in stomach cancer. Gann Monogr Cancer Res 1994; 42:163.

CHAPTER 6

Angiogenesis and GI Cancer David A. Litvak

T

he cellular mechanisms contributing to the growth of cancers include the loss of tumor suppressor genes, the activation of oncogenes, and the effects of selected trophic factors and intracellular polyamines which stimulate or support cancer cell proliferation.1-4 In addition, for tumors to reach clinically relevant sizes they must evade the host immune system5 and, more importantly, develop an adequate blood supply to support tumor progression and metastasis which is referred to as either developing an angiogenic phenotype or turning on the angiogenic switch.6 Angiogenesis is the process of new blood vessel formation which occurs in both normal physiologic processes (e.g., wound healing, embryogenesis, and organogenesis) and abnormal physiologic processes (e.g., arthritis and tumor progression) (Fig. 6.1).7 New blood vessels are formed from mature blood vessels in a process referred to as sprouting angiogenesis in which endothelial cells proliferate and migrate as long chains of cells from preexisting vessels in response to trophic and chemotactic factors (e.g., angiogenic factors) and changes in the local environment (e.g., proteolysis of components of the extracellular matrix surrounding the blood vessels).8-10 Following cell migration and proliferation, a new vessel lumen is formed, and these immature vessels subsequently undergo a complex process of remodeling and maturation.8 However, the mechanisms by which tumors stimulate quiescent blood vessels to undergo angiogenesis are not completely understood and appear to depend on several factors, including the effects of genetic alterations (e.g., the loss of tumor suppressor activity), tissue-specific signals, the induction of angiogenic factors or downregulation of antiangiogenic factors, and the effects of both stromal cells and components of the extracellular matrix (e.g., cell adhesion molecules).8,9,11 Although the exact mechanisms involved in tumor angiogenesis have not been entirely defined, investigation in this area has led to the identification and development of several potentially clinically significant prognostic indicators (e.g., angiogenic factor tissue and serum levels)12 and therapeutic agents.13,14

Development of the Angiogenic Phenotype in Tumors Although angiogenesis is essential to both tumor progression and the development of metastases, many of the molecules and mechanisms that are involved in the angiogenic process still have not been elucidated. Recently, in vivo models of carcinogenesis have helped to define better the temporal relationship between development of the angiogenic phenotype and tumor progression. Three models have been successful in investigating this relationship and involve overexpression of either the SV40 T antigen oncogene under control of the insulin gene promoter in RIP-Tag transgenic mice which develop pancreatic islet cell cancers, the bovine papillomavirus genome in BPV1.69 transgenic mice which develop dermal fibrosarcomas, or the human papillomavirus-16 oncogene under control of the Molecular Mechanisms in Gastrointestinal Cancer, edited by B. Mark Evers. ©1999 R.G. Landes Company.

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Fig. 6.1. The process of tumor angiogenesis. In a complex series of events, angiogenic factors provide a signal for quiescent mature blood vessels to develop new vascular sprouts that subsequently undergo remodeling and maturation.

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In Vivo Models of Tumorigenesis Hyperplastic Islets (CIS)

Angiogenic Islet

A

Normal Islets (onc+)

B

Normal Dermis

Fibromatosis

Aggresive Fibromatosis

C

Normal Squamous Epithelium

Hyperplasia

Dysplasia

Tumor

Fibrosarcoma

Carcinoma

Fig. 6.2. Angiogenesis precedes tumor development in three in vivo cancer models in which the degree of angiogenesis present parallels the progression of tissues from normal to malignancy. A) RIP-Tag transgenic mice that develop pancreatic islet cell tumors. B) BPV1.69 transgenic mice that develop fibrosarcomas. C) K14-HPV16 transgenic mice that develop squamous cell carcinoma. [Reprinted with permission from Hanahan D and Folkman J. Cell 1996; 86:353-364. ©1996 Cell Press Publishers].

keratin 14 gene regulatory region in K14-HPV16 transgenic mice which develop epidermal squamous cell carcinoma.11,15 In each of these three models, angiogenesis was detected before the emergence of solid tumors (Fig. 6.2). However, there are differences in the cellular mechanisms that are activated during angiogenesis in each of these cancer models. For example, during epidermal squamous cell carcinogenesis in K14-HPV16 transgenic mice, there is induction of the angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).16 However, in the RIP-Tag transgenic mice, both VEGF d bFGF are constitutively expressed in normal and premalignant lesions, and the mechanisms leading to angiogenesis in this particular model are not clear. Furthermore, additional studies have demonstrated the correlation between angiogenesis and downregulation of angiogenesis inhibitors, such as thrombospondin (TSP-1) in transformed fibroblasts and mammary epithelial cells.17,18 In fact, tumors may secrete both angiogenic factors, such as VEGF and bFGF, and antiangiogenic factors, such as angiostatin,17

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Fig. 6.3. Turning on the angiogenic switch in tumors represents a change in the relative balance between activators and inhibitors of angiogenesis. [Adapted with permission from Hanahan D and Folkman J, Cell 1996; 86:353-364. ©1996 Cell Press Publishers].

suggesting that the development of an angiogenic phenotype in tumors may be a complex process which involves a balance of expression of both angiogenic and antiangiogenic factors (Fig. 6.3).11 The complex process of tumor angiogenesis is mediated by multiple signals which may arise from either tumor cells, stromal cells (e.g., macrophages or mast cells) infiltrating the tumor, or the local tissue environment (Fig. 6.4).6,11 In addition to the direct effects of tumor cells on angiogenesis, tumor cells may recruit stromal cells that secrete angiogenic factors (e.g., bFGF) or chemotactic factors for additional stromal cells (e.g., tumor necrosis factor-α [TNF-α]), thus amplifying the angiogenesis signal.6 The local tissue environment also may contribute to the signal for angiogenesis. Hypoxia that is frequently present in the center of tumors is a potent stimulus for both the secretion of angiogenic factors and the activity of macrophages.19 In addition, changes in the turnover of extracellular matrix proteins, such as collagen, laminin, and fibronectin, in proximity to preexistent blood vessels may contribute to the regulation of new capillary formation.9 Furthermore, finetuning of the angiogenic signal may involve both the secretion of angiogenic mediators as inactive pro-forms (e.g., angiostatin that is cleaved from a larger parent molecule,

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Angiogenesis

Macrophage

Mast Cell

Basement Membrane

Chemolactic Molecules

Tumor

Angiogenic Molecules Angiogenic Inhibitors Plasminogen Activators Collagenases Heparanases

Fig. 6.4. The complex process of tumor angiogenesis is mediated by multiple molecules which are secreted from tumor cells or infiltrating stromal cells, such as macrophages and mast cells. [Adapted with permission from Steiner R, Weisz PB, and Langer R (eds) 1992 Angiogenesis. Key Principles-Science-Technology-Medicine, Birkhaeuser Verlag, Basel Publishers].

plasminogen) or the sequestration of certain angiogenic factors (e.g., bFGF) in the extracellular matrix, rendering them inactive.20,21

Mediators of Angiogenesis Angiogenic factors secreted by tumor and stromal cells are essential to tumor angiogenesis and include both peptides, such as bFGF, VEGF, hepatocyte growth factor (HGF), and small molecules, such as platelet activating factor (PAF) and nitric oxide (NO).22-24 On the other hand, transforming growth factor-beta (TGF-β) appears to be important to the process of angiogenesis, but its role as an angiogenic or antiangiogenic factor, to date, remains unclear. bFGF, a heparin-requiring peptide growth factor, has been shown to induce cell proliferation, migration, and tube formation in collagen matrix gels in vitro.22,25 A number of tumors and tumor-derived cell lines express bFGF mRNA or protein,26 and bFGF has been detected in the urine of a number of patients with selected cancers.27 These findings suggest that bFGF may be essential to tumor formation and is supported by the findings of Kandel et al28 in which development of an angiogenic phenotype in their fibrosarcoma model was temporally related to the increased secretion of bFGF.

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Perhaps the best known of the angiogenic factors, VEGF, is a potent mitogen for endothelial cells in vitro and in vivo29 that is stimulated by both the presence of hypoxic conditions and the effects of certain growth factors, such as epidermal growth factor (EGF) and keratinocyte growth factor.30 In addition, VEGF mRNA levels are induced by activation of the oncogenes, ras31,32 and c-Src,33 and by mutations in the p53 gene in certain cell types,31 suggesting that VEGF is essential to turning on the angiogenic switch during tumor progression. Moreover, increased VEGF expression has been noted in certain human gastrointestinal tumors.24,34 HGF (i.e., scatter factor), is a cytokine that acts through a specific receptor tyrosine kinase encoded by the c-met oncogene to induce endothelial cell proliferation and migration.35,36 HGF induces formation of tube-like structures from endothelial cells in vitro37 and is an important chemotactic factor for macrophages. HGF also induces secretion of other angiogenic factors (e.g., VEGF and PAF).38 Moreover, like VEGF, high levels of HGF are expressed in certain cancers,35 but, to date, the role of HGF in tumorogenesis has not been entirely defined. Nitric oxide and PAF are small molecules that contribute to angiogenesis; however, their roles in tumor angiogenesis are still being defined. PAF, a molecule derived from cell membrane phospholipids, may be important in mediating a number of biologic events of the inflammatory process,39 but its role in tumor angiogenesis is less clear. PAF directly stimulates migration of endothelial cells40 and may mediate TNF-α-induced angiogenesis. The administration of a PAF-receptor antagonist has been shown to reduce TNF-α-dependent angiogenesis.41 However, most of PAF’s effects on angiogenesis may be mediated by other angiogenic factors, such as VEGF, bFGF, and HGF.42 This is suggested by studies that have shown that PAF induces expression of these angiogenic factors at the gene level.42 Nitric oxide, a potent vasodilator, also may be important to angiogenesis, and investigators have demonstrated that nitric oxide-generating compounds stimulate endothelial cell proliferation while nitric oxide-synthase inhibitors inhibit angiogenesis.43 However, the role of nitric oxide in tumor progression has not been entirely defined. Nevertheless, overexpression of nitric oxide-synthase in colon cancer cells is associated with increased vascularity and growth of tumors in vivo,44 and the administration of nitric oxide-synthase inhibitors have been shown to slow the growth of xenotransplanted tumors in mice.24 The role of TGF-β in angiogenesis is ambiguous. While in vitro studies have shown that TGF-β inhibits endothelial cell proliferation, migration, and branching tube formation, suggesting an antiangiogenic role, TGF-β does not appear to inhibit endothelial cell tube formation entirely.45,46 Furthermore, in vivo, TGF-β appears to promote angiogenesis above a certain threshold tissue level which may be an indirect effect of recruiting stromal cells which secrete angiogenic factors.46,47

Genetic Alterations and Angiogenesis The close temporal relationship between the progression of the normal epithelium to cancer and the development of an angiogenic phenotype in tumors suggests that the accumulation of genetic alterations (i.e., the activation of oncogenes such as k-ras or the loss of tumor suppressor genes such as p53 noted in many cancers) may be causally linked to the differential expression of selected angiogenic factors (e.g., VEGF) (Fig. 6.5).32,48 In support of this hypothesis, studies have demonstrated that specific genetic alterations are associated with both increased expression of angiogenic factors or decreased expression of antiangiogenic factors.49,50 In vitro experiments with intestinal epithelial (IEC-18) cells and fibroblasts, have shown that overexpression of the ras oncogene is associated with a transformed phenotype and increased VEGF secretion.31,32 In addition, mutations in a number of other tumor suppressor genes, such as vhl and lod2, and oncogenes, such as raf, fos, and

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Fig. 6.5. The temporal association between known genetic alterations, as occurs in colorectal carcinoma, and the differential expression of angiogenic factors, as has been proposed, in various stages of tumorogenesis, tumor progression, and metastasis. [Adapted with permission with Rak et al, Eur J Cancer 1996; 32A:2438-2450. ©1996 Elsevier Science Ltd Publishers].

Src also are associated with the increased secretion of angiogenic factors from tumor cells,32,51-53 and Ellis et al54 have shown that transfecting HT-29 colon cancer cells with antisense c-Src results in decreased expression of VEGF. However, the regulation of specific angiogenic factors may depend on the type of cancer, as well as the specific genetic alteration involved. In cultured fibroblasts, loss of the tumor suppressor, p53, is associated with both decreased secretion of the antiangiogenic protein, TSP-1, and increased secretion of VEGF.31,55 However, in a human fibrosarcoma-derived cell line, loss of p53 activity is associated with increased expression of VEGF and bFGF, but TSP-1 levels remain unchanged.31 The cellular mechanisms by which different genetic alterations affect the expression of angiogenic factors, and thus contribute to tumor angiogenesis, still have not been completely defined and currently are the focus of intensive investigation.

Angiogenesis and Cancer Metastasis The process of cancer metastasis is a multi-stage and multi-factorial process (Fig. 6.6)5 in which cancer cells from the primary tumor must proliferate, migrate, and invade blood vessels and lymphatic channels. These same cells also must be able to adhere to and invade through blood vessels, proliferate in the new tissue environment, and, as for the primary tumor, develop a competent vascular supply to become clinically relevant (i.e., reach sizes greater than 1-2 mm3). Increased expression of angiogenic factors may contribute to the development of metastasis in several types of tumor models, but a particular tumor’s metastatic potential also may depend on the local tissue environment. The expression of VEGF has been shown to correlate with the vascular content and metastatic capability of some

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The Process of Metastasis Transformation

Angiogenesis

Motility & Invasion

Capillaries, Venules, Lymphatics

Responses to Microenvironment & Proliferation

Extravasation into Organ Parenchyma

Angiogenesis

Adherence to Vessel Wall

Embolism & Circulation

Multicell Aggregates (Lymphocyte, platelets)

Arrest in Capillary Beds

Metastasis of Metastases Metastases

Fig. 6.6. In order to produce clinically significant metastases, tumor cells must promote an angiogenic phenotype, invade blood vessels, travel to and infiltrate new tissues, evade the host immune system, proliferate, and induce angiogenesis again. [Reprinted with permission from Fidler IJ, Molecular biology of cancer: invasion and metastasis. In DeVita JR, Hellman S, Rosenberg SA eds. Cancer Principles and Practice of Oncology 1997:135-152. ©1997 Lippincott-Raven Publishers.]

tumors,56 and a study by Kitadai et al57 demonstrated an association between increased bFGF levels and the presence of lymphatic or more distant metastases and predicted recurrent disease based, in part, on bFGF levels in patients with colon cancer. In addition, the local tissue environment appears essential to the progression of metastatic lesions. Human renal cancer cells implanted in the kidney, colon cancer cells implanted in the cecum, or gastric cancer cells implanted in the stomach of mice have been shown to have greater growth, vascularization, and expression of bFGF or VEGF compared to cell-type matched controls that were implanted in the subcutaneous tissue.5,58,59 The development of tumor metastases also depends on the expression of antiangiogenic factors secreted from cancer cells. In the study by O’Reilly et al,20 the angiogenesis inhibitor factor, angiostatin, was identified as a secretory product from tumor cells that may contribute to tumor dormancy (i.e., stable, nonexpansive, and clinically undetectable disease). Based on their findings involving Lewis lung cancer tumors in mice, these authors concluded that downregulation of angiostatin occurring with primary tumor resection contributed to the rapid growth of previously dormant metastases.20 Furthermore, angiostatin may contribute to tumor dormancy by increasing the rate of cancer cell apoptosis, as has been shown for various tumors in mice;13 however, the mechanisms by which angiostatin exerts its pro-apoptotic effects on cancer cells is still being investigated.

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Angiogenesis as a Prognostic Indicator for Cancers The angiogenic activity of certain tumors, as determined by the microvessel content and the expression of angiogenic factors, may correlate with both cancer progression and patient prognosis. Studies of patients with either colon or gastric cancer have noted the correlation between microvessel counts (i.e., determined by histologic sections) and both the presence of hepatic or lymphatic metastases and patient survival.60-63 A number of studies also have reported the correlation between microvessel content, expression of angiogenic factors in tumor specimens, and the presence of advanced disease in certain cancers,60,61,63 suggesting that the determination of angiogenic factor levels may provide prognostic information in selected cancers. Elevated serum or tumor tissue levels of certain angiogenic factors are associated with the occurrence of advanced disease in several different types of cancer. Investigators have shown the correlation between tumor levels of bFGF or VEGF (by immunohistochemistry) and advanced disease in breast,64 gastric,65 colon,56 and hepatocellular cancers.66 In addition, serum levels of angiogenic factors may be of clinical prognostic value in cancer patients. Elevated serum levels of bFGF are associated with certain kidney and cervical cancers,67,68 and elevated urine levels of bFGF have been identified in patients with bladder cancer.69 Increased serum levels of both VEGF and bFGF have been noted in patients with aggressive colorectal cancers,12 and serum levels of HGF may better correlate with the presence of recurrent breast cancer than do other tumor markers, such as CEA and CA15-3.12

Novel Angiogenesis-Based Chemotherapeutic Agents The inhibition of tumor angiogenesis is a novel treatment strategy for cancer, and a number of antiangiogenic compounds have been investigated as potential chemotherapeutic agents. In addition, several of these compounds have entered early phase clinical trials with promising results. These investigational compounds have been developed to target one or more of the components of the angiogenesis process, such as endothelial cell proliferation and migration, extracellular matrix proteolysis, and the effects of angiogenic or antiangiogenic factors (Table 6.1). Furthermore, recent studies have demonstrated the efficacy of introducing antiangiogenic genes into the tumor cells (i.e., gene therapy) in experimental cancer models.70,71

Compounds that Inhibit Endothelial Cell Proliferation and Migration: TNP-470 (AGM-1470) TNP-470 (AGM-1470), a synthetic derivative of the anti-amebic and anti-angiogenesis agent, fumagillin, is one of the first angio-inhibitors to enter clinical trials.14 Early in vitro studies demonstrated that TNP-470 inhibits endothelial cell proliferation by producing cell cycle arrest at the first gap (G1) phase.72 At high concentrations, TNP-470 is directly cytotoxic to cancer cells, but also may adversely affect the host immune system.73 A number of in vivo studies have demonstrated that TNP-470 inhibits the growth of various tumors transplanted subcutaneously in mice;74,75 however, the inhibitory effects of TNP-470 on tumor growth appear variable and tumor-specific, demonstrating less effect on tumor growth in models of pancreatic and hepatocellular carcinoma.73,76 Nevertheless, each of these studies clearly demonstrated that TNP-470 effectively inhibits tumor metastasis.73,76 Recent studies suggest that TNP-470 combined with standard chemotherapeutic agents is an effective treatment against various types of cancer.77

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Table 6.1 Investigational angiogenesis-based chemotherapeutic agents TARGET Endothelial cell proliferation Endothelial cell migration Extracellular matrix metalloproteases Cellular adhesion molecules Angiogenic factors

AGENT TNP-470, angiostatin, suramin, genestein Linomide Batimastat, marimastat Anti-integrin αvβ3 antibodies CLM-609 Suramin, anti-VEGF antibodies, tecogalan linomide, SR25989, retinoic acid

Compounds that Inhibit Extracellular Matrix Proteins: Batimastat, Marimastat, and Anti-αvβ3 Integrin Antibodies Batimastat (BB-94) and marimastat (BB-2516) are two structurally similar synthetic matrix metalloprotease inhibitors which have proven successful in both in vivo tumor models and phase I/II clinical trials.78-81 Batimastat preceded marimastat in development and demonstrated its effectiveness in the treatment of malignant ascites.78,80 However, the usefulness of barimastat as a chemotherapeutic agent was limited by both its poor oral bioavailability which necessitated intraperitoneal administration and its potential for inducing acute bowel toxicity.78,80 On the other hand, marimastat, although structurally similar to batimastat, is administered orally and may be useful in the treatment of advanced stage lung cancer, but limited by a dose-cumulative toxicity characterized by severe inflammatory polyarthritis.79 The development of neutralizing antibodies to the cell adhesion molecule, integrin αvβ3, which contributes to endothelial cell migration, proliferation, and invasion, is another strategy to block tumor angiogenesis. In vivo studies have demonstrated that the administration of antibodies to αvβ3 integrin (e.g., LM609 or 17E6) in breast cancer- or melanomabearing mice inhibits angiogenesis, growth of primary tumors, and metastasis.82,83

Compounds that Modulate the Effects of Angiogenic Factors: Angiostatin, Suramin, and Anti-VEGF Antibodies The antiangiogenic factor, angiostatin, which inhibits endothelial cell proliferation and may play a role in tumor metastasis dormancy also has been shown both to inhibit tumor growth and to prevent metastases in several animal models when administered exogenously.13,84 Furthermore, recent studies have shown that transfer of the angiostatin gene to tumor cells (i.e., gene therapy) effectively induces glioma, breast cancer, and fibrosarcoma cell death in vivo.70,71 The efficacy of angiostatin gene transfer in inhibiting tumor growth may be a useful alternative to the expensive preparation of recombinant angiostatin. Early clinical trials with angiostatin may be initiated in the near future. Suramin, an anti-trypanosomal drug and a protein-tyrosine phosphatase inhibitor, appears to block the trophic and angiogenic effects of several growth factors (e.g., EGF and insulin-like growth factor-I [IGF-I]) on selected cancers, including breast and lung carcinoma in vivo.85,86 In addition, although the anti-tumor effects of suramin have not been fully elucidated, this compound, in combination with standard chemotherapeutic agents, produced good response rates with reversible hematological toxicity in a phase II trial for advanced prostate cancer.87 However, early phase trials for advanced nonsmall cell lung cancer and breast cancer were not successful.86

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The administration of neutralizing antibodies to the potent angiogenic factor, VEGF, is another attractive treatment strategy to inhibit tumor angiogenesis. Early studies demonstrated that anti-VEGF antibodies were capable of blocking both proliferation of human umbilical vein endothelial cells in vitro and growth of a human hepatoma in vivo.88 In addition, more recent studies have demonstrated that the administration of VEGF-neutralizing antibodies completely inhibits both angiogenesis and growth of human rhabdomyosarcoma and prostate tumors transplanted to athymic mice.89,90 Furthermore, anti-VEGF antibodies have been shown to block the growth and fluid production of MM2 breast adenocarcinoma ascites tumors in mice,91 suggesting that neutralizing-VEGF antibodies potentially may be an effective clinical strategy for patients with ascites-producing solid tumors.

Conclusions The recent explosion of research in the field of angiogenesis has contributed significantly to a better understanding of how tumors switch to an angiogenic phenotype, thereby leading to the development of both clinically significant lesions and metastases. In particular, there has been substantial progress in four active areas of angiogenesis research: the role of genetic alterations in the activation of angiogenesis, the angiogenic effects provided by proteins of the extracellular matrix, the identification and characterization of angiogenic factors, and the concept of metastasis dormancy. Investigation in these areas has contributed to the development of novel cancer treatment strategies which target angiogenesis in solid tumors and which also may provide prognostic or diagnostic information for certain solid tumors. Future studies may identify which angiogenesis-based treatments are most effective for cancer patients, but it is anticipated that these novel therapies will be a part of a multi-modality approach to the treatment of solid tumors.

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Index A

G

Adenomatous polyposis coli (APC) 42-44, 55, 83, 84, 88 Angiogenesis 4, 40, 43, 68, 98, 99, 100-108 Apoptosis 25, 39, 41, 42, 49, 50, 57-71, 84, 85, 90, 105 Arachidonic acid 37-39 Autocrine growth 4, 12, 56

G-proteins 21 Gastric cancer 2-4, 9, 10, 22, 28, 54, 57, 64-66, 69, 80, 82, 88, 89, 105, 106 Gastrin 7-12, 22, 30

B Bax 41, 60-63, 66, 70 Bcl-2 39, 41, 59-69

C CCK 10, 30 Cdk inhibitors 55 Cell adhesion molecules 98 Cell cycle 49-63, 68-71, 79, 81, 87, 106 Colon cancer 5, 6, 8, 11, 12, 23-25, 27-30, 39-44, 55, 61, 63-65, 68, 69, 72, 84, 103-105 Cyclic AMP 21, 32 Cyclin-dependent kinases (Cdks) 50-52, 54, 56, 69 Cyclins 50-53, 57, 86 Cyclooxygenase 37-40, 44 Cytokines 37, 38, 49

D

H Hepatocyte growth factor (HGF) 2, 6, 89, 100, 102-108 Her2/neu (c-erbB2) 1, 2, 4, 6, 79, 89

M Mismatch repair genes 84 Mitogen-activated protein kinases 30 Mutations 21, 41-44, 53-57, 61-63, 65-68, 70, 71, 79-90, 103

N Neurotensin 6, 10, 30 NSAIDs 38, 41-44

O Oncogenes 53, 55, 57, 63, 66, 68, 79-81, 89, 90, 93, 98, 103

P

E2F 51-54, 86, 87 Epidermal growth factor 1, 29, 56, 89, 103 Esophageal cancer 54, 56, 57, 66

p53 44, 53-67, 69-71, 81, 82, 85, 86, 88, 90, 103, 104 Pancreatic cancer 3, 4, 10, 11, 23, 29-32, 57, 58, 67-69, 85, 86, 89 Platelet activating factor (PAF) 102,103 Prostaglandins 37, 38 Protein kinase A 21 Protein kinase C 21, 25 Protein tyrosine kinases 28

F

R

Familial adenomatous polyposis (FAP) 35, 43, 55, 56, 82, 83, 88 Fibroblast growth factor (FGF) 1, 3, 4, 6, 40, 54, 100-106

Ras 21, 28-32, 44, 79, 80 Receptor antagonists 3, 9, 10 Retinoblastoma (Rb) protein 86

DCC 84, 86, 88 DPC4/Smad4 86, 87

E

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S Signal transduction 11-13, 21-23, 27, 29, 30, 32, 56, 79, 87 Somatostatin 4, 8, 13 Src 21, 28, 29, 31, 103, 104

T Transforming growth factor 1, 39, 56, 65, 102 Tumor suppressor proteins 81, 86, 87

V Vascular endothelial growth factor (VEGF) 40, 100, 102-106, 108