Apoptosis in Carcinogenesis and Chemotherapy: Apoptosis in cancer

Apoptosis in Carcinogenesis and Chemotherapy George G. Chen · Paul B.S. Lai Editors Apoptosis in Carcinogenesis and ...

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Apoptosis in Carcinogenesis and Chemotherapy

George G. Chen · Paul B.S. Lai Editors

Apoptosis in Carcinogenesis and Chemotherapy Apoptosis in Cancer

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Editors Dr. George G. Chen Chinese University of Hong Kong Prince of Wales Hospital Department of Surgery Shatin, New Territories Hong Kong/PR China

ISBN 978-1-4020-9596-2

Dr. Paul B.S. Lai Chinese University of Hong Kong Prince of Wales Hospital Department of Surgery Shatin, New Territories Hong Kong/PR China

e-ISBN 978-1-4020-9597-9

DOI 10.1007/978-1-4020-9597-9 Library of Congress Control Number: 2008942032 c Springer Science+Business Media B.V. 2009 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Foreword

Although research on carcinogenesis has focused more on cellular proliferation than on cell death, yet understanding the mechanism of apoptosis may have important implications for cancer therapy. This book brings together experts from around the world who will discuss the common cancers encountered in clinical practice in the laboratory setting. During the induction of these common cancers, the role of apoptosis in cellular and molecular changes is emphasized, critically highlighting possible anti-cancer strategies. For those who are interested in carcinogenesis and for those who are seeking new approaches to anti-cancer therapy, this book is an important reference. It serves not only as a reference of the current understanding of apoptosis in common cancers but also an important bridge between the laboratory and clinical practice. The editors and contributors are to be congratulated in bringing together an important pool of up-to-date knowledge to light and further our interest in this exciting and expanding field. Arthur K. C. Li Emeritus Professor of Surgery The Chinese University of Hong Kong

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Preface

The role of apoptosis in cancer development and emerging treatment strategies has rapidly expanded over the past few years. The novel discovery in the apoptotic pathways and their relevant molecules provides us not only the knowledge how tumors develop but also the opportunity to design new therapeutic tools to prevent or inhibit the growth of tumors with minimal side-effects. Undoubtedly, understanding the events involved at a molecular level can permit the manipulation of apoptosis for therapeutic purposes. In healthy subjects, apoptosis is a normal and continuous process with complex physiological controls. However, due to various environmental and endogenous factors this process becomes out of control or develops in a manipulated direction in cancers. The imbalance between the pro-apoptosis and anti-apoptosis is often a two-side coin. With a shift in favour of the latter, cells may growth uncontrollably. In contrast, with a shift in favour of the former, cells may die or sensitive to cell death stimuli. There may be some common points of the apoptotic process in tumors of different tissue/cell types. However, different cancers often possess their own specific and dedicated molecules that regulate apoptosis. These specific and dedicated molecules or pathways are truly reflected in the volume of this book which critically describes and summarizes the up-to-date research on an emerging topic of “Apoptosis in Carcinogenesis and Chemotherapy” in 15 chapters, each of them focusing on a particular tumor, including breast, bladder, cervical, colorectal, cutaneous, esophageal, gastric, hematologic, laryngeal, liver, lung, nasopharyngeal, pancreatic, prostate and thyroid cancers. The book, a collection of cutting-edge reviews by established leaders in the field, will be of great interest to not only clinicians interested in molecular approaches of apoptosis in chemotherapy but also basic scientists working in the field of cancer research and apoptosis. Hong Kong, PRC Hong Kong, PRC

George G. Chen Paul B.S. Lai

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Contents

1 Apoptotic Signaling Pathway and Resistance to Apoptosis in Breast Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prasanthi Karna and Lily Yang

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2 Anti-Cancer Strategy of Transitional Cell Carcinoma of Bladder Based on Induction of Different Types of Programmed Cell Deaths . . 25 Jose A. Karam and Jer-Tsong Hsieh 3 Apoptosis in Carcinogenesis and Chemoherapy of the Uterine Cervix Sakari Hietanen

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4 Apoptosis in Colorectal Tumorigenesis and Chemotherapy . . . . . . . . . 75 Shi Yu Yang, Kevin M. Sales and Marc C. Winslet 5 Apoptosis in Cutaneous Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Michael B. Nicholl and Dave S.B. Hoon 6 Apoptosis in Carcinogenesis and Chemotherapy – Esophageal Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Yan Li and Robert C.G. Martin 7 Molecular Targets in Gastric Cancer and Apoptosis . . . . . . . . . . . . . . . . 157 Elizabeth K. Balcer-Kubiczek and Michael C. Garofalo 8 Apoptosis and the Tumor Microenvironment in Hematologic Malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 Danielle N. Yarde and Jianguo Tao ix

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Contents

9 Bcl-2 Family Members in Hepatocellular Carcinoma (HCC) – Mechanisms and Therapeutic Potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Shihong Ma, George G. Chen and Paul B.S. Lai 10 Apoptosis in the Development and Treatment of Laryngeal Cancer: Role of p53, Bcl-2 and Clusterin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 Maximino Redondo, Rafael F´unez and Francisco Esteban 11 Cyclooxygenase 2 and its Metabolites: Implications for Lung Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Kin Chung Leung and George G. Chen 12 Roles of Negative and Positive Growth Regulators in Nasopharyngeal Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Mong-Hong Lee, Huiling Yang, Ruiying Zhao and Sai-Ching J. Yeung 13 Cellular Signaling Mechanisms in Pancreatic Apoptosis . . . . . . . . . . . . . 295 Nawab Ali, Stewart MacLeod, R. Jean Hine and Parimal Chowdhury 14 Strategies to Circumvent Resistance to Apoptosis in Prostate Cancer Cells by Targeted Necrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Richard D. Dinnen, Daniel P. Petrylak and Robert L. Fine 15 Carcinogenesis and Therapeutic Strategies for Thyroid Cancer . . . . . 347 Zhi-Min Liu and George G. Chen Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

Contributors

Nawab Ali Graduate Institute of Technology, University of Arkansas at Little Rock, Little Rock, AR 72205, USA, [email protected] Elizabeth K. Balcer-Kubiczek Department of Radiation Oncology, Radiation Oncology Research Laboratory, University of Maryland School of Medicine, 655 W. Baltimore Street, BRB, Baltimore MD 21201, USA, [email protected] George G. Chen Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, [email protected] Parimal Chowdhury Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA, [email protected] Richard D. Dinnen Experimental Therapeutics, Division of Medical Oncology, College of Physicians and Surgeons, Columbia University, 650 West 168th Street, BB 20-05, New York, NY 10032, USA, [email protected] Francisco Esteban Department of Otolaryngology, Hospital Virgen del Rocio, Avda. Manuel Siurot s/n CP 41013, University of Sevilla, Sevilla, Spain, [email protected] Robert L. Fine Experimental Therapeutics, Division of Medical Oncology, College of Physicians and Surgeons, Columbia University, 650 West 168th Street, BB 20-05, New York, NY 10032, USA, [email protected] ´ Rafael Funez Department of Pathology, CIBER Epidemiologia y Salud Publica (CIBERESP), Hospital Costa del Sol, Carretera de C´adiz Km 187, 29600 Marbella, University of Malaga, Malaga, Spain, [email protected] Michael C. Garofalo The Marlene and Stewart Greenebaum Cancer Center, Department of Radiation Oncology, University of Maryland School of Medicine, 22 S. Greene St., Baltimore MD 21201, USA, [email protected] Sakari Hietanen Department of Obstetrics and Gynecology, Turku University Central Hospital, Kiinamyllynkatu 4-8, 20520 Turku, Finland, [email protected] xi

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Contributors

Jean Hine Department of Nutrition, Health Policy and Management, College of Public Health Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA, [email protected] Dave S.B. Hoon Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404, USA, [email protected] Jer-Tsong Hsieh Department of Urology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9110, USA, [email protected] Jose A. Karam Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA, [email protected] Prasanthi Karna Department of Surgery, Winship Cancer Institute, Emory University School of Medicine, Clinic C, Room C-4038, 1365 C Clifton Road NE, Atlanta, GA 30322, USA, [email protected] Paul B.S. Lai Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, [email protected] Mong-Hong Lee Department of Molecular and Cellular Oncology, Anderson Cancer Center, The University of Texas M.D., 1515 Holcombe Blvd, Houston, TX 77030, USA, [email protected] Kin Chung Leung Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, [email protected] Yan Li Division of Surgical Oncology, Department of Surgery, University of Louisville School of Medicine, 511 S Floyd ST, MDR Bld, Rm 326A, Louisville, KY 40202, USA, [email protected] Zhi-Min Liu Department of Biochemistry and Molecular Biology, The School of Basic Medical Sciences, Chongqing Medical University, Chongqing, China, [email protected] Shihong Ma Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, [email protected] Stewart MacLeod Department of Obstetrics and Gynecology, Arkansas Children’s Hospital, Little Rock, AR 72205, USA, [email protected] Robert C.G. Martin II Department of Surgery, Division of Surgical Oncology, University of Louisville School of Medicine, 511 S Floyd ST, MDR Bld, Rm 326A, Louisville, KY 40202, USA, [email protected] Michael B. Nicholl Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404, USA, [email protected]

Contributors

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Daniel P. Petrylak Experimental Therapeutics, Division of Medical Oncology, College of Physicians and Surgeons, Columbia University, 650 West 168th Street, BB 20-05, New York, NY 10032, USA, [email protected] Maximino Redondo Department of Biochemistry, CIBER Epidemiologia y Salud Publica (CIBERESP), Hospital Costa del Sol, Carretera de C´adiz Km 187, 29600 Marbella, University of Malaga, Malaga, Spain, [email protected] Kevin M Sales University Department of Surgery, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, UK, [email protected] Jianguo Tao Hematopathology and Laboratory Medicine, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida College of Medicine, 12901 Magnolia Drive, MCC 2071F, Tampa, FL 33612, USA, [email protected] Marc C. Winslet University Department of Surgery, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, UK; Royal Free Hampstead NHS Trust Hospital, London, UK; University College Hospital, London, UK, [email protected] Lily Yang Departments of Surgery and Radiology, Winship Cancer Institute, Emory University School of Medicine, Clinic C, Room C-4088, 1365 C Clifton Road NE, Atlanta, GA 30322, USA, [email protected] Shi Yu Yang University Department of Surgery, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, UK, [email protected] Huiling Yang Department of Pathophysiology, Sun Yat-Sen University Medical School, Guangzhou, China, [email protected] Danielle N. Yarde Hematopathology and Laboratory Medicine, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida College of Medicine, 12901 Magnolia Drive, MCC 2071F, Tampa, FL 33612, USA, [email protected] Sai-Ching J. Yeung Endocrine Neoplasia and Hormonal Disorders, General Internal Medicine, Ambulatory Treatment and Emergency Care, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA, Syeung @mdanderson.org Ruiying Zhao Departments of Molecular and Cellular Oncology, Ambulatory Treatment and Emergency Care, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA, [email protected]

Abbreviations

1-BI 5-FU 5-LOX AA AC AdPC AEF AI AIF Akt/PKB ALL AML AMPK Ant Apaf-1 APC Apoptosis APRIL ASO ATC ATG ATP BAD BAFF BAG BAK1 BAX BBC3 BC BCG Bcl-2 Bcl-XL

1-Benzylimidazole 5-Fluorouracil 5-lipoxygenase Arachidonic acid Adenyl cyclase Adenomatous polyposis coli Alternative-reading frame protein Allelic imbalance Apoptotic inducing factors A serine/threonine protein kinase/Pritein kinase B Acute lymphoblastic leukemia Acute myelocytic leukemia AMP-activated protein kinase Antennapedia homeobox domain Apoptotic protease activating factor-1 Anaphase-promoting complex Programmed cell death A proliferation-inducing ligand Antisense oligonucleotides Anaplastic thyroid carcinoma Autophagy-related gene Adenosine tri-phosphate Bcl-2-associated death promoter B cell-activating factor of the tumor necrosis factor family Bcl-2-associated athanogene BAK; BCL-2 antagonist/killer Bcl-2-associated X protein PUMA; Bcl-2 binding component 3 Biochemotherapy Bacillus Calmette-Guerin B-cell lymphoma 2 Basal cell lymphoma-extra large

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BE BER bFGF BH domains Bik Bim BIR BIRC4 BIRC5 Bmf BMI1 BMSC BN Bnip Bok/Mtd B-RAF BV CAM-DR cAMP CARD CASP Caspase CCCG CCND2 CCNE1 CDC CDH1 CDK CDKN1A CDKN1B CDKN2A CDKN2B CDKs CDNA CEA c-FLIP CIN Cisplatin CLL CLU CML COX COX-2 CP

Abbreviations

Barrett’s Esophagus Base excision repair Basic fibroblast growth factor Bcl-2 homology domains Bcl-2 interacting killer Bcl-2 interacting mediator of cell death Baculoviral IAP repeat XIAP; X-linked inhibitor of apoptosis Survivin; baculoviral IAP repeat-containing 5 Bcl-2-modifying factor Polycomb group ring finger Bone marrow stromal cell Bombesin Bcl-2/adenovirus E1B 19 kDa interacting protein Bcl-2-related ovarian killer v-raf murine sarcoma viral oncogene homolog B1 Bee venom Cell adhesion mediated drug resistance Cyclic adenosine monophosphate Caspase recruitment domain Caspase; apoptosis-related cysteine peptidase Cysteine aspases Colorectal Cancer Collaborative Group G1/S –specific cyclin D2 Cyclin E Cell cycle division E-cadherin Cyclin-dependent kinase p21; WAF1/CIP1; cyclin-dependent kinase inhibitor 1A p27; kip1; cyclin-dependent kinase inhibitor 1B p16; INK4A; cyclin-dependent kinase inhibitor 2A p15; INK4B; cyclin-dependent kinase inhibitor 2b Cyclin-dependent Kinases Complementary DNA Carcinoembryonic antigen Cellular FLICE-like inhibitory protein Cervical intraepithelial neoplasia cis-diamminedichloroplatinum Chronic lymphocytic leukemia Clusterin Chronic myelocytic leukemia Cyclooxygenase Cyclooxygenase 2 Ceruloplasmin

Abbreviations

CRC CRT CSC CTNNB1 Cyto c DAP4 DAPK DCC DCF DCIS DcR3 DD DED DEDs DIABLO DISC DITC DNA DNMT1 DOX DR DR4/DR5 E E2F1 EAC EBV ECF ECM EDAR EGCG EGF EGFR EGFR-KI EM-DR EMT EP1, EP2, EP3, EP4 ER ERBB2 ERK ESCC FA FADD FAP

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Colorectal cancer Chemoradiation Cigarette smoking condensate β-catenin; cadherin-associated protein Cytochrome c Dipeptidyl-aminopeptidase Calmodulin (CaM)-regulated Ser/Thr kinase Deleted in colorectal cancer Docetaxel, cisplatin, 5-fluorouracil Ductal carcinoma in situ Fas decoy receptors Death domains Death effecter domains Death effector domains Direct IAP binding protein with low pI Death-inducing signaling complex Dacarbazine Deoxyribonucleic acid DNA methyl trasferase Doxorubicin Death receptor Death receptor 4/Death receptor 5 HPVs encode six early E2F transcription factor 1; retinoblastoma-associated protein Esophageal adenocarcinoma Epstein-Barr virus Epirubicin, cisplatin, 5-fluorouracil Extracellular matrix Ectodysplasin-A receptor Epigallocatechin-3-gallate Epidermal growth factor Epidermal growth factor receptor Epidermal growth factor receptor kinase inhibitor Environment mediated drug resistance Epithelial-mesenchymal transition PEG2 receptors Estrogen receptor HER-2, erbB-2; v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 Extracellular signal-regulated kinase Esophageal squamous cell carcinoma Follicular adenoma Fas-associated death domain Familial adenomatous polyposis

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FAP-1 FasL FAS-R FC FGF FHIT FLIP FLIPs FN FP FVPTC GAP GAST GERD GI GPR30 GPx GRP GSI GSK3 Gsp GSTP1 HA HAT HC HCC HDAC HDI HGF/SF HIT-T15 HMGB1 HMGI HNPCC HPV Hrk/DP5 HSC HSP hTERT IAP Id1 IGFBP IGF-I R IGF-I IHC

Abbreviations

Fas-associated phosphatase-1 Fas ligand Fas receptor Follicular carcinoma Fibroblast growth factor Fragile histidine triad Fas-associated death domain-like interleukin1β-converting enzyme inhibitory protein FADD-like ICE inhibitory proteins Fibronectin PGF2α receptors Follicular variant of PTC GTPase-activating protein GAS; gastrin Gastroesophageal reflux disease Gastrointestinal G protein-coupled receptor 30 Glutathione peroxidases Gastrin-releasing peptide Gamma-secretase tripeptide inhibitor Glycogen synthase kinase 3 G protein subunit alpha Glutathione S-transferase π Hurthle cell adenoma Histone acetyltransferase Hurthle cell carcinoma Hepatocellular carcinoma Histone deacetylase Histone deacetylase inhibitor Hepatocyte growth factor/scatter factor Pancreatic B-cell line High-mobility group B1 High mobility group I Hereditary non-polyposis colonic cancer Human papillomavirus Harakiri Hematopoietic stem cell Heat shock protein Human telomerase reverse transcriptase Inhibitor of apoptosis proteins Inhibitor of differentiation Insulin-like growth factor binding protein Insulin-like growth factor receptor Insulin-like growth factor Immunohistochemistry

Abbreviations

IKB IKK IL-1 IL1A IL1F6 IL1F8 IL-6 IL-8 Ink4a iNOP IRF-3 ITF KIT KRAS K-sam L LCM LEEP LF LIN LMP1 LNA LOH LV MAD2 MADDH MALT MAP MAPK MCC Mcl-1 MDM2 MEN MET MGMT MINPP MINT2 miR MITF MLH1 MM MMP mPGES

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Inhibitor of NFkB IκB kinase Interleukin Interleukin 1, α Iinterleukin 1 family member 6, ε Interleukin 1 family member 8, ξ Interleukin-6 Interleukin-8 Inhibitor of CDK4 Interfering nanoparticles Interferon regulatory factor-3 Intestinal trefoil factor c-kit; v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog Kirsten rat sarcoma viral oncogene homolog KATO III cell-derived stomach cancer amplified 2 late Laser capture microdissection Loop electrosurgical excision procedure Lactoferrin Laryngeal intraepithelial neoplasia Latent membrane protein 1 Locked nucleic acid Loss of heterozygosity Leucovorin Mitotic arrest deficient 2 Mothers against decapentaplegic (MAD) Drosophila homolog Mucosa-associated lymphoid tissue. MYH- associated polyposis Mitogen-activated protein kinase Mutated in colon carcinoma Myeloid cell leukemia-1 Mouse double minute 2 homolog Multiple endocrine neoplasia c-met; hepatocyte growth factor receptor O-6-methylguanine-DNA methyltransferase Multiple inositol polyphosphate phosphatases Munc18-1-interacting protein 2 MicroRNA Microphthalmia-associated transcription factor MutL human homolog DNA mismatch repair Multiple myeloma Mitochondrial membrane permeabilization Microsomal prostaglandin E synthase

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MRD mRNA MSH MTC mTOR MUC MYC MYH NAC N-CAM NF-KB NFKB1 NF-κB NGFR NHL NIS Nix NNK NO NOTCH Noxa NPC NSAID NSCLC OMM ORF P53 P73 PAC PAC-1 PALA PANC-1 PARP PCD PCNA PDGF PGE2 PGES PGH2 PGI2 PGIS PI3K PI3K

Abbreviations

Minimal residual disease Messenger RNA Mismatch repair ATPase MutS family Medullary thyroid cancer Mammalian target of rapamycin Mucin; oligometric mucus/gel-forming) c-myc; v-myc avian myelocytomatosis viral oncogene homolog MutY human homolog base excision repair a nucleotide binding domain and CARD Neural cell adhesion molecule Nuclear factor-kappaB NF-κB; Nuclear factor kappa light polypeptide gene enhancer in B-cells Nuclear factor kappa B Nerve growth factor receptor Non-Hodgkin’s lymphoma Sodium iodide symporter Nip3-like protein X 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone Nitric oxide Neurogenic locus notch NADPH oxidase activator Nasopharyngeal carcinoma Non-steriodal anti-inflammatory drugs Non-small cell lung cancer Outer mitochondrial membrane Open reading frame Tumor protein p53 Tumor protein p73 Paclitaxel Procaspase-activating compound-1 N-(phosphonacetyl)-L-aspartic acid Human pancreatic cancer cell line-1 Poly-ADP ribose polymerase Programmed cell death Proliferation cell nuclear antigen Platelet-derived growth factor Prostaglandin E2 Prostaglandin synthase Prostaglandin H2 Prostacyclin I2 Prostacyclin synthase Phosphatidylinositol 3-kinase Phosphoinositide 3-kinase

Abbreviations

PKB PLC PML PPAR PPARdelta PPARγ PR PTC PTEN PTGS2 PTHrP PUMA Rassf RASSF1A Rb RIPK1 RNA RNAi ROS RT RTK RUNX3 SAHA SCCL Se SHH shRNA siRNA SMAC SMAD SP Spike SSAT STAT T3 T4 tBid TC TCC TCF TERT TFF1 TG TGFB1 TGFBR

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Protein kinase Phospholipase C Promyelocytic leukemia tumor suppressor Peroxisome proliferator activated receptor Peroxisome proliferator-activated receptor delta Peroxisome proliferator-activated receptor-gamma Progesterone Papillary thyroid carcinoma Phosphatase and tensin homolog Cyclooxygenase-2; COX2; prostaglandin endoperoxide synthase) Parathyroid hormone related peptide p53 upregulated modulator of apoptosis Ras-association domain family of protein Ras association RalGDS/AF-6 domain family 1 Retinoblastoma Receptor-interacting protein kinase 1 Ribonucleic acid RNA interference Reactive oxygen species Radiotherapy Receptor tyrosine kinase Runt-related transcription factor 3) Suberoylanilide hydroxamic acid Squamous cell carcinoma of larynx Selenium Sonic hedgehog Short hairpin RNA Short interfering RNA Second mitochondria-derived activatior of caspase Small mothers against decapentaplegic Side-population Small protein with inherent killing effect Spermidine/ spermine N1-acetyltransferase Signal transducers and activators of transcription Triiodothyronine Thyroxine Truncated Bid Thyroid cancer Transitional cell carcinoma T-cell factor Telomerase reverse transcriptase pS2; gastrointestinal trefoil protein 1 Thyroglobulin TGFβ; transforming growth factor beta 1 Transforming Growth Factor-β receptors

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TGF-β THXA2 THXB2 THXS TIL TNF TNFR TNFR1 TNFα TPO TRADD TRAF1/2 TRAF2 TRAIL TRID TRUNDD TRx TSH TSLC TTF-1 TUNEL TVPTC TXR VEGF wt XIAP

Abbreviations

Transforming growth factor-beta Thromboxane A2 Thromboxane B2 Thromboxane synthase Infiltration by lymphocytes Tumor necrosis factor Tumor necrosis factor receptor Tumor necrosis factor receptor 1 Tumor necrosis factor α Thyroid peroxidase TNFR-associated death domain TNF receptor associated factors 1 and 2 TNF receptor–associated factor 2 TNF-related apoptosis inducing ligand Decoy receptors 1 (DcR1 or TRAIL-R3) Decoy receptors 2 (DcR2 or TRAIL-R4) Thioredoxin reductases Thyroid stimulating hormone Tumor suppressor in lung cancer Thyroid transcription factor 1 Terminal deoxynucleotidyl transferase-dUTP nick end labeling Tall cell variant of PTC Thromboxane receptor Vascular endothelial growth factor wild type X-linked inhibitor of apoptosis protein

Chapter 1

Apoptotic Signaling Pathway and Resistance to Apoptosis in Breast Cancer Stem Cells Prasanthi Karna and Lily Yang

Abstract A major challenge in the treatment of human breast cancer is the development of resistant mechaims to apoptosis in cancer cells that leads to a low senstivity to therapeutic agents. Recent advances in investigation of the cellular origin of breast cancer showed that breast cancers can be derived from a few tumor initiating cells or cancer stem cells. Increasing evidence supports the notion that cancer stem cells are highly aggressive and resistant to conventional therapies, leading to the progression of breast cancer. Therefore, understanding the molecular mechanisms of differential regulation of the apoptitic signaling pathway in normal mammary epithelial cells, breast cancer stem cells, and breast cancer cells representing different stages of the disease should allow for the development of novel therapeutic approaches targeting dysfunctional apoptotic signaling pathways in breast cancer cells and/or cancer stem cells. Keywords Apoptosis resistance · Breast cancer stem cells · Molecular targeted therapy

Introduction Apoptosis is a highly regulated, energy-dependent programmed cell death in which the cell activates a signaling cascade that leads to cell death without triggering an inflammatory response. Apoptosis plays a critical and natural physiological role in tissue homeostasis as well as in elimination of abnormal cells that are superfluous, diseased or otherwise had served their useful purpose. Apoptosis can be initiated by a variety of stimuli, including developmental signals, cellular stress and disruption of cell cycle. In contrast, execution of apoptosis is a relatively unformed signal process, involving characteristic morphological and biochemical changes. The morphologic hallmarks include membrane blebbing, cell shrinkage, chromatin P. Karna (B) Departments of Surgery and Radiology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 1,

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P. Karna and L. Yang

condensation and DNA fragmentation (Kerr et al., 1972). A number of the key factors involved in the regulation, coordination and execution of these events have been identified (Deveraux and Reed, 1999; Igney and Krammer, 2002; Okada and Mak, 2004; Peter and Krammer, 2003; Stroh and Schulze-Osthoff, 1998). Apoptosis plays a major role in various stages of normal breast development, including the formation of the intraductal lumen during morphogenesis of breast ducts, at the end of the menstrual cycle, and in the involution of mammary glands after cessation of lactation (Anderson, 1999; Debnath et al., 2002; Hahm and Davidson, 1998). For example, mammary epithelial cells proliferate to develop additional ductal branching and lobuloalveolar growth in mammary ductal glands during pregnancy. After lactation, breast ducts undergo an involution stage with massive apoptosis and reconstruction of the ducts leading to the return of the primary breast duct structure (Hahm and Davidson, 1998). Apoptosis also occurs in the lobular unit of terminal duct with proliferation of the gland epithelial cells several days before the menstrual cycle and a peak in apoptosis close to the end of the cycle (Anderson, 1999). During these processes, cell proliferation and apoptosis is highly regulated to maintain structural and functional characteristics of normal breast ductal glands. Based on the established interactions among the known mediators of apoptosis, two classic pathways of apoptotic signaling in mammalian cells have emerged. The first one is extrinsic pathway, which involves signaling by interaction of apoptotic inducing ligands with their cell surface death receptors, which then activates the caspase cascade through adapter molecules. The second intrinsic pathway is mediated by mitochondria, which is initiated by the withdrawal of growth factors and treatment with some chemotherapy drugs. The activation of this pathway is initiated by the release of cytochrome c from mitochondria, activation of Apaf-1 and triggering of the activation of caspases. Increasing evidence shows that the apoptosis is a dynamic process that is tightly regulated by several signal pathways. It is clear that activation of caspases, dynamic changes in the location and levels of the Bcl-2 family of proteins, levels of the inhibitor of apoptosis family (IAP) of proteins play key roles in execution and regulation of the apoptotic cell death induced by both pathways (Reed, 1998; Reed, 2000; Tamm et al., 1998). Development of drugs that target and directly switch on the cell death machinery in tumors constitutes a novel way of cancer therapy. Induction of apoptosis by chemotherapy drugs, radiation and blocking growth factor signaling pathways has been used for the treatment of breast cancer. However, it is known that some breast cancers are highly resistant to conventional therapy. The evasion of apoptosis is a critical component of oncogenic transformation and resistance to chemotherapy. To develop the novel therapeutic approaches for the apoptosis resistant breast cancer cells, it is crucial to understand the regulation of the apoptotic pathways and the molecular mechanisms of resistance to apoptosis in these breast cancer cells. Many of the chemotherapeutic drugs target cancer cells at multiple molecular and cellular levels. Cancer cells may escape from apoptosis in response to chemoand radiotherapy, by downregulation of the death receptor pathway, over-expressing anti-apoptotic Bcl-2 protein family, and upregulation of the anti-apoptotic factors (Cory and Adams, 2002; Deveraux and Reed, 1999; Lowe and Lin, 2000; Peter

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Apoptotic Signaling Pathway and Resistance to Apoptosis

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and Krammer, 2003). Moreover, the development of breast cancer is a multistage process involving various genetic alternations and cellular abnormalities that provide advantages for the growth and progression of tumors. Defects in the apoptotic signaling pathway not only promote the progression of breast cancer from ductal carcinoma in situ (DCIS) to invasive and then to metastatic stage, but also reduce sensitivity of the cancer cells to commonly used chemotherapy drugs, hormonal therapy and growth factor receptor inhibitors or blocking antibodies. At present, several mechanisms are found to be responsible for chemoresistance in cancer cells, such as modification of drug-target interactions, decreased uptake or increased elimination of active molecule, defects in the apoptosis, and dysfunction in other cell death pathways including necrosis and autophagy. Presently, resistance to apoptosis is recognized as one of the major problems for cancer therapy. The effects of therapy on genetically unstable, rapidly dividing groups of tumor cells usually leads to only a temporary relief of the tumor burden, because this is usually followed by the outgrowth of a subpopulation of cells that carry advantageous genetic mutations or alternations that make them non-responsive to therapy. Loss of ability to undergo cell death might be one of the key factors in the selection leading to treatment resistance. This review outlines the basic pathways of apoptosis, and discusses mechanisms of apoptosis resistance, the concept of cancer stem cells and their role in resistance to treatment. Finally, potential approaches for targeting apoptosis resistant cancer stem cells are described.

Apoptotic Signal Pathway During the last decade, many cellular factors involved in apoptosis have been identified and their roles in apoptotic signaling have been elucidated. Apoptosis is initiated when the cells receive negative signaling, and proceeds through an extrinsic (death receptor pathway) or an intrinsic (mitochondria-mediated pathway) pathway (Reed, 2000). The extrinsic pathway is triggered by ligation of cell surface death receptors with their specific ligands, whereas the intrinsic pathway is set off when the cells are under severe stress and is characterized by leakage of cytochrome c from mitochondria. There is also some evidence implying a crosstalk between cell death receptors and mitochondrial pathways under certain conditions. These distinct pathways converge with the activation of the caspase cascade, but their relative contribution is not fully understood. Recent studies reveal a close cooperation between both pathways in maintaining homeostasis, preventing autoimmunity and terminating an immune response by using mice deficient for both Fas and Bim (Hughes et al., 2008; Hutcheson et al., 2008).

The Extrinsic Pathway The extrinsic pathway is a receptor-mediated and regulated by the members of tumor necrosis factor (TNF) receptor superfamily namely, Fas (CD 95) and TNF-related apoptosis inducing ligand (TRAIL) receptors. The binding of the corresponding ligands results in receptor trimerization and clustering of the receptor death domains.

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The cytosolic domains of the death receptors form a death-inducing signaling complex (DISC) that links up with the adaptor molecule, Fas-associated death domain (FADD) or TNFR-associated death domain (TRADD). The DISC–FADD/TRADD complex, binds to the initiator caspases 8 and 10 through its death effector domains (DED, D4/D5), thereby causing the autocatalytic cleavage of procaspase-8 and activation of downstream executioner caspases (Hengartner, 2000; Krammer, 2000; Thorburn, 2004). The ligands activating the death receptor pathways include TNFα, Fas ligand, and TRAIL. TNFα is a cytokine produced by macrophages/monocytes during acute inflammation. It regulates inflammation, survival, proliferation and apoptosis of cells. Tumor necrosis factor receptor 1 (TNFR-1) induces both death and survival signals. TNFα binds to TNFR-1 or TNFR-2 and trimerizes the receptors, resulting in formation of a complex between FADD and procaspases 8/10 to activate apoptotic signals (Basile et al., 2001; Chinnaiyan et al., 1996; Sheikh and Huang, 2003). However, depending on the type of stimulus, TRADD may stimulate NF-κB activity, leading to the recruitment of TNF receptor associated factors 1 and 2 (TRAF1/2), ribosome interacting protein and c-IAP1, which interact with anti-apoptotic proteins to prevent apoptosis. Since the level of TRAF2 is elevated in numerous tumors, this may cause the formation of TNFR, TRADD and TRAF2 complex and activate the cell survival pathway leading to apoptosis resistant tumors. Fas (APO-1 or CD95), a member of the TNF superfamily, is a widely expressed transmembrane protein in cell membranes of normal and malignant cells. Fas/CD95 receptor/ligand system is an important signaling pathway in the regulation of apoptosis. Commonly used chemotherapeutic drugs may induce apoptosis by increasing Fas expression. Four distinct TNF-related apoptosis inducing ligands (TRAIL-R1-4) have been identified on the surface of cells. TRAILs induce apoptosis in a variety of transformed or tumor cells by binding to DR4 and DR5, leading to the recruitment of adaptor proteins and activation of caspases 8, 9, 7 and 3 (Kim et al., 2000). It has been shown that TRAIL selectively induces apoptosis in a variety of tumor cells and transformed cells, but not in most normal cells, and therefore has garnered immense interest as a promising agent for cancer therapy (Kim et al., 2000; Walczak et al., 1999). This selectivity could be due to a higher level of TRAIL receptors in cancer cells compared to normal cells. Furthermore, TRAIL also interacts with ‘decoy’ receptors, DcR1 and DcR2, which lack functional death domains and do not induce apoptosis, which may contribute a low sensitivity of normal cells to TRAIL-induced apoptosis (Kim et al., 2000).

The Intrinsic Pathway In this pathway, the mitochondria play a central role in the integration and execution of a wide variety of apoptotic signals (e.g., loss of growth factors, hypoxia, oxidative stress or DNA damage) and provide the energy required for execution of the apoptotic program and release of pro-apoptotic proteins such as cytochrome c, endonuclease G and apoptosis-inducing factor. The Bcl-2 family of proteins plays

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critical roles in regulating the intrinsic pathway. Activated pro-apoptotic Bcl-2 proteins attach to the mitochondrial outer membrane to form conducting channels, allowing cytochrome c to translocate from the intermembrane matrix into the cytoplasm. In the cytosol, cytochrome c combines with ATP, Apaf-1 and procaspase 9 to form an apoptosome, which activates caspase 9 and subsequently caspase 3 leading to cell death. The mitochondrial permeability transition pore (PTP) and Bax play important roles in this process (Crompton, 1999; Reed and Kroemer, 2000).

Caspase Activation The caspases are cysteine–aspartic acid-proteases that cleave proteins with specific amino acid sequences. Caspases are synthesized as inactive precursors (procaspases), which upon proteolytic cleavage to become activated caspases to cleave various protein substrates. Of the 14 caspases identified in humans, two-thirds play an important role in apoptosis. Caspases are grouped into initiators or effectors of apoptosis, depending on their point of entry into the apoptotic pathway. The initiator caspases 2, 8, 9 and 10 are activated by proximity-induced dimerization, and they in turn proteolytically cleave the inactive pro-forms of the effector caspases 3 and 7. The effector caspases in turn sequentially cleave protein substrates within the cell, which results in apoptosis (Boatright and Salvesen, 2003; Hengartner, 2000; Motadi et al., 2007).

The p53 Pathway In addition to extracellular signals, cells can undergo apoptosis in response to internal signals including genetic abnormality. Defective or inappropriate cell cycle progression caused by a variety of genotoxic injuries can result in cell cycle arrest, and subsequent induction of apoptosis. The tumor suppressor gene p53 has been clearly linked to these pathways leading to its designation as the ‘guardian of the genome’. DNA strand breaks induce rapid p53 upregulation. The upregulation of p53 is mostly post-transcriptional, involving both, an increase in translation and prolonged half-life (Sherr and McCormick, 2002). Increase in Bax transcription may be in part responsible for p53-induced apoptosis following DNA damage (Chipuk et al., 2004; Miyashita and Reed, 1995). The fact that Bcl-2 acts downstream of p53 signal points out that there are additional, parallel and p53-independent pathways regulating DNA damage induced apoptosis in mammalian cells.

Mechanisms of Resistance to Apoptosis Investigations into the mechanisms of emergence of the chemoresistant phenotypes have involved traditional studies using unicellular models exposed to incremental doses of chemotherapeutic agent, with consequent selection for resistant clones and cell populations. Such studies have led to the identification and characterization of

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many resistance mechanisms, such as decreased drug uptake, increased drug extrusion, alterations in the drug target, alterations in drug metabolism, repair of DNA damage, alteration of cell cycle checkpoint control, and changes in downstream mediators of apoptosis. Recent advances in molecular analysis of the apoptotic signal pathways have revealed some dysfunctional apoptotic signals that confer a low sensitivity to apoptosis and therapeutic agents. The following are a list of some representative changes in the apoptotic signal pathways.

Death Receptors Because of the physiological role of death receptors in normal cells, downregulation or loss of death receptors contributes to a malignant phenotype. Death receptor activated apoptosis is negatively regulated by cellular FLICE-like inhibitory protein (c-FLIP). c-FLIP is structurally related to caspase 8 and can bind to FADD, but lacks enzymatic activity. It thus prevents apoptosis by blocking association of caspase 8 with the DISC. Downregulation of c-FLIP renders cells sensitive to all known death receptors-mediated cell death including Fas, TNF-R and TRAIL-Rs. PS-341, a proteasome inhibitor, can induce a substantial reduction in c-FLIP, and has been successfully combined with the death ligand TRAIL to promote tumor cells’ apoptosis synergistically (Sayers et al., 2003). Recombinant TRAIL alone or in combination with chemotherapy resulted in sensitization of p53 wild type, mutant, and null cell lines to TRAIL-mediated apoptosis by downregulating expression of c-FLIP (Galligan et al., 2005). Further investigations indicated that human cancers have developed mechanisms to avoid Fas-mediated apoptosis since many tumor cells are shown to be resistant to FasL or Fas antibody induced apoptosis (Barnhart et al., 2004). Somatic deletions and mutations of Fas receptor were identified in several types of human cancers (Beltinger et al., 1998; Boldrini et al., 2002; Landowski et al., 2001). In addition, some tumor cells produce a high level of soluble Fas to block interactions between cell surface Fas receptor and FasL (Lee et al., 1999; Liu et al., 2002). A reduced level of expression of cell surface Fas receptor is common in many tumor types, including breast cancer, due to downregulation of Fas gene expression or decreased cell surface transportation (Bullani et al., 2002; Mullauer et al., 2000; Viard-Leveugle et al., 2003). However, increasing evidence shows that downregulation of Fas may be a cause for resistance to apoptosis only in a small percentage of human tumors since many tumor cells that are resistant to Fas-mediated apoptosis do not carry Fas mutations and also exhibit an adequate level of Fas expression (Elnemr et al., 2001; Muschen et al., 2001). In human normal and breast cancer cell lines and tissues, Fas and FasL are co-expressed in normal mammary epithelial and breast cancer cells. FasL is weakly expressed in normal breast ductal cells but is strongly expressed in most breast cancer cell lines and tissues. However, Fas receptor is found to be abundant in normal breast epithelial cells but is low in breast cancer cells with heterogeneous expression levels ranging from weak to strong (Mullauer et al., 2000). A low sensitivity to death receptor mediated apoptosis in the presence of a high level

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of FasL and a moderate level of Fas receptor suggests that breast cancer cells may have developed anti-apoptotic mechanisms downstream of the death receptor activation, which block apoptotic signaling pathway. Co-expression of Fas and FasL in tumor cells that are resistant to Fas-mediated apoptosis supports the notion that the presence of downstream inhibitory factors that block the apoptotic signal pathway (Abrams, 2005; Mullauer et al., 2000; Yang et al., 2003a). TRAIL-induced apoptosis has a significant clinical potential. However, different human tumors have a wide range of sensitivities to TRAIL-mediated apoptosis and some human tumor cells display a low level of TRAIL expression or activity (Ibrahim et al., 2001). It has been shown that most human breast cancer cells are highly resistant to TRAIL treatment (Bockbrader et al., 2005; Singh et al., 2003). Some tumor cells have completely lost the expression of TRAIL receptor. Furthermore, some TRAIL resistant cells express high levels of both TRAIL receptor and ligand (Jin et al., 2004; Singh et al., 2003). It has been shown that over-expression of Bcl-2 blocked activation of TRAIL-mediated apoptosis by inhibiting the release of mitochondrial cytochrome c and the cleavage of caspase-7 (Sun et al., 2001). However, treating TRAIL resistant tumor cells with subtoxic concentrations of chemotherapeutic drugs sensitizes them to apoptosis (Bockbrader et al., 2005; Odoux et al., 2002; Shankar and Srivastava, 2004; Singh et al., 2003).

Caspases Resistance of cancer cells to apoptosis may also be due to failure of initiator caspases to activate the caspase cascade. Deficiency in the levels of expression of procaspase genes was detected in some tumors. In one instance, deletion or silencing of the caspase 8 gene was discovered in neuroblastoma and non-small cell lung cancer (Hopkins-Donaldson et al., 2003; Iolascon et al., 2003; Teitz et al., 2002). Downregulation of the initiator caspase 8 may be responsible for resistance to apoptotic signaling. Silencing of caspase 8 expression by DNA methylation in cancer cells correlated with resistance to rhTRAIL (Eramo et al., 2005; van Noesel et al., 2003). Suppression of caspase 8 expression was shown to occur during the development of neuroblastoma metastases in vivo, and reconstitution of caspase 8 expression in deficient neuroblastoma cells suppressed metastases formation (Iolascon et al., 2003). Deficiency in caspase 3 was also found in human breast cancer as well as in several other tumor types (Fujikawa et al., 2000; Iolascon et al., 2003; Kolenko et al., 1999). It was also demonstrated that despite the absence of caspase 3 in breast cancer cells, the apoptotic pathway was able to proceed via sequential activation of caspase 9 followed by that of caspases 7 and 6, and cells exhibited all the morphological changes associated with apoptosis (Liang et al., 2001). It is now generally agreed that the presence of processed or active caspase 3 does not always coincide with the presence of cleaved substrates and apoptosis since downstream caspase inhibitors in the apoptotic pathway that are upregulated in human cancer cells can block the apoptotic process (Yang et al., 2003a). Examination of levels

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of pro-apoptotic and/or active caspases in breast carcinoma tissues from 440 breast cancer patients at different stages of the disease showed high levels of procaspases and/or active caspases in most human breast cancer tissues. A high level of procaspase 3 expression is found in 58% of DCIS and 90% of invasive breast cancer tissues (Bodis et al., 1996). A strong expression of procaspase-3, 6 and 8 is significantly associated with the extent of apoptosis and high grade DCIS lesions. A strong positive correlation was found between active caspase 3, 6 and XIAP, suggesting the presence of a negative feed back in breast cancer tissues. Results of paired or non-paired normal and breast cancer tissues by Western blot analysis also demonstrated the presence of active caspase 3 fragments in most breast cancer but not in normal breast tissues. Coincidentally, the breast cancer tissues with active caspase 3 fragments also express a high level of another IAP protein, survivin (Yang et al., 2003a). At present, the significance of co-existing active caspase and IAP proteins in breast cancer tissues is still under investigation. Detection of active caspases 3 and 6 indicates that the effectors upstream of the apoptotic signaling pathway are functional and the apoptotic pathway is activated in breast cancer tissues. It is possible that abnormalities generated by genetic changes in breast cancer cells activate the apoptotic signaling pathway and induce apoptosis in the majority of the cancer cells. The cells that have developed apoptotic resistance, such as those expressing high levels of IAP proteins XIAP and survivin, are able to block the caspase activity and grow into tumor masses that are highly resistant to apoptosis.

IAP Family of Proteins IAPs are a family of proteins containing one or more conserved, cysteine and histidine-rich baculoviral IAP repeat (BIR) N-terminal domains and a C-terminal RING domain. Members of the IAP family of proteins, including NAIP, XIAP, c-IAP1, c-IAP2, survivin, Livin and Ts-IAP, have been identified and their roles in inhibiting caspase activity have been elucidated (Deveraux and Reed, 1999; Roy et al., 1997). The BIR domains of the IAPs form the zinc-figure-like structures that bind to active caspases to block caspase activity. The RING domain acts as an ubiquitin ligase to facilitate proteasomal degradation of caspases as well as regulation of the IAP themselves (Morizane et al., 2005; Suzuki et al., 2001; Vaux and Silke, 2005). Specific interactions of BIR domains with different caspases have been determined by studying the structures of caspases and IAPs. For example, the proximal link region of BIR2 of XIAP protein binds and blocks the active site of caspase-3 and -7 while the BIR3 domain binds and inhibits active caspase-9 (Huang et al., 2001). XIAP is the most potent endogenous caspase inhibitor (Shin et al., 2003; Tamm et al., 2003) and its upregulation is found in many breast cancer cell lines and tissues. The pro-apoptotic protein XAF1 binds to XIAP and releases active caspases from XIAP inhibition and promotes degradation of another IAP family of protein, survivin (Arora et al., 2007; Liston et al., 2001). Several studies demonstrated the

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association of the a high level of XIAP with resistance to chemotherapy in human caners (Amantana et al., 2004; Cheng et al., 2002; Notarbartolo et al., 2004; Yang et al., 2003b). Unlike other IAPs, survivin is expressed broadly in embryonic and fetal tissues but is undetectable in normal adult differentiated tissues (Adida et al., 1998; Altieri, 2003). Survivin is a structurally unique member of the IAP family that acts as a suppressor of apoptosis and plays a central role in cell division. Although a structural basis for a direct interaction between survivin and caspase-3 has not been defined, inhibition of caspase-3 and -7 activities has been demonstrated in survivin protein (Shin et al., 2001). Increasing evidence suggests that survivin is closely associated with mitochondria-dependent apoptosis. Downregulation of survivin expression or function results in the activation of caspase-9 (Mesri et al., 2001; Yang et al., 2003b). It has also been shown that survivin associates with XIAP through the BIR domain to form a survivin-XIAP complex that promotes XIAP stability and synergistic inhibition of apoptosis (Dohi et al., 2004). Survivin is overexpressed in most human tumor types including 70% of human breast cancer tissues but not in normal breast tissues and its high expression is associated with poor survival (Ambrosini et al., 1997; Li, 2003; Tanaka et al., 2000). Survivin counteracts apoptotic stimuli induced by Fas, Bax, caspases, and anticancer drugs (Tamm et al., 1998). Therefore, Survivin may contribute significantly to the development and progression of cancer, and could become an effective therapeutic target.

The Bcl-2 Family The Bcl-2 family of genes encodes proteins that inhibit or promote apoptosis by regulating the mitochondrial pathway. This family of proteins shares up to four conserved regions known as Bcl-2 homology (BH) domains. Pro-apoptotic proteins include Bax, Bak, Bad, and Bcl-xs , whereas Bcl-2 and Bcl-XL are antiapoptotic (Cory and Adams, 2002; Reed, 1998). Overexpression of Bcl-2 protects cells from a variety of apoptotic stimuli, such as growth-factor withdrawal, exposure to chemotherapy agents or toxins, viral infection and inappropriate oncogene expression. A number of Bcl-2 related proteins have been subsequently identified (Krajewski et al., 1999; Reed, 1998) and characterized. The relative levels of proand anti-apoptotic Bcl-2 family of proteins have been suggested to function as a ‘rheostat’ regulating the apoptotic threshold of the cell (Yang and Korsmeyer, 1996). The antagonistic function of some of the members of the family is at least in part explained by their ability to form heterodimers. The anti-apoptotic proteins Bcl-XL and Bcl-2 form heterodimers with pro-apoptotic Bax (Oltvai and Korsmeyer, 1994). An excess of Bax promotes cell death, but co-expression of Bcl-XL or Bcl-2 can neutralizes this effect. Interestingly, under certain circumstances, Bcl-2 and Bcl-XL are targets of caspases, and cleavage of these proteins converts them from pro-survival to pro-apoptotic molecules that are able to induce cytochrome c release from the mitochondria (Cheng et al., 1997; Clem et al., 1998).

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The pro-apoptotic members can be separated into two structurally distinct subfamilies: (1) The ‘multi-domain’ proteins (BAX and BAK) share three BH regions and lack the BH4 domain; (2) ‘BH3-only’ proteins (Bnip3, Nix/Bnip3L, Bid, Noxa, Puma and Bad) share only the BH3 domain (Kelekar and Thompson, 1998). The BH3-only proteins initiate apoptosis through the activation of Bax and Bak. Studies using knockout mice have demonstrated that Bax and Bak are essential for apoptosis initiation via the intrinsic pathway. Cells lacking both Bax and Bak were completely resistant to apoptosis stimuli, while the cells lacking only Bax or Bak were not resistant to these stimuli. This suggests a functional redundancy between Bax and Bak (Tobiume, 2005). Under normal conditions, Bax is present in the cytosol. However, in response to apoptotic stimuli, it undergoes a conformational change that triggers its translocation to and insertion into the outer mitochondrial membrane leading to permeabilization of the outer mitochondrial membrane and release of pro-apoptotic proteins. On the contrary, Bak is always localized to the mitochondria as an integral membrane protein and has been reported to be maintained in an inactive conformation by anti-apoptotic Bcl-2 family proteins (Cheng et al., 2003; Willis et al., 2005). Interaction of pro- and anti-apoptotic Bcl-2 family of proteins in regulation of the apoptotic signaling pathway has been an intensive area of research. Although the roles of the Bcl-2 family of proteins in this dynamic process have yet to be elucidated, it is known that anti-apoptotic Bcl-2 proteins interact with pro-apoptotic proteins such as Bax and Bak to regulate their activity. Once the apoptotic signal is received, BH3-only proteins bind to and neutralize the anti-apoptotic Bcl-2 proteins, thereby releasing BAX and BAK (Kelekar and Thompson, 1998). It has been shown that overexpression of Bcl-2 or Bcl-XL sequester BH3-only proteins and prevents Bax translocation and activation (Cheng et al., 2001; Van Laethem et al., 2004). There is an ever increasing body of literature showing that a high level of Bcl-2 not only promotes resistance to apoptosis but also increases the recurrence rate and enhances chemo- and radio-resistance in many types of human cancers (Miyake et al., 1998, 1999). However, although Bcl-2 protein is detected in over 80% of breast cancers, the level of Bcl-2 expression is correlated well with the presence of estrogen- and progesterone (PR)-receptor positive breast cancer cells (Gee et al., 1994; Krajewski et al., 1995; Villar et al., 2001). The level of its expression is surprisingly associated with a good prognosis of the breast cancer patients (Krajewski et al., 1999; Schorr et al., 1999). On the other hand, the presence of high levels of apoptotic cells in the breast cancer tissues for the same group of patients is an indication of poor prognosis factors including larger tumor sizes, more aggressive tumor types, high proliferation rates and lymph node metastases (Joensuu et al., 1994). Therefore, the role of Bcl-2 in regulating apoptosis in breast cancer cells is still controversial. Results of recent studies show that Bcl-2 is expressed mostly in the luminal and low grade breast cancer type that are positive for estrogenand PR- receptors, which are known to have a better prognosis compared to other subtypes of breast cancers (Alsabeh et al., 1996; Megha et al., 2002; Meijnen et al., 2008). It is possible that differential regulation of the key apoptotic regulators in breast cancer cells that are initiated from cellular origins along different stages of

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breast stem/progenitor cells contributes the apoptotic response in breast cancer cells. However, the mechanism of the differential role of Bcl-2 in regulating apoptosis in different subtypes of breast cancer cells, such as basal and luminal types, has yet to be determined.

Cancer Stem Cells Identification of Cancer Stem Cells Cancers originally develop from normal cells by accumulating oncogenic mutations and gaining the ability to proliferate aberrantly, and eventually turning to malignant. The concept of initiation of many tumor types from transformed adult stem cells has been around for a long time (Reya et al., 2001). Stem cells may be preferential targets of initial oncogenic mutations because in most cancer originating tissues, they are the only long-lived populations and are therefore exposed to more genotoxic stresses than their shorter-lived, differentiated progeny (Pardal et al., 2003). Like normal adult stem cells, cancer stem cells can divide indefinitely, giving rise to both more cancer stem cells and progeny that ultimately differentiate into different cell types in a tumor. The presence of breast cancer stem cells were firstly demonstrated in a cell population isolated from breast cancer patients that strongly expresses CD44 but is negative or has a low level of CD24 (CD44+ /CD24− ). It has been shown that tumor xenografts can grow in mice that receive as little as 100 of CD44+ /CD24− cancer cells, while CD44− /CD24+ cells do not exhibit tumor growth, even at a very high cell numbers, leading to the assumption that CD44+ /CD24− cells contain breast cancer stem cells (Al-Hajj et al., 2003). Examination of gene expression profiles of CD44+ /CD24− cells isolated from human normal breast ducts and breast cancer tissues shows that those cells are estrogen negative but express high levels of genes that are involved in cell motility, invasion, apoptosis, and extracellular matrix remodeling. Breast cancer patients with a high percentage of CD44+ cells have a poorer clinical outcome compared to the patients whose tumors mainly composed of CD24+ cancer cells (Shipitsin et al., 2007). However, extensive research on the CD44+ /CD24− cell population in human breast cancer cells and tissues suggests that CD44+ /CD24− cell population contains the stem-like/progenitor cells for breast cancer but it is not a precise biomarker for breast cancer stem cells. A recent report from studying normal and cancer human mammary epithelial cells shows that breast cancer cells that have both CD44+ /CD24− and aldehyde dehydrogenase activity (ALDH) biomarkers are highly tumorigenic and produce tumor xenografts in mice when the number of injected cells is as little as 20 CD44+ /ALDH+ cells (Ginestier et al., 2007). The level of expression of ALDH1 detected in 577 breast carcinomas by immunostaining correlated with poor prognosis of the breast cancer patients (Ginestier et al., 2007). Recently, several groups also reported identification of cancer stem cells from brain, prostate, head and neck, liver, and pancreatic cancers. Results of these

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studies show that cancer stem cells of each tumor type display distinct surface biomarkers, such as human leukemia (CD34+ /CD38− ) (Bonnet and Dick, 1997), breast (CD44+ /CD24− /ESA− ) (Al-Hajj et al., 2003), head and neck (CD44+ ), prostate (CD44+ /alpha2beta1hi/CD133+ ) (Collins et al., 2005; Prince et al., 2007), brain and colon (CD133+ ) (O’Brien et al., 2007; Singh et al., 2004), pancreatic (CD24+ /CD44+ /ESA+ ) (Li et al., 2007) liver (CD90+ ) (Yang et al., 2008) and lung cancers (Sca1+ /CD45− /Pecam− /CD34+ ) (Kim et al., 2005).

Apoptosis Resistance in Cancer Stem Cells Increasing evidence shows that cancer stem cells are highly resistant to traditional chemo- and radio-therapy. It has been shown that subpopulation of the cancer cells with stem cell-like properties can be selected from drug-treated cancer cells (Eramo et al., 2006; Kang and Kang, 2007; Wei et al., 2006). Although high levels of ATP-binding cassette (ABC) drug transporters found in stem cells contribute, in part, to drug resistance, several other factors also play roles in resistance to therapy such as higher levels of DNA repair and a lowered ability to enter apoptosis (Keshet et al., 2008; Lou and Dean, 2007). For example, cancer stem cells isolated from primary glioblastoma Multiforme (GBM) are resistant to treatment of many drugs. It seems that drug resistance in these cells is not the result of extrusion of the drug by (ABC) transporters since a high concentration of fluorescent chemotherapy drug, doxorubicin, is found inside the nucleus of the cells. It is likely that intrinsic anti-apoptotic function of the stem cells play a role in drug resistance (Eramo et al., 2006). Studies on the colorectal tumors indicate that apoptosis is lower in CD44+ tumor cells as compared to CD44− cells and this suppression of apoptosis in CD44+ cells is due to a highly activated Wnt pathway (Schulenburg et al., 2007). At present, it is still unclear whether the apoptotic signal pathway in cancer stem/progenitor cells significantly differs from other cells and what are mechanisms conferring their insensitivity to apoptosis and becoming self-renewal and long-lived cells. Therefore, understanding the mechanisms of apoptosis resistance in cancer stem cells should allow for the identification of molecular targets for the development of new therapeutic approaches targeting cancer stem cells for overcoming resistance to apoptosis. Result of a previous study suggests that breast cancer stem/progenitor cells have different control mechanisms of cell proliferation and apoptosis and mutation in p53 may further reduce sensitivity of the cancer stem cells to apoptosis. Immunohistochemical examination of human breast cancer tissues from pre-invasive DCIS to invasive ductal carcinoma cases showed that breast cancer cells with a stem cell phenotype were estrogen/PR negative, p53 positive and Bcl-2 negative. In contrast, breast cancers with more differentiated luminal cell phenotype were estrogen/PR positive, p53 negative and Bcl-2 positive (Megha et al., 2002). Using dye-effluxing feature of stem cells, called side-population (SP), and CD55 as markers, mammary tumor stem cells are isolated from two mammary carcinoma cell lines and those cells are resistant to serum depletion- or ceramide-inducing

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apoptosis (Xu et al., 2007). Recent studies have shown that multiple signaling pathways and cellular factors are involved in the development of resistance to apoptosis. For example, CD133 (+) colon cancer stem cells produce and utilize IL-4 to protect themselves from apoptosis. On the other hand, anti-IL-4 neutralizing antibody strongly enhances the effect of chemotherapeutic drugs in these cells (Todaro et al., 2007). MCF7 SP cells have breast cancer stem cell markers, but do not express multiple drug resistance transporters. However, these stem cells have high levels of TNFα receptors, TNFR-p55 and TNFR-p75, which interact with wild type TNFα and increase cell survival by activation of NFκB (Li et al., 2007). Treatment with recombinant mutant TNFα results decreased self-renewal ability and an increased apoptosis in the cells through a mechanism that the mutant TNFα mainly binds to TNFR-p55, resulting in induction of apoptosis of SP cells. A recent study also shows that epidermal growth factor receptor (EGFR) signaling is required for the proliferation and survival of human brain tumor stem cells. Treatment of the cells with tyrosine kinase inhibitors inhibit proliferation and induce apoptosis (Soeda et al., 2008). In breast cancer cells, EGFR signaling-induced apoptosis resistance is mediated by upregulating an important transcriptional factor, HIF-1α, which is usually activated under hypoxic conditions. HIF-1α then increases the level of survivin gene transcription (Peng et al., 2006; Wang and Greene, 2005; Weant et al., 2008). Interestingly, a high level of HIF-1α is found in the breast stemlike CD44+ cell populations isolated from both normal human mammary ductal epithelial cells and invasive breast cancer tissues (Shipitsin et al., 2007). At present, the role of survivin in cell proliferation and survival of cancer stem cells has yet to be elucidated. In addition to survivin, upregulation of HIF-1α in cancer cells also transcriptionally activates the expression of stem cell factor gene in breast cancer cells, which may play critical roles in self-renewal and survival of the cancer stem cells (Han et al., 2008). Our recent study using a breast cancer cell line (MCF-10DCIS) that gives rise heterogeneous DCIS and invasive breast cancer xenografts in nude mice shows that CD44+ MCF-10DCIS cells express a high level of survivin and are resistant to therapeutic agents (Karna et al., 2008). However, the functional roles of survivin in breast cancer stem cells have yet to be elucidated. Additionally, survivin is found in several other normal stem cell populations, such as hematopoietic, mesenchymal, skin keratinocyte and colon stem cells (Leung et al., 2007; Kestendjieva et al., 2008; Marconi et al., 2007; Zhang et al., 2001; Fukuda and Pelus, 2006). It has been shown that conditional deletion of the survivin gene from the hematopoietic compartment of mice leads to ablation of the bone marrow with widespread loss of hematopoietic progenitors and rapid mortality. Therefore, survivin is essential for steady-state hematopoiesis and survival in the adult mice (Leung et al., 2007). A previous study reported that survivin regulates the proliferation and apoptosis of normal hematopoietic cells through a p21 WAF1/Cip1-dependent pathway as well as a p21 independent mechanism (Fukuda et al., 2004). In murine embryonic stem (ES) cells, Oct-4 may be involved in survival of undifferentiated ES cells under stress by activating survivin gene expression through interaction with STAT3 since the apoptotic cell death is significantly increased in response to all stress situations

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in Oct-4 knocked-down ES cells (Guo et al., 2008). Upregulation of survivin may also be one of the mechanisms for resistance to radiation in stem cells. It has been shown that treatment of primary BALB/c mouse mammary epithelial cells with clinically relevant doses of radiation enriches in normal progenitor cells. Irradiated mammary epithelial stem cells display increased levels of β-catenin and survivin expression, and maintain the numbers of colony formation compared to the cells without expressing a stem cell marker (Woodward et al., 2007). Results of recent studies indicate that interaction of cancer stem cells with extracellular matrix promotes survival, invasion and resistance to drug treatment (Toole and Slomiany, 2008). Hyaluronan is a large, linear glycosaminoglycan that is elevated in the extracellular matrix of many tumor types. CD44, a cellular receptor for hyaluronan, has been used as a biomarker to identify breast cancer stem cells. It has been shown that blocking the interaction of hyaluronan with CD44 interaction by various antagonists, such as small hyaluronan oligomers suppresses the growth of glioma in vivo by enhancing apoptosis and inhibition of cell survival pathways (Gilg et al., 2008). Thus, such hyaluronan antagonists may have potential to be developed into therapeutic agents targeting cancer stem cells to enhance the response to chemotherapy.

Development of Therapeutic Approaches Targeting Cancer Stem Cells There is an increasing recognition that treatment failure in cancer may be associated with the failure to kill a small subpopulation of tumor cells that have been characterized as tumor stem cells, which are considered resistant to chemotherapy. Understanding the critical parameters for effective cancer cell killing and demonstrating substantial efficacy gains by targeting a smaller number of cancer stem cells as opposed to the entire tumor-cell population is very important in fulfilling the therapy. Recent advances in understanding tumor biology and molecular mechanisms of apoptosis resistance of cancer stem cells provide great opportunity for the development of molecular targeted therapy for drug resistant cancer stem cells. CD44 is highly expressed in cancer stem cells of several tumor types and its signal has been associated with proliferation, survival and invasion of the cancer stem cells. Development of small molecular inhibitors or antagonists for CD44 may block the key cellular signaling pathways activated by the interaction of hyaluronan with CD44, resulting sensitization of the cancer stem cells to apoptosis induced by chemotherapy drugs and reduction of metastatic potential of tumor cells. Since the importance of EGFR signaling in aggressive tumor biology and resistance to apoptosis has been demonstrated in brain and breast cancer stem cells, the combination of EGFR inhibitors that are currently used in clinic with chemotherapeutic drugs or other molecular targeted agents is a very promising approach (Farnie et al., 2007). High levels of expression in breast cancer tissues, together with potential functional importance in maintenance of cancer stem cells and conferring resistance

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to therapeutic agents make IAP proteins attractive targets for developing novel cancer therapies that target cancer stem cells. Several types of small molecule mimics of Smac, a pro-apoptotic protein that bind to IAPs to release their suppression on caspase activity, have been developed and their effects on apoptosis induction and tumor growth inhibition have been demonstrated (Li et al., 2004; Sun et al., 2007). Smac protein in its dimeric form effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. A non-peptide smac mimetic binds to both BIR domains of XIAP with an IC50 value of 1.39 nM and induces apoptotic cell death (Sun et al., 2007). SMAC peptide mimitics can bind to XIAP, cellular cIAP-1, and IAP 2 (cIAP-2) and synergizes with both TNFα and TRAIL to increase caspase activation and induce apoptosis in human cancer cells (Li et al., 2004). The binding of the small-molecule IAP antagonists to the BIR domains also results in induction of ubiquitination proteasomal degradation of c-IAPs (Varfolomeev et al., 2007). A recent study reports that an Her-2 positive inflammatory breast cancer line, SUM190PT, does not respond well with trastuzumab (an Her-2 blocking antibody) treatment and upregulation of the levels of both survivin and XIAP is found in the resistant tumor cells, while another trastuzumab responsive breast cancer cell line, SKBR-3, shows a decrease in the levels of XIAP and survivin expression. Furthermore, a combination of inhibition of XIAP using RNA interference (RNAi) or a small-molecule XIAP inhibitor with trastuzumab significantly increases the apoptotic cell death in trastuzumab resistant SUM190PT cell line (Aird et al., 2008). Although CD44+ cells isolated from normal breast epithelial cells or the early DCIS stage of breast cancer tissues do not express Her-2 gene, the CD44+ cells from some of invasive breast cancer tissues are Her-2 positive. Therefore, it is feasible to apply above combination therapy for the treatment of CD44+ invasive or inflammatory breast cancers. Inhibition of expression levels or function of survivin using siRNAs or small molecule inhibitors is also a promising approach for enhancing the response to therapeutic agents in apoptosis resistant cancer cells. A recent study reports that a small-molecule survivin inhibitor (YM155) suppresses expression of survivin and induces apoptosis in human prostate cell lines, and inhibits the growth of tumors in vivo in nude mice, while it has little effect on expression levels of other IAP- or Bcl-2-related proteins (Nakahara et al., 2007). Currently, the compound YM-155 has entered phase II trials in the United States and Europe (Astellas Pharma, Inc.). Stably knockdown survivin expression using a survivin siRNA expressing vector in several cancer cell lines leads to increased apoptotic rate after treatment with doxorubicin or TNFα in vitro and decreased tumor formation and reduced angiogenesis in vivo (Li et al., 2006). XIAP and survivin antisense oligonucleotides cause induction of apoptosis, and significant cell death in vitro. Inhibition of tumor growth in vivo were observed when this antisense treatment is combined with irradiation or chemotherapeutics (Hu et al., 2003). This strategy is currently the most advanced modality of targeting IAP proteins in cancer, and is evidenced by several phase I/II clinical trials that are under way for targeting XIAP and survivin (Cummings et al., 2006; Vucic and Fairbrother, 2007).

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Weant AE, Michalek RD, Khan IU, Holbrook BC, Willingham MC, Grayson JM (2008) Apoptosis regulators Bim and Fas function concurrently to control autoimmunity and CD8+ T cell contraction. Immunity 28:218–230 Wei C, Guo-min W, Yu-jun L (2006) Apoptosis resistance can be used in screening the markers of cancer stem cells. Med Hypotheses 67:1381–1383 Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI, Adams JM, Huang DC (2005) Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 19:1294–1305 Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, Rosen JM (2007) WNT/betacatenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci USA 104:618–623 Xu JX, Morii E, Liu Y, Nakamichi N, Ikeda J, Kimura H, Aozasa K (2007) High tolerance to apoptotic stimuli induced by serum depletion and ceramide in side-population cells: high expression of CD55 as a novel character for side-population. Exp Cell Res 313:1877–1885 Yang E, Korsmeyer SJ (1996) Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 88:386–401 Yang L, Cao Z, Yan H, Wood WC (2003a) Coexistence of high levels of apoptotic signaling and inhibitor of apoptosis proteins in human tumor cells: implication for cancer specific therapy. Cancer Res 63:6815–6824 Yang L, Mashima T, Sato S, Mochizuki M, Sakamoto H, Yamori T, Oh-Hara T, Tsuruo T (2003b) Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide. Cancer Res 63:831–837 Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, Chu PW, Lam CT, Poon RT, Fan ST (2008) Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 13: 153–166 Zhang T, Otevrel T, Gao Z, Gao Z, Ehrlich SM, Fields JZ, Boman BM (2001) Evidence that APC regulates survivin expression: a possible mechanism contributing to the stem cell origin of colon cancer. Cancer Res 61:8664–8667

Chapter 2

Anti-Cancer Strategy of Transitional Cell Carcinoma of Bladder Based on Induction of Different Types of Programmed Cell Deaths Jose A. Karam and Jer-Tsong Hsieh

Abstract Current treatment for advanced malignancy of the bladder cancer relies on combination chemotherapeutic agent regimens. In these patients chemotherapeutic resistance emerges in the form of an aggressive subpopulation of tumor cells even if there has been a good initial response. The majority of chemotherapeutic agents target mitotic cancer cells by inducing DNA damage pathways or altering cell cycle regulation. Drug resistance often arises from the remaining post-mitotic cancer cells, which can be further targeted by triggering the intrinsic apoptotic, autophagic, or necrotic pathways. If the death pathway could be activated extrinsically, there would be the potential for a synergistic toxic effect on tumor cells. In addition, if the death pathway was identified as being nonfunctional for any of the reasons outlined, the restoration of function could reactivate the immune system anti-tumor response. Current strategy is to decipher the complexities of death pathways in order to identify key effector molecules and their function. As their mechanisms have been elucidated, so has the knowledge that aberrations can occur at many points along the pathway, interfering with normal function. This has fueled exciting new research attempts to engineer novel therapeutic options. Keywords Apoptosis · Autophagy · Bladder Cancer · Necrosis

Introduction Transitional cell carcinoma (TCC) of the bladder is the second most common malignancy of all genitourinary cancers and the second leading cause of death from cancer of the urinary tract. In U.S., the incidence of bladder cancer has increased during the past two decades. The American Cancer Society estimated that 68,810 new cases and 14,100 cancer related deaths will occur in 2008. Many epidemiologic studies have clearly demonstrated that the incidence of bladder cancer is associated with J.-T. Hsieh (B) Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 2,

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occupational or environmental exposure to several known chemical carcinogens (O’Donnell, 2007). It is also known that smoking is a major risk factor (Pelucchi et al., 2006). An estimated 100,000 youth begin smoking everyday. If the current epidemic continues at the present rate, tobacco will be by far the largest cause of disease burden worldwide. One hundred million deaths from tobacco occurred in the 20th century and 900 million are projected for the 21st century if smoking patterns continue unabated. It is estimated that tobacco related mortality could rise from 5 million to over 10 million by the year 2025. It is known that tobacco use and exposure to second-hand smoke are responsible for over 30% of all cancer deaths. The incidence of bladder cancer in both male and female is on the rise from the recent years and smoking has been shown to be a major risk factor. Seventy to 80% of bladder cancer patients present with low grade, noninvasive tumors or with superficial papillary tumors confined to mucosa (Hall et al., 2007). Administration of intravesical chemotherapy or immunotherapy often can prolong the progression-free interval after initial transurethral resection; however, bladder cancer will recur in 60% of these patients. As many as 20% of these recurrent tumors may present with a higher grade and invasive properties (Hall et al., 2007). On average 15 to 30% of all patients with bladder cancer are diagnosed with invasive tumors, and half of those treated with a local modality will relapse with metastatic disease within 2 years (Hall et al., 2007). In general, bone, live and lung are the three most common metastatic sites for bladder cancer. Like other cancer types, the mortality of this disease is due to metastatic disease. The phenotypic heterogeneity of bladder cancer in large part reflects underlying differences in molecular characteristics and it is now clear that bladder cancer develops along complex molecular pathways. Intensive research over the last decade includes both cellular and molecular studies of bladder cancer. This has provided great insight into the biology of this cancer and is now beginning to shape clinical practice. One of the most exciting developments in molecular and cell biology has unveiled molecular defects in cancer cells. Targeting those defects using molecular biology technologies becomes an active research area of cancer therapy. Technologies such as high-throughput transcript profiling, genomic microarray technologies, and proteomics, have facilitated the comprehensive identification and understanding of molecular pathways and targets that are active in bladder cancer. The ultimate goal of cancer research is to develop effective regimens to achieve cure of disease. Despite of many chemotherapeutic developed from past decades most of them are targeting the machinery of DNA replication in malignant cells, which is not always effective. Further understanding of the processes that contribute to cell death and survival in cancer cells has opened options for developing new class of therapeutic agents, which are critical in reducing morbidity and mortality from cancer. The extensive characterization of the regulation of programmed cell death such as apoptosis has allowed the development of targeted therapies designed to induce apoptotic cell death and biomarkers that can measure therapy-induced apoptosis in patients. Given the wide variety of genetic and epigenetic defects that can suppress apoptosis in most cancers, recent discoveries of non-apoptotic mechanisms in cancer cells offer

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new targets for cancer therapy. Thus, the goal of this chapter is to discuss the regulation and significance of programmed cell death in bladder cancer and the potential application for future therapeutic development.

Different Types of Programmed Cell Deaths Apoptosis Apoptosis is a form of programmed cell death in multi-cellular organisms. It is one of the main types of programmed cell death (PCD) and involves a series of biochemical events leading to a characteristic cell morphology and death, in more specific terms, a series of biochemical events that lead to a variety of morphological changes, including blebbing, changes to the cell membrane such as loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation (Kroemer et al., 2005). Processes of disposal of cellular debris whose results do not damage the organism differentiate apoptosis from necrosis (Table 2.1). In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis, in general, confers advantages during an organism’s life cycle. For example, the differentiation of fingers and toes in a developing human embryo occurs because cells between the fingers apoptose; the result is that the digits are separate. Between 50 billion and 70 billion cells die each day due to apoptosis in the average human adult. For an average child between the ages of 8 and 14, approximately 20 billion to 30 billion cells die a day. In a year, this amounts to the proliferation and subsequent destruction of a mass of cells equal to an individual’s body weight. Thus, agent(s) with the ability of inducing apoptosis in cancer could be considered a front-line therapeutic agent. Currently, apoptosis is known to be initiated by the interplay of intrinsic and extrinsic signals that converge into a common downstream effector pathway (McKnight et al., 2005). Signals for apoptosis arising from within the cell (intrinsic) largely depend on the release of molecules that are normally sequestered in the mitochondria, including cytochrome-c. In contrast, the extrinsic pathway involves Table 2.1 Comparison between apoptosis, autophagy and necrosis Apoptosis Morphology

Chromatin condensation, Nuclear and cytoplasmic blebbing, Apoptotic body Key regulators caspase cytochrome C Bcl-2 family Pathway Irreversible Immune reaction Suppression

Autophagy

Necrosis

Cytoplasmic vesicles, Intact nuclear and cytoplasmic membrane

Swollen organelles, Cytoplasmic membrane rupture

Beclin/ATG6 LC3/ATG8 ATG1, 5, 7 Reversible Inflammatory

PARP RIPK1 TRAF2 calpains Irreversible Inflammatory

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interaction of a class of cell surface molecules known as death receptors with their respective ligands. This mechanism is one of the ways by which cytotoxic immune cells can eliminate tumor cells and protect the host against the development of new cancers. Alterations in apoptosis pathways are important not only in ontogenesis but also tumorigenesis, as they allow cancerous cells to survive longer, resist normally harmful stresses, and become more invasive (Reed, 1999). Bladder cancer has been shown to resist programmed cell death both with altered expression of pro- and anti-apoptotic proteins (McKnight et al., 2005; Shariat et al., 2003, 2004a).

Autophagy Autophagy (or autophagocytosis) is a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery (Table 2.1). It is a highly regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. It is a major mechanism by which a starving cell reallocates nutrients from unnecessary processes to more-essential processes. In general, cytoplasmic material, including organelles, is segregated into a double membrane-bound vesicle and then delivered to the lysosomal compartment for degradation (Eskelinen, 2005). There are three major forms of autophagy, i.e. macro-autophagy, micro-autophagy and chaperone-mediated autophagy (Cuervo, 2004). Two main functions have been proposed for the autophagic process. Firstly, autophagy is a short-term stress response in nutrient-limited conditions or aminoacid deficiency. It was coined to describe how a cell, facing starvation, degrades intracellular components to obtain nutrients for survival. Secondly, it is suggested that autophagy plays a role in type II, or autophagic, cell death (Gozuacik and Kimchi, 2004). Autophagy has been observed in cancer cells faced with a variety of metabolic and therapeutic stresses. In general, several conditions have been demonstrated to induce autophagy, such as interruption of growth factor signaling pathways (Lum et al., 2005), activation of mitogen-activated protein kinase signaling (Corcelle et al., 2006), inhibition of proteasomal degradation (Pandey et al., 2007), the accumulation of intracellular calcium (Hoyer-Hansen et al., 2007), and endoplasmic reticulum stress (Ogata et al., 2006). Although the number of stress-activated pathways linked to autophagy seems to be increasing rapidly, direct biochemical associations between these stress signals and the known mammalian autophagy genes, which direct the complex structural changes associated with autophagy, have not been fully characterized (Table 2.1). One recent report suggests that the accumulation of reactive oxygen species (ROS), which may be a common result of many of these cellular responses to stress, can directly activate autophagy. It has been demonstrated that accumulated ROS following nutrient withdrawal lead to inactivation of the cysteine protease (i.e., ATG4), leading to accumulation of the LC3-phosphoethanolamine precursor that is required for the initiation of autophagosome formation (Scherz-Shouval et al., 2007). Further characterization

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of the interface between stress signals and the autophagy machinery may unearth new targets for drug development in cancer. Once activated, persistent autophagy, which depletes the cell of organelles and critical proteins, can lead to a caspase-independent form of cell death (Kim et al., 2006). In cells undergoing persistent autophagy, however, hallmarks of apoptosis, such as caspase activation (Gonzalez-Polo et al., 2005), necrotic cell death, organellar swelling, and plasma membrane rupture, can often be observed (Bursch et al., 1996), suggesting that autophagy is a dynamic that could further lead to apoptosis or necrosis. In contrast to other irreversible modes of cell death, autophagy can be activated in a more transient fashion in cancer cells and can paradoxically contribute to cell survival. Autophagy promotes survival by serving as an intracellular mechanism by which cells dispose of damaged organelles and proteins (Gu et al., 2004; Iwata et al., 2006) and recycle macromolecules to maintain bioenergetics (Lum et al., 2005). Using cell lines deficient in apoptosis, several studies have shown that autophagy activated in response to growth factor withdrawal, hypoxia, or insufficient tumor angiogenesis allowed tumor cells to survive these stresses (Lum et al., 2005; Degenhardt et al., 2006). Genetic depletion of essential autophagy genes in the face of these stresses enhanced cell death. However, in these cases, once the stress was removed autophagy was down regulated, which indicate that, unlike apoptosis or necrosis, autophagy is a reversible phenomenon. Current research efforts are focused on identifying critical regulators that control the down-regulation of autophagy because inhibition of these regulators may enhance autophagic cell death and convert a cell survival response into a cell death process.

Necrosis Necrosis plays a critical role in tissue/organ injury produced by a variety of insults. This type of cell death occurs in numerous human diseases, such as acute myocardial infarction, acute renal and liver failure, and stroke. Necrotic cell death is often referred to as unscheduled cell death, implying that within a multi-cellular organism it is an unregulated process. The disruption of plasma membrane that is characteristic of necrotic cell death leads to the spillage of intracellular proteins that activates a damage response from the host immune system (Zeh and Lotze, 2005). This brisk inflammatory response and immune amplification of the damage signal is in sharp contrast to apoptotic cells that are silently removed by tissue macrophages. Thus, necrosis was viewed as strictly a pathologic form of cell death, rather than a physiologically programmed process. Increasing evidence suggests that, much like apoptosis, specific genes have evolved to regulate necrotic cell death (Golstein and Kroemer, 2007; Festjens et al., 2006). It has been difficult to characterize the essential regulators of necrotic cell death in the absence of apoptosis. Much of the literature describing necrotic cell death consists of experiments done in cell lines subjected to death receptor ligation (Eguchi et al., 1997; Goossens et al., 1995; Boone et al., 2000) or in neurons subjected to ischemia or glutamate excitotoxicity (Ankarcrona et al., 1995; Zhu et al., 2005).

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Because these interventions can also activate apoptosis, concomitant treatment of cells with pan-caspase inhibitors pushes cells into necrotic cell death (Vercammen et al., 1998). Genetic studies have identified death receptor adaptors, including receptor-interacting protein kinase 1 (RIPK1) and the tumor necrosis factor (TNF) receptor–associated factor 2 (TRAF2), as essential regulator of death receptor– induced necrotic cell death (Holler et al., 2000; Chan et al., 2003; Lin et al., 2004). Cells deficient in RIPK1 or TRAF2 are protected from necrotic cell death when treated with Fas ligation and caspase inhibitors or hydrogen peroxide alone (Shen et al., 2004). Activation of RIPK1 leads to its translocation to the inner mitochondrial membrane disrupting the association between the cyclophilin D and adenosine nucleotide translocator, resulting in ATP depletion and the accumulation of ROS (Temkin et al., 2006).

The Relationship Between Apoptosis, Autophagy and Necrosis In tumor cells genetically deficient in the ability to undergo apoptosis, autophagy was activated and protected the cell from metabolic compromise and death. Autophagy can often be observed in cells undergoing necrosis. These findings suggest that, within the same tumor necrosis, autophagy and apoptosis may coexist, and the relative contributions of these three processes can dictate the trajectory of tumor growth or regression and the host response. For example, metabolic and therapeutic stresses lead to acute NAD+ and ATP depletion (Buzzai et al., 2005) accompanied by increased intracellular calcium and ROS. Cells that do not adapt to these changes undergo necrotic cell death. The activation of stress regulators, such as AMP-activated protein kinase (AMPK), allows cells to acutely survive these changes. AMPK-dependent phosphorylation results in the inhibition of mammalian target of rapamycin (mTOR) (Feng et al., 2005), which inhibits autophagy. AMPK-dependent phosphorylation also activates p53, which can lead to autophagy or apoptosis in different cells within the same tumor, through the activation of Bax and Bak, the cytoplasmic release of cytochrome c, and the activation of caspases. Unlike apoptosis or necrosis, stress-induced autophagy can lead to autophagic cell death or to cell survival. Taken together, these three deaths appear to interlink even though each death mechanism has its unique pathway.

Signaling Pathways to Death Fas (CD95) Fas is an established death receptor. When activated by Fas ligand (FasL), Fas activates downstream signals that induce apoptosis (Nagata, 1997). Svatek et al. reported that soluble form of Fas was present in the cell lysate and supernatant of high-grade bladder cancer cell lines suggesting that it is likely to be produced and released by bladder cancer cells (Svatek et al., 2006). In vivo, urinary soluble Fas was an independent predictor of bladder cancer presence and invasiveness

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in patients with a past history of non-muscle-invasive disease. Urinary soluble Fas outperformed NMP22 for both bladder cancer detection and staging. These observations suggest that soluble Fas may play a role in the local immunosuppression in bladder cancer associated with tumor development, biologic aggressiveness, and/or progression. Immunohistochemical analysis of the Fas system on 123 bladder cancer specimens and 30 benign bladders, Fas was expressed in 90% of benign specimens compared to 58% of cancer specimens (Yamana et al., 2005). In addition, decreased Fas expression was associated with higher tumor stage, grade, and disease-specific mortality in univariate analysis (Yamana et al., 2005).

Caspase-3 Caspase-3 is a downstream apoptosis effector molecule that causes cellular disassembly (Arai et al., 2005). Giannopolou et al. (2002) studied caspase-3 expression in 53 patients with bladder cancer and did not find a correlation between caspase3 expression and tumor grade or stage. Burton et al. (2000) evaluated caspase-3 expression in 34 patients with carcinoma in situ, of which 41% developed invasive bladder cancer. They reported that activated caspase-3 over-expression was associated with higher rates of disease invasiveness. Conversely, a more recent study by Karam et al. (2007a) involving 226 consecutive patients treated with radical cystectomy specimens reported that 49% of the patients had loss of caspase-3 expression, which was associated with higher pathologic grade and stage, and presence of lymph node metastasis. Moreover, loss of caspase-3 was an independent predictor of bladder cancer-specific survival after radical cystectomy (Karam et al., 2007a).

Bcl-2 Bcl-2 is an anti-apoptotic protein present in intracellular membranes, and controls cytochrome c location, caspase status, and ion channels involved in apoptosis (Kluck et al., 1997). Over-expression of bcl-2 was found in 32% of radical cystectomy specimens, and correlated with higher pathologic stage, and disease recurrence and cancer-specific mortality rates (Karam et al., 2007a). In agreement with these findings, two other groups reported that over-expression of bcl-2 was associated with worse all-cause survival (Kluck et al., 1997; Ong et al., 2001) and lower response rates to chemotherapy (Ong et al., 2001).

Survivin Survivin is a newly discovered member of the Inhibitor of Apoptosis family, and inhibits apoptosis, at least partly, by blocking downstream caspase activity (Kong et al., 1998; Ambrosini et al., 1997). Survivin also controls mitotic progression and induces changes in gene expression that are associated with tumor cell invasiveness (Akhtar et al., 2006; Salz et al., 2005). Survivin mRNA is selectively expressed during embryonic and fetal development (Altieri, 2003) becomes undetectable or expressed at low levels in most differentiated normal adult tissues, and

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is over-expressed in humans cancers (Adida et al., 1998). In bladder cancer, urinary levels of Survivin gene activation, both at the protein and the mRNA level, have been shown to be associated with cancer presence, higher tumor grade, and advanced pathologic stage (Smith et al., 2001; Shariat et al., 2004b; Schultz et al., 2004). Survivin over-expression was present in 63% of bladder cancer specimens, and was associated with higher pathologic stage, presence of lymphovascular invasion and lymph node metastasis, bladder cancer recurrence and bladder cancer-specific survival in patients treated by radical cystectomy (Karam et al., 2007a). In addition, the proportion of specimens with survivin over-expression increased gradually from non-muscle invasive bladder cancer to advanced bladder cancer to metastatic lymph node tissue (Shariat et al., 2007).

EGCG [(-) epigallocatechin-3-gallate] Green tea is a commonly used beverage worldwide. It is believed to have chemopreventive potential. EGCG is the major catechin in green tea (Khan et al., 2006). EGCG was studied in T24 bladder cancer cell line (Qin et al., 2007). EGCG inhibits bladder cancer cell viability in a dose and time dependent manner. In addition, EGCG induces apoptosis in T24 cells. This was evident through the effect of EGCG on cell morphology, and flow-cytometry with annexin V and Propidium Iodide staining. Furthermore, EGCG results in activation of caspase-3 and cleavage of PARP (poly-ADP ribose polymerase). EGCG also affects the PI3K/Akt pathway. It down-regulates the expression of phospho-PDK1 and phospho-Akt without affecting total Akt expression. The effects of EGCG extend to the bcl-2 family of proteins resulting in decrease of Bcl-2 and Bcl-xL, and increase in Bax and Bad. Activation of the PI3K/Akt pathway has been implicated in bladder cancer pathogenesis (Wu et al., 2004). The ability of EGCG to influence PI3K/Akt and apoptosis make this compound an attractive candidate for bladder cancer chemoprevention and treatment.

Curcumin Curcumin (diferuloylmethane), a curry pigment extracted from Curcuma longa, suppresses NF-κB activation (Singh and Aggarwal, 1995). NF-κB is a heterodimer of p50, p65, and IκBα. NF-κB is inactive when cytoplasmic, but when IκBα becomes phosphorylated and subsequently degraded, this complex translocates to the nucleus, binding to specific DNA sequences, resulting in transcription of multiple genes involved in apoptosis, proliferation and metastasis (Aggarwal, 2004). Curcumin has been investigated in vitro in 2 bladder cancer cell lines (RT4V6 and KU-7) (Kamat et al., 2007). The former is sensitive to, while the latter is resistant to IFN-α and TRAIL. At low concentrations, curcumin alone inhibited the growth of both cell lines and resulted in DNA fragmentation. When combined with gemcitabine, paclitaxel, TNF, and TRAIL, curcumin improved the effects of these

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agents, resulting in potentiation of apoptosis, as indicated by the Annexin V assay. Curcumin mediates its apoptotic effects through caspase-3 activation and PARP cleavage. Curcumin also suppresses NF-κB activation resulting from treatment on bladder cancer cell lines with TNF, gemcitabine, and cigarette smoke.

Silibinin Silibinin, a flavonolignan, is a natural product that induces apoptosis in bladder cancer cell lines in vivo and in vitro. Silibinin activates p53 using the ATM-Chk2 pathway, resulting in caspase-dependent apoptosis in bladder cancer cell line RT4. Silibinin increased levels of cleaved caspase-3, caspase-9, and PARP; it also activates JNK, resulting in caspase-2 activation, cytochrome c release, and subsequent apoptosis. In addition, silibinin results in p21 cleavage mainly via a caspase-2 dependent mechanism (Tyagi et al., 2006). Silibinin was recently evaluated in a mouse preclinical model of bladder cancer. A bladder cancer mouse model was developed using the tobacco carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine. Silibinin given orally prevented development of bladder cancer in some mice, while in others it arrested tumor progression at the level of mucosal dysplasia, resulting in a low number of invasive lesions. In this animal model, the effects of Silibinin were mediated by increased apoptosis. A decrease in Erk1/2 phosphorylation, survivin, cyclin D1, and NF-κB p65 levels was also noted with Silibinin treatment (Tyagi et al., 2007). Oral Silibinin was also studied in RT4-human bladder cancer mouse xenografts. Silibinin decreased tumor growth, accompanied by tumor cell apoptosis in vivo with increasing p53 and activated caspase-3 levels. In addition, it caused p53independent decrease in survivin tissue protein expression and prevented survivin nuclear localization. Interestingly, silibinin also decreased circulating tumor-related plasma survivin levels (Singh et al., 2008).

Vitamins Vitamins C and K3 have been studied in bladder cancer cell lines (UMUC-14, 253 B-J, and RT4) in vitro and in vivo in a mouse model. Vitamins C and K3, used separately, induces modest decrease in cell growth and apoptosis. When combined, a synergistic effect was noted without a decrease in the mouse weights. In addition, the use of vitamins C and K3 potentiated the anti-tumor effects of gemcitabine, a known chemotherapeutic agent against bladder cancer. In a mouse model of bladder cancer using UMUC-14, the combination of vitamins C and K3 resulted in improved tumor growth inhibition when combined with gemcitabine. In vivo however, the inhibitory effects of these agents resulted mainly from an antiproliferative mechanism, rather than apoptosis (Kassouf et al., 2006). These results suggest that vitamins C and K3 could be used to improve the activity of chemotherapeutic agents, and could allow the use of lower concentrations of these potentially toxic agents.

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Epigenetic Modifiers Epigenetic mechanisms induce changes in gene expression without affecting the DNA sequence itself. These changes included DNA methylation and histone modifications (e.g. acetylation, methylation, phosphoryation and sumoylation)-both of which could be reversed with drug treatment (Herman and Baylin, 2003). Genes silenced by DNA hypermethylation can be reactivated by small molecule– DNA hypomethylating agents (also known as DNMT inhibitors) (Goffin and Eisenhauer, 2002). These agents are basically structural analogs of the nucleoside deoxycytidine. After being phosphorylated, these compounds can be incorporated into DNA or RNA and then inhibit DNMT activity by forming a covalent bond. Currently, five DNMT inhibitors have been used in preclinical animal models/clinical trials, namely 5-azacytidine (5-Aza), 5-aza-2 -deoxycytidine (decitabine), dihydro5-azacytidine (DHAC), arabinofuranosyl-5-azacytosine (fazarabine) (Goffin and Eisenhauer, 2002) and zebularine with oral administration (Cheng et al., 2003). In general, these compounds can elicit a profound demethylation of the gene promoter of cyclin kinase inhibitor such as p16 gene, which is able to cause cell cycle arrest or apoptosis (Srivastava et al., 2007). Histone deacetylase inhibitors (HDACIs) can increase acetylation of histones and various other proteins by inhibiting histone deacetylase activity. HDAC, along with their counterparts, histone acetyl transferases, regulate the status of histone acetylation and thus are involved in transcriptional regulation. It is known that HDAC are over-expressed in many cancers, and the death-inducing capability of different HDAC inhibitors correlates with their HDAC-inhibitory potency, it is widely accepted that the cell death-inducing function of HDAC inhibitors is due to their ability to inhibit HDAC activity. HDACIs can be classified into several groups, including (i) short-chain fatty acids, such as sodium butyrate; (ii) hydroxamic acids, such as suberoylanilide hydroxamic acid (SAHA) (Richon et al., 1996); (iii) cyclic tetrapeptides, such as FK228; and (iv) benzamides, such as MS-275. HDACIs have been documented to cause Gl and/or G2 phase cell-cycle arrest, cell differentiation and/or apoptotic induction and proliferation inhibition in virtually all cancer types, including epithelial (neuroblastoma, glioma, melanoma, lung, breast, pancreas, ovary, colon, prostate and bladder) and hematological (lymphoma, leukemia and multiple myeloma) tumors (Kelly et al., 2002). Shao et al. (2004) reported that SAHA induced not only apoptosis but also autophagy in cancer cells, suggesting that epigenetic modifiers could have multiple functions in modulating different cell deaths. With respect to a reciprocal interaction between different epigenetic machineries, it is very likely that the combination of epigenetic modifiers can achieve better effect than single agent. Our laboratory has reported on the DNA demethylating agent 5-Azacytidine, and the histone deacetylase inhibitors Trichostatin A and FK228 (Depsipeptide, also known as Romidepsin) in vitro and in vivo in subcutaneous and orthotopic models of bladder cancer. We found that the combination of FK228 and 5-Azacytidine results in a dose and time-dependent G2/M cell cycle arrest as well as apoptosis, with FK228 being the most potent agent.

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In vivo, intravenous injection of FK228 resulted in significant decrease in tumor growth without affecting mouse weights (Karam et al., 2007b). In a more recent study, we screened a chemical library and identified a tranilast analogue, DMCA (2,3-dimethoxycinnamoyl azide), which could potentiate the effects of FK228. We investigated these compounds as single agents and in combination in T24, UMUC3 and TCC-SUP bladder cancer cell lines. The combination results in profound cell death, mainly through apoptosis as evident by PARP cleavage. This process appeared to be p53-independent. FK228, but not DMCA, up-regulated p21 expression, which correlated with the cell cycle arrest induced by these drugs. Interestingly, FK228, but not DMCA, increase the levels of acetylated H3, while the combination results in higher levels of acetylated H3 compared to FK228 alone. This elevation of acetylated H3 levels resulted was responsible for the induction of p21 expression. When used in vivo in a subcutaneous bladder cancer mouse model, DMCA alone did not cause any decrease in tumor growth, but when combined with FK228, the reduction of tumor growth was more significant than that produced by FK228 alone. Again, the combination did not result in significant toxicity in mice (Fan et al., 2008).

Arsenic Compound Arsenic compounds or arsenicals are well-known toxic and carcinogenic agents (Wang et al., 2002). The genotoxic and co-genotoxic effects of inorganic arsenicals are well documented in mammalian systems, both in vitro (Jha et al., 1992) and in vivo (Tice et al., 1997). A number of hypotheses have been formulated to explain the genotoxicity of arsenicals, and it appears that a variety of mechanisms may be involved in the toxicity of these compounds (Raisuddin and Jha, 2004). The toxic effects of arsenic that are of most concern to humans are those that occur from chronic, low-level exposure. Arsenic is primarily associated with various human malignancies, including bladder cancer. In a recent study, sodium arsenite can cause calmodulin (CaM)-regulated serine/threonine kinase (DAPK) promoter gene hypermethylation and decrease of DAPK protein expression in an immortalized normal urothelial cell line (i.e., SV-HUC-1) (Chai et al., 2007). DAPK is a 160-kD protein kinase that mediates cell death. The activated forms of DAPK are capable of inducing two distinct cytoplasmic events characteristic of programmed cell death, including membrane blebbing and the formation of autophagic vesicles (Inbal et al., 2002). A survey of cancer cell lines derived from various human tumors has shown that the mRNA and protein expression of DAPK were frequently lost (Kissil et al., 1997). DAPK mRNA and protein expression levels were below detection limits in 80% of B cell lymphoma and leukemia cell lines and in 30–40% of cell lines derived from bladder carcinomas, breast carcinomas, and renal cell carcinomas (Kissil et al., 1997; Katzenellenbogen et al., 1999), suggesting that loss of DAPK expression provides a positive selective advantage during carcinogenesis. This study provides a new insight for the understanding of the role of PCD in arsenic-induced carcinogenesis of urothelial cells.

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Bacillus Calmette-Guerin (BCG) Intravesical bacillus Calmette-Guerin (BCG) is considered a very effective immunotherapy for superficial TCC. Based on its well-defined activity as an immune adjuvant, it has long been hypothesized to exert anti-tumor activity by helping to generate a tumor specific immune response, which in turn eliminates tumor. Studies from a number of investigators have demonstrated that intravesical BCG results in a significant cell mediated response in the bladder characterized by the recruitment and expansion of T lymphocytes, and the elicitation of a series of immune associated cytokines. However, studies of BCG anti-tumor activity have also suggested a direct cytotoxic effect of BCG on tumor cells (Fontana et al., 1999). It appears that BCG treatment of bladder cancer cell lines such as T24 and 253J does not induce apoptosis (Chen et al., 2007). Instead, BCG may induce autophagy in bladder cancer cells because the induction of autophagy has been shown to modulate the immune response to BCG (Gutierrez et al., 2004), as well as enhance antigen presentation to CD4 effector and perhaps regulatory T cells (Munz, 2006).

Beclin-1/ATG6 The proteins involved in the autophagic process in yeast have been isolated and characterized (Mizushima, 2007), which has been named as autophagy-related gene (ATG). Many mammalian homologues of yeast ATG genes have been identified and characterized as well, suggesting that the molecular mechanisms of autophagy have been conserved from yeasts to mammals. The beclin-1 (60-kDa coiled-coil protein) is a mammalian homologue of yeast ATG6/Vps30 with a role in mediating autophagy (Liang et al., 1999). The protein appears to be essential for autophagosome formation and it binds to class III PI3K (i.e., Vps34) and other protein such as Vps15 and ATG14, which regulate autophagosome formation (Kihara et al., 2001). Moreover, it has been shown that this PI3K is required for macroautophagy in nutrient-starved cells (Eskelinen et al., 2002), for normal lysosomal enzyme sorting and protein trafficking in the endocytic pathway (Petiot et al., 2003). Beclin-1 is mono-allelically deleted in human various cancers and is expressed at reduced levels in tumors (Liang et al., 1999; Aita et al., 1999). The deletion of this region is found in up to 40% of human prostate cancers, 50% of human breast cancers, and 75% of human sporadic ovarian cancers (Liang et al., 1999; Aita et al., 1999). In the early stages of carcinogenesis, activation of autophagy may block tumor growth, while in the late stages; it favors survival of cells in lowvascularized tumors and removal of damaged intracellular macromolecules after anticancer treatments (Cuervo, 2004). Increased expression Beclin-1 expression or knocking-down endogenous Beclin-1 expression in cancer cell has been correlated with the number of autophagy vesicles (Ding et al., 2007). The enforced expression of this autophagy gene not only promotes nutrient deprivation-induced autophagy in human cancer cells but also inhibits their tumor-forming potential, indicating that autophagy may be a fundamental mechanism for preventing

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the deregulated growth of tumor cells (Liang et al., 1999). On the other hand, ectopic over-expression of Beclin-1 in breast cancer cells with monoallelic deletions of the beclin-1 gene can inhibit cell proliferation, colony formation in soft agar and tumorigenesis in nude or SCID mice (Furuya et al., 2005), indicating that Beclin-1 can function as a tumor suppressor. However, Beclin-1 accumulation in autophagosomes in the initial stages of type II PCD (Yu et al., 2004) suggests that its role as a tumor suppressor is most probably related to regulation of macroautophagy.

LC3/ATG8 During the autophagic process, a cup-shaped structure, also referred to as an isolation membrane or preautophagosome, forms in the cytosol. It engulfs cytosolic components, including organelles, and later become enclosed to form an autophagosome. The autophagosome subsequently fuses with a lysosome, enabling the intraautophagosomal components to become degraded by lysosomal hydrolytic enzymes. Most of the characterized ATG gene products, including ATG3, ATG5, ATG7, ATG10, ATG12, and LC3, are involved in two ubiquitylation-like modifications of target proteins. LC3 was first isolated as a microtubule-associated protein. ATG12conjugation and LC3-modification (ATG8-lipidation in yeast), which are essential for the dynamic process of autophagosome formation. Pro-LC3 is processed to its cytosolic form, LC3-I, exposing a carboxyl terminal glycine (Kabeya et al., 2000). LC3-I is also activated by ATG7, transferred to ATG3, a second E2-like enzyme, and modified to a membrane-bound form, LC3 (Tanida et al., 2001, 2002). LC3-II is localized to preautophagosomes and autophagosomes, which makes this protein an autophagosomal marker in cells by examining the re-localization pattern of LC3 using GFP-LC3 transfection or the presence of conjugated form of endogenous LC3 using western blot analysis (Li et al., 2008). In addition, a recent study demonstrates that reduced LC3 expression in cancer cells sensitizes autophagic cells to undergo apoptosis in presence of endoplasmic reticulum stress inducers (Ding et al., 2007), indicating that the critical role of LC3 in maintaining autophagy status. The outcome of this study provides a strong rationale for developing a new strategy of cancer therapy.

mTOR The mammalian TOR (mTOR), a serine/threonine protein kinase, is a key regulator of cell growth, proliferation, cell motility and cell survival since this kinase is able to regulate protein translation by phosphorylating S6K1 and 4E-BP1. Now, increasing evidence suggests that its deregulation is associated with human diseases, including cancer and diabetes. The mTOR pathway integrates signals from nutrients, energy status and growth factors to regulate many processes, including autophagy, ribosome biogenesis and metabolism. In general, inducers such as growth factors, amino

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acids, glucose, energy status, and many forms of stress (e.g. osmotic stress, DNA damage) have been well documented for mTOR induction (Kotoulas et al., 2006; Botti et al., 2006). Under conditions of energy deprivation that increase the AMP/ATP ratio, AMPK becomes active and phosphorylates TSC2 (tuberin) to stimulate its GAP activity (Inoki et al., 2003) mTOR form a heterodimer with the TSC1 (hamartin) and TSC2 (tuberin) (Long et al., 2005). Both are known as tumor suppressors with GTPaseactivating protein (GAP) for the Ras-family GTP-binding protein Rheb (Saucedo et al., 2003; Stocker et al., 2003; Zhang et al., 2003; Tee et al., 2003; Garami et al., 2003) that is an upstream integrator for mTOR pathway (Tapon et al., 2001; Gao et al., 2002). Thus TSC1/2 activation leads to inhibition of Rheb and mTOR as well. Rapamycin induces autophagy in yeast and metazoans, and TOR appears to regulate autophagy through several genes that directly mediate autophagosome formation, such as the autophagy-specific kinase ATG1, and the ubiquitin-related LC3, a molecule that localizes to and promotes the expansion of the growing autophagosome (Reggiori and Klionsky, 2005). Finally, the assembly and activity of a multi-protein complex containing hVps34 and Beclin-1, required for autophagosome induction, is regulated by nutrient conditions and, by inference, TOR activity (Pattingre et al., 2005). In this moment, the mTOR status in bladder cancer is still largely unknown. However, deletions in chromosome 9 have long been considered the most frequent chromosomal aberration in bladder cancer. Various laboratories have reported loss of microsatellite markers across the entire length of chromosome 9 in 30 to 70% of the studied cases (Cairns et al., 1993a, b, 1994; Linnenbach et al., 1993; Miyao et al., 1993; Devlin et al., 1994). Loss of chromosome 9 has been the only chromosome loss at the early tumor stages Ta and T1 while at later stages loss of other chromosomes was detected concomitantly with loss of chromosome 9. In addition, several mutations have also been associated with TSC1 gene. Based on these studies, very likely, mTOR is presumably over-activated in bladder cancer.

Calpains Calpains are Ca2+ -dependent cysteine proteases that play an important role in cell differentiation and in apoptosis/necrosis. At least two well-known genetic disorders and one form of cancer have been linked to tissue-specific calpains. For example, the mammalian calpain 3 (also known as p94) is the gene product responsible for limb-girdle muscular dystrophy type 2A (Richard et al., 1995; Ono et al., 1998), calpain 10 has been identified as a susceptibility gene for type II diabetes mellitus, and calpain 9 has been identified as a tumor suppressor for gastric cancer. Calpains have been implicated in the mediation of necrosis in different cellular models subjected to diverse insults and in a limited number of in vivo models (Harriman et al., 2000; Schumacher et al., 1999; Liu and Schnellmann, 2003; Waters et al., 1997). Although these studies suggest that calpains play a critical role in necrosis, it is not clear which calpain isoform(s) are involved, how calpains are activated, which intracellular targets are modified by calpains, and how proteolysis of calpain targets leads to cell

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death. In bladder cancer, the role of calpain in necrosis or apoptosis is still largely unknown. However, calpain has been suggested to play an important role in mediating cell motility, suggesting that calpain can be a potential therapeutic target in bladder cancer.

Poly-ADP Ribose Polymerase (PARP) PARP cleavage has been characterized a hallmark of apoptosis. However, cells deficient in the pro-apoptotic Bcl-2 genes Bax and Bak that served as model systems to study necrotic cell death because of their inability of undergoing apoptosis when treated with alkylating chemotherapy, an immediate metabolic crisis develops characterized by NAD+ depletion followed by ATP depletion. A key mediator of this process is poly-ADP ribose polymerase (PARP), a nuclear protein whose activation by DNA damage rapidly depletes the cell of NAD+ , the essential cofactor for aerobic glycolysis. This imposes a transient inhibition of glycolysis, and cells dependent on glycolysis for ATP production, such as cancer cells, die by necrosis. This may explain how alkylating chemotherapy is selective for cancer cells even when given at very high doses (Zong et al., 2004). PARP-dependent necrosis is also associated with TNF receptor–independent activation of RIPK1, TRAF2, and downstream effector c-Jun NH2-terminal kinase (Xu et al., 2006). The bioenergetic crisis that occurs with acute NAD+ and ATP depletion in glycolytic cells is associated with the accumulation of high concentrations of intracellular calcium (Xu et al., 2001) and ROS (Lin et al., 2004), which in turn cooperate to activate Ca2+-dependent cytosolic proteases calpains (Syntichaki et al., 2002). Activated calpains cleave Ca2+ extrusion channels (Bano et al., 2005) and permeabilize lysosomes (Yamashima et al., 2003), leaking executioner cathepsins (Yamashima et al., 1998). High intracellular calcium concentrations and ROS also activate phospholipase A2 (El Mahdani et al., 2000; Hayakawa et al., 1993). Proteolysis and lipid peroxidation ensues, leading to widespread membrane permeabilization and irreversible necrotic cell death.

Therapeutic Strategies Based on Different Programmed Cell Deaths Current strategies to use the death receptor pathway and restore its functionality are under investigation as putative novel treatment modalities. Although there is a large number of phase 1 trials of TRAIL receptor targeting, most approaches are not yet clinically relevant. Administration of the recombinant death receptor ligands FAS-L and TRAIL have been shown in vitro to induce apoptosis in bladder cancer cell lines (Micheau et al., 1997). TRAIL demonstrates preferential apoptosis induction in tumor cells, while sparing most normal cells. A synergistic effect was evident when TRAIL was co-administered with various chemotherapeutic agents, sensitizing previously resistant cells to a responsive phenotype following chemotherapeutic pretreatment (Shankar et al., 2004). Radiotherapy followed by rTRAIL resulted in complete

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eradication of established tumor with prolonged survival (Shankar et al., 2004). Unfortunately systemic administration of FAS-L has been shown to induce severe liver toxicity in rats, limiting its introduction into clinical trials. However, rTRAIL appears to lack these severe side effects and it has been shown to be relatively safe when administered to nonhuman primates (Ashkenazi et al., 1999). In addition to rTRAIL administration, another approach has involved the development of monoclonal antibodies to the TRAIL receptors DR4 and DR5, which may be more specific, avoiding competitive interaction with decoy receptors (Yagita et al., 2004). Using an alternative approach to death ligand administration FAS-L gene therapy has recently shown success, restoring FAS mediated apoptosis to previously resistant bladder cancer cell lines (Sudarshan et al., 2005) which has been tested clinically in combination with doxorubicin for bladder cancer patients (Mizutani et al., 1997). Currently the only death ligand-based treatment in clinical practice is recombinant TNF, which is administered regionally for the treatment of limb sarcoma. Higher systemic doses cannot be used because they are ineffective and induce severe, systemic sepsis-like toxicity. The introduction of other death ligand therapies into clinical trials has been slow to date, largely due to concerns regarding potentially severe systemic and organ specific toxicity. However, a number of phase I trials of treatment for advanced solid malignancies are now in progress. Interestingly immunotherapeutic treatments currently in use, including bacillus Calmette-Guerin for superficial bladder cancer, may already be operating via a TRAIL mediated pathway (Ludwig et al., 2004). Certain immune mediating cytokines, such as the interferon family, have been shown to induce TRAIL expression on immune effecter cells. This indicates that an indirect role for TRAIL is already in place in interferon treatments for renal and other carcinomas. A better understanding of death ligand pathways could potentially lead to the development of less toxic alternative therapies. Strategies to circumvent the toxic side effects could include more localized administration, such as intravesically in the bladder, or the development of more specific receptors that would only target death receptors expressed on tumor cells. Efforts to induce the apoptotic pathway by directly activating tumor expressing death receptors may prove to be futile or ineffective in the presence of over expressed, downstream apoptotic inhibitors such as cFLIP and IAPs. Attempts to restore functionality to death receptor pathways inhibited by the over-expression of anti-apoptotic proteins has centered around trying to decrease their expression through the administration of inhibitory antisense or siRNA molecules. Using antisense oligonucleotides down-regulation of Bcl-2 sensitized renal cancer cells to FAS mediated apoptosis (Kelly et al., 2004) and down-regulation of survivin inhibited cell growth in bladder cancer cell lines (Fuessel et al., 2004). siRNA molecules have down-regulated cFLIP expression and increased sensitivity to TRAIL induced apoptosis in many tumor types, including renal tumors (Brooks and Sayers, 2005). siRNA molecules to other IAPs such as XIAP have also been used effectively in other tumors and a number have already entered phase I trials (Schimmer, 2004).

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Using a pre-clinical model to evaluate experimental therapy, inhibition of therapyinduced autophagy either with short hairpin RNA against the autophagy gene ATG5 or with the antimalarial drug chloroquine enhanced cell death and tumor regression of Myc-driven tumors in which either activated p53 or alkylating chemotherapy was used to drive tumor cell death (Amaravadi et al., 2007). Although numerous other mechanisms for the anti-tumor activity of chloroquine have been proposed (Langer et al., 1999; Kwiek et al., 2004; Schneider et al., 2006), in vitro studies at low micromolar doses achievable in patients have shown that chloroquine causes a dose-dependent accumulation of large autophagic vesicles and enhances alkylating therapy–induced cell death to a similar degree as knockdown of ATG5 (Amaravadi et al., 2007). These and other studies (Katayama et al., 2007; Abedin et al., 2007) suggest that autophagy can promote resistance to DNA-damaging therapy. The regulation of damage-induced autophagy may be different than the regulation of autophagy that is activated in response to bioenergetic compromise or inhibition of growth factor signaling. Although changes in gene expression have been associated with both forms of autophagy (Crighton et al., 2006; Rose et al., 2006), the demonstration that starvation-induced ROS can directly impair ATG4-dependent inhibition of autophagy raises the possibility that autophagy secondary to bioenergetic compromise can be activated in a more rapid energy-efficient fashion using chronically expressed basal autophagy machinery (Scherz-Shouval et al., 2007). Because autophagy is a reversible damage response unlike apoptosis and necrosis, it is not surprising that nearly all therapeutic insults currently used in cancer therapy, including cytotoxic chemotherapy (Amaravadi et al., 2007; Kanzawa et al., 2004; Rez et al., 1996), radiation (Paglin et al., 2001; Yao et al., 2003), kinase inhibitors (Ertmer et al., 2007; Takeuchi et al., 2004) that disrupt growth factor signaling, or hormonal therapy (Bursch et al., 1996), can activate this process. Combining autophagy-inducing therapies with autophagy inhibitors is currently being tested pre-clinically and clinically in several malignancies. Necrosis is always an inflammatory cell death, in contrast to apoptosis and autophagy, in which the balance of anti-inflammatory and pro-inflammatory signals dictates the immune outcome. Extracellular high-mobility group B1 (HMGB1) can bind to RAGE receptors (Dumitriu et al., 2005) and Toll-like receptors (Park et al., 2004) on macrophages, stimulating the secretion of pro-inflammatory cytokines (Scaffidi et al., 2002). The binding of HMGB1 to the RAGE receptor is not sufficient to activate macrophages but instead amplifies the production of proinflammatory cytokines in macrophages activated by other environmental factors, such as DNA-protein complexes (Tian et al., 2007). The necrosis-induced recruitment of macrophages and the subsequent macrophage-associated production of angiogenic and growth factors may be the explanation of how cancers often arise at the site of chronic inflammation (Vakkila and Lotze, 2004), and investigators have suggested that inflammation promoted by tumor necrosis can accelerate tumorigenesis (Iwata et al., 2006). In the context of anticancer therapy aimed at established tumors, however, necrosis-associated damage signals, such as HMGB1, may also be capable of initiating anti-tumor immunity. HMGB1 is an effective immune adjuvant that promotes the migration and maturation of dendritic cells and clonal selection,

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and the expansion and survival of naive T cells. In vivo studies suggest that immunization with HMGB1 can enhance anti-tumor immunity against otherwise poorly immunogenic apoptotic tumors (Rovere-Querini, 2004). The link between necrosis and adaptive immunity may be an explanation for how alkylating chemotherapy can induce long-term remissions and cures in indolent and aggressive lymphomas with well-characterized apoptotic defects.

Conclusions Deciphering the complexities of death receptor pathways has led to a greater understanding of their molecular structure and function. As their mechanisms have been elucidated, so has the knowledge that aberrations can occur at many points along the pathway, interfering with normal function. This has fueled exciting new research attempts to restore functionality to these important receptors or use this deadly inbuilt machinery as a novel therapeutic option.

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Chapter 3

Apoptosis in Carcinogenesis and Chemoherapy of the Uterine Cervix Sakari Hietanen

Abstract The development of cervical cancer is tightly linked to Human papillomavirus (HPV) infection. The expression of HPV oncogenes, notably E6 and E7 disturbs the coordinate regulation of cell cycle and apoptosis. By interfering with these cell processes HPV oncoproteins cause cancer precursor lesions that may progress to invasive cancer. Treatment of invasive cancer is successful with surgery or chemoradiotherapy in early stage disease. If the cancer is metastatic or the cancer recurs after primary therapy, the patients are usually treated with cisplatin based chemotherapy, but the impact to survival is poor. Chemotherapy represses the transcription of E6/E7 and this DNA damaging stress can activate p53 tumor suppressor in cervical cancer cells, where its function is abrogated by E6 in non-stress conditions. p53 can trigger apoptosis as a result of chemotherapy or it can induce DNA repair and inhibit proliferation by arresting cell cycle progression. Due to its central role in the development of cancer, maintenance of transformed phenotype or response to therapy, E6/E7 oncogenes are unique targets for novel therapeutic strategies. RNA interference has evolved from the recent studies as the most promising targeted therapy approach. This review will summarize the present central knowledge of the role of HPV and apoptosis in the carcinogenesis and responses to current treatment modalities of cervical cancer and will view perspectives for therapies aiming to downregulate HPV oncogene expressions. Keywords Apoptosis · Cervical cancer · Chemotherapy · Human papillomavirus · p53

Introduction Cervical cancer is the second most common malignancy in women worldwide after breast cancer. Each year, approximately 480 000 new cases are diagnosed, and the death toll is nearly 300 000 lives (Bosch and de Sanjose, 2003). Cervical cancer is S. Hietanen (B) Department of Obstetrics and Gynecology, Turku University Central Hospital, Kiinamyllynkatu 4-8, 20520 Turku, Finland e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 3,

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a major cause of cancer-related morbidity and mortality among women, especially in developing countries that lack efficient screening programs and adequate health care system. In the last three decades a substantial body of evidence has accumulated showing that genital human papillomavirus (HPV) infection plays a critical role in the pathogenesis of both squamous and adenocarcinoma of the cervix (zur Hausen, 2002). The development of the cancer is a multistep process that involves cervical mucosal cell transformation by oncogenic E6 and E7 proteins. E7 inactivates the cell cycle regulator pRb inhibiting cell cycle arrest while E6 inactivates the tumor suppressor protein p53, the main regulator of apoptosis (Dyson et al., 1989a; Hubbert et al., 1992; Huibregtse et al., 1993). HPV infection alone is unable to cause cancer. Only a small fraction of the patients will develop invasive disease (Steben and Duarte-Franco, 2007). The host immune defense plays a pivotal role in the clearance from the virus. This is compellingly shown in the recent studies using preventing vaccines developed against the L2 capsid protein. IN these studies nearly a 100% prevention against cervical cancer precursor lesions was achieved (Stanley, 2007). In case of viral persistence and possible viral integration to the host DNA, genomic instability with chromosomal alterations occur leading to severe cell cycle perturbation, clonal outgrowth and ultimately invasive cancer. The research efforts during the last two decades have paved the way for the efforts to develop targeted therapy particularly against high risk HPV E6. Still today, surgery, radiation and chemotherapy remain the mainstays of the treatment. It appears that the genotoxic treatment in the foreseeable future will remain in the treatment in different combinations like chemoradiation or different chemotherapy drug combinations, but an interesting possibility will be to use them together with E6 silencing therapies.

Cervical Cancer Carcinogenesis and Apoptosis Initial Events in HPV Carcinogenesis Human papillomaviruses are small, double-stranded DNA viruses that infect mucosal or cutaneous surfaces, causing warts or epithelial tumors. About one third of the over 100 virus types identified thus far are specific for epithelia of the lower anogenital tract. These can be divided into two groups: the “low risk” HPVs such as types 6 and 11, which are rarely found in malignant tumors but induce benign genital warts, and the “high risk” HPVs such as 16, 18, 31 and 45, which are frequently found in cervical carcinoma and are regarded as etiologic agents for both cervical cancer and its precursors. The cancer develops from well-defined precursor lesions referred to as squamous intraepithelial lesions. The first step in an HPV infection appears to be the access of the HPV to basal or parabasal cells as a consequence of microtrauma (Giroglou et al., 2001). For an active infection to take place, the virus must have entry to the generative compartment of the epithelium, capable of mitotic division. This is thought to be the principal reason why the squamocolumnar junction of the uterine cervix is so often involved in HPV infections (Fig. 3.1). In this

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Fig. 3.1 Multistep cervical carcinogenesis through precursor lesions. Progression of cervical cancer is dependent on interplay between high-risk HPV and host events. The initial infection is usually cleared by host defense, but if the virus persists it can progress to higher grades. The progression is characterized with increasing expression of E6/E7 resulting in deranged cell cycle regulation and differentiation. This results in increased cell proliferation, which is normally counteracted by apoptosis induced by p53 and other pro-apoptotic proteins. This is counteracted mainly by E6 which allows continued proliferation. This leads to genomic instability and accumulation of genomic changes that render the cells capable for invasion

transformation zone, basal and parabasal cells are constantly exposed to the environment. HPVs induce hyperplasia of the intermediate cells, and in the more superficial cells nuclear degeneration and cytoplasm vacuolization (termed koilocytosis). New viral particles are produced in these superficial cells, which are also the only site for assembly of the viral particles (Koss, 1987). All HPVs encode six early (E) proteins and 2 late (L) proteins according to the time when these genes are activated in a productive infection. The E region genes have a function in viral replication and cellular transformation. Whereas the expression of early genes occurs within the proliferative part of the infected lesion, late-gene expression as well as viral DNA replication is restricted to the differentiating part. The late-gene region contains the L1 and L2 genes which code for the structural proteins (Zheng and Baker, 2006). Productive infection requires a squamous epithelium in which keratinocyte differentiation takes place and results in the formation of large numbers of viral particles. The viral DNA is maintained as a stable episome and the E2 protein is required for the initiation of viral DNA replication, genome segregation and regulation of early viral promoter to control the expression of oncogenic E6 and E7

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proteins (Zheng and Baker, 2006). In productive infections, the epithelium always displays the typical cytopathic effect of HPV (Purola and Savia, 1977). These usually manifest as condylomas or low-grade cervical intraepithelial neoplasia (CIN). In contrast, high grade dysplasia and invasive carcinoma usually contain integrated viral DNA and do not show signs of productive infection. As a result of high-risk HPV DNA integration, E2 open reading frame (ORF) is disrupted and its regulatory action on E6 and E7 is lost. The major consequence is the constitutive expression of these two oncoproteins which are considered to be the main effectors of the malignant transformation in the cervical mucosal cells. The integration of HPV has been considered a critical step in the carcinogenesis, but recent data showing that 62% of the cancers contain integrated HPV suggests that this might not be an absolute requirement for the carcinogenesis. HPV 18 in contrast to many other types seem to be integrated practically always (Corden et al., 1999; Pett and Coleman, 2007)

Regulation of Cell Cycle and Apoptosis by HPV Proteins In the episomal state the E5 mRNA is the most abundant viral transcript (Stoler et al., 1992). It has been shown that E5 interferes with TRAIL signaling and inhibits Fas-induced apoptosis (Kabsch et al., 2004). The E5 also modulate EGF receptor signaling (Crusius et al., 1998) and down-regulate the expression of MHC class I molecules (Ashrafi et al., 2006). Therefore, E5 seems to protect the HPV from the host immune defense in the early stages of the infection. However, when HPV integrates into the host DNA the E5 ORF is often deleted (Corden et al., 1999). Immune surveillance is also hampered by E6 binding to interferon regulatory factor-3 (IRF-3). The inhibition of IRF-3 transactivation function deregulate death signaling (Ronco et al., 1998b). Recognition of various microbes occurs through toll-like receptor signaling. Recently, it was shown that E6 can block transcription of TLR9 gene rendering the cells resistant to apoptosis (Hasan et al., 2007). All these interactions give rise to viral persistence, which is a well known risk factor for progression in HPV infected cervical cancer cells. Both the E6 and E7 proteins are required for the cellular transformation and for the maintenance of the transformed phenotype (DeFilippis et al., 2003). E6 binds to the key tumor suppressor protein p53 and directs it to ubiquitin-mediated degradation (Scheffner et al., 1990), while E7 inactivates the cell cycle regulator pRb in a similar fashion (Dyson et al., 1989b; Scheffner et al., 1992).

E7 Interactions with Cellular Targets The efficacy of E7 binding and inactivation of pRb is related to the transforming capacity of the respective HPV type (Heck et al., 1992). Thus, E7 proteins of the high-risk HPVs have a ten-fold greater affinity for Rb and its relatives p107 and p130 than the low-risk types (Berezutskaya et al., 1997). HPV E7, binds pRb blocking

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Table 3.1 HPV oncoprotein interactions with cellular pathways E5

E6

E7

Fas receptor signaling TRAIL DISC EGF receptor signaling c-jun

p53 E6AP BAK BAX c-myc TNF-R1 FADD Paxillin E6BP/ERC-55 E6TPI PDZ proteins IRF-3 CBP/p300 Mcm7 hTERT XRCC1

Rb p130 p107 p600 p300 p21 p27 E2F1 HDACs BRG1 hTID-1 TBP

Highlighted targets are directly involved in apoptosis

its transcription factor E2F-regulatory function resulting in constitutive activation of E2F responsive genes (Imai et al., 1991). Normally, keratinocytes differentiate and become senescent towards the upper layers of the epithelium. The E7 protein is important in the viral life cycle, because it interferes with the differentiation and enables the cell reenter s-phase where it exploits cellular replication factors (Wise-Draper and Wells, 2008). Repression of E2F-mediated transcription by Rb is partially dependent on histone deacetylase (HDAC) binding to Rb, and high-risk E7 is also associated with HDACs, independently of Rb binding (Brehm et al., 1998). E7 associates with several other proteins involved in cell cycle regulation, e.g., with cyclins A and E and cyclin-dependent kinase inhibitors p21 and p27, but these interactions are not considered to be as central as the binding to Rb (Longworth and Laimins, 2004) (Table 3.1). Apart from its ability to interfere with cell cycle regulation, E7 can induce genomic instability (Hashida and Yasumoto, 1991), probably by inducing inappropriate centrosome duplication (Duensing et al., 2000a). Keratinocytes are sensitized to apoptosis by E7, but this activity is blocked by co-expression of E6 (Stoppler et al., 1998)

E6 Interactions and Inhibition of Apoptosis Transforming property of the HR-HPV E6 proteins involve the degradation of p53 in association with cellular ubiquitin ligase E6AP (Huibregtse et al., 1991). Due to the increase of proliferation induced by E7 the p53 expression is triggered. The host attempt to block the abnormal proliferation is thwarted by degradation of p53 by ubiquitin proteasome pathway. E6 proteins of both oncogenic and benign HPV types associate with p53 in vitro, but only binding by E6 proteins can target

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p53 for degradation (Crook et al., 1991). Nonetheless, also E6 which retain the capacity to bind p53, without promoting its degradation, interfere with the ability of p53 to up-regulate the transcription of target genes (Crook et al., 1994; Pim et al., 1994). The loss of p53 function results in disordered differentiation, less controlled cell cycle progression, altered response to stress, accumulation of mutations and decreased sensitivity to apoptotic signals, which all can promote malignant transformation. Although inactivation of p53 is considered to be crucial for E6-induced keratinocyte transformation, certain p53-independent functions of E6 may also be involved. High-risk E6 protein interacts with numerous other cellular proteins, e.g., with paxillin, interferon regulatory factor-3, transcriptional coactivator CBP/p300 and proapoptotic factor Bak (Ronco et al., 1998a; Patel et al., 1999; Zimmermann et al., 1999; Thomas and Banks, 1998a) (Table 3.1). Bak, which is directly connected to apoptosis was identified to bind E6 irrespective to the HPV type (Thomas and Banks, 1998b). Suppression of Bax activity by E6 has been observed in HeLa cells and this activity was found to be necessary for the anti-apoptotic function of E6 (Vogt et al., 2006). Several other E6 activities contribute to the inhibition of apoptosis: E6 degrades pro-apoptotic c-myc (Gross-Mesilaty et al., 1998), increases anti-apoptotic survivin transcription (Borbely et al., 2006) and binds to tumor necrosis factor receptor 1 (TNFR1) (Filippova et al., 2002), death effector domains (DEDs) of FADD (Filippova et al., 2004) and procaspase 8 (Filippova et al., 2007). E6 interferes also with proteins involved in transcription control like E6TP1 (Gao et al., 2002) and ADA3 (Zeng et al., 2002). High-risk, but not low-risk, E6 can also induce degradation of several proteins that contain multiple PSD95/Dlg/ZO-1 (PDZ) domains. One of the targets of HR-HPV:s is Dlg (Kiyono et al., 1997). Loss of Dlg correlates with increased proliferation and dysplasia. E6 binding to PDZ proteins is mediated by a particular PDZ-binding motif within the carboxyl-terminus of E6, and E6 proteins rendered defective in PDZ-binding appear to be less effective in transforming immortalized keratinocytes than their intact counterparts (Watson et al., 2003).

HPV Induced Chromosomal Instability One of the key events in cervical carcinogenesis is the evolvement of numerical and structural chromosome changes. Integration of HPV DNA has been found in most cervical cancers but very rarely in initial stages of the HPV-induced lesions (Pett and Coleman, 2007). The absence of HR-HPV DNA integration in some carcinomas implies that integration is not always required for malignant progression (Vinokurova et al., 2008) and it has been suggested that viral integration may not be a cause for genomic instability, but rather its consequence. In many cancers and cancer- or precursor-derived cell lines both viral episomes and integrated sequences coexist, but the episomes are often deleted during the progression. Nevertheless, it is clear that HPV proteins can induce genomic instability by e.g. interfering with centrosomes (Duensing et al., 2000b), amplifying integration gene locus

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(Kadaja et al., 2007) or modulating telomerase activity (Kiyono et al., 1998). Additionally, E6 can interact with the DNA repair mechanism through inhibiting p53 function, but also directly by binding e.g. to proteins involved in single-strand break repair (Iftner et al., 2002).

Cervical Cancer Therapy and Apoptosis Current Therapy for Cervical Cancer and Its Precursor Lesions Cervical cancer precursor lesions, CIN and carcinoma in situ are treated by local excision or ablation by conisation, loop electrosurgical excision procedure (LEEP), laser- or cryosurgery. Early-stage (stages IA, IB, small IIA tumors) invasive cancer with no parametrial involvement is managed by radical hysterectomy or high-dose radiotherapy. The surgery results in 80–90% 5-year survival rates showing that these cancers can often be permanently cured by surgery (Cannistra and Niloff, 1996). Randomized studies have recently shown that concurrent chemotherapy and radiotherapy have significantly reduced the risk of death of patients with locally advanced cervical cancer (Rose, 2003). This combined treatment is more efficient against locally advanced cervical cancer than either radiotherapy or chemotherapy alone (Green et al., 2005). For the metastasized or recurrent cancer platinum analogues are the mainstay of chemotherapy. Cisplatin has been studied most and so far is the most effective single agent and it induces tumor responses in 20–40% of patients (Moore, 2008). Despite the often good initial antitumor effect however, treatment with cisplatin alone prolongs overall survival only marginally of patients with the most advanced stages of cervical cancer or recurrent cancer after genotoxic treatment. Less than 20% of the patients with stage IV disease survive beyond 5 years (Long, 2007). Better treatments are obviously needed, but the task based on conventional cytotoxic therapy has proven to be extremely challenging. Recent reports show that combining certain newer chemotherapy drugs, like topotecan, with cisplatin produces higher response rates than treatment with cisplatin alone although at the expense of increased general toxicity (Ackermann et al., 2007; Long, 2007). Several other chemotherapy combinations have been studied resulting only to a modest effect.

Genotoxic Treatment and p53 Mediated Response to DNA Damage Despite the recent advances in the HPV research and cancer research in general to develop target specific drugs against cervical cancer, DNA damaging therapies, either by chemotherapy drugs or radiotherapy remain the cornerstone for the treatment of this disease. This is due to the fact that in general cervical cancer is sensitive

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to the genotoxic treatment, and e.g. radiotherapy alone or together with concurrent chemotherapy can be often curative in early stages of invasive cervical cancer. The earliest in vitro studies focusing on p53 and chemosensitivity strongly suggested that loss of wt p53 function is a major factor explaining chemoresistance in various human cancers, e.g., in hematologic malignancies and in breast cancer (Wattel et al., 1994; Aas et al., 1996). Collective data from numerous subsequent studies demonstrate that p53 may play a proapoptotic, neutral or even an antiapoptotic role after treatment with chemotherapy drugs, depending on the cancer cell type and, on other factors, such as the type of chemotherapy drug. A metaanalysis performed on 356 studies found conflicting conclusions on p53 status and its affect on apoptosis in several cancer cell types (Cimoli et al., 2004). In general, chemotherapy-induced p53 activity tends to promote apoptosis in hematological malignancies, but in solid tumors the outcome of the p53 response varies considerably (Brown and Wouters, 1999). Therefore, p53:s position in conveying either apoptotic or cell cycle arrest and repair signals is ambivalent. Both cell type and different molecular background determines the outcome together with p53. Consequently, p53 can act either as a sensitizer to the chemotherapy or a resistance factor. In contrast to most other cancers, the p53 gene is usually not mutated in cervical cancer. Owing to the fact that cervical cancer cells carry almost always wild type p53 but is degraded by HR-HPV, it was formerly regarded completely non-functional in cervical cancer cells. However, it has become evident from the recent works by several groups that p53 inactivation may be reverted in HPV E6 carrying cells and the p53 status in cervical cancer cells is not equal to that of cancer cells with a mutated p53 gene. The opportunity to modulate p53 activity by repressing the E6 mediated degradation offers a unique possibility to modulate conventional treatment responses in cervical cancer and design completely new ones based on target specific interference.

Radiotherapy Induced DNA Damage and Cervical Carcinoma The relationship between p53 status and radiosensitivity has been extensively studied in various in vitro and in vivo models, and in clinical samples. Certain cancer cell types, such as malignant lymphocytes, are very sensitive to radiation, and in these cells the predominant mode of cell death after γ-IR is p53-dependent apoptosis that can be attenuated by abrogating the p53 response (Schmitt et al., 2002). Most cancer cells of epithelial origin are relatively resistant to radiotherapy. They do not seem to undergo apoptosis readily in response to irradiation, but rather are prone to cellular senescence or mitotic catastrophe, and the disruption of p53 function often does not markedly affect cellular susceptibility to γ-IR (Roninson, 2003). Radiotherapy in the presence of mutant p53 has usually been associated with decreased effect (Cuddihy and Bristow, 2004). In non-HPV related cancer, ionizing radiation evokes protein kinases including ATM and DNA-PK that phosphorylate p53, which prevents the interaction with its major regulator MDM2. This activated form of p53

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upregulates downstream targets such as the cyclin dependent kinase inhibitor p21 and the pro-apoptotic molecules PUMA and Noxa. These two pro-apoptotic proteins then upregulate the Bcl family member Bax. Depending on the severity of the DNA damage the cell undergoes apoptosis induced by Bax or growth arrest by p21 (Harris and Levine, 2005). In cervical cancer cells the major regulator of p53 is not MDM2, but E6 (Hietanen et al., 2000; Hengstermann et al., 2001) (Fig. 3.2a). It has been shown that HPV 16 E6 abrogates the cellular response to γ-IR in the epidermal cells of transgenic mice, and this takes place not only by p53-dependent, but also by p53-independent mechanisms (Song et al., 1998). Restoring wt p53 activity in cervical cancer cells through adenovirus-mediated gene transfer has been reported to increase cellular sensitivity to γ-IR in vitro (Huh et al., 2003). Irradiation can activate p53 responsive reporter even in HPV positive cervical cancer cells (Koivusalo et al., 2002) and radiation induced apoptosis can be inhibited by increasing E6/E7 expression (Kamradt et al., 2000). Increased expression of the p53 downstream target gene p21 was frequently detected in cervical cancer specimens obtained from patients after

Fig. 3.2 (a) The feedback loop of p53-HDM2 is switched off in cervical cancer cells due to p53 degradation by E6. MCF-7 breast cancer cells are HPV negative with wild type p53 and the p53 tumor suppressor can be stabilized by an inhibitory peptide binding to HDM2 (MDM2). This STIP peptide is expressed in thioredoxin (Trx) scaffold and the construct can be detected with anti-thioredoxin antibody. When the peptide is modified with three alanine substitutions (STIPala), it cannot stabilize p53. In HPV positive cells inhibition of HDM2 binding to p53 cannot stabilize p53 due to presence of E6 (Hela + STIP). Modified from Hietanen et al. (2000). (b) By repressing the transcription of E6 by chemotherapy drugs, here with doxorubicin or degrading the E6 mRNA by RNA interference the p53 can be stabilized, but this effect is significantly enhanced when these two methods are combined. Modified from Koivusalo et al. (2005)

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radiotherapy suggests that radiation may evoke p53 activity also in vivo although p21 accumulation may occur by means independent of p53 (Oka et al., 2000). In another clinical study p53 gene mutations were associated with local tumor recurrence after radiotherapy in patients with FIGO stage IIIB cervical cancer, suggesting that cervical cancers carrying wt p53 are more sensitive to γ-IR than their p53 mutated counterparts (Ishikawa et al., 2001). Taken together, wt p53 activity can be revived in cervical cancer cells with radiation and it may play a significant role in determining the fate of cells that have been exposed to γ-IR. Also p73, a p53 homologue, has been implicated in the cellular response to γ-IR in cervical cancer: in a study increased p73 expression in cervical cancer samples was associated with a favorable response to radiotherapy and better patient survival (Liu et al., 2004).

Chemotherapy and Apoptosis in Cervical Carcinoma At present more than fifty chemotherapy drugs are used to treat malignancies. The exact mechanisms of action of most of these drugs are still inadequately understood at the molecular level, but in general, the ability to cause DNA damage and DNA replication inhibition, appears to be a central property of conventional chemotherapy compounds. Chemotherapy agents can induce death in the cancer cells by apoptosis. However, inhibition of apoptosis does not necessarily protect the cells from anti-cancer agents (Brown and Attardi, 2005).Therefore it has become evident, like for radiotherapy, that in solid tumors other modes of cell death occur during chemotherapy and these include mitotic catastrophe, autophagy, necrosis and senescence. Regardless the mode of death, the function of p53 or lack of it may play a significant role in the cellular response to these drugs. Cervical cancer cells have previously been considered as harbouring permanently non-functional p53 due to E6. However, chemotherapy compounds can stabilize p53 protein in cervical cancer cells and this occurs partly because of repression of E6 transcription (Butz et al., 1995, 1996; Wesierska-Gadek et al., 2002; Koivusalo et al., 2002; Koivusalo and Hietanen, 2004; Hietanen et al., 2000). Chemotherapy-activated p53 also transactivates its downstream target genes in various HPV positive cervical cancer cells (Butz et al., 1999; Hietanen et al., 2000; Wesierska-Gadek et al., 2002). Therefore, both p53 and the downstream effectors of the p53 pathway are functional in cervical cancer cells after chemotherapy although the amount of E6 is reduced, but not completely abolished. In a panel of 31 chemotherapy drugs 28 were found to decrease the transcription of E6 mRNA levels suggesting that the viral oncogene transcription is sensitive to cytotoxic compounds. In the same study, antracyclins, topoisomerase I and II inhibitors and platinum compounds activated the p53 reporter significantly in cervical cancer cells, whereas microtubule inhibitors showed only a modest effect (Koivusalo and Hietanen, 2004). After treatment with these chemotherapy compounds, no direct correlation between the E6 mRNA levels and p53 activity could be observed. However, no significant increase in the reporter activity was observed unless the E6 mRNA was first reduced

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suggesting that the activity of p53 may have been a consequence of the extent of the genotoxic damage, but could not be activated unless relieved from the degradation by E6 (Koivusalo and Hietanen, 2004). The role of p53 in response to chemotherapy has been sparsely studied in cervical cancer cells most likely, due to the false assumption that these cells are equivalent to p53 null cells. In terms of clinical response to chemotherapy an important question is how does the p53-E6 interplay affect the outcome of genotoxic treatment? In a recent study Liu et al. found that ectopic E6 expression in non-transformed keratinocytes sensitized the cells to many chemotherapy drugs and the increase in apoptosis was attributed to activation of Cdk1 (Liu et al., 2007). In light of the fact that E6 targets to a number of proapoptotic proteins, especially in the early stages of the neoplasia, these findings are unexpected. Ectopic E6 expression in non-transformed keratinocytes differs from the conditions of cervical cancer cells carrying integrated E6. Nevertheless, we have also found caspase activation and morphological changes typical for apoptosis correlating with the cytotoxic effect in cancer cells treated with chemotherapy compounds. However the responses to different therapies after modulation of E6 transcription were variable (Koivusalo et al., 2005). We also observed that the functional status of the p53 protein in the cervical cancer cells may either enhance or inhibit the cytotoxicity of the chemotherapy drug, even within a same drug category: with treatment of platinum compounds the p53 response may be sensitive to the special carrier ligand (Koivusalo et al., 2002;Koivusalo and Hietanen, 2004). The influence of p53 to the cytotoxicity depends to a large extent on the concentration of the drug. In high concentrations the dose response curves of p53 null and p53 active cells overlap. It is also likely that the concentration and the exposure time to the drug will have an impact on the form of cell death (Havelka et al., 2007).Very little is known whether the chemotherapy effect seen in clinic is also result of apoptotic death. Increased apoptosis has been observed in CIN in form of increased caspase 3 expression in high grade CIN compared to low-grade, but a reduction of Bax, Caspase 3, and Caspase 6 in invasive carcinoma (Chung et al., 2002). There is some evidence that cisplatin together with radiation may induce apoptotic cell death that can be observed with in situ detection methods from post treatment tumor biopsies (Iwakawa et al., 2007). Apoptosis defective cells have also been linked to aggressiveness of cervical tumors (Hockel et al., 1999). There is clearly a need for more studies addressing the chemotherapy death in the tumors, but it seems that apoptosis occurs also clinically in cervical cancer after genotoxic treatment. The problem with the vast majority of studies dealing with chemotherapy responses in vitro is that the concentrations of the drug and the exposure times vary extremely and are often incomparable. It is also difficult to define a “clinically relevant dose” used in cell culture experiments. We may know drug plasma concentrations in patient treatment schemes and calculate corresponding concentrations for the in vitro growth conditions, but we simply don’t know enough of the drug tumor kinetics in complex clinical treatment schedules (El Kareh and Secomb, 2005). A number of chemotherapy drugs can activate p53 in cervical cancer cells despite HPV E6 expression. In some cases the activated p53 promotes cell death, but

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in others p53 can counteract the effect of the anti-cancer drug. It is also clear that the p53 effect to the death of the cells is sometimes significant, but even then p53-independent mechanisms prevail (Koivusalo et al., 2002; Koivusalo and Hietanen, 2004). Nevertheless, studies addressing the effects of E6 together with chemotherapy provide a rationale basis for attempts to improve treatment efficacy with viral oncogene modulation. Profiling of individual cancer tumors may ultimately allow us tailor a specific treatment for the patient (Garman et al., 2007) and taking into account the status of p53 may become important when designing therapies against cancer.

Chemoradiotherapy – Radiosensitization by Chemotherapy Compounds Concomitant administration of cisplatin-based chemotherapy and radiotherapy (chemoradiotherapy) has dramatically improved pelvic control and the survival of patients with locally advanced cervical cancer compared to either treatment alone, and chemoradiotherapy is now the standard treatment for high-risk early stage and locally advanced cervical cancer. In fact, the combination of radiotherapy with chemotherapy is now considered the standard treatment for a number of tumors of different origin. Most radiosensitizing agents are selected based on empiric laboratory or clinical observations, without much knowledge of the molecular basis of these agents’ interaction. This is due to the fact that our understanding of the true mechanisms of these interactions is poor. The general mechanisms for chemoradiation effect are: modification of DNA damage, decreased DNA repair, increased apoptotic cell death resulting from DNA injury, cytokinetic co-operation caused by cell-cycle redistribution like arrest of G2/M (Wilson et al., 2006). Cervical cancer cells in the S phase are the most radioresistant, and cells in the G2–M phase of the cell cycle are the most radiosensitive (Terasima and Tolmach, 1961). Cisplatin exerts its antitumor effects by forming various DNA adducts (Reed, 1998) resulting in interstrand and intrastrand crosslinks and inhibition of DNA replication and transcription. Depending on the extent of the damage these lesions can be repaired, cause mutations or activate apoptosis cascade. These cross-links of cisplatin can directly impair repair (Eastman and Barry, 1987). Cisplatin and radiation induce DNA damage with different mechanisms which is a prerequisite for synergistic interaction (Wilson et al., 2006). There are more than 20 proteins involved in the recognition of the DNA distortion (Lieberman, 2008). Cisplatin adducts are mainly repaired by nucleotide excision repair (Reed, 1998). These DNA adducts are repaired at a slower rate in the absence of functional p53 (Fan et al., 1997; Pestell et al., 2000). If the repair fails sustained JNK/MAPK activation leads to apoptosis (Mansouri et al., 2003). p53 is centrally involved in the regulation of several NER-associated genes such as p48DDB2 and XPC which participate in DNA damage recognition, and p53R2, a gene encoding a catalytic subunit of ribonucleotide reductase that provides deoxynucleotides for

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NER (Adimoolam and Ford, 2002; Hwang et al., 1999; Tanaka et al., 2000). p53 also regulates the helicase activity of the NER-associated transcription factor IIH (Wang et al., 1995). The NER status appears to be a key determinant of cisplatin sensitivity in testicular and ovarian cancer cells (Koberle et al., 1999; Selvakumaran et al., 2003), and abrogation of wt p53 function by HPV E6 or dominant negative p53 expression results in reduced NER activity (Smith et al., 1995). Thus, it is possible that the cisplatin-induced p53 activity enhances NER-mediated repair of the DNA adducts and that this promotes cell survival in the presence of wt p53. The mismatch repair, MMR proteins hMSH2 and hMLH1 bind with high affinity to cisplatin adducts but not to oxaliplatin adducts (Fink et al., 1996), which might explain why loss of MMR confers resistance to cisplatin, but not to oxaliplatin (Fink et al., 1997). Carboplatin and oxaliplatin are often used instead of cisplatin against various tumors although not cervical cancer. Concurrent treatment with cisplatin, carboplatin and oxaliplatin with γ-irradiation all have a synergistic effect on HPV positive cervical cancer cell lines (Koivusalo et al., 2002).In the same study, the dual modality treatment reduced E6 mRNA expression and led to stabilization and activation of p53. However, the platinum treatment without γ-irradiation differed significantly with respect to p53: in p53 null cells (either by ectopic E6 or dominant negative p53) the effect of cisplatin was greater than in the native cancer cells with induced p53. This suggests that the cisplatin-induced p53 activity may enhance NER-mediated repair of cisplatin-DNA adducts and this results in reduced apoptosis in the presence of wt p53.

Activation of Apoptosis with HPV Targeting Molecules Marked improvement of patient survival rates might not be achievable with conventional chemotherapy drugs and radiation and cure with the conventional approaches is an unrealistic goal in advanced cervical cancer. Research efforts should focus on approaches based on specific targeting of cervical cancer cells, as these approaches could enable selective killing of cancer cells and result in a better antitumor effect with less side effects. The last decades have provided us with a wealth of information on the basic biology of cervical cancer specifically on the interactions of viral oncogenes and host proteins. This has formed the basis for attempts to interfere with these processes in order to induce selective kill of the cells that have escaped normal growth control.

Downregulation of HPV Oncoproteins by RNA Interference The recognition of HPV E6 and E7 oncoproteins as key factors in transformation and apoptosis inhibition has made them as attractive candidates for targeted therapy. The recently introduced short interfering RNA (siRNA) technology, based on a phenomenon called RNA interference (RNAi), has enabled much more sustained silencing of target gene expression compared to antisense and ribozyme

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techniques. SiRNAs are short duplexes which are designed to trigger specific enzymatic destruction of specific transcripts via the RNA interference pathway. The cellular components of the RNAi machinery are highly conserved throughout evolution: they are present and functional in plants, lower organisms, as well as in mammalian cells (Elbashir et al., 2001). In RNAi, long double-stranded RNA is first degraded to approximately 22-nucleotide RNA duplexes by the RNAse III enzyme Dicer (Bernstein et al., 2001), and the resulting effector molecules termed siRNA are involved in the sequence-specific targeting and subsequent cleavage of target mRNA as part of the RNA-induced silencing complex RISC (Hammond et al., 2000). siRNA molecules convert their target mRNAs into double-stranded RNAs that are, in turn, degraded into new siRNA molecules (Lipardi et al., 2001). The generation of new siRNA molecules could explain why even nanomolar concentrations of siRNA can induce efficient and relatively long-lasting repression of target mRNA levels. Many siRNAs can induce substantial target specific degradation, but unmodified synthetic siRNA constructs themselves are susceptible to degradation by RNases. A number of efforts trying to modify the oligonucleotides have been presented, but only some nuclease-stabilizing modifications are compatible with the RISC, a requirement for gene silencing. From these studies have evolved siRNA constructs with alternating 2 -O-methyl and 2 -fluoro nucleotides which are very potent and extremely stable with minimal off-target effects. These siRNAs have proved their efficacy in vivo (Soutschek et al., 2004). Although there are still a number of obstacles in the proper delivery for the oligonucleotides, it seems probable that RNAi drugs will eventually make it to the market. At present a pubmed search for studies on HPV 16 and 18 E6/E7 siRNA effects on cervical cancer cells both in vitro and in mouse tumor models yields over 30 original papers. All these studies show that the oncogenic viral transcripts can be successfully targeted with RNA interference resulting to activation of p53 and its downstream targets. In the majority of these studies reactivation of p53 has been the main goal of the approach and little attention was directed towards other E6 or E7 targets. All of the studies come to the same conclusion that RNA interference is a promising new technology that may provide lead molecules for drug design against cervical cancer. In the majority of the studies the biological outcome of the RNAi has been inhibition of growth/senescence. The studies showing apoptosis in the target cells have primarily used vector delivered siRNAs. This discrepancy may likely to result from the different transfection conditions. It is well known that p53 is extremely sensitive to even low levels of damage (Vousden and Lane, 2007). Moreover, transfection per se can induce p53 stabilization (Lipinski et al., 2001). Using synthetic oligonucleotides alone for E6/E7 RNAi for marked apoptosis induction in pure nonstress conditions has so far not been shown. Similarly targeting E6 with neutralizing antibodies alone or even together with E6siRNA stabilizes p53 and restores its transactivation properties does not induce apoptosis (Courtete et al., 2007). However, combining RNAi to DNA damaging agents in cervical cancer cells induces

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augmented p53 response and induces enhanced apoptosis (Koivusalo et al., 2005; Putral et al., 2005) There are some problems related to the stability and delivery of the oligonucleotides as depicted above, but there may be other factors that affect the duration of the desired effect in RNAi of HPV induced cancer. The kinetic studies of p53 activation after E6 mRNA downregulation with siRNA revealed that after reaching the peak activation after about 72h after oligonucleotide transfection, the activity declines. This occurs despite E6mRNA levels still being repressed (Koivusalo et al., 2005, 2006). Recently it was reported that the target cells may become siRNA-resistant over time in a vector delivered siRNA system (Tang et al., 2006). The development of resistance to SiRNA occurs during passaging of the cells and manifests in degradation resistance of the target. After E6 oligonucleotide siRNA transfection the p53 activity declines within 1–2 days. We have observed that this extinguishing of p53 can be attributed to host p53 antagonists, notably MDM2 (Koivusalo et al., 2006). It has been previously shown that MDM2 is inactive in E6 expressing cervical cancer cells and the protein cannot be stabilized by inhibiting the interaction between MDM2 and p53 protein by small peptides (Hietanen et al., 2000) (Fig. 3.2). This is due to E6 that switches off the p53-MDM2 pathway. When E6 is downregulated and p53 becomes active this pathway is restored (Hengstermann et al., 2001; Hietanen et al., 2000). The p53 decline after E6 RNAi can be partially counteracted by HDM2 knockdown by siRNA, but only additional inhibition of other known p53 ubiquitin ligases COP1 and Pirh2 together with c-jun kinase, JNK a sustained p53 activation can be achieved (Koivusalo et al., 2006). This leads to enhanced growth suppression, but not apoptosis. At present, little is known about COP1 and Pirh2 and practically nothing in HPV related disease due to their very recent discovery (Leng et al., 2003; Dornan et al., 2004). Neither it is known whether they play different role in different cells, and in different phases of development. One intriguing possibility is that they act as a fail-safe control. In cervical cancer cells, knocking down one by one of them after E6 downregulation increases p53 activity stepwise suggesting that they may indeed act as backup for each other. The backup system may exist, because it is of vital importance to keep p53 in a very strict control in normal cells in order to avoid its deleterious effects (Vousden and Lane, 2007).

DNA Damage Induction by Chemotherapy and Concurrent E6 Downregulation After DNA damage with anti-cancer agents MDM2 undergoes several covalent modifications which prevent its binding and subsequent degradation of p53 (Appella and Anderson, 2001). When E6 is downregulated in cervical cancer cells and the stabilized p53 becomes active after treatment with chemotherapy compounds, typical morphological and biochemical characteristics of apoptosis appear (Putral et al., 2005; Koivusalo et al., 2005). Many of the chemotherapy compounds can

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stabilize p53 to some extent but further reduction of E6 mRNA levels by RNAi increases the protein content even more (Fig. 3.2b). Combining most cytotoxic drugs to E6 siRNA results in sustained and synergistic activation of p53 with doxorubicin, gemcitabine, mitoxantrone, paclitaxel, topotecan, and additively with carboplatin, oxaliplatin and mitomycin.However, p53 activity after cisplatin and etoposide counteracts the toxic effect indicating that p53 role may be drug dependent. Although E6 siRNA increases the death together with these compounds the resultant dose response is subadditive. In contrast, Putral et al. reported that E6 siRNA enhances sensitivity to cisplatin due to p53 activity. This should mean that the two compounds act synergistically. However, the concept synergism is more complex and should be based on analyses on treatment interactions (Tallarida, 2000). Moreover, short term survival experiments (<2–3 days) are inferior to clonogenic assays in terms of observing a meaningful effect (Havelka et al., 2007; Brown and Wouters, 1999). Enhanced p53 activation by e.g. E6 silencing might oppose the cytotoxic action induced by some drugs and therefore more data is needed on the p53 DNA damage responses in cervical cancer cells.

MicroRNA as RNAi Targets in Cervical Carcinoma After the discovery of RNA interference, the researchers began to search for endogenous small non-coding RNAs. Today more than 500 human genes are found to encode such microRNA (miR) that are predicted to control the expression of one third of human coding genes. miR regulate the expression of many messenger RNAs (mRNA) through suppression of protein translation degradation of their target RNAs (Dalmay, 2008). These miR play important roles in regulating cellular proliferation, development and viral defence and 30% of all genes are subject to regulation by multiple miR (Lim et al., 2005). In cancers different patterns of miR expression has been found implying miR expression profiles as means to distinguish cancers into new prognostic categories (Lu et al., 2005). Recently in a mouse model a cholesterol linked 2 -O-methyl modified oligonucleotide effectively reduced the target miR and cholesterol biosynthesis which they control (Krutzfeldt et al., 2005). These so called antagomirs target miR as siRNA target mRNA. Recently it was shown that members of the miR-34 family are direct p53 targets and ectopic expression of miR-34 causes cell cycle arrest in the G1 phase, senescence or apoptosis in different contexts see review by Hermerking 2007 and references therein (Hermeking, 2007). This suggests that p53 may regulate already synthesized transcripts by mir-34. Very few papers have so far addressed miR in cervical cancer. Martinez et al. Reported that mir-218 was underexpressed in cervical cancer and its precursor lesions compared to the normal cervix (Martinez et al., 2007). Lui et al instead found reduced expression of miR-143 and increased expression of miR-21 in cancer samples (Lui et al., 2007). Although at very early stages miR profiling may present new insights to carcinogenesis and may provide us with new level of interfering with the process and induce apoptosis or senescence in tumor cells.

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With clear miR profiles of various cancers, one potential treatment approach would be to introduce back the miR that have been lost during carcinogenesis or then knock down miR with aberrant downstream function by introducing antagomirs. Furthermore, accumulating evidence indicates that miR are functioning coordinatively in gene regulation. It is possible to revert abnormal cells back to normal by multiple synthetic miR.

Concluding Remarks Cervical cancer is exceptional compared to most other cancers because it is so strongly associated with a single etiological factor, HPV. Since the discovery of the connection between HPV and cervical cancer an intense research has revealed exciting interactions of HPV encoded proteins and host growth control mechanisms, which are interesting in cancer biology even in broader sense. Prophylactic vaccines have shown a very high efficacy against HPV infection and cancer precursor lesions. However, these vaccines don’t prevent the development of cancer in women already infected with the virus. Even if the efficacy of the vaccines would remain at the very high level that is reported today, we will have cervical cancer still for decades if not longer. Therefore, the search for new treatment strategies should go on with high priority. The conventional DNA damaging therapies are still the mainstay of the treatment of advanced cervical cancer. Novel targeted therapies, both small molecule approaches and RNA interference based strategies are emerging where the aim is to combat the interaction of HPV with apoptosis and cell cycle regulation. There are still a number of open questions related to the delivery, efficacy and stability of these compounds. Even if these obstacles are overcome, DNA damaging therapy will probably be combined to the targeted therapies for a foreseeable future.

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Chapter 4

Apoptosis in Colorectal Tumorigenesis and Chemotherapy Shi Yu Yang, Kevin M. Sales and Marc C. Winslet

Abstract Colorectal cancer is the second most common cancer in the western world. At least 40% of colorectal cancer patients develop metastases; chemotherapy alone or in combination with radiotherapy is usually used as adjuvant treatment for advanced disease. Unfortunately adjuvant treatments are often ineffective due to the development of resistance. A major contributor to chemo-resistance is the inhibition or avoidance of apoptosis. This chapter reviews the genetic mutations in colorectal tumorigenesis; the alterations of apoptosis in colorectal cancer progression; and the relationship between mutations and apoptotic changes. The factors which affect and regulate apoptosis in colorectal cancer development are evaluated. The dysfunction in different apoptotic pathways through which colorectal cancer cells develop resistance to chemotherapies is discussed. Finally the potential molecular targets and therapeutic strategies designed against these targets are proposed. Keywords Chemo-resistance · Colorectal cancer · Dysfunction of apoptosis · Genetic mutations

Introduction Colorectal cancer (CRC) is the second most common cancer in the western world. Despite advances in the management of this condition, including improved surgical technique, the use of chemo or radiotherapy and more recently the use of screening, the mortality has not changed for decades. Colorectal cancer is presently treated by surgical ablation, but many tumours are detected at a late stage when surgery cannot cure the disease. At least 40% of patients with colorectal cancer develop metastases; chemotherapy alone or in combination with radiotherapy can be used as an adjuvant therapy to surgery for more advanced disease (Labianca et al. 1997).

S.Y. Yang (B) Department of Surgery, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, UK e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 4,

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However, these approaches are not highly effective against disseminated colorectal metastases (Magnuson et al. 1995). New therapeutic strategies are needed for treatment of advanced or metastatic colorectal cancer.

Epidemiology of Colorectal Carcinoma Colorectal carcinoma is primarily a disease of the western world, with the highest reported incidence of about 40 cases per 100,000 people occurring in west Europe closely followed by the USA. It is rare in Asia, Africa and parts of South America. Although no age group is immune, it occurs maximally in the 7th decade of life. The sex incidence of colorectal cancer is almost equal but rectal tumours are more common in men and colonic carcinoma more common in women giving male to female ratio for death of 6:5 for rectal and 7:11 for colonic carcinomas respectively.

Aetiology of Colorectal Carcinoma Aetiologically colorectal cancers can be divided into three categories: sporadic (a single case of CRC), familial (two or more first-degree relatives with CRC) and hereditary CRC. Aetiological studies have shown that the incidence of colorectal carcinoma differs between countries and between different cultural groups within a given country. The finding that migrants show the cancer incidence of their adopted country within a generation supports environmental and cultural factors in the aetiology of colorectal cancer. The most strongly implicated environmental and cultural factor is a high fat, high protein, low fibre diet (Willett 1989). A high fat diet stimulates choleresis increase the amount of primary bile acids in the faeces. The bile acid are then metabolised by colonic bacterial enzymes leading to the production of lithocholic and chenodeoxycholic acid, both known mutagens. Bacterial action on protein breakdown products entering the large bowel may produce known carcinogens such as the N-nitrosamines. Dietary fibre consists of various undigested polysaccharides, mainly celluloses and some resistant starches. Its protective effects result from increasing the bulk of stool, retaining faecal water, diluting and absorbing luminal toxins and reducing colonic transit time hence decreasing exposure of colon to carcinogens. Apart from environmental and diet factor, inflammatory bowel disease also predisposes to colorectal carcinoma. Ulcerative colitis increases the risk of developing colorectal cancer with tumours starting to appear 5–8 years after the onset of the disease. Following this the risk increase almost exponentially with time, about 5% of patient developing tumours after 10 years of disease, 20% at 20 years and almost 50% at 30 years. The risk for patients with Crohn’s colitis developing colorectal cancer is less than ulcerative colitis, but still is 4–20 times higher than the general population. Some medical treatments including implantation of ureter into colon, cholecystectomy gastric surgery and terminal ileal resection may also be associated

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with an increase risk of colonic cancer probably as a result of increasing delivery of bile salts and protein breakdown products to the colon. There are several hereditary conditions with a high risk of colorectal cancer. The main hereditary colon cancers include familial adenomatous polyposis (FAP); hereditary non-polyposis colonic cancer (HNPCC or Lynch syndrome) and MYH-associated polyposis (MAP). FAP is a disease leading to the development of numerous colonic cancers. It is inherited in an autosomal dominant fashion with high penetrance but variable expression, and account for about 1% of colorectal cancer (Rustgi 2007). The genetic abnormality has been localised to the FAP locus on the long arm of chromosome 5 (Bodmer et al. 1987; Groden et al. 1991; Kinzler et al. 1991). A recent investigation discovered a FAP founder mutation (a mutation that has been traced from many individuals in the present-day population back to a common ancestor). It was found that a married couple who sailed from England to America around 1630 may be the ancestors of hundreds of people alive today who are at high risk of FAP in the United States (Neklason et al. 2008). The most compelling feature of this condition is the onset and progression of a large numbers of small adenomatous polyps throughout the colon. These polyps begin to appear at puberty and become apparent until age 40. The inevitable course is the development of colorectal cancer, unless interrupted by surgical intervention. HNPCC was originally described in the early 20th century; subsequently it was further elaborated by Henry Lynch (Lynch 1974) and refined in 1990s (Vasen et al. 1991, 1999). This syndrome shows a preference for the right colon and accounts for 3–4% of all colorectal cancer cases. MAP is an autosomal recessive inheritance condition that has been found in association with multiple colorectal adenomatous polyps (Sieber et al. 2003). MAP is defined as involving bi-allelic inactivation and patient harbouring multiple colorectal adenomatous polyps without evidence of FAP (Sieber et al. 2003). Mono-allelic carriers do not carry an increase risk of colorectal cancer (Balaguer et al. 2007). MAP accounts for less than 1% of all colorectal cancers.

Colorectal Carcinoma Pathology Colorectal cancers usually grow circumferentially and radially. Continued radial growth leads to penetration of tumour though the bowel wall that may lead to invasion of neighbouring structures depending on the tumour site. At the time of presentation, approximately 40% patients already have distant metastases. The most common sites for spread of colorectal cancer are in order: liver, lung, retroperitoneal, ovary, peritoneal cavity and rarely adrenals. The most common form of spread is along its lymphatic drainage which occurs in about 40% of colorectal cancer. Invasion of colonic veins may occur in 15–50% cases and lead to hepatic metastases via the portal system or to lung metastases via vertebral venous plexi. Histologically about 95% of colorectal cancers are adenocarcinomas. They may initially appear as polypoidal or ulcerative, circumferential growth then leading to the formation of annular tumour.

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Genetic Changes of Colorectal Carcinoma Advances in molecular biology have led to a great understanding of the genetic changes involved in colorectal cancer. It has been known that the genetic changes parallel the sequences from adenoma to carcinoma and are involved with activation of oncogenes and inactivation of tumour suppressor genes. These changes not only involve alterations in synthesis of cellular proteins such as cell adhesion molecules, but also engage with the genetic control of cellular function such as apoptosis and DNA repair. Oncogenes are transforming genes that lead to carcinogenesis. Normal cells always contain homologous genes named proto-oncogenes. Proto-oncogenes are part of the cell normal genetic machinery and are expressed during cell proliferation. Abnormal activation or mutation of proto-oncogenes leads to oncogenesis. The later then causes malignant transformation by overproduction of normal or mutant oncoproteins which result in uncontrolled continuous proliferation. Tumour suppressor genes act in a contrary fashion to oncogenes by preventing tumorigenesis. It was found that some epithelial cancers that had missed or translocated a chromosome would be malignant, however when these malignant cells were experimentally fused with normal cells, they reverted toward their benign counterparts. When the fused hybrid cell ejected the normal chromosome, malignancy occurs again. It was later found that these missed or translocated chromosomes are tumour suppressor genes. One of most common tumour suppressor gene is p53 which codes for p53 protein. The normal p53 protein binds to chromosomal DNA hence inhibiting cell proliferation. It is apparent that loss of normal p53 by point mutation or chromosomal deletion will predispose to cell proliferation, especially if already stimulated by oncogenic changes. So far the most important genetic alteration identified in the development of CRC include adenomatous polyposis coli (AdPC), mutated in colon carcinoma (MCC) and many genes related to HNPCC such as hMLH1, hMSH2, hPMS1, hPMS2 and hMLH3 (Bronner et al. 1994; Fishel et al. 1993; Leach et al. 1993; Liu et al. 1994; Nicolaides et al. 1994; Palombo et al. 1994; Papadopoulos et al. 1994; Parsons et al. 1993). These will be discussed in more detail in the following related sections.

Current Treatment of Colorectal Cancers The three most common types of CRC treatment are surgery, chemotherapy and radiotherapy. The current mainstay of treatment for CRC is surgery which may be curative or palliative. Curative surgery aims to excise the tumour with an adequate margin of surrounding tissue and its regional lymphatics. Usually appropriate treatment is tailored to the individual patient depending on presentation, fitness for surgery and extent of disease but even in the presence of distance spread the primary tumour may be resected as an effective form of palliation. In order to improve survival rate; chemotherapy and radiotherapy are also used as neo-adjuvant or adjuvant treatment.

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CRC is a common cancer; hence many chemotherapeutic agents have been used either alone or in combination with surgery for the systemic treatment of this disease. The most common drug used is 5-fluorouracil (5-FU). 5-FU is an antimetabolic agent that acts during DNA synthesis (S phase of cell cycle). Although 5-FU has the best single agent activity, the results when used alone are general disappointing with response rate of 15–20% and medium duration of response that is short at less than 6 months. This has not significantly increased survival. The limited efficiency of 5-FU may be due to a combination of its short half life and the fact that only around 3% of cancer cells are in S phase at any moment. Other agents used alongside 5-FU include methotraxate, cisplatin, thymidine, intereron, N-(phosphonacetyl)-Laspartic acid and folinic acid. These agents are used in tandem to modulate 5-FU effects. Radiotherapy has little place in the treatment of gastrointestinal cancers which are generally radio-resistant as the radiation dose required leads to significant side effects such as radiation enteritis and small bowel adhesions. A major clinic study included 8507 patients from 22 randomised trials was carried out by the Colorectal Cancer Collaborative Group (CCCG) to compare the outcome of adjuvant preoperative or postoperative radiotherapy with those of surgery alone (Colorectal Cancer Collaborative Group 2001). It has been found that both pre and post operative radiotherapies significantly reduced the yearly risk of local recurrence compared to surgery alone, although they failed to improve overall survival as deaths from other causes increased. The safety of the pre-radiotherapy requires improvement. If the side effects can be limited without compromising effectiveness, the preoperative radiotherapy would benefit young, high risk patients (Colorectal Cancer Collaborative Group 2001). As radiotherapy reduces the risk of recurrence, if patients have stage II or III rectal cancer; the risk of cancer recurrence is great enough to justify the use of radiotherapy in addition to surgery. There remains some debate about whether it is best to give radiotherapy to people with rectal cancer before or after surgery, but evidence suggests a long or short course in a neo-adjuvant setting is optimal.

Colorectal Tumorigenesis The majority of colonic cancers originate as adenomata although some may pass through the adenoma phase quickly. There are parallel epidemiological studies on the prevalence of colonic adenomata and carcinoma to support this hypothesis. It has been shown that the incidence of polyp disease peaks 5 years before the incidence of colorectal cancer.

Adenoma Adenoma is a benign glandular epithelium tumour found throughout the large bowel. Although they are benign, they are usually considered true neoplasms because of their enlarged and elongated nuclei. The dysplastic cells in early

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adenomata are located at the luminal surface of the crypt while the cells at the base of crypts appear morphologically normal (Shih et al. 2001). Some studies have indicated that the adenomatous polyps grow inward, this means that dysplastic proliferating cells migrate from the top of the crypt to the base of the crypt (Lightdale et al. 1982; Moss et al. 1996a). This suggests that dysplastic process is initiated at the luminal surface of the crypt. There are three growth forms of adenoma. Tubular adenomata have a tube-like appearance consisting of branching of glandular epithelium and account for about 70% of all adenomata. Villous adenomata are pointed or blunt finger-like processes covered by epithelium. About 10% of all adenomata are villous adenomata. Tubulovillous adenomata have a mixed pattern of tubular and villous structures. The Tubulovillous adenoma seems to be an intermediate form between tubular and villous adenoma (Peipins and Sandler 1994) and accounts for about 20% of adenomata. Although it is generally believed that adenoma is pre-carcinoma, the site distribution of adenoma throughout the large bowel does not correlate with the distribution of colorectal carcinoma. Carcinomas occur most frequently in the distal part of the bowel (the rectum and sigmoid colon) (de Jong et al. 1972) whereas adenomata are more evenly distributed throughout the whole large bowel (Correa et al. 1977; Arminski and McLean 1964). There appears to be a relationship between the distributions of large adenomata and that of colorectal cancer (Peipins and Sandler 1994). The variation in the site distribution of adenoma and cancer suggests that the risk factors for adenomata are different from those for colorectal cancer. The risk factors for adenoma may operate throughout the large bowel, while the risk factors for promoting adenoma to carcinomas may only operate or operate more effectively in the distal portion of the bowel. It is difficult to investigate the expected progress from adenoma to carcinoma without interruption as adenomata are usually removed when they are discovered. Although it is difficult to determine the time required for the transition from adenoma to carcinoma, it has been estimated that for fast growing tumours it would require 6–8 years to grow to a 6 cm diameter cancer (Welin et al. 1963). The malignant potential of adenomata varies with size, histological type and grade of epithelial atypia. The adenomatous polyp is usually small and has a low malignant potential, whereas tumours with a villous structure are usually large and have much higher cancer incidence. Nevertheless, it estimated that the period of growth from an adenoma to a cancer is around 5 to 20 years (Muto et al. 1975).

Adenoma-Carcinoma Sequence As the most, if not all, CRC arise from adenomatous polyps the neoplastic process is actually a progression from adenoma to carcinoma. This process has been termed as “adenoma-carcinoma sequence” (Jackman and Mayo 1951). Evidence from autopsy, clinical, epidemiologic and molecular genetic studies has contributed to the development of this theory. Although there have been arguments about

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adenoma-carcinoma sequence (Jass 2004) the following points favour such an evolution. A. Histological similarity between adenomata and carcinomas: Histological aspects of adenomata are frequently observed in specimens of colorectal carcinoma (Kyzer et al. 1992; Pollard et al. 1992). Careful microscopic examination of colorectal tumours revealed contiguous histological transformation from benign to malignant tissue changes in the same tumour (Morson 1974). Immunhistochemical studies that identify the number of tumour associated antigens in tumour cells have detected a similarity between adenomata and carcinomas (Skinner and Whitehead 1981). B. Specific genetic changes have been associated with the progression from adenoma to carcinoma: Molecular genetic studies have associated specific genetic alteration with the cellular progression of hyper proliferation of the adenomacarcinoma sequence (Fearon and Vogelstein 1990). For example the loss of a large portion of chromosome in 17p has been seen in more than 75% of CRCs; but such loss has a different frequency in small, intermediate and large adenomata and carcinomas. The frequency of such loss is related to the progression of individual tumours from adenomata to carcinomas (Fearon et al. 1987). The loss of the wild type p53 allele has also been found to be connected with the progression from adenomata to carcinomas (Fearon and Vogelstein 1990). C. Epidemical investigations have shown the similar age distribution of adenoma and carcinoma: Comparing the age distribution of adenoma patients with that of carcinoma patients has shown that the peak age for the adenomata lies about 7–8 years earlier than that of carcinomas, approximately the time course for adenoma to develop to carcinoma (Enterline 1976). The epidemiological age distribution between adenoma and carcinoma also occurred in familial adenomatous polyposis (Muto et al. 1975). D. Surgical removal of adenoma reduces the incidence of carcinomas: Several cohort studies have shown that colonoscopic polypectomy substantially reduces the incidence of colorectal cancer in the cohort compared with that expected in the general population (Citarda et al. 2001; Meagher and Stuart 1994; Thiis-Evensen et al. 1999; Winawer et al. 1993). These observations not only strengthen the theory that colorectal adenoma progress to colorectal carcinoma, but also support the concept that colonoscopic polypectomy is one of the practical options to prevent CRC. E. Large adenomata have a similar distribution predilection to carcinomas: Generally, the distribution of adenomata throughout the large bowel does not correlate with the distribution of CRC. However, when comparing the distribution of large adenomata with that of CRC (O’Brien et al. 1990; Peipins and Sandler 1994) there is correspondence between the distribution of large adenomata and that of CRC. In summary, the concept of the adenoma-carcinoma sequence is gaining increasing support as an important factor in the development of colorectal cancer. However, not all adenomata undergo malignant changes; and some carcinomas may develop from flat mucosa.

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Gene Mutations and Colorectal Tumorigenesis Normal human colon consists of about 107 crypts; and each crypt contains several thousand differentiated cells and a small number of stem cells. Stem cells reside at the bottom of the crypts and divide slowly and systemically, whereas differentiated cells divide rapidly and travel to the top of the crypt, where they undergo apoptosis. Each day a total of approximately 1010 cells are shed by the colon and have to be replaced (Rajagopalan et al. 2003). During cell division, gene mutations can occur as a result of DNA replication and chromosome segregation (Loeb et al. 1974). This cascade of gene mutations has been known as chromosome instability. Chromosome instability is believed to be responsible for most colorectal cancer. The AdPC gene is a tumour-suppressor gene on chromosome 5q. It is generally believed that colorectal cancer is initiated by inactivation of the AdPC gene. The AdPC gene was first identified in families with a hereditary predisposition to develop colorectal cancer. In this condition, family members are born with an error in one of their two AdPC gene copies. Patients with a mutation in one copy of the gene are usually well as long as the other gene copy is normal. However, due to chromosome instability colon cells develop a second defective gene. As a result, by their teens and twenties these individuals develop hundreds to thousands of polyps. Some of these polyps invariably progress to colorectal cancer. Subsequently it has been found that these types of polyps are not limited to hereditary colon cancer. By the age of 70 years, about 35% general population will develop at least one polyp with mutations in the AdPC gene (Williams et al. 1982). Initially the crypt in which the AdPC-mutant cell resides becomes dysplastic as abnormal cells accumulate to slowly produce a polyp. The development of a large polyp probably requires the acquisition of further mutation, for example, in the KRAS gene (Fig. 4.1). The KRAS gene lies on chromosome 12 and is a proto-oncogene. Mutation in the KRAS gene may either diminish or increase its function. Mutations that lead to an increase in function convert this proto-oncogene into an oncogene. The majority of KRAS gene mutation occurs in codons 12, 13 and 64 (Capella et al. 1991; Ishii et al. 2004; Toyooka et al. 2003). KRAS proteins activate a variety of growth pathways including RAF/MAPK, JNK and PI3-K. Their downstream gene targets include cyclin D1 and vascular endothelial growth factor (VEGF). If a KRAS mutation occurs without a preceding AdPC mutation, the result is a tiny aberrant crypt

Fig. 4.1 The multistage progress of a normal colorectal epithelium to colorectal cancer through a series of genetic mutations

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focus that is non-neoplastic. When it occurs in polyp cells in which the AdPC gene is inactive, the development from polyp to a large adenoma occurs. An effective mutation on only one allele of the KRAS gene will trigger this accelerated growth (Fig. 4.1). DCC/SMAD: DCC (Deleted in Colorectal Cancer) is allelic losses on chromosome 18q. DCC has been identified in approximately 70% of primary colorectal cancers, particularly in advanced colorectal cancer with metastasis (Fearon and Vogelstein 1990) and assumed to be a tumour-suppressor gene encoding a protein with sequences similar to the cell adhesion molecules such as N-CAM (Fearon and Vogelstein 1990). Previous studies have suggested that DCC plays a role in both epithelial and neuronal cell differentiation (Hedrick et al. 1992; Lawlor and Narayanan 1992). In vivo experiments have shown that inactivation of the mouse homologue of DCC does not increase tumour predisposition. These observations fail to support a tumour-suppressor function for DCC (Fazeli et al. 1997a). The role of DCC in colorectal cancer requires further investigation. Subsequently several tumour suppressor genes including SMAD4 have been identified in the same chromosome 18q (Eppert et al. 1996; Hahn et al. 1996). P53 gene (located on chromosome 17) is the most commonly mutated tumour suppressor gene in various kinds of malignant tumours (Olivier et al. 2002). Its product, p53 protein, is a transcription factor that regulates the cell cycle. P53 has been described as “the guardian of the genome” (Bouchet et al. 2006) because of its role in conserving stability by preventing genome mutation. So far it has been found that p53 has three significant actions to stabilise the genome. Firstly p53 can activate DNA repair proteins when DNA has sustained damage. Secondly p53 triggers cell growth arrest at the G1 (Lane 1992) or G2 (Agarwal et al. 1995; Stewart et al. 1995) phase of the cell cycle when DNA damage occurs. Thirdly p53 initiates cell apoptosis if the DNA damage proves to be irreparable (Lane 1992). In this manner, p53 protects the normal cell from proceeding to replicate damaged DNA which usually leads to cell malignancy. As the most commonly altered gene in human, p53 gene has a mutation frequency in excess of 50% (Harris and Hollstein 1993). Most mutations are mis-sense mutations within the evolutionarily conserved DNA binding domain (Hollstein et al. 1994). Mutational induced inactivation of p53 gene is usually critical in the transformation from adenoma to carcinoma, and p53 is inactivated in more than 75% of all colorectal cancer (Boland 1997). Colorectal cancer patients with p53 mutation have a worse outcome and shorter survival time than patients whose cancers do not have mutations in the p53 genes (Russo et al. 2005). P53 mutation rarely occur in human colorectal adenomata with a low or intermediate degree of dysplasia but it occurs much more frequently in high grade of dysplasia (Bronner et al. 1994). In vivo studies have shown that p53 gene mutation plays a role in colon cancer progression; such a role becomes important only in the late stages of disease (Fazeli et al. 1997b) (Fig. 4.1). One significant action of p53 protein is its action on the induction of apoptosis when the DNA damage becomes irreparable. Apoptosis is the “last resort” to avoid proliferation of cells containing abnormal DNA. Apoptotic cell death is a critical element to eliminate the aberrant cells and the failure of apoptosis is decisive in

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Fig. 4.2 Balance changes between cell proliferation and apoptosis along with the progression from normal colorectal epithelium to colorectal cancer

the development of a malignant clone during adenoma-carcinoma transition (Plentz et al. 2003) (Fig. 4.2). The roles of apoptosis in the development of colorectal carcinomas will be discussed in more detail in a later section.

Apoptosis in Colorectal Tumorigenesis Definition of Apoptosis Apoptosis is a strictly regulated cell death in multi-cellular organisms. It involves a series of regulated processions such as morphological changes, loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation. Therefore apoptosis is sometime called “programmed cell death”. There are at least two distinct types of cell death in multi-cellular organisms: apoptosis and necrosis. Necrosis is a form of traumatic cell death which usually results from acute cellular injury and can elicit an inflammatory reaction. In contrast to necrosis, apoptosis usually confers advantages during an organism life cycle. During apoptosis, cellular debris from apoptotic cells is removed in an inconspicuous way via phagocytosis by neighbour cells or specialized macrophage-like cells without damage to the organisms. It usually takes several hours for the full course of apoptosis from initiation to the final fragment of the cell. At the initiation stage, the chromosome condenses around the edge of the nucleus; chromatin is segregated and compacted. At the same time, the cell begins to shrink and cytoplasm begins condensing. Afterward, the nucleus

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becomes fragmented; the whole cell breaks up into membrane-bound apoptotic bodies. Morphologically, the special feature of apoptosis is that the contents of the cell are never released but remain membrane bound. Finally the membrane-bound apoptotic bodies are phagocytosed by surrounding cells. In contrast to apoptotic cells, the necrotic cell plasma membrane becomes permeable in the early stages; the cell starts swell and burst; the contents of the cell are released which often trigger an inflammatory response. Apoptosis may proceed in two pathways: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is called the mitochondrial pathway as it begins with the release of cytochrome C from the mitochondria. It is also called the “damage pathway” because DNA damage is the usual cause of the release of cytochrome C. It is usually triggered by UV irradiation, γ-irradiation, some chemotherapeutic drugs, reactive-oxygen species (ROS: superoxide anion, hydrogen peroxide, organic peroxide) and radicals generated by the cell as a by-product of normal metabolism. The intrinsic pathway is regulated by the Bcl family proteins that are comprised of pro-apoptotic and anti-apoptotic proteins. The extrinsic pathway is caused by the direct interaction between so called “death ligands” and “death receptors”, in which caspase-8 is activated. When death ligands such as tumor necrosis factor α (TNFα), TNF-related apoptosis-inducing ligand (TRAIL), Fas bind to their receptors; a multi-molecular complex of protein is then formed. This complex is called the death-inducing signaling complex (DISC). DISC formation is followed by the activation of caspase 8 which in turn activate caspase 3. The activated caspase 3 executes apoptosis.

Function of Apoptosis in Gastrointestinal Tract Apoptosis is a basic biological procession and plays very important roles in development, growth, morphogenesis, tissue remodelling, immune responses and removal of unnecessary, aged or damaged cells. Throughout adult life several millions cells in the human body undergo apoptosis every second helping to maintain homeostasis in self-renewing organs. In the normal mammalian gastrointestinal tract, apoptosis is inconspicuous histologically; it accounts for the bulk of cell loss and is a central feature of cell number regulation in adult gut (Hall et al. 1994). The physiological functions of apoptosis in gastrointestinal tract can be defined in the following three aspects. A. Apoptosis helps maintain normal cell balance in gastrointestinal tract: The epithelial cells of the gastrointestinal tract proliferate, mature and recycle constantly throughout the life of an individual. The intestinal villus consists of differentiated absorptive cells that originate from the intestinal crypts. The crypt stem cells proliferate to produce the epithelial cells, which migrate upward and proliferate several times before reaching the villus as differentiated epithelia cells. Epithelial cells in the top of villus will then undergo apoptosis and eventually extrusion into the intestinal lumen (Cheng and Leblond 1974a,b,ca; Traber 1994). Normal human colon consists of about 107 crypts; each crypt contains several thousand

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differentiated cells and a small number of stem cells. Stem cells reside at the bottom of the crypts and divided slowly and systemically, whereas differentiated cells divide rapidly and travel to the top of the crypt. Each day a total of approximately 1010 cells are shed into colon lumen through apoptosis (Rajagopalan et al. 2003). Apoptosis, therefore, is crucial for maintenance of normal morphology and function in the gastrointestinal tract. B. Apoptosis removes damaged cells in the gastrointestinal tract: The Gastrointestinal tract is very sensitive to radiation. Various studies have shown an increase in apoptosis in crypts after radiotherapy. A very small dose of radiation can elevate the level of apoptosis in crypt stem cells. The apoptosis plays an important role in terms of protection epithelial cells against carcinogenesis by removal of potential carcinogenic damaged cells (Potten et al. 1994). Epithelial cells in the intestinal mucosa are the initial site of host invasion by viruses. After 6 hours of infection by a virus apoptosis occurs in human colon epithelial cells (Kim et al. 1998). The apoptosis help to prevent the spread of viral infection and prevents aberrant cells from developing into tumours. Several previous studies have found that during the transformation of normal colonic epithelium to benign adenoma or carcinomas, apoptosis markedly decreases in colorectal tissues. The results of these studies demonstrate that adenoma malignant transformation to carcinomas is associated with prolonged cell survival and sustained inhibition of apoptosis (Bedi et al. 1995; Moss et al. 1996b). C. Apoptosis help to archive homeostasis in the gastrointestinal tract: In order to maintain the GI tract homoeostasis the number of cells needs to be kept relatively constant through cell death and cell proliferation. Homeostasis is achieved with the rate of cell mitosis (proliferation) being balanced by apoptosis. If cells divide slower than they die the organism will suffer cell loss. If cells divide faster than they die, the body will effectively develop a tumour. A recent in vivo study with transgenic mice has shown that the balance between proliferation and apoptosis is critically important to maintain homeostasis, when the balance is disturbed by decreased apoptosis advanced tumours and metastasis occur (Guasch et al. 2007).

Apoptosis and Tumorigenesis Spontaneous Apoptosis In normal physiological conditions small intestinal mucosa undergoes a process of continual cell turnover that is essential for maintenance of normal function. Cell proliferation is confined to the crypt, while differentiation occurs during a rapid, orderly migration up to the villus. The differentiated enterocytes, which make up the majority of the cells in the intestinal mucosa, then undergo a process of spontaneous apoptosis. In the small intestine a higher frequency of apoptosis was found in the positions 4 and 5 from the base of the crypt, the apparent location of the stem cells (Potten et al. 1997). Therefore spontaneous apoptosis in crypt may be responsible for eliminating extra stem cells. A high level of pro-apoptosis protein-Bax in the same position (Potten et al. 1997; Potten 1997) further supports this hypothesis.

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It has been estimated that an extra stem cell in a crypt will lead 60–120 additional epithelial cells per crypt (Potten et al. 1997). It makes sense for crypt to keep a stable stem cell pool in order to maintain a stable cell number in the crypt. Spontaneous apoptosis has also been observed in the low and mid region of villi and the incidence increases with increasing distance from crypt. Normal colonic mucosa stem cells, located at the bottom of a crypt, proliferate to form daughter cells that proliferate rapidly and migrate up the crypts, differentiating into columnar epithelial, goblet and enteroendocrine cells (Bach et al. 2000). In normal physiological conditions cell proliferation occurs mainly at the lower half part or two thirds of the crypt. As the daughter cells migrate towards the upper crypt, they lose the ability to proliferate, but still differentiate. A few days later these differentiated enterocytes undergo apoptosis and are shed by the exfoliating action of the gut contents (Strater et al. 1995). Colonic apoptosis of epithelial cell mainly occurs at the luminal surface (Moss et al. 1996a; Strater et al. 1995). It is interesting to note that spontaneous apoptosis in the colonic epithelium occurs independently of p53 and Bax, but is related to the anti-apoptosis protein Bcl-2. It has been found that when the Bcl-2 gene is knocked out, the rate of spontaneous apoptosis increases at positions 1 and 2 from the bottom of colonic crypt, the location of stem cell in colonic crypt (Merritt et al. 1995). Therefore, Bcl-2 gene is believed to play an important role in the regulation of cell number in the colonic epithelium. High level of Bcl-2 proteins present in colonic mucosa is an explanation why colon epithelium is susceptible to cancer. Apoptosis in Early Stage of Adenoma In contrast to normal colonic crypt proliferation and apoptosis distribution patterns; in adenomatous polyps cellular proliferation mainly occurs in the luminal surface and apoptosis in the lower part of crypt (Aotake et al. 1999; Arai and Kino 1995; Moss et al. 1996a,b; Partik et al. 1998; Regitnig and Denk 2000; Sinicrope et al. 1996; Strater et al. 1995). Generally speaking the level of apoptosis in adenoma crypts increases compared to surrounding normal crypts (Shiff and Rigas 1997). An in vivo study using rats has shown that adenoma cells have cell cycle time half that of normal colonic epithelium cells; indicating adenomata have higher growth rates (Sunter et al. 1980) (Fig. 4.2). The increase in apoptosis in adenomata is an attempt to limit the expansion of the tumour cell population and most colorectal adenomata are stable for a long time before transforming to carcinomas. After 3–5 years of adenoma diagnosis 70% have not changed in size (Stryker et al. 1987). One study demonstrated there was a strong correlation between cellular proliferation and apoptotic index in low grade adenoma. However when low grade adenomata developed into high grade dysplasia or carcinoma this correlation was lost (Aotake et al. 1999). The ratio of apoptotic cells to proliferating cells has been found to be higher in small adenomata and lower in large ones with high grade dysplasia (Baretton et al. 1996; Koike 1996). This suggests that the imbalance between cell growth and cell death plays a critical role for the development of adenoma into carcinoma (Fig. 4.2).

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Apoptosis in the Transition of Adenoma to Carcinoma An increasing accumulation of evidence demonstrates that the frequency of apoptotic death in carcinomas is higher than that in adenomata (Baretton et al. 1996; Carr 2000; Hao et al. 1998; Hawkins et al. 1997; Ikenaga et al. 1998; Koike 1996; Nomura et al. 2000; Takano et al. 1996; Ward et al. 1997; Yamamoto et al. 1998). This means that the proportion of epithelial cells undergoing apoptotic cell death increases in the course of the adenoma-carcinoma sequence. This seems to contradict the general idea that decreased apoptosis is associated with cancer progress. However, tumour progression is associated with an imbalance between cellular proliferation and apoptosis (Anti et al. 2001; Melen-Mucha and Niewiadomska 2002), not necessarily with decreased apoptotic cell death (Fig. 4.2). The increased cell growth and proliferation activity in tumours might directly increase apoptosis due to lack of nutrients, competition for growth factors and oxygen supply (Evan and Littlewood 1998). Many studies have shown that the level of cellular proliferation correlates with apoptotic cell death in adenomata and carcinomas (Arai and Kino 1995; Carr 2000; Evertsson et al. 1999; Hao et al. 1998; Ikenaga et al. 1998; Sinicrope et al. 1999; Tatebe et al. 1996). It might well be that the increased cell proliferation corresponds with an increased proportion of apoptotic cell death during the adenoma-carcinoma sequence. The prolonged period of transition of adenoma to carcinoma might well help tumour tissue to be selected for the survival of mutant cells or cells with a survival phenotype. The selection of advantageous mutations is as important as an increased mutation rate in carcinogenesis (Tomlinson et al. 1996) and the selective pressure would reverse the increased apoptosis. A higher level apoptosis in the progress of the adenoma-carcinoma sequence also helps explain the slow growth of adenomatous polyps and why there was prolonged period (5– 20 years) for an adenoma to grow to a carcinoma (Muto et al. 1975). The level of apoptosis in tumour tissues is changed along with the progress of carcinogenesis during the adenoma-carcinoma sequence. The incidence of apoptosis was found lower in carcinomas than in the adenomata (Aotake et al. 1999; Bedi et al. 1995; Partik et al. 1998; Valentini et al. 1999); while other studies found that apoptosis level was the same in both (Kawasaki et al. 2001; Kikuchi et al. 1997; Moss et al. 1996b; Sinicrope et al. 1998). All of these studies, however, found that as long as there was imbalance between proliferation and apoptosis with higher rates of proliferation compared to the lower incidence of apoptosis, there was progress from adenoma to carcinoma.

Relation of Apoptosis and Gene Mutations AdPC gene mutations and apoptosis: In addition to being a tumour-suppressor, AdPC also can act as a pro-apoptotic protein. The normal AdPC protein can accelerate apoptosis-associated caspase activity without increasing caspase expression and translation (Chen et al. 2004; Steigerwald et al. 2005). This is abolished by addition of caspase-8 inhibitor suggesting caspase-8 is an essential component of AdPC-mediated apoptosis (Chen et al. 2004; Steigerwald et al. 2005). Mutations in

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AdPC correlate with a decrease in apoptosis (Bernal et al. 2005; Venesio et al. 2003) but is tissue-dependent (Hasegawa et al. 2002). Loss of AdPC in cell culture results in a slower apoptosis (Nathke 2006). Transgenic mouse studies showed that inactivation of AdPC decreases apoptosis in differential cells near the villus tip (Sansom et al. 2004). In the crypt loss of AdPC initially led to an increase in apoptosis followed by a decrease (Sansom et al. 2004) suggesting that tissue compartments might have a different response to AdPC loss (Nathke 2006). AdPC has been shown to be expressed in normal colorectal epithelial cells as they migrate from the base to the top of crypts (Senda et al. 1996; Smith et al. 1993). Disruption of AdPC would disturb the balance between new cell formation at the base of the crypts and the cell death at the top of the crypts, leading to a relative expansion (tumour) of the progeny of AdPC-mutant cells. Taken together, these studies demonstrate that apoptosis is involved in the very earliest stages of colorectal neoplasia. KRAS gene mutations and apoptosis: The KRAS is an oncogene, approximately 40–50% of colorectal carcinomas have KRAS gene activating mutations (Andreyev et al. 1998; Bos 1989). There are several inter-connected pathways initiated by KRAS. KRAS activated MAPK up-regulates the multi-drug resistancemediating p-glycoprotein, leading to cancer cell resistance to several commonly used anti-neoplastic drugs (Fujita et al. 2005). KRAS activated P13-K pathway promotes cell survival though the induction of pro-survival genes and inhibition of pro-apoptotic Bcl-2, leading to a decrease in mitochondria dependent apoptotic activity (Cardone et al. 1998). So far the mechanism through which KRAS mediates progression of colorectal cancer is unclear. Activation of RAS in mouse models does not cause colorectal cancer and little is known about the role of the RAS-MAPK pathway in the regulation of cell number on the crypt/villus axis. However, several studies have shown that suppression RAS by a RAS kinase suppressor induces an anti-apoptosis effect (Yan et al. 2004). P53 and apoptosis: Tumour suppressor gene p53 is mutated in 75% of colorectal cancer patients (Fearon and Vogelstein 1990). Wild-type p53 can induce apoptosis in a human colon tumour-derived cell line (Shaw et al. 1992). It has been found that p53 has profound effects on cellular responses to chemotherapeutic drugs used in colorectal cancer (Bunz et al. 1999). A study using 4 different colorectal cancer cell lines (HCT116, RKO, RW2982 and SW403) showed that p53 played an important role in the apoptotic cascade induced by oxaliplatin. Inactivation of p53 induced significant resistance to oxaliplatin (Arango et al. 2004). The impact of p53 protein on human colorectal adenoma/carcinoma apoptosis remains controversial. Many studies have demonstrated that adenoma and/or carcinoma with a high rate of cellular p53 expression are more likely to have a low apoptotic index (De Angelis et al. 1998; Kikuchi et al. 1997; Kobayashi et al. 1995; Koike 1996; Langlois et al. 1997; Schwandner et al. 2000; Sinicrope et al. 1996; Tanimoto et al. 1998). Other studies have shown that the frequency of apoptosis in colorectal neoplasia increases in the course of tumour progression. This is associated with a disturbed balance of proliferation and apoptosis, but is not influenced by p53 gene mutations (Hao et al. 1998; Hawkins et al. 1997; Takano et al. 1996; Tatebe et al. 1996). This controversial phenomenon may be due to the diversity of human colorectal cancer

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samples used in these studies. P53 mutation rarely occurs in human colorectal adenomata with a low or intermediate degree of dysplasia but it was seen much more frequently in high grade dysplasia (Bronner et al. 1994). In vivo studies also showed that p53 gene mutation plays a role in colon cancer progression; such a role becomes important only in late stages (Fazeli et al. 1997b) (Fig. 4.1). SMAD 4 and apoptosis: Smads are a novel class of proteins that function as transcription regulators. They bind directly to cis regulator of gene promoters or interact with the transcription regulators (Akhurst and Derynck 2001; Derynck et al. 2001). Smad proteins play a pivotal role in intracellular TGF-β signalling (Heldin et al. 1997; Massague 1998). Eight Smads have been found in mammals and divided into three groups according to their functions in signalling transduction. Inactivated mutations of smad proteins have been found in human cancers. Smad 4 mutations have been observed in up to 30% of CRC (MacGrogan et al. 1997; Takagi et al. 1996; Thiagalingam et al. 1996) and smad 2 mutations found in 4% (Eppert et al. 1996; Riggins et al. 1997) of human CRC.

Factors Effecting Apoptosis in Colorectal Cancer TGF-ββ Transforming Growth Factor-beta (TGF-β) is a member of a growth factor superfamily that regulates cellular proliferation, differentiation, apoptosis and extracellular matrix formation (Shi and Massague 2003; Derynck and Feng 1997; Massague 1998). TGF-β serves as a tumour suppressor in the normal colon by inhibiting cell proliferation and inducing apoptosis (Markowitz and Roberts 1996). Disruption of TGF-β allows colon epithelial cell to escape growth suppression and is consistent with the transformation of human colon adenomata to malignant carcinomas (Grady et al. 1998; Markowitz and Roberts 1996). The most disruption of TGF-β signalling results from mutational inactivation of the TGF-β receptor (TGFBR2) that occurs in approximately 30% of CRC (Grady et al. 1999). Using defined cell line systems it has been shown that both genomic instability and clonally selection of TGF-β resistant cells contribute to the high frequency of TGFBR2 mutations in microsatellite unstable (MSI) colon cancers (Biswas et al. 2008). Following binding to its receptors, TGF-β is capable of activating SMAD transcriptional factors to regulate the expression of target genes (Heldin et al. 1997). However, the mechanism of TGF-β inducing apoptosis is largely unknown. It has been indicated that the TRAIL system is critically involved in TGF-β induced cell death (Herzer et al. 2005). TGF-β signalling in human CRC has not been fully characterized owing to its high mutational frequency (Ijichi et al. 2001), TGF-β-induced apoptosis is an active field of research. As TGF-β-induced apoptosis plays a critical role in opposing unscheduled increases in cell proliferation (Wakefield and Stuelten 2007), the TGF-β pathways and its mediated proteins such as SMAD is becoming an attractive therapeutic target for the treatment of CRC.

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Survivin Survivin is a small anti-apoptosis protein that is expressed strongly in embryonic and foetal tissues. In the normal differentiated tissues it has only been found in the thymus and basal colonic epithelium (Altieri 2003). Using immunohistochemistry techniques 53% of 171 human CRC samples were found to express survivin (Kawasaki et al. 1998). Survivin-positive cases were strongly associated with reduced apoptotic index and its presence in colorectal cancer has become an important predictive parameter for poor outcome in CRC (Kawasaki et al. 1998). It is believed that survivin can bind caspase-3 and/or caspase-7 and prevent them acting as executors of apoptosis thus suppressing apoptosis (Shin et al. 2001; Tamm et al. 1998). Survivin regulates the cell cycle by promoting microtubule formation in the mitotic spindle. Disruption of survivin-microtubule interactions results in loss of survivin anti-apoptosis function during mitosis (Li et al. 1998). This suggests that survivin may counteract a default apoptosis in cell cycles (Prabhudesai et al. 2007).

IGF-I Insulin-like growth factor I (IGF-I) has been reported to have the potential to protect a broad range of cells from a variety of apoptosis challenges. IGF-I receptors are present in the primary cell masses of human colon carcinomas and in CRC cell lines (Pollak et al. 1987). Colorectal carcinomas have a 10 to 50-fold increases in the level of IGF-I and IGF-II when compared to adjacent uninvolved colonic mucosa (Freier et al. 1999; Hakam et al. 1999; Michell et al. 1997). IGF-I stimulates growth of HT-29, LS411N LS513, SW480 and WiDr human colorectal carcinoma cell lines (Lahm et al. 1994). Accumulated data from laboratory experiments demonstrate that IGF-I and IGF-II are able to stimulate the growth of wide variety of cancer cells and to suppress apoptosis. The IGF system has become an attractive molecular target for anticancer therapies. Inhibition of the IGF-IR pathway has not been successfully exploited as a therapeutic strategy due to the lack of clinically applicable inhibitors of IGF-IR. Although some positive results have been obtained in recent in vivo studies using anti-IGF-IR antibodies to treat prostate cancer (Wu et al. 2006), the adverse effects of this therapy cannot be ruled out as it interferes with the systemic IGF system. Functionally IGF-I has metabolic and mitogenic actions (which include antiapoptosis and cellular survival functions). It has been shown that IGF-I regulates cellular proliferation, differentiation (Drucker 1997) and apoptosis (Remacle-Bonnet et al. 2000) of intestinal epithelium cells. IGF-I fully protected HT-29-D4 colon carcinoma cells undergoing apoptosis induced by tumour necrosis factor-α (RemacleBonnet et al. 2000). Disruption of the interaction between IGF-I and its receptor with a IGF-I receptor antagonist increases colon cancer cell caspase 3/7 activity 2–7 times; caspase 8 activity 2–5 times and caspase 9 1.2–1.6 times (Yang et al. 2008). The efficiency of this IGF-I receptor antagonist offers a potential novel therapy for CRC in the future (Yang et al. 2008). Over-expression of IGF-I inhibitory protein,

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IGF binding protein 4 (IGFBP-4), in an in vivo CRC model elevated cancer tissue apoptosis and decreased cancer cellular mitosis (Durai et al. 2007a,b).

COX-2 Cyclooxygenase (COX) is an enzyme that converts arachidonic acid to prostaglandin H2 . The latter is subsequently converted to a large number of other structurally related prostaglandins. There are three COX isoforms: COX-1, COX-2 and COX-3. COX-3 is a splice variant of COX-1 which retains intron one and has a frameshift mutation, therefore it is also called as COX-1b or COX-1v (Chandrasekharan et al. 2002). COX-1 is a constitutive enzyme being found in most mammalian cells. COX-2 is undetectable in most normal tissues, but becomes abundant in activated macrophages and other cells at sites of inflammation. COX-2, but not COX-1, is induced by a wide range of growth factors and cytokines (Gupta et al. 2002). In terms of their molecular biology, COX-1 and COX-2 have the similar molecular weight (approximately 70 and 72 kDa respectively), 65% amino acid sequence homology and near-identical catalytic sites. COX-2 is over-expressed in 40% of colorectal adenomata and 85% of CRC (Eberhart et al. 1994). This occurs in many types of cells within the tumour; including the neoplastic epithelial cells, endothelial cell, infiltrating host fibroblasts and inflammatory cells (Williams et al. 2000a). Disruption of either COX-1 or COX-2 reduces the development of colonic tumour in mice (Chulada et al. 2000; Oshima et al. 1996). Accumulating evidence from both clinical trials and animal studies has shown that non-steroidal anti-inflammatory drugs (NSAIDs) which inhibit either COX-1 or COX-2 reduce the development of CRC (Hawk et al. 2003) and inhibition of COX-2 can treat established cancers without toxicity to the gastrointestinal tract (Williams et al. 2000b). The capability of COX inhibitors to reduce cancer development is due to the induction of apoptosis in adenoma (Martin et al. 2002; Watson 2004). The fact that over-expression of COX-2 in HCT-15 colorectal cancer cells attenuates NSAIDs and 5-FU-induced apoptosis, up-regulates Bcl-2 protein and inhibits the cytochrome c-dependent apoptotic pathway (Sun et al. 2002) suggests that COX-2 anti-apoptosis is due to increased anti-apoptotic proteins, Bcl-2 and Bcl-XL , and decreased pro-apoptotic protein, BAX.

ITF Intestinal trefoil factor (ITF) is a compact protease-resistant peptide secreted by goblet cells in both small and large bowel. The ITF biological functions include enhancing cell migration, promoting wound healing and protecting intestinal epithelial barrier from damage (Kanai et al. 1998; Mashimo et al. 1996). ITF deficiency has increased in apoptosis in the colon and an ITF-expressing colonic cell line is resistant to apoptosis induced by serum starvation. Exogenous ITF protects human colonic carcinoma-derived cell line (HCT116) and a non-transformed rat intestinal

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epithelial cell line (IEC-6) from apoptosis (Kinoshita et al. 2000). Because goblet cell apoptosis is often a colonic response to radiation and chemotherapy it was proposed that radiation and chemotherapy reduced ITF expression in goblet cell leading impaired healing for damaged colon (Beck et al. 2004).

Apoptosis and Colorectal Cancer Chemotherapy Some Chemotherapies Inducing Cancer Apoptosis CRC is presently treated by surgical ablation, but many colorectal cancers are detected at a later stage when surgery cannot cure the disease. At least 40% of patients with colorectal cancer develop metastases; chemotherapy alone or in combination with radiotherapy can be used as an adjuvant therapy to surgery for more advanced disease (Labianca et al. 1997). Chemotherapeutic agents usually induce a serial of cellular responses which affect tumour cell proliferation and survival. Apoptosis is one of these responses. To this day the most studied and major chemotherapeutic agent in colorectal cancer is 5-fluorouracil (5-FU). Combining 5FU with agents such as oxaliplatin has significantly improved response rates up into the 40-50% range in metastatic colorectal cancer patients (Giacchetti et al. 2000). It has been found that both 5-FU and oxaliplatin act in advanced colorectal cancer by the selective induction of apoptosis in colon cancer cells (Arango et al. 2004; Rigas et al. 2002). The Combination of 5-FU with irinotecan also induces apoptosis in various colon carcinoma cell lines (Grivicich et al. 2005). Increased numbers of in vitro and in vivo studies have demonstrated that activation of apoptosis contributes to the cytotoxicity action of most chemotherapeutic agents (Hannun 1997; Johnstone et al. 2002). It has been shown that both intrinsic and extrinsic apoptosis pathways are activated in all chemotherapy induced apoptosis (Ferreira et al. 2000; Friesen et al. 1999; Fulda et al. 1997, 1998a,b,c, 2001).

Apoptosis and Cancer Cell Resistance to Chemotherapy Even after chemotherapy metastases still spread due to cell resistance. This is a major contributor to the limited effectiveness of current chemotherapeutic drugs. Cancer cells utilize many mechanisms to protect against the damaging affects of chemotherapeutic agents. For instance, some cancer cells can reduce intracellular drug accumulation by increasing drug efflux from cells and decreasing drug influx into cells. Some cancer cells can reduce drug damage by modifying drug targets or activating DNA repair mechanisms. Perhaps a very important mechanism for cancer cell resistance to chemotherapy is the inhibition of apoptosis. The apoptotic process includes the initiation, transduction, amplification and execution stages. Disruption or dysfunction at any of these stages would disable apoptosis leading to cancer cell resistance to chemotherapy. Defects of apoptosis can be divided into dysfunction in the intrinsic and the extrinsic pathway.

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Dysfunction in the intrinsic pathway: Disruption of the intrinsic apoptotic pathway is very common in colorectal cancers. The p53 tumour suppressor gene is the most frequently mutated gene in human CRC (Fazeli et al. 1997b). As being discussed previously, loss of p53 function can both disable apoptosis and accelerate tumour development. Because Bcl-2 family members play an important role in regulating the intrinsic apoptotic pathway; mutations or altered expression of Bcl-2related proteins can dramatically change sensitivity in human cancers (Reed 1999) or animal cancer models (Reed 1999; Schmitt et al. 2000; Wei et al. 2001; Zhang et al. 2000). Release of cytochrome c from mitochondria is also a critical step of the intrinsic apoptotic pathway and is named as one of post-mitochondrial events (Johnstone et al. 2002). In the case of defects in post-mitochondrial events; even if the outer mitochondrial membrane has been permeated, the cytoplasmic cytochrome c can still be relocated to the mitochondria to restore trans-membrane potential and maintain ATP production, thus preventing apoptosis from happening (Waterhouse et al. 2001). Dysfunction in the extrinsic pathway: The extrinsic pathway of apoptosis is caused by the direct interaction between so called “death ligands” and “death receptors”, in which caspase-8 is activated. When death ligands such as TNFα, TRAIL, Fas etc. bind the death receptors such as TRAIL receptor and CD95; a multi-molecular complex of protein is then formed. This complex is called the death-inducing signaling complex (DISC). DISC formation is followed by the activation of caspase 8 which in turn activates caspase 3. The activated caspase 3 initiates apoptosis. This pathway can be regulated by c-FLIP, which inhibits upstream activator caspases and inhibitor of apoptosis proteins (IAP). Mutations in cancer cells often target regulators of the intrinsic apoptotic pathway; tumourigenic disruption in extrinsic pathway occurs less frequently. Nevertheless, CD95 and TRAIL receptors are all involved in the procession of lymphocytes killing tumour cells. Dysfunction of the death receptor could allow cancer cell to escape from immune responses and provide an advantage to the developing cancer cells. Previous studies have shown that loss of CD95L or TRAIL function can promote tumour growth and spread (Rosen et al. 2000; Takeda et al. 2001).

Challenges for CRC Treatment Despite advances in treatment of colorectal cancers, the prognosis for patients, especially for those with metastatic colorectal cancers, remains poor. In reality, the causes of the limited effectiveness of current chemotherapeutic drugs include drugs systemic toxicity due to a lack of specificity; rapid drug metabolism and both intrinsic and acquired drug resistance. It is therefore important to start looking for targeted therapeutic or tailored treatment strategy. Although not every conventional agent was designed to induce apoptosis directly, in fact most of these agents kill cancer cells though apoptotic pathways directly or indirectly. Drugs that directly induce apoptosis have several advantages over those that do not directly cause

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apoptosis. Firstly, apoptosis removes apoptotic cells in an inconspicuous way with phagocytosis by neighbour cells or specialized macrophage-like cells without producing inflammation and damage to normal boundary tissue and cells. Secondly, drugs that induce apoptosis directly are less mutagenic. Finally directly inducing apoptosis would eliminate cancer cell totally and avoid tumour recurrence, unlike cytostasis which usually leads to tumour relapse.

Strategies for Colorectal Cancer Treatments Inducing apoptosis is an ultimate objective of anticancer treatments. New colorectal cancer therapeutic strategies should target apoptosis regulators thus improving response rates to chemotherapy in colorectal cancer. Manipulation of p53 Several studies have shown that the inactivation of p53 can significantly increase colorectal cancers resistance to 5-FU (Bunz et al. 1999; Longley et al. 2002) and oxaliplatin (Arango et al. 2004). Considering 75% colorectal cancer patients exhibit p53 mutations, drugs that restore p53 activity in p53 null tumours or that activate p53 downstream pathway may have clinical benefits. There are several ways to manipulate p53 function. The first approach is to restore or increase p53 function in cancer cells that would then become more susceptible to chemotherapeutic agents. In an animal model it has been demonstrated that intra-tumour injection of adenovirus-mediated wild type p53 gene significantly increased colorectal cancer apoptosis and suppressed tumour growth (Spitz et al. 1996). Using a replication-deficient virus carrying wild type of p53 gene (Ad5CMV-p53) to replace mutated p53 in tumour cells suppressed tumour growth and increased survival of nude mice bearing human colon cancer expressing mutant p53 (Baek et al. 2004; Harris et al. 1996). The second way to correct mutated p53 gene is to kill p53-deficient cells selectively. This approach requires the introduction of genetically modified viruses which take advantage of dysfunctional p53 in cancer cells. It therefore selectively kills p53 dysfunctional cancer cells. A human adenovirus E1B gene encodes a 55-KD protein that inactivates p53 protein. It has been found that E1B gene-attenuated adenoviruses (Onyx-015) selectively lysed p53-deficient human tumour cells but not cells with functional p53 (Bischoff et al. 1996). An in vitro experiment (Heise et al. 1997) has shown that Onyx-015 selectively lyse colon cancer cell lines (SW620, HT-29, DLD-1 and SW480). Administration of Onyx-015 to patients with metastatic colorectal cancers by hepatic artery infusion has been found to kill tumour cells selectively; no dose-limiting toxicities were observed in this phase I/II study. Patients had failed extensive first-line prior chemotherapy therapy and the median survival of patients was improved after Onyx-015 treatment (Reid et al. 2005).

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The final method is to manipulate p53 function using small-molecule modulators of the p53 family signalling pathways. Using a cell-based chemical library screening strategy a number of small molecules that activate p53, or increase expression of p53 downstream protein, have been identified (Wang et al. 2006). It has been shown that some of these small molecular compounds have potent anti-tumour effects in either HCT116/p53 (-/-) or DLD1 human colon tumour xenografts (Wang et al. 2006). Many of these compounds act at relatively high doses which are unsuited with their pharmacological use. Therefore these compounds need to be optimized with appropriate pharmacological properties.

Down-Regulation of Anti-Apoptotic Proteins The majority of colon carcinomas are known to over-express anti-apoptotic proteins such as survivin, Bcl-2, Bcl-xL and X-IAP which are involved in the regulation of apoptosis. Using antisense approaches to decrease expression of these proteins would restore or increase cancer cell apoptotic potential. Applying Bcl-xL antisense oligonucleotide to Caco-2 colon cancer cells reduces the Bcl-xL protein level by almost 50% and results in a 300% increase in apoptosis after IR treatment (Wacheck et al. 2003). Combinational inhibition of bcl-2, bcl-XL and EGF receptor by antisense oligonucleotides results in a strong ant-proliferate and pro-apoptotic response in colon cancer cells and tumour xenografts in nude mice (Tortora et al. 2003). Induction of a natural antisense of survivin in a human colon cancer cell line resulted in a down-regulation of survivin expression and an increase in apoptosis and subsequently increased in the sensitivity to anticancer agents (Yamamoto et al. 2002). Increasing evidence from in vitro and in vivo experiments indicate that such approaches may work; but efficient delivery of the antisense DNA to tumour cells for clinical use is still a challenge.

Targeting Factors Which Affect Apoptosis IGF-1: Insulin-like growth factor I (IGF-I) has been reported to have the potential to protect a broad range of cells from a variety of apoptotic challenges. IGFI receptors are present on the primary cell masses of human colon carcinomas (Pollak et al. 1987). Colorectal carcinomas have a 10 to 50-fold increase in the level of IGF-I and IGF-II when compared to adjacent colonic mucosa (Freier et al. 1999; Hakam et al. 1999; Michell et al. 1997). IGF-I stimulates growth of HT-29, LS411N LS513, SW480 and WiDr human colorectal carcinoma cell lines (Lahm et al. 1994). Accumulated data demonstrates that IGF-I is able to stimulate the growth of wide variety of cancer cells and to suppress apoptosis. Experimentally the IGF system has become an attractive molecular target for anticancer therapies. Inhibition of the IGF-IR pathway has not been successfully exploited as a major anticancer therapeutic strategy due to the lack of clinically applicable inhibitors. Although some positive results have been obtained in recent in vivo studies using anti-IGF-IR antibodies to treat prostate cancer (Wu et al. 2006), the adverse effects

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of this therapy cannot be ruled out as it interferes with the systemic IGF system. We recently designed and synthesised a novel antagonist of IGF-I receptor. The effect of this antagonist on human colon cancer cell proliferation was examined. The results showed that the antagonist induced colon cancer cell apoptosis and inhibited cancer cell proliferation. The different changes of Caspase 3/7, 8 and 9 activities suggested that the extrinsic pathways may play a major role in the antagonist peptide induced apoptosis (Yang et al. 2008). Using a gene transfer approach an IGF-I inhibitory binding protein- IGFBP-4 was over-expressed around the tumour in colorectal animal model. This resulted in a decrease in the expression of Bcl-2 and increase in Bax and subsequently increased tumour apoptosis (Durai et al. 2007a,b,c). Survivin: 53% of human colorectal cancer samples are survivin-positive. Strong expression of survivin is strongly associated with a reduced apoptotic index and its presentation in colorectal carcinoma has become an important predictive/prognostic parameter for poor outcome (Kawasaki et al. 1998). Therefore survivin, as a suppressor of apoptosis, has been proposed as an attractive target for new anticancer interventions (Pennati et al. 2007). Several approaches have been used to counteract survivin in tumour cells. Anti-sense oligonucleotides that inhibit survivin mRNA translation have triggered spontaneous apoptosis in human melanoma cell lines (Grossman et al. 1999). Small molecule antagonists such as CDK inhibitor prevent survivin phosphorylation (Pennati et al. 2005) and Hsp90 inhibitors counteract survivin-Hsp90 interaction (Meli et al. 2006). Both increased apoptosis in multiple tumour cell lines including human CRC cells. Survivin has an especially

Fig. 4.3 Potential targets for the new approaches to the colorectal cancer chemotherapy (targets have been highlighted with red)

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immunological property; therefore it can be used for a survivin-directed immunotherapeutic approach. Several phase I trials have been carried out based on this approach (Tsuruma et al. 2004a,b).

Activating an Alternative Apoptosis Mutations in the extrinsic apoptotic pathway are not as frequent as those in the intrinsic apoptotic pathway. Over-expression of Bcl-2 or Bcl-XL or other intrinsic proteins don’t affect TRAIL induced apoptosis (Walczak et al. 2000). TRAIL specifically target tumour cells and is relatively non-toxic to untransformed cells. Recently an in vitro and in vivo experiment using human monoclonal antibodies (HGS-ETR1 and 2) to treat colorectal tumour cell lines (Colo 205, HCT 116 and HCT-15) and colorectal xenograft in a mouse model has been carried out (Marini et al. 2006). It demonstrated that HGS-ETR1 and HGS-ETR2 induced apoptotic cell death in a dose-dependent fashion and the efficacy of the treatment seems to be at least partially Bax-dependent. Similarly to the results from cell culture experiments, in vivo experiments demonstrated a dose-dependent delay in tumour growth following the treatment with both antibodies (Marini et al. 2006). Interestingly we recently designed and synthesised a novel antagonist of IGF-I receptor. Treating colorectal carcinomas cell line (HT-29) with this antagonist induced predominantly extrinsic apoptotic cell death (Yang et al. 2008) (Fig. 4.3). This may have the potential to be developed as a novel therapy for CRC in the future.

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Chapter 5

Apoptosis in Cutaneous Melanoma Michael B. Nicholl and Dave S.B. Hoon

Abstract Cutaneous melanoma is in general highly refractory to chemotherapy and radiotherapy. Melanoma has been resistant to therapeutics that induce apoptosis leading to a poor outcome in development of effective therapeutics. Over the years countless experimental drugs have been tested to induce apoptosis to melanoma cells in patients but very few have gone beyond phase III treatment. As melanoma progresses it develops stronger anti-apoptotic properties, thus the management of disease beyond surgical resection is very difficult. In this review we examine some of the key factors that have been shown to play a role relating to anti-apoptosis events in melanoma. These factors include p53, APAF-1 (apoptotic protease activating factor-1), Bcl-2, and IAP (inhibitors of apoptosis). The review presents how several of these factors, such APAF-1 and IAPs, can be used as surrogate biomarkers for disease outcome. APAF-1 loss or downregulation was demonstrated to be an important factor in melanoma’s aggressive behavior. The use of apoptosis related factors as biomarkers can be applied to assess both tumor tissue and blood. By assessing tumors and identifying their potential of resistance to therapeutic intervention, alternative treatment strategies may be considered. Effective therapeutics to melanoma may be developed to target agents that cause apoptosis-independent death. It is highly important to further understand the mechanism of melanoma cell apoptosis resistance in order to develop more effective new therapeutics. Keywords APAF-1 · Melanoma · Survivin · Apoptosis · IAP

D.S.B. Hoon (B) Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404, USA e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 5,

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Introduction Overview of Cutaneous Melanoma Care Melanoma is an aggressive form of skin cancer and leads to the largest number of deaths from skin cancer annually. Early stages of cutaneous melanoma have an excellent prognosis; in advanced stages the prognosis is poor with few patients expected to survive beyond five years (Balch et al. 2001). Fortunately, the majority of melanoma is diagnosed in the early stages. Treating the disease early in its course lessens its lethal capacity as it is the development and growth of widespread metastasis which are responsible for melanoma-specific deaths (Eton et al. 1998; Lee et al. 2000). Estimates predict 62,480 new cases and 8420 deaths from melanoma in the United States in 2008 (Jemal et al. 2008). These numbers are representative of an ever increasing rate of melanoma diagnosis as reflected in the increase in melanoma incidence over the past few decades (Jemal et al. 2000). From 1986 to 2001, the incidence is estimated to have increased 2.4 fold (Welch et al. 2005). The reasons are likely multifactorial and include better surveillance by dermatologists and increased skill of pathologists to diagnose the disease. Unfortunately, the mortality from melanoma has remained largely unchanged over the same time period (Jemal et al. 2001). Effective treatment of melanoma consists of surgical excision of the primary tumor and metastatic lesions, when feasible (McKinnon et al. 2005; Wood et al. 2001). Improved overall survival can be achieved in patients with metastatic disease, but only if the tumor lesion can be completely removed with surgery (Barth et al. 1995). Despite aggressive surgical intervention, the majority of high-risk patients will have melanoma recurrence. Poor outcomes result for those patients with disease unamenable to surgery because effective adjuvant therapy is lacking, regardless of the treatment strategy. Melanoma is known to be in general not very responsive to radiation, chemotherapy or immunotherapy. Many believe this is strongly associated with strong anti-apoptosis mechanisms in melanoma cells. Unfortunately there has not been any new effective therapeutic drug approved by the USA FDA in the last 10 years that has significant long-term effects on AJCC stage III and IV patients. Chemotherapy, immunotherapy, and biochemotherapy offer marginal improvements in survival, although some treatments do transiently halt disease progression (Tawbi and Kirkwood 2007; Lui et al. 2007; Agostino et al. 2007; Terando et al. 2007; Kim-Schulze et al. 2007). Melanoma is recalcitrant to many chemotherapeutic mechanisms. Although dacarbazine (DTIC) is FDA approved for adjuvant melanoma therapy, treatment with this drug and others including, alkylating agents, taxanes, vinca alkaloids, alfa-interferon and platinum compounds, show little or no benefit to overall survival when used alone or in combination (Tawbi and Kirkwood 2007; Whitehead et al. 2004; Einzig et al. 1996; Luce et al. 1970; Schilcher et al. 1984). It is observed that as a primary melanoma lesion grows it has a high

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propensity to metastasize and become increasingly resistant to apoptosis. Inhibition of apoptosis is one of the most common means of chemotherapy resistance in melanoma (Serrone and Hersey 1999; Glinsky et al. 1997). This has been a continual challenge in developing new effective chemotherapeutics to melanoma. Melanoma exhibits high resistance to radiotherapy (RT) (Mendenhall et al. 2008). Prospective trials show adjuvant radiation to decrease local recurrence rates in highrisk melanoma, but RT does not improve overall survival. RT often is associated with a high complication rate in patients who have had regional lymphadenectomy. Significant numbers of patients who undergo RT for extremity melanoma will suffer from intractable and activity-limiting lymphedema. Thus RT treatment to control melanoma progression is not effective and its side effects are unfavorable. A major paradox surrounds immunotherapy for melanoma. Although human melanoma is considered to be one of the most significant immunogenic cancers, it is very resistant to host immune attacks. Host adaptive immune responses such as cytotoxic T-cells and antibodies are known to be generated during early tumor development; however the tumor eventually becomes resistant to these immune responses. Similarly, innate immune responses have been shown to be induced in melanoma development but eventually become inefficient in controlling tumor progression. Immunotherapeutic strategies such as monoclonal antibody administration and adoptive immunotherapy have had some success but these treatments are still not validated in randomized trials (Agostino et al. 2007; Terando et al. 2007). Surgical resection still remains to date the most significant intervention to cure and control melanoma.

Mechanisms of Apoptosis in Melanoma Apoptosis is programmed cell death in which activation of a specific enzyme cascade ultimately leads to characteristic cell shrinkage, plasma membrane blebbing, nuclear condensation, and endonucleolytic cleavage of DNA into fragments as originally described by Kerr et al. (1972). Although the mechanism of apoptosis has been well described, it is continually being updated with new pathways and factors. As systemic biology analysis develops, apoptosis pathways will be better understood; this is inevitable as the human genome is being further deciphered and more detailed analysis of gene expression and regulatory mechanisms involving mRNA, miRNA, proteomics, transcriptional factors, etc are being carried out. An extrinsic (death receptor) pathway and intrinsic (mitochondrial-mediated) pathway governs the activation of apoptosis. p53 is a significant factor involved in apoptosis however its mechanistic role in melanoma cell is complex and not fully deciphered. A normally functioning apoptotic mechanism requires adaptor molecules and regulators of apoptosis, besides the signals necessary to activate the extrinsic and intrinsic pathways, and the proenzymes which are activated as the cascade progresses (Hussein et al. 2003; Sartorius et al. 2001). The interaction of adaptor protein Apaf-1 with mitochondrial cytochrome c, caspase 9, and ATP is essential to the extrinsic pathway function (Hajra and Liu 2004). Control of the intrinsic pathway is regulated by

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the Bcl-2 family of proteins, which consists of both pro-and anti-apoptotic regulators. The inhibitors of apoptosis proteins (IAP), which include XIAP, survivin and livin, regulate both upstream and downstream components of the apoptosis pathway (Nachmias et al. 2004). Increased expression of apoptotic inhibitors or impairment of a pro-apoptotic pathway can lead to resistance to apoptosis in melanoma (Igney and Krammer 2002).

Bcl-2 Bcl-2 are a family of genes that govern the permeablization of the outer mitochondria membrane (Vaux et al. 1988). Bcl-2 genes can be pro- or anti- apoptosis whereas Bcl-2 proper is anti- apoptotic. Chemotherapeutic resistance of melanoma has been linked to the anti-apoptotic mechanisms of Bcl-2 protein (Tas F et al. 2004). Interestingly Bcl-2 and MITF, a key transcriptional regulator of the melanogenesis pathway and other pathways, have linkage that may relate to melanoma cell resistance to chemotherapy (Cartlidge et al. 2008). Recent studies have suggested B-RAF regulates Bcl-2 and Bim proteins thus influencing apoptosis. B-RAF V600E is one of the most common genetic aberrations in melanoma cells (Shinozaki et al. 2004) and is suggested to regulate apoptosis (Boisvert-Adamo and Aplin 2008). In recent clinical trials, an antisense oligonucleotide (oblimersen sodium) to Bcl-2 has been used in combination with systemic chemotherapy of dacarbazine in advanced melanoma patients (Bedikian et al. 2006). The trial demonstrated improved clinical outcomes. These approaches to block anti-apoptotic mechanisms and reduce melanoma cell resistance to chemotherapeutic agents are encouraging developments in the treatment of melanoma.

p53 Chemotherapy damages cancer cells function by inducing apoptosis through intracellular structural damage (Martin et al. 1997). Dacarbazine (DTIC) and cisplatin, commonly used in chemotherapy regimens for melanoma, effect DNA methylation and cross-linking, respectively. The cellular response to such overwhelming injury is the initiation of apoptosis. Chemotherapy-induced apoptosis is usually associated with activation of p53, but it is unclear how p53 facilitates apoptosis in the setting of melanoma treated with chemotherapy (Li et al. 1998b; Weller 1998). TP53 mutations are uncommon in primary melanoma, but are more frequent in metastatic melanoma lesions (Albino et al. 1994). Melanoma expressing mutant p53 is much more resistant to chemotherapy in vitro than cell lines expressing the wild type p53 (Li et al. 1998b, 2000), therefore its influence is anticipated to be strong in vivo when it is present. But loss of p53 and mutant p53 is not common in melanoma specimens; therefore aberration in p53 does not fully explain resistance to chemotherapy, although interactive pathways may also be involved. In

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general, p53 mutation in cutaneous melanoma is 15% or less (Essner et al. 1998). Recently, studies have suggested the resistance level of melanoma to DNA damaging chemotherapy agents such as cisplatin may be through p53 isoforms such as p53beta and Delta40p53 being present in the nuclear and cytosolic compartments of melanoma cells (Avery-Kiejda et al. 2008). The identification of p53-independent apoptotic pathways may provide new approaches for chemotherapeutics. One potential drug is the gamma-secretase tripeptide inhibitor (GSI) which is capable of inducing apoptosis in melanoma cells and not normal melanocytes (Qin et al. 2004). The drug can induce upregulation of BH3 protein-members (Bim and Noxa) that activated apoptosis. Further studies are needed to determine the clinical efficacy of this therapy in patients.

Apaf-1 The intrinsic pathway of apoptosis is activated in response to stimuli such as DNA damage, hypoxia and cell detachment (Danial and Korsmeyer 2004). In the presence of appropriate stimulus, pro-apoptotic Bcl-2 family members are activated and insert into the mitochondrial outer membrane, leading to mitochondrial outer member permeabilization (Green, 2005). Cytochrome-c is released from the inter-mitochondrial membrane space into the cytosol. Here cytochrome-c binds to apoptotic protease activating factor-1 (Apaf-1), which is found in the region of chromosome 12q22.1. Apaf-1 is a major effector protein in the initiator portion of the extrinsic apoptosis pathway which is required along with cytochrome c and ATP to activate caspase 9 (Anichini et al. 2006). The structure formed by Apaf-1 and cytochrome c, the apoptosome, is a wheellike complex consisting of seven spokes and a central hub (Zou et al. 1999). In the absence of cytochrome c, Apaf-1 in the apoptosome lies in an inhibitory conformation (Acehan et al. 2002). ATP is also necessary to transform Apaf-1 into an active state (Riedl et al. 2005). Binding of caspase-9 to the central hub and conversion from the pro-enzyme state is the final phase of apoptosome function. Apoptosome function is modified by positive and negative regulators. A novel protein containing a NB domain and CARD (NAC) and Nucling have been found to promote the activation of caspase-9 by the apoptosome (Chu et al. 2001; Sakai et al. 2004). NAC interacts selectively with the CARD (caspase recruitment domain) of Apaf-1 to enhance the activation of caspases. Nucling expression is induced by pro-apoptotic stimuli and its function is to upregulate the expression of both cytochrome c and Apaf-1. NAC and Nucling have not yet been shown to play a role in chemotherapy induced apoptosis or as a survival mechanism in melanoma treatment. Both IAPs and heat shock proteins have been found to negatively regulate apoptosome function (Lindholm and Arumae 2004). Recently, these factors have been under intense investigations as potential targets of reversing anti-apoptosis. X-chromosome linked IAP (XIAP) inhibits the activity of activated caspase-9 in the apoptosome (Denault et al. 2007). XIAP role in apoptosis is poorly understood

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and may have importance regulating survival in melanoma cell. Heat shock proteins have been found to bind either cytochrome-c or Apaf-1 to inhibit their binding to the other components of the apoptosome thereby downregulating the apoptosis cascade (Bruey et al. 2000; Pandey et al. 2000; Saleh et al. 2000). Apaf-1 and caspase 9 are necessary components of the p53-dependent apoptosis pathway (Soengas et al. 1999). Loss or inactivation of either Apaf-1 or caspase 9 results in inactivation of p53-dependent apoptosis. Examination of Apaf-1 expression levels in melanoma tumors revealed a significant difference in Apaf-1 cytoplasmic expression (Baldi et al. 2004). Metastatic deposits were shown to have weak or negative immunohistochemistry (IHC) staining for Apaf-1 compared to primary lesions. Primary melanomas which later developed nodal metastasis expressed less Apaf-1 than those which did not develop metastasis. Another group found a significant decrease in Apaf-1 mRNA levels between Clark level II and III melanomas, lending evidence that Apaf-1 may play an important role early in the course of melanoma development (Niedojadlo et al. 2006). Apaf-1 expression does not seem to correlate with primary tumor site, subtype, or ulceration status (Dai et al. 2004). Additionally, Apaf-1 protein expression level determined by IHC does not predict 5-year patient survival (Dai et al. 2004). This suggests that its expression is not the only critical factor for melanoma cell survival. Loss of an Apaf-1 allele was found to be common in melanoma (Soengas et al. 2001). Tumors with loss of heterozygosity (LOH) for Apaf-1 demonstrate low levels of Apaf-1 expression (Fujimoto et al. 2004a). Decrease in expression may be also regulated through epigenetic mechanisms; however the frequency of this event is low and likely not a significant factor (Schwabe and Lubbert, 2007). Our group demonstrated that Apaf-1 LOH is found to increase with tumor progression and more commonly found in metastatic melanoma than in primary tumors (Fujimoto et al. 2004a). To evaluate whether loss of the Apaf-1 locus influences tumor progression, we assessed LOH of microsatellites on the Apaf-1 locus (12q22–23) in 62 primary and 112 metastatic melanomas. It was discovered that frequency of allelic imbalance was significantly higher in metastatic tumors (n = 36 of 98; 37%) than in primary melanomas (n = 10 of 54; 19%; P = 0.02). The study demonstrated that in metastatic melanomas, Apaf-1 loss significantly correlated with a worse prognosis (P < 0.05) in the patients. The study suggested that the correlation between Apaf-1 loss and tumor progression may make it a candidate tumor suppressor gene. The LOH was frequent in the 12q22–23 chromosome region centromeric to the APAF-1 locus suggesting that other tumor-related genes may also be present in the 12q22–23 region. This finding suggests an important role for Apaf-1 in development and survival of melanoma metastasis. Clinical translational analysis suggests Apaf-1 LOH and expression correlate with decreased survival in AJCC stage III/IV disease, with survival difference being more pronounced in stage III melanoma (Fujimoto et al. 2004a) (Fig. 5.1). Low levels of Apaf-1 inhibit melanoma cells from undergoing chemotherapyinduced apoptosis (Soengas et al. 2001); similarly, transient transfection of Apaf-1

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Fig. 5.1 (A) Correlation of allelic imbalance (AI) on 12q22-23 in serum with overall survival. AI (+) group showed significant decrease of overall survival compared with AI (−) group in prebiochemotherapy serum (log-rank test P < 0.05). (B) Correlation of biochemotherapy response with overall survival on patients associated for serum AI. Non-responder group showed significant decrease of overall survival compared with the responder group (log-rank test P < 0.0001). LOH, loss of heterozygosity

into melanoma cell lines increases cytoplasmic Apaf-1 expression and results in susceptibility of these cell lines to chemotherapy-induced apoptosis (Dai et al. 2004). Studies have demonstrated downstream caspase activation and apoptosis induction by chemotherapeutic agents in Apaf-1 negative cell lines (Zanon et al. 2004). Previously our group and others have demonstrated that circulating tumor-related DNA can be detected in cancer patients and can be used as diagnostic and prognostic biomarkers. Both serum and plasma can be used for assessment of free circulating DNA such as microsatellite LOH (Taback et al. 2004; Hoon et al. 2004a). Our group has demonstrated the utility of assessment of Apaf-1 gene region with allelic imbalance (AI) in tissues can also be detected as free circulating DNA in melanoma patients (Fujimoto et al. 2004b). Circulating DNA with AI of 12q22-23 in serum was evaluated as a surrogate biomarker to predict biochemotherapy (BC) treatment response in melanoma patients (Fujimoto et al. 2004b). Sera were assessed from 49 AJCC on Cancer stage IV melanoma patients treated with BC. Serum AI of the 12q22-23 region was demonstrated to be present before and/or after BC with variance among different patients. The BC responders showed a significantly lower frequency of AI (5 of 24, 21%) compared with nonresponders (11 of 20, 55%; Fisher’s exact test, P < 0.029). It was demonstrated that serum AI on 12q22-23 was associated with worse prognosis (log-rank test, P < 0.046). DNA from tumor cells has been suggested to be released by tumors and circulating tumor cells in the blood through non apoptotic mechanisms such as shedding, necrosis and physical destruction (Koyanagi et al. 2006). Death through apoptosis is thought to degrade genomic DNA into small fragments at specific sites which are further destroyed in the tissue microenvironment. Patients responding to therapy and undergoing apoptosis would have less circulating DNA in the blood. These findings indicate that serial serum genetic analysis of tumor-related AI on 12q22-23 has clinical utility in predicting tumor response to therapy. This is one of the first circulating DNA serum

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assays whereby patient responses were correlated to a specific loss of expression of a gene (Taback and Hoon 2004). Melanoma patients with a good clinical response to biochemotherapy (BC) have been shown to have a significantly lower frequency of allelic imbalance at the Apaf1 locus than those patients with a poor response to biochemotherapy (Fujimoto et al. 2004b). (Fig. 5.1)

Inhibitors of Apoptosis (IAP) The inhibitors of apoptosis proteins (IAP) interact with caspases to downregulate function (LaCasse et al. 1998). There are multiple members of the IAP family including survivin, livin and XIAP. Survivin is the most widely studied in melanoma. Survivin (chromosome 17q25) is the shortest member of the IAP family and varies structurally from other IAPs as it lacks a RING-finger domain (Ambrosini et al. 1997; Zaffaroni et al. 2005). Survivin is regulated in a cell cycle dependent manner, and its expression is markedly increased in the G2 M phase (Li and Altieri 1999). Not surprisingly, survivin plays a critical role in microtubule assembly and normal mitotic progression (Zaffaroni et al. 2005). During mitosis, survivin binds to the mitotic spindle and may inhibit caspases from inducing apoptosis (Temme et al. 2003; Li et al. 1998a). Survivin is expressed normally in fetal tissues and is generally not found in adult differentiated tissues (Ambrosini et al. 1997). Survivin is strongly expressed in the majority of cancer types, including colon, lung, breast, brain and melanoma (Altieri 2003). The presence of survivin in many premalignant lesions suggests it may have influence in early tumor development and progression (Zaffaroni et al. 2005). Survivin overexpression may allow malignancies to overcome inhibition of cell cycle checkpoints and permit cancer cell progression (Sah et al. 2006). Low expression of survivin in normal tissues suggests targeted treatment of survivin would generate low toxicity in normal tissues making survivin an attractive target for cancer therapies (Li and Brattain 2006). Survivin may be considered as a universal tumor antigen that may serve as a widely applicable target for anticancer immunotherapy. Use of a survivin-based dendritic cell vaccine in melanoma patients resulted in little toxicity in a phase 1 trial (Otto et al. 2005). A strong immune response to a survivin epitope was induced (Otto et al. 2005). Survivin expression in cancer cells induces resistance to chemotherapy through inhibition of apoptosis (Zaffaroni et al. 2002). Transfection of survivin protects fibroblasts and cancer cell lines from taxol induced apoptosis (Li et al. 1998a; Zaffaroni et al. 2002; Giodini et al. 2002). Cancer cells accumulate survivin in mitochondria (Dohi et al. 2004). Survivin inhibits the mitochondrial pathway of apoptosis by interacting with caspase-9 and SMAC and disrupting apoptosome function (Song et al. 2003; Tamm et al. 1998). Inhibition of survivin in melanoma cells by ribozyme transfection increased chemotherapy-induced apoptosis in cell lines and in xenografts implanted in nude mice (Pennati et al. 2004). In the setting of

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Fig. 5.2 Correlation of survivin mRNA copies to overall survival in patients receiving only postoperative melanoma cell vaccine (n = 37). Survivin/GAPDH copy ratio < 1.0 × 10−3 (n = 22) versus survivin/GAPDH copy ratio ≥ 1.0 × 10−3 (n = 15)

melanoma cell vaccine treatment, survivin correlates with poor response to therapy (Takeuchi et al. 2005). Evaluation of polyvalent melanoma vaccine treated stage IV melanoma patients in an adjuvant setting showed a significant survival advantage for those patients with low copy numbers of survivin in metastatic specimens (Fig. 5.2) (Takeuchi et al. 2005). Melanoma cells with high survivin expression may have more aggressive proliferation as well as resistance to apoptosis-related death. This aggressive phenotype is likely to override the host immunity and impair the efficacy of active-specific immunotherapy such as with melanoma vaccine. This could explain why higher survivin expression levels correlated with poorer prognosis of patients with stage IV melanoma in our study. Survivin expression may be a valuable surrogate indicator of disease outcome and treatment resistance in advanced-stage melanoma patients. Like other IAP members, livin can inhibit apoptosis by preventing the activation of caspase-9 by APAF-1 and cytochrome c. Melanoma cell lines have been reported to express livin mRNA. Although survivin and livin may block apoptosis of melanoma cells in primary and metastatic sites, the functional and target differences between these two proteins remain unclear. Analysis of livin demonstrated that it was well expressed in metastatic melanoma however the expression did not correlate to overall treatment response with the melanoma vaccine (Takeuchi et al. 2005). Melanoma can be very aggressive at early stages of development. In general a small primary tumor (3–4 mm) or micrometastasis (2–3 mm) in the draining lymph node can be very lethal within a short follow-up time. For most carcinomas these size lesions are generally not as lethal. This suggests melanoma can acquire the apoptosis resistant phenotype early in development, thus leading to such aggressive behavior. A simplistic model cartoon is shown in Fig. 5.3. Although apoptosis resistance is one major factor contributing to the success of metastasis, other events of metastasis are also involved (Hoon et al. 2006).

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Fig. 5.3 A simplistic cartoon model is presented of early melanoma progression and apoptosis. Primary cutaneous melanoma often first spreads to the sentinel lymph node (SN) and then to non SN (NSN). These lymph nodes are draining nodes of the primary melanoma tumor whereby the SN is the first site of likely metastasis. As the tumor lesion progresses and apoptosis resistant clones survive there is a greater likelihood of successful tumor development in the SN and NSN, and then eventual metastasis to distant organs. As the tumor progresses the apoptosis (−) phenotype becomes more dominant resulting in a highly aggressive tumor refractory to apoptosis targeted chemotherapy

Conclusion Overall, melanoma aggressive behavior and resistance to therapies appear to be related to the strong anti-apoptotic mechanisms the cells develop during tumor progression. This is in general more pronounced in melanoma than any other solid tumor. We provide evidence from our studies of how significant apoptosis related functions such as downregulation and upregulation of key apoptosis pathway proteins occur in melanoma cells. Melanoma cell refractoriness to therapeutics is a ongoing and challenging problem whereby, resistance to apoptosis is not well understood. Therapeutic strategies for melanoma may require the addition of inhibitors to anti-apoptotic mechanisms to the targeted therapies. Other treatment approaches may be to develop therapeutics that cause non apoptotic cell death (Sheridan et al. 2008). The resistance to apoptosis in melanoma cells may be inherent to its melanocyte origin. Melanocytes are resistant to excessive sunlight, heat, and chemical exposure on the skin. These attributes may be enhanced during transformation

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and progression during development of a melanoma lesion. It is clear new targets of anti-apoptosis mechanisms need to be identified to develop more counter-effective therapeutics.

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Whitehead RP, Moon J, McCachren SS, Hersh EM, Samlowski WE, Beck JT, Tchekmedyian NS, Sondak VK (2004) A Phase II trial of vinorelbine tartrate in patients with disseminated malignant melanoma and one prior systemic therapy: a Southwest Oncology Group study. Cancer 100:1699–1704 Wood TF, DiFronzo LA, Rose DM, Haigh PL, Stern SL, Wanek L, Essner R, Morton DL (2001) Does complete resection of melanoma metastatic to solid intra-abdominal organs improve survival? Ann Surg Oncol 8:658–662 Zaffaroni N, Pennati M, Colella G, Perego P, Supino R, Gatti L, Pilotti S, Zunino F, Daidone MG (2002) Expression of the anti-apoptotic gene survivin correlates with taxol resistance in human ovarian cancer. Cell Mol Life Sci 59:1406–1412 Zaffaroni N, Pennati M, Daidone MG (2005) Survivin as a target for new anticancer interventions. J Cell Mol Med 9:360–372 Zanon M, Piris A, Bersani I, Vegetti C, Molla A, Scarito A, Anichini A (2004) Apoptosis protease activator protein-1 expression is dispensable for response of human melanoma cells to distinct proapoptotic agents. Cancer Res 64:7386–7394 Zou H, Li YC, Liu HS, Wang XD (1999) An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 274:11549–11556

Chapter 6

Apoptosis in Carcinogenesis and Chemotherapy – Esophageal Cancer Yan Li and Robert C.G. Martin

Abstract Esophageal cancer is the sixth most common cause of cancer-related death. Esophageal carcinoma can be divided in squamous cell carcinoma and adenocarcinoma by histopathology, and these two main histologic types of esophageal cancer are quite different in many aspects. Apoptosis in esophageal cancer has been extensively studied, and a recognized mechanism for the development of esophageal cancer is an imbalance between cell renewal and cell death, with apoptosis being inhibited. The inability of esophageal cancer to undergo apoptosis when presented with an insult is an important indicator of the propensity for the esophageal malignant transformation. Many of the molecular events necessary for activation, amplification and execution of the apoptotic process in esophageal cancer, and it is evident that diverse drugs can kill esophageal tumor cells through activating common apoptotic pathways. In the last decade, it has been demonstrated that resistance to apoptotic stimuli is a hallmark feature of esophageal cancer and a complex network of pro- and anti-apoptotic proteins that governs the tight regulation along apoptotic pathways in regards of esophageal carcinogenesis and chemotherapy is revealed, and these findings could lead to the identification of several new molecular targets for chemoprevention and chemotherapy in esophageal cancer. Keywords Apoptosis · Chemoprevention · Esophageal Carcinoma

Introduction Overview of Esophageal Cancer Esophageal cancer, one of the most malignant gastrointestinal cancers, is the sixth most common cause of cancer-related death, and 462,000 new cases were diagnosed during 2002 (Parkin et al. 2005). Esophageal cancer forms, by definition, in

Y. Li (B) Department of Surgery, Division of Surgical Oncology, University of Louisville School of Medicine, 511 S Floyd ST, MDR Bld, Rm 326A, Louisville, KY 40202, USA e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 6,

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tissues lining the esophagus, and the dominant presenting symptom of esophageal carcinoma is dysphagia, which is present in approximately 70% of all patients at diagnosis (Enzinger and Mayer 2003). Esophageal cancer is usually detected at an advanced stage, and numerous patients develop locally recurrent tumors or distant metastases within a short period of time after curative surgery. Despite advances in diagnosis and staging, (neo-) adjuvant therapy and surgical technique, the overall survival of the esophageal carcinoma remains lower than other solid tumors (Enzinger and Mayer 2003, Jemal et al. 2005). Esophageal carcinoma can be divided into squamous cell carcinoma and adenocarcinoma by histopathology. Although some studies do not differentiate between these two carcinoma entities, there is no doubt that esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) are two different diseases with different pathogenesis, epidemiology, tumor biology and prognosis requiring different therapeutic strategies. Actually, these two main histologic types of esophageal cancer are quite different in many aspects. First, the location within the esophagus differs. The majority of squamous cell carcinomas located in the upper two thirds of the thoracic esophagus, whereas the majority of adenocarcinomas originated in the lower third or bridged the gastroesophageal junction (Clark et al. 1994, Tytgat 1995). Second, the epidemiology of these two types of carcinoma is quite distinct. ESCC has been stable or decreasing in incidence and is associated with tobacco and alcohol abuse, male sex, and black race (Holmes and Vaughan 2007, Siewert and Ott 2007), whereas EAC is the most rapidly increasing type of tumor in many Western nations and is associated with gastroesophageal reflux disease, Barrett’s esophagus, obesity, male sex, and white race (el Serag 2002, Jemal et al. 2005, Pera et al. 2005). Third, these two carcinomas also have differing paths of histopathologic progression from premalignancy to invasive cancer. ESCC progresses, like most upper digestive squamous cell carcinomas, from atypia originating in epithelial cells through carcinoma in situ to invasive carcinoma, whereas EAC usually progresses in a stepwise sequence through Barrett’s metaplasia to dysplasia to adenocarcinoma. Last, the differences between ESCC and EAC are also reflected in different molecular pathways to carcinogenesis. Although both histologies of ESCC and EAC had shared some same carcinogenetic pathways such as upregulation of genes involved in cell cycle regulators, extracellular matrix genes, and immune response genes and down-regulation of genes related to calcium ion binding and gap junctions, other aspects of the gene expression profile of ESCC were more closely related to normal esophagus, whereas EAC was more closely related to the profile identified in Barrett’s esophagus (Greenawalt et al. 2007, Kan et al. 2001). Because of the evident differences between ESCC and EAC, we will discuss these two histopathological cancers separately in this chapter as a conceptual basis to allow a clear evaluation of the different apoptotic pathways and chemotherapeutic strategies.

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Overview of Apoptotic Pathways The ability of tumor cell populations to expand in number is determined not only by the rate of cell proliferation but also by the rate of cell death, while programmed cell death –apoptosis represents a major source of this cell death. The concept of apoptosis as a form of cell death being distinct from necrosis was introduced by Kerr et al. in 1972 (Kerr et al. 1972). Apoptosis is a genetically controlled physiological cell death program that occurs in all eukaryotic organisms and controls normal cell numbers during development and disease. Presently, it is generally agreed that dysfunction of the apoptotic process can result in an uncontrolled cell growth, and loss of control of the balance between cell growth and cell death is central to the development of cancer. Recent advances in studying the molecular mechanisms related to the carcinogenesis have provided further understanding of the apoptotic signalling transductions. The apoptotic machinery can be broadly divided into two classes of components – sensores and effectors. The first class of components are responsible for monitoring the extracellular and intracellular environment for conditions of normality or abnormality to determine whether a cell should be eliminated, whereas the second class of components function as effectors of apoptotic death (Hanahan and Weinberg 2000). Although it may be too simple, apoptotic signalling that triggers apoptosis within the cell is classified generally into two molecular pathways: (1) the death receptor pathway (also called the extrinsic pathway) and (2) the mitochondrial pathway (also called the intrinsic pathway) (Guicciardi and Gores 2005). The extrinsic pathway is a receptor mediated and regulated by the members of the tumor necrosis factor (TNF) receptor superfamily namely: Fas and TNF-related apoptosis inducing ligand (TRAIL) receptors. Binding of these receptors with their ligands such as Fas ligand (FasL) and TRAIL respectively, activates this pathway and results in oligomerization of the receptors and recruitment of the adaptor proteins, i.e., Fas-associated death domain (FADD), TRAIL associated death domain (TRADD respectively and caspase-8, forming a death-inducing signaling complex (DISC). The activated form of caspase-8 at the DISC in turn cleaves and activates effector caspases (caspase-3, -6 and -7), resulting in apoptotic cell death (Ashkenazi and Dixit 1998). The intrinsic pathway is a mitochondrial mediated death process regulated by the Bcl-2 family of proteins which comprise of the pro-apoptotic proteins such as Bax, Bak, Bad and Bid; and the anti-apoptotic proteins such as Bcl-2 and BclXL. DNA damage induced by chemicals, growth factor deprivation and oxidative stress initiates this pathway resulting in release of mitochondrial proteins including cytochrome-c and second mitochondria-derived activator of caspase. Mitochondrial membrane permeabilization (MMP) is facilitated by caspase-8 which mediates the proteolytic maturation of pro-apoptotic Bcl-2 family protein Bid. Caspase8 truncated Bid (tBid) translocates from the cytosol to the outer mitochondrial

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membrane(OMM) (Lutter et al. 2000). This tBid triggered Bax- dependent MMP results in full insertion of Bax in the mitochondrial membrane and its oligomerization to form a pore like structure, thereby promoting release of mitochondrial proteins (Cheng et al. 2001). Once released from the mitochondria, cytochrome-c along with apoptosis protease activating factor-1 (Apaf-1) induces activation of caspase-9, resulting in initiation of the apoptotic caspase cascade (caspase-3, -6 and -7). Actually, the intrinsic and extrinsic are two interlinked pathways that can induce apoptosis by activation of caspases. Based on their function, the caspases are classified into three subtypes, inflammatory caspases, apoptotic initiator caspases and apoptotic effector caspases. Apoptotic initiator caspases possess long prodomains containing either a death effector domain (caspase-8 and -10) or a caspases activation and recruitment domain (caspase-2 and -9) which mediates the interaction with upstream adaptor molecules. Apoptotic effector caspases (caspase-3, -6 and -7) that are typically processed and activated by upstream caspases and perform the downstream execution steps of apoptosis by cleaving multiple cellular substrates (Degterev et al. 2003). In regards of malignance, accumulating evidence suggests that apoptosis is important not only for the spontaneous elimination of potentially malignant cells but also for tumor progression. Acquisition of ability to evade apoptosis is a hallmark of cancer, with both the loss-of-function of pro-apoptotic signals and gain-of-function of anti-apoptotic mechanisms contributing to tumorigenesis and the cancer phenotype (Hanahan and Weinberg 2000). The process of apoptosis is a dynamic interplay of several molecules with upregulatory and downregulatory properties that is largely dependent on the cell type and the form of insult. No single factor in the machinery of apoptosis operates in isolation, and it is unlikely that the activation or inactivation of a single component will alter the ultimate fate of the cell and lead to programmed cell death. Therefore, we will discuss, in this chapter, individually the components of apoptotic machinery in the apoptotic signalling pathways as a conceptual basis in regards of the carcinogenesis of ESCC and EAC.

Apoptosis and Esophageal Squamous Cell Carcinoma Esophageal Squamous Cell Carcinoma ESCC is the most common type of malignance within the esophagus, and for most of the modern era, ESCC is accounted for 85% of esophageal cancer. Indeed, worldwide, it is the case; there are regions in the world where the incidence of ESCC is strikingly high, including certain part of China, Iran, and Southern Africa, and this likely reflects an interaction between genetics and environment. The principal precursor lesion of ESCC is epithelial dysplasia, characterized by an accumulation of atypical cells with nuclear hyperchromasia, abnormally clumped chromatin and loss of polarity (Stoner et al. 2007). ESCC exhibits three types of growth patterns: (1) fungating, in which the tumor appears as an exophytic mass with intraluminal

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growth; (2) ulcerating, characterized by a large ulcer that may penetrate into the mediastinum; and (3) infiltrating, in which the tumor exhibits noncohesive growth in both horizontal and vertical planes of the esophagus. Microscopically, ESCC range from well-differentiated tumors that exhibit keratinization, intracellular bridges, moderate nuclear atypia, and minimal necrosis, to poorly differentiated lesions that can be difficult to categorize as squamous in origin, are mitotically active, and contain substantial necrosis. ESCC develops through a progressive sequence from mild to severe dysplasia, carcinoma in situ and, finally, invasive carcinoma (Anani et al. 1991, Kuwano et al. 1993). Multiple factors lead to the occurrence and development of ESCC. Molecular studies of human esophageal squamous cell carcinoma have identified numerous genetic abnormalities, the summarized genetic changes observed in ESCC include: (1) alterations in tumor suppressor genes leading to altered DNA repair, cell proliferation and apoptosis; (2) disruption of the G1/S cell cycle checkpoint and loss of cell cycle control; and (3) alterations in oncogene function leading to deregulation of cell signaling pathways (Stoner and Gupta 2001). Although therapeutic strategies have been improved, the prognosis of patients with ESCC is still poor. Moreover, ESCC cells are known to develop resistance to chemotherapeutic drugs, thus resulting in a dramatic decrease in the 5-year survival rate for ESCC (Enzinger and Mayer 2003). Obviously, a better understanding of the molecular mechanisms in carcinogenesis and progression of ESCC helps to improve the prognosis of patients with ESCC.

Apoptotic Signaling Pathways in ESCC Carcinogenesis Apoptosis in ESCC has been extensively studied, and a recognized mechanism for the development of ESCC is an imbalance between cell renewal and cell death, with apoptosis being inhibited. The inability of esophageal squamous cell carcinoma to undergo apoptosis when presented with an insult is an important indicator of the propensity for this malignant transformation. While the discoveries in the field of apoptotic pathway and the molecular aspects are still being delineated, the existence of the apoptotic signaling from the studies of past several years is becoming clear regarding the components of apoptotic machinery as carcinogenetic mechanism in ESCC. Death Receptors and Ligands in ESCC The eight known TNF receptor superfamily members include TNF receptor-1 (TNFR1 or death receptor 1[DR1]), Fas (CD95 or DR2), DR3, TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 (TRAIL-R1 or DR4), TRAIL receptor 2 (TRAIL-R2 or DR5), DR6, ectodysplasin-A receptor (EDAR), and nerve growth factor receptor (NGFR) (Ashkenazi 2002, Cleveland and Ihle 1995, Nagata 1997, Rowinsky 2005). All these cell surface receptors are thought to mediate death signals. These receptors contain an intracellular region of homology designated as

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the death domain. Two additional membrane antagonist decoy receptors that belong to the above family of death receptors have also been identified. These decoy receptors known as TRID (also known as DcR1 or TRAIL-R3) and TRUNDD (also known as DcR2 or TRAIL-R4) either lack the death domain or contain a mutated version of the death domain, and consequently protect cells from the cytotoxic effects of TRAIL (MacFarlane et al. 1997, Pan et al. 1998). TRAIL Pathway in ESCC TRAIL pathway could play an important role in ESCC carcinogenesis, however, to date, there has been no report dealing specifically with TRAIL pathway in the initiation of ESCC. The in vitro studies by Kondo et al. provided indirect evidence that TRAIL and its receptors play roles in the carcinogenesis of ESCC. Although the ESCC cell lines expressed both DR4 and DR5, showing a quintessential pattern of TRAIL receptors expression these cell lines are generally resistant to TRAIL. They found that high incidences of decoy receptor expression in these ESCC cell lines, and this may be one of the unique molecular characteristics reflecting TRAIL resistant nature of ESCC (Kondo et al. 2006b). Sheikh et al. provided further evidence that decoy receptor TRID is expressed at higher levels in ESCC, five of seven ESCC exhibited higher TRID expression levels when compared to their matching normal tissue samples. It is likely that ESCC cells may gain a growth advantage by overexpressing TRID in order to protect themselves against TRAIL-mediated apoptosis (Sheikh et al. 1999). Furthermore, Chang et al. also demonstrated that TRAIL showed the highest positivity among apoptosis-related proteins, and it was considered the putative marker involved in the progression from non-invasive neoplasm to invasive ESCC. TRAIL positivity was significantly correlated with patient survival in their study (Chang et al. 2005). Fas/ Fas Ligand (FasL) System in ESCC Despite the presence of tumour specific cytotoxic T cells and natural killer cells, numerous ESCC patients develop locally recurrent tumors or distant metastases after curative surgery (Alderson et al. 1995, O’Connell et al. 1999). The battle between the host and tumor cells is a two-way mechanism, in which the tumor cells subvert the host attack by down-regulation of Fas expression and fight back by upregulating FasL expression. The majority of ESCC had up-regulation of FasL, which helped the tumor cells to counterattack the immune system by killing Fas-sensitive cytotoxic T cells. Also in the majority of tumors, the function of Fas was subverted by the down-regulation of expression, suggesting that this may be a self-defense mechanism for ESCC cells to evade Fas-mediated killing from the host cytotoxic and/or killer cells (Gratas et al. 1998). In human ESCC, lower expression of Fas and higher expression of FasL have been found compared to that in the corresponding normal tissue, indicating an association between the aberrant expression of Fas and FasL and ESCC (Xue et al. 2007). In addition, aberrant expression of Fas and FasL occuring early in

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dysplasia and in ESCC in situ has been associated with differentiation, invasiveness, and metastasis of ESCC cells and with patients’ survival (Bennett et al. 1999, Shibakita et al. 1999). Spontaneous apoptosis has been observed in ESCC patients, while enhanced Fas expression is responsible for high apoptotic index in ESCC cells. In addition, ESCC patients with a low apoptotic index showed a poor outcome compared with those with a high apoptotic index (Shibakita et al. 2000). Chan et al. investigated expression of Fas in ESCC patients, and found that Fas expression was detected in 89.7% of ESCC patients and the higher Fas expression correlated with better tumor differentiation but not with other patients or tumor variables, and higher Fas expression was also associated with better survival (Chan et al. 2006). These findings suggest that aberrant expression of Fas related inhibition of apoptosis plays an important role in the carcinogenesis of ESCC. Sun et al. performed genetic polymorphisms in Fas and FasL. Their analysis of 588 ESCC patients and 648 frequency-matched control subjects demonstrated that polymorphisms in the promoter region of the death receptor Fas and FasL were associated with an increased risk of the development of ESCC in a Chinese population (Sun et al. 2004). Polymorphisms in the Fas and FasL genes are functionally important, and Fas –1377G/A and FasL –844T/C polymorphism found both located in the promoter region of the gene have given the role of Fas and FasL in the development of cancer (Houston and O’Connell 2004, Kanemitsu et al. 2002, Wu et al. 2003). It might expect individuals who carry the Fas –1377AA and/or FasL –844CC genotype, and thus have decreased expression of Fas and/or increased expression of FasL over a lifetime, to be at a higher risk for developing ESCC. It has emerged that ESCC can also counterattack the immune system. Expression of FasL by ESCC cells in vivo enables them to induce Fas-mediated apoptosis of activated tumor infiltrating lymphocytes. This counterattack model suggests that the FasL may offer a survival advantage to ESCC cells (Bennett et al. 1998, Kozlowski et al. 2007). A particular study carried out by Bennett et al. have demonstrated that ESCC cell lines express FasL and can kill lymphoid cells by Fas-mediated apoptosis (Bennett et al. 1999). The ESCC derived-FasL acts upon surrounding tumor infiltration by lymphocytes (TIL) to induce apoptosis as a mechanism of host immune evasion. In addition, it has been also demonstrated that the Fas/FasL system significantly affects the survival of ESCC patients, with decreased survival seen in ESCC patients of Fas down-regulation. These results provide the direct evidence to support the Fas counterattack as a mechanism of immune privilege in vivo in ESCC (Gratas et al. 1998, O’Connell et al. 1999). It should mention here that the serum FasL concentration was also investigated in the ESCC patients by Kozlowski et al. They found that the level of serum FasL in ESCC patients was significantly higher than in healthy volunteers, furthermore, the serum levels of sFasL in stage III to IV ESCC patients were significantly higher than in stage II ESCC patients. These results provided further evidence that the increase in serum FasL in patients with ESCC might be reflective of their tumor burden insofar as the tumor may release FasL (Kozlowski et al. 2007).

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Bcl-2 Family Proteins in ESCC Among of the most important regulators of apoptosis are the proteins of the Bcl-2 family. Bcl-2 family proteins, such as Bcl-X, Bax, Bad and Bak, share significant homology and play a critical role to control apoptosis. Overexpression of Bcl-2 prevents carcinoma cells from apoptosis induced by various stimuli, therefore, Bcl2 may play a critical role in the development and progression of human tumors (Ruoslahti and Reed 1994). Bcl-X is another important apoptosis regulator and has two forms, Bcl-XS and Bcl-XL through alternative splicing. Bcl-XL has an antiapoptotic function and shows great homology to Bcl-2, but Bcl-XS acts to promote apoptosis (Kroemer 1997). The other members such as Bax, Bad and Bak also promote apoptosis. Proapoptotic Bcl-2 family proteins regulate the permeability of the mitochondrial membrane. Increased mitochondrial permeability allows leakage of cytochrome C from mitochondria into cytoplasm, triggering caspases activation and apoptosis (Yin 2000). Given the importance of Bcl-2 family members in regulating the apoptotic pathway, it is not surprising that these genes are altered in ESCC. A reciprocal expression pattern of Bcl-2 during terminal differentiation of normal esophageal epithelium has been obviously detected and Bcl-2 is predominantly expressed in the basal cell layer (Takayama et al. 2001). In regard of carcinogenesis, it has been shown that the Bcl-2 protein is overexpressed in approximately one-third of ESCC as well as in a significant portion of preneoplastic lesions of ESCC (Sarbia et al. 1997). High Bcl-2 expression correlates inversely with the apoptotic index, and it is proposed that this down regulation of apoptosis contributes to the pathogenesis of ESCC (Azmi et al. 2000). A correlation between Bcl-2 expression and survival has been reported, but remains controversial. Some authors found the correlation between Bcl-2 expression and clinical outcome but others not (Sarbia et al. 1996, Sarbia et al. 1997, Torzewski et al. 1998). Bcl-XL is also detectable in normal esophageal epithelium, and predominantly expressed in the suprabasal cell layer (Takayama et al. 2001). Although Bcl-XL expression has been found in the large majority of all preneoplastic lesions and neoplastic lesions of ESCC, a downregulation of Bcl-XL during loss of differentiation in ESCC is observed together with a tendency of decreasing levels of Bcl-XL protein expression toward unfavorable outcome. The evidence of a decreasing expression of Bcl-XL protein during the progression of ESCC could be interpreted against the backdrop of apoptosis as a result of the interaction of many cell death promoting and protecting proteins (Torzewski et al. 1998). Bax protein has been established as a tumor suppressor, Bax inactivation leads to rapid tumor growth and to a decrease in the extent of spontaneous apoptosis of ESCC. Kurabayashi et al. demonstrated that the survival of patients with Baxpositive tumors was significantly poorer than that of patients with Bax-negative tumors. This finding supports the hypothesis that Bax protein expressed in cancerous regions loses death-promoting apoptotic activity and cannot contribute to suppression of the tumor progression leading to poor prognosis in some cases of human ESCC (Kurabayashi et al. 2001), however it is controversial with others.

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Hsia et al. and Natsugoe et al. used antibodies specific for the human Bax proteins to examine the expression in specimens of patients with ESCC. Their results indicate that the overall expressions of Bax were 100% and 38.7%, respectively, however no significant correlations were found between the expression of Bax and survival rate (Hsia et al. 2001, Natsugoe et al. 2001). Inactivation of p53 Gene in ESCC The p53 tumor suppressor gene is involved in the regulation of apoptosis. It is generally accepted that p53 is not required for normal cellular development, but in certain environments such as DNA damage and cellular stress, and its expression is stimulated. In response to cellular DNA damage, p53 protein can stop the cell cycle at phase G1, mainly by transactivating the WAF1/CIP1 gene that encodes p21 protein, a potent inhibitor of cyclin-dependent kinases (El Deiry et al. 1993). Another important effect of p53 being reported is to regulate the expression of death receptors (Fridman and Lowe 2003). The function of p53 is inactivated during the development of most human tumors, and the main mechanism of p53 inactivation is due to p53 gene mutation which occurs in more than 50% human tumors (Levine et al. 1991). Mutation of p53 is a frequent event in ESCC, the frequencies vary from 26% to 87% that occur in multiple sites throughout the open reading frame, which are mostly limited to DNA binding domain that spans exon 5 through exon 8 (Mir et al. 2005). Shao et al. investigated the p53 genotypes by polymerase chain reaction-restriction fragment length polymorphism among 673 patients with ESCC and 694 healthy controls in a Chinese mainland population. They found that the p53 gene codon 72 Pro/Pro genotype was significantly associated with the increased risk of ESCC and it may be an independent factor in susceptibility to ESCC (Shao et al. 2008). The frequency of p53 gene mutations in Japanese population is 47.4% and there are three prominent features: (1) a predominance of transversions, in particular the G:C to T:A transversion; (2) a relatively low frequency of transitions; and (3) a relatively high percentage of frame shift mutations (Egashira et al. 2007). In addition, p53 is also believed to have a critical role in angiogenesis of ESCC, it has been reported that expression of p53 correlated well with the depth of tumor invasion, lymphatic invasion and lymph node metastasis (Han et al. 2007). The staining pattern in biopsies and radical tumor tissues and multifocal accumulation of p53 protein in ESCC also suggest that p53 gene alterations facilitate esophageal carcinogenesis. However, in the literature, the prevalence of p53 protein accumulation reported varies from 50% to 87.2% in ESCC (Coggi et al. 1997, Lam et al. 1997, Muro et al. 1996, Puglisi et al. 1996, Ribeiro et al. 1998, Seitz et al. 1995). Immunohistochemistry (IHC) detection of P53 protein accumulation remains an indirect method without certain biologic significance, while the functional assay could be a useful tool to test the transcriptional competence of the p53 alleles expressed in ESCC tissues (Michel et al. 2002). Robert et al. showed that 47/56 ESCC tested by functional assay had an inactivated p53, and, these mutations were further confirmed by DNA sequencing (Robert et al. 2000). The

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main advantage of functional assay is to detect the functional consequences of gene mutations, in addition, functional assay also can be used to determine if one or both alleles are inactivated. Another important mechanism of p53 inactivation is that p53 protein interacts with other proteins causing either promote p53 protein degradation or inhibit its ability to transactivate the downstream genes involved in apoptosis (Lane 1994). Li et al. used non-peptidic small molecular inhibitors to release p53 protein and demonstrated that the apoptotic rate is consistent with the accumulation of reactivated wild-type p53 protein in the tested ESCC cell lines (Li et al. 2005). Caspases in ESCC Although mutations in cancer cells often target regulators of the upstream of apoptotic pathway such as p53 mutation, alterations that disrupt apoptosis downstream have been reported. Given the important role of caspases as downstream effective molecules in apoptosis, caspases activation appears to be a critical determinant of sensitivity or resistance to cytotoxicity in the ESCC carcinogenesis. Hsia et al. reported that caspase-3 positive staining was a significant prognostic factor in predicting survival in primary resected ESCC. There was a 25% 5-year survival in caspase-3 positive patients compared with a 6% 5-year survival in caspase-3 negative patients (Hsia et al. 2003). This could imply that downstream defects in apoptosis contribute to ESCC carcinogenesis. Nevertheless, it is generally accepted that post mitochondrial mutations appear less frequently than those targeting upstream components of the apoptotic program because it is difficult in maintaining cell viability following damage to the mitochondria.

Apoptosis in ESCC Chemotherapy Chemotherapy for the treatment of cancer was introduced into the clinic more than fifty years ago. Although this form of therapy has been successful for the treatment of some tumors such as certain leukemias, its success for the treatment of common epithelial tumors has been less than spectacular and overall mortality rates for most epithelial origin cancers such as ESCC have not declined in the last 30 years (Hong and Sporn 1997). Perhaps the best target of the insensitivity of cellular response is apoptosis, while single mutations that disable apoptosis can produce multiple chemotherapeutic resistances. It is now believed that apoptotic pathways contribute to the cytotoxic action of most chemotherapeutic drugs (Lowe and Lin 2000), while the resistance of tumor cells to drug-induced apoptosis is emerging as a major category of cancer treatment failure (Schmitt 2003). Many of the molecular events necessary for activation, amplification and execution of the apoptotic process, and it is evident that diverse drugs can kill tumor cells by activating common apoptotic pathways. Resistance to apoptotic stimuli is a hallmark feature of ESCC. In the last decade, a complex network of pro- and anti-apoptotic proteins that governs the tight regulation

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along apoptotic pathways in regards of ESCC carcinogenesis and chemotherapy is revealed, and these findings could lead to the identification of several new molecular targets for ESCC prevention and therapy. Both clinical trials and experimental studies had been conducted with candidate chemopreventive agents that could induce caner cell apoptosis in the ESCC. Although the results of these studies have not been too encouraging, it provides therapeutic strategies that could help improve response rates to chemotherapy in patients with ESCC. There are multiple points at which the chemotherapeutic agents is effective on ESCC cells, but a full consideration of all these is beyond the scope of this chapter. Therefore we only discuss that the activation of apoptosis can be trigged in regards of ESCC chemotherapy and ESCC chemotherapeutic resistances in this chapter. Anticancer Drugs and the Apoptotic Pathway Anticancer Properties of Enhancing TRAIL-Induced Apoptosis TRAIL is a promising agent for development as a cancer-specific therapeutic agent because it induces apoptotic cell death in a wide variety of transformed cell lines and tumors in animal models but not in most normal cells or tissues (Ashkenazi et al. 1999, Walczak et al. 1999). TRAIL is known to induce apoptosis in target cells via activation of the caspase cascade, and it has also been disclosed not only to initiate the mitochondrial (intrinsic) pathway but also the death receptor (extrinsic pathway). Inhibition of apoptosis because of TRAIL resistance has been found in ESCC. Well-differentiated ESCC tended to be more resistant to the combination treatment compared to moderately and poorly differentiated ESCC. One of the possible explanations for this is that well differentiated ESCC retains more characteristics of normal esophageal epithelial cells. Kondo et al. established ESCC cell lines to test the TRAIL-mediated apoptosis pathway in ESCC. They found that all ESCC cell lines expressed both DR4 and DR5, showing a quintessential pattern of death receptor expression in malignant cell lines. However, the decoy receptors such as DcR1/2, thought to play a cytoprotective role against TRAIL, express in ESCC cell lines higher than expected, with only 1 decoy-receptor negative cell line. The high incidences of decoy receptor expression may be one of the unique molecular characteristics reflecting TRAIL resistant nature of ESCC (Kondo et al. 2006a,b). When the cisplatin was selected to break TRAIL resistance, they found that the cisplatin-dependent death receptor upregulation is as a critical factor in augmenting TRAIL-mediated apoptosis in ESCC. The results indicate that the combination of TRAIL and cisplatin was effective against ESCC cell lines (Kondo et al. 2006a). These findings are clinical importance because the combination therapy may overcome TRAIL resistant thereby break the cisplatin resistant in ESCC. Sequential administration of cisplatin and TRAIL might be the better way of treating the ESCC patients. Another interesting study was carried out by Teraishi et al. to investigate the anticancer effects of ZD1839 in combination with TRAIL against human ESCC

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lines. They used ESCC cell line TE8, resistant to TRAIL, tested whether ZD1839 combined with TRAIL could induced apoptosis, and found that ZD1839 inhibited the phosphorylation of Akt, and enhanced TRAIL-induced apoptosis via activation of caspase-3 and caspase-9, and inactivation of Bcl-XL. These results indicated that ZD1839 can augment the anti-cancer activity of TRAIL, even in TRAIL-resistant ESCC. The anti-cancer propertie of enhancing TRAIL-induced apoptosis suggests that combination treatment with ZD1839 and TRAIL may have potential in the treatment of ESCC patients (Teraishi et al. 2005). Anticancer Properties of Enhancing Fas/FsaL-Mediated Apoptosis Decreased expression of Fas could impair apoptosis of ESCC cells not only in response to antitumor T cells, but also in response to autocrine suicide via tumorexpressed FasL. This could account in part for the apoptotic resistance of ESCC with low Fas expression. Up to data, there is no report in regard of the chemotherapeutic agent via ESCC Fas/FsaL signaling, however Fas expression playing a key role in apoptosis of ESCC may help in designing new therapeutic approaches based on reinforcement of Fas/FasL-induced tumor apoptosis. Anticancer Properties of p53-Mediated Apoptosis Mutations in p53 or in the p53 pathway can produce multiple chemoresistance in vitro and in vivo, and it has been found that reintroduction of wild-type p53 into p53 null tumor cells can re-establish chemosensitivity (Wallace-Brodeur and Lowe 1999). Some studies have been shown that the p53 gene up-regulation is closely related to sensitivity to chemoradiotherapy. A recent in vitro anti-cancer effect of isorhamnetin on human ESCC cell line (Eca-109) was investigated by Ma et al. They found that isorhamnetin appears to be a potent drug against ESCC due to its potential to upregulate p53 along with inhibition of proliferation in ESCC cells (Ma et al. 2007). Shimoyama et al. investigated the roles of p53 and p21 expression in response to the chemoradiation therapy for human ESCC. They found that wild type p53 or p21 expression could be altered by the chemoradiation therapy, which could influence the therapeutic efficacy (Shimoyama et al. 1998). However, p53 status is not a universal predictor of treatment response, because not all drugs absolutely require p53 for their apoptotic function (Herr and Debatin 2001). This could be an explanation for those studies with confirmed observation that p53 protein is frequently expressed in ESCC but no significant association is found between p53 protein expression and response to chemo/radiotherapy (Lam et al. 1997, Puglisi et al. 1996). In some settings, p53 loss can even enhance drug-induced cell death (Bunz et al. 1999). For instance, Nakashima et al. evaluated 30 patients with ESCC received preoperative chemotherapy then underwent esophagectomy with lymph node dissection. Preoperative chemotherapy against primary lesions was ineffective in all the patients who expressed p53, but not p21. On the other hand, chemotherapy was effective against metastatic lymph nodes which were p53 negative but p21 positive. These findings suggest that p21 positive expression in the

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absence of p53 is associated with favorable effects of preoperative chemotherapy in ESCC patients (Nakashima et al. 2000). Sarbia et al. examined retrospectively the correlation between the outcome of patients with locally advanced ESCC after multimodal treatment (radiochemotherapy +/− surgery) and the expression of apoptosis-regulating proteins in pretherapeutic biopsies. They also found that p53negative tumors showed a significantly better outcome than p53-positive tumors in patients treated by radiochemotherapy and surgery (Sarbia et al. 1998). Another example for regarding an effective treatment for ESCC linked to p53 is a recently identified ribonucleotide reductase, p53R2. p53R2 is directly regulated by p53 in the supply of nucleotides for repairing damaged DNA. Yokomakura et al. investigated the improvement in radiosensitivity of human ESCC cells using p53R2 small interfering RNA (siRNA). They demonstrated that p53R2 is associated with the radiosensitivity of ESCC cell lines, and that p53R2 expression is reduced after X-ray irradiation following the transfection of p53R2 siRNA (Yokomakura et al. 2007). Nevertheless, loss of p53 function could still correlate with multidrug resistance in ESCC because the properties of p53 gene family regulate cell-cycle arrest, apoptosis and DNA damage repair. Anticancer Properties of Bcl-2 Family Mediated Apoptosis Alteration of Bcl-2 family proteins can drastically alter drug sensitivity in experimental models (Wei et al. 2001), and are associated with multidrug resistance in human cancers (Schmitt et al. 2000). Puglisi et al. investigate the immunohistochemical expression bcl-2 proteins along with p53 in ESCC and to assess the sub groups of patients with a response to preoperative chemo/radiotherapy. They found that bcl2 expression was no longer detected in the residual neoplastic cells of a previously bcl-2 positive tumour after treatment. Interestingly, of the three non-responsive cases in which bcl-2 protein expression was evaluated, two are bcl-2 positive biopsy specimens. The tumour cells might be resistant to the action of radiation and cytotoxic drugs, which depends on bcl-2 anti-apoptotic activity that bypasses the induction of apoptosis by p53. However, the number of cases in the present study was too few to permit testing of this hypothesis (Puglisi et al. 1996). Szumilo et al. also investigated the expressions of p53 and bcl-2 to estimate the pathological response to prechemotherapy in endoscopic biopsy ESCC specimens. Although the pathological response to treatment had no significant associations with the p53 and bcl-2 proteins in ESCC in their study, they found that the patients in complete response were bcl-2 protein-negative (Szumilo et al. 2000). Ikeguchi et al. retrospectively investigated the prognostic significance of the mmunoreactivities of Bax in 141 surgically resected ESCC patients. They found that loss of Bax expression was detected more frequently in p53-positive tumors, and Bax expression correlated with favorable prognosis in 57 patients with postoperative chemoradiotherapy (Ikeguchi et al. 2001). Natsugoe et al. also investigated immunohistochemically the expressions of p53, Bax, and Bcl-XL investigated in 111 patients with advanced ESCC. In 44 patients who underwent chemotherapy and/or radiation therapy after

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surgery, Bax and Bcl-XL expression was not related to patients’ survival (Natsugoe et al. 2001). Because the mechanisms of controlling apoptosis in ESCC are complex, it is not easily described on the basis of immunohistochemical results for these molecules even though all the investigations above imply that bcl-2 apoptotic pathways may be associated with chemoresistance in ESCC. However, an interesting investigation closely related to Bcl-2 family was performed by Faried et al., in which chemically synthesized sugar-cholestanols were introduced to possess potential multi-target anticancer activity against human ESCC cell lines because the apoptotic cell death was induced through modulating the Bcl-2 family, caspase cascade and PARP. Although they did not detect the altered expression levels of anti-apoptotic proteins such as Bcl-2 and Bcl-xL, they found that expression level of Bax protein was significantly enhanced in ESCC cells, which result in the disruption of a balance between pro-apoptotic and anti-apoptotic molecules in the apoptosis machinery, leading to release of cytochrome c and then apoptosis (Faried et al. 2007). Anticancer Properties of Caspases Mediated Apoptosis The ability of anticancer agents to trigger caspases activation appears to be a critical determinant of sensitivity or resistance to cytotoxic therapies. As a consequence, inhibition of caspases activation may be an important factor in chemoresistance. Paclitaxel is a highly effective chemotherapy agent against ESCC. Faried et al. demonstrated that human ESCC cell lines (TE-13 and TE-14 cells) arrested at G2/M phase and eventually undergo apoptosis in response to paclitaxel. A high number of apoptosis cells in these two ESCC cell lines were accompanied by an increased level of active cleaved fragment of downstream caspase effector and executor that cleaves specific substrate such as PARP, suggesting that the mitochondrial (intrinsic) signaling pathway may play a role in mediating cell apoptosis by paclitaxel (Faried et al. 2006). Inhibitor of Apoptosis Proteins Family Mediated Apoptosis Resistance An important group of factors, which consists of structurally related proteins known as the inhibitor of apoptosis proteins (IAP) family, possesses two unique features. First, they are the only cellular factors that can act both on initiator and effector caspases (Deveraux et al. 1999). Second, the biological effect of these proteins can be converted from anti-apoptotic to pro-apoptotic (Clem et al. 2001, Nachmias et al. 2003). To date, eight human IAPs have been identified: c-IAP1, c-IAP2, NAIP, Survivin, XIAP, Bruce, ILP-2, and Livin (Liston et al. 2003). Imoto et al. identified high copy-number amplification at 11q21-q23 in cell lines derived from ESCC. The target gene within an amplicon at 11q22 in ESCC has been suggested as cIAP1, and the expression of cIAP1 was consistently up-regulated in ESCC cell lines that showed amplification at 11q21-q23. This study revealed overexpression of cIAP1 in the majority of ESCC samples (Imoto et al. 2001). Nemoto et al. investigated the potential roles of IAP family proteins in the homeostasis of

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normal esophageal epithelia as well as the pathogenesis of ESCC and found that patterns of IAP expression were not uniform in ESCC samples. Some cases exhibited very strong expression of particular IAPs, while the others revealed an almost normal level of expression. However, survivin expression revealed a very high frequency in many of the esophageal cancer cells in the majority of cases (Nemoto et al. 2004). In addition, survivin expression have been reported by Kato et al. to correlate with the response of patients to chemotherapy (Kato et al. 2001). Ngan et al. investigated the effect of oxaliplatin on two esophageal cancer cell lines, ESCC (TE3) and EAC (TE7). They found that ESCC cells died via apoptosis following cell-cycle arrest at G (2) phase after oxaliplatin treatment. Further investigations indicated that survivin promoter activity was also inhibited by oxaliplatin, and oxaliplatin-induced apoptosis in the TE3 cells was caspase dependent. However, downregulation of Bad, Bid, Puma, and Noxa, lack of cytochrome c release. Limited loss of mitochondrial membrane potential in early phase implied possible different initiation pathways other than the mitochondrial pathway (Ngan et al. 2008).

Apoptosis and Esophageal Adenocarcinoma Gastroesophageal Reflux Disease, Barrett’s Esophagus and Esophageal Adenocarcinoma EAC is one of the most rapidly increasing cancers in Caucasian males in the US (Blot et al. 1991, Pera et al. 1993). Nearly all EAC arises from BE, a premalignant disease caused by gastroesophageal reflux disease (GERD). GERD is one of the most prevalent gastrointestinal disorders affecting patients’ quality of life (Nebel et al. 1976, Thompson and Heaton 1982). Barrett’s Esophagus (BE), a GERD-associated complication, is defined as the replacement of normal esophageal squamous mucosa by specialized intestinal columnar mucosa with appearance of goblet cells. The presence of BE is associated with an increased risk of developing EAC, with the well established histological sequence of chronic GERD to esophagitis to metaplasia to dysplasia to adenocarcinoma (Jankowski et al. 1999). BE is the only known precursor lesion for EAC (Altorki et al. 1997, Cameron 1997, Isolauri et al. 1997), and EAC is considered a life-threatening complication of GERD with approximately 15% probability of surviving for 5 years after a diagnosis of EAC (Blot et al. 1991, Enzinger and Mayer 2003, Farrow and Vaughan 1996, Pera et al. 1993, 2005, Tew et al. 2005). The histologic changes leading to EAC are accompanied by alterations at the cellular and molecular levels. Abnormalities in apoptosis, proliferation and differentiation are common cellular events in BE and EAC (Hong et al. 1995, Ouatu-Lascar et al. 1999, Whittles et al. 1999). Acquired genetic abnormalities, such as gene mutation, gene deletion, loss of heterozygosity, aberrant methylation and aberrant gene expression, have been reported (Beer and Stoner 1998, Casson 1998, Fitzgerald and Triadafilopoulos 1998). The accumulation of genetic and epigenetic aberrations

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produces one or more clones with malignant potential (Chen and Yang 2001, Jankowski et al. 1999). Although it is generally accepted that EAC progresses in a stepwise fashion: esophagitis to metaplasia to dysplasia to EAC, however, it is unclear why some BE patients develop EAC and some do not. Gastroduodenal contents exposure of the esophageal mucosa is considered to be an important risk factor for developing of esophagitis, BE and EAC, but the carcinogenesis of EAC are not fully understood. Knowledge about EAC as a distinct clinical entity has been accumulating because of the rapid increase in incidence and consequent change in research focus. We will summarize the current understandings of EAC regarding apoptosis related carcinogenesis and the chemotherapies.

Apoptosis in EAC Carcinogenesis Death Receptor TRAIL Pathway in BE and EAC TRAIL and its receptors are functional in the carcinogenesis of EAC. A study carried out by Younes et al. has demonstrated that TRAIL-R1 (DR4) and/or TRAIL-R2 (DR5) are expressed in both human EAC tissue and EAC cell lines (Bic-1 and Seg1). They further investigated the function of these two receptors in EAC cell lines and found that targeting of TRAIL-R1 and/or TRAIL-R2 by agonist monoclonal antibodies can induce apoptosis in a time and dose dependent manner (Younes et al. 2006). Popnikolov et al. investigated TRAIL expression in Barrett’s esophagus with dysplasia and adenocarcinoma. They found that the TRAIL was rarely and weakly expressed in Barrett’s esophagus with dysplasia and EAC. The results show that the downregulation of TRAIL is associated with development of dysplasia in Barrett’s esophagus, and the immunohistochemically detected downregulation of TRAIL expression appears to be a promising indicator for dysplasia in Barrett’s esophagus (Popnikolov et al. 2006). It is generally accepted that TRAIL specifically kills malignant cells but spares the normal tissues, however a study performed by Kim et al. using soluble TRAIL preparations demonstrated that apoptosis can be induced by TRAIL in primary human esophageal epithelial cells. They demonstrated that soluble TRAIL preparations bind with death receptors in cell membrane and trigger apoptosis through a mitochondria independent signal transduction pathway (Kim et al. 2004). Although it is largely unknown regarding the role of TRAIL pathway played in the progression of BE and EAC, these investigations provided indirect evidences that proapoptotic gene TRAIL might be very useful in gene therapy for apoptosis resistant EAC. Fas/FasL System in BE and EAC It has been reported that Fas is not expressed normal esophageal mucosa (van der Woude et al. 2002, Vollmers et al. 1997). Younes et al. investigated status of Fas expression in BE with and without dysplasia or EAC in tissue sections from

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esophageal biopsies and esophagectomy specimens. They found that Fas is usually undetectable or is expressed at a low level in BE with or without dysplasia or EAC. Fas expression in goblet cell-containing glands is less frequent than in glands with gastric cardia phenotype in the same specimens (Younes et al. 2000). In contrast, Hughes et al. demonstrated that wild-type Fas protein is expressed abundantly but not distributing into cell surface in EAC. Their study describes Fas expression in Barrett’s esophagus, EAC, and three esophageal adenocarcinoma cell lines. Immunohistochemical analysis demonstrated 30.5% of EAC with faint cytoplasmic staining, and 69.5% were negative for Fas. Similar levels of Fas mRNA were identified in EAC compared to that in esophageal squamous mucosa or BE. The EAC cell line Seg-1 was negative for Fas expression by immunohistochemical analysis, but Western blot analysis demonstrated abundant, appropriately sized Fas protein. In agreement with the immunohistochemical analysis, flow cytometry of Seg-1 showed minimal amounts of Fas on the cell surface. They concluded that wild-type Fas protein is expressed abundantly in EAC but is retained within the cytoplasm, resulting in decreased Fas cell-surface expression, which could correlate with resistance to Fas-mediated apoptosis (Hughes et al. 1997). Younes et al. reported that overexpression of Fas ligand during malignant transformation of Barrett’s metaplasia, they used tissue sections from esophageal biopsies and resection specimens to immunostain for FasL. The results indicated that FasL was detected in 55% of cases of BE without dysplasia, however all cases of BE (100%) with low- or high-grade dysplasia were FasL-positive. They concluded that FasL overexpression is acquired early during malignant transformation of BE (Younes et al. 1999). bcl-2 Family in BE and EAC Accumulating evidence indicated that bcl-2 family play an important role in the transformation from BE to EAC. A progressive reduction of bcl-2 expression through low- to high-grade dysplastic Barrett’s epithelium and adenocarcinoma has been observed. Katada evaluated the alteration of apoptosis in the esophageal epithelium during the esophagitis-Barrett esophagus-adenocarcinoma sequence, and found that inhibition of apoptosis by overexpression of bcl-2 protein occurs early in the dysplasia-carcinoma sequence. They also found that bcl-2 was not widely overexpressed in Barrett high-grade dysplasia and adenocarcinoma despite the absence of apoptosis (Katada et al. 1997). The observations by Raouf et al. suggest loss of bcl-2 regulation is a factor in the multistep progression to EAC, consistent with the reports of reduced apoptosis early in the metaplasia-dysplasiaEAC sequence, which is agreed with Katada et al.’s finding (Raouf et al. 2003). Unfortunately, the role of bcl-2 played in EAC carcinogenesis is quite controversial. Goldblum et al. and van der Woude et al. also observed the negative immunoreactivity of bcl-2 in the dysplastic BE and EAC epithelium, however their conclusions are disagreed with Raouf and Katada because either only lamina propria immune cells showed positive staining but not the EB epithelium or no regenerative crypt compartment positive staining was found (Goldblum and Rice 1995, van der Woude et al. 2002). The observations above implied that prolongation of cell survival may promote neoplastic progression but the cells

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could acquire bcl-2 expression or other ways of avoiding apoptosis as malignancy appearing. The study carried out by van der Woude also showed that Bcl-xl, another antiapoptotic Bcl-2 family member, was highly positive in BE with dysplasia and tumor cells but not in BE intestinal metaplasia. They found that Bax, a proapoptotic member of the Bcl-2 family, was positive in all samples, and there was a significant negative correlation between the intensity of Bax staining and the transformation of BE to EAC (van der Woude et al. 2002). Chatzopoulos et al. investigated Bax and Bcl-2 expressions in 31 patients with BE and 21 GERD controls in Greece population for the reason that mortality from esophageal cancer in Greece is among the lowest in the world. They found that increased expression of Bax was noticed in the total BE patients compared with GERD controls while Bcl-2 expression did not differ between the two groups. The findings indicate Bax protein overexpression in BE could promote BE cell apoptosis, and this may partly contribute to the explanation why the incidence of EAC is so low in Greece (Chatzopoulos et al. 2007).

Alteration of p53 Protein in BE and EAC p53 gene mutation in BE and EAC has been widely reported, however the reported p53 mutation by immunohistochemistry as an indirect method to detect p53 mutation has been reported with variable frequencies in EAC and in BE with high-grade and low-grade dysplasia (Bian et al. 2001, Casson et al. 1991, Djalilvand et al. 2004, Doak et al. 2003, Dolan et al. 2003, Gleeson et al. 1995, 1998, Gonzalez et al. 1997, Hamelin et al. 1994, Ireland et al. 2000, Neshat et al. 1994, Schneider et al. 1996, Schneider et al. 2000, Taniere et al. 2001). Djalilvand et al. used laser capture microdissection (LCM), an accurate isolation for different genotype cells, to study the incidence and types of p53 mutations in BE with and without dysplasia and in EAC, however, they show that p53 gene mutations are relatively rare in esophageal preneoplastic and neoplastic conditions. The abnormalities occurred in exons 7 and 8 in the form of point mutations (Djalilvand et al. 2004). Chung et al. also used LCM and direct sequencing to maximize to investigate the frequency of p53 mutation. IHC was also performed in their study to evaluate EAC and adjacent BE with dysplasia, and the results were correlated with mutation status to determine whether IHC can be a valid alternative in detecting p53 changes for this setting (Chung et al. 2007). They found that the frequency of p53 gene mutation is 75% (30/40) of EAC, and it is higher than the 43% (150/350) calculated from a total of 14 studies in the literatures above. In addition, they reported that twenty-seven percent of the mutations were found in exon 4, which is different from previous data that showed >90% of p53 mutations in exons 5–8 in solid tumors such as that in ESCC (Hollstein et al. 1991, Mir et al. 2005). Although these discrepancies exited, using LCM to study a pure population of tumor cells and mutations should be more accurate because of its elimination of DNA contamination by non-neoplastic cells and further study is needed to clarify their results.

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Although the presence of a p53 mutation increases the risk of neoplastic progression, a number of studies indicate that the absence of this mutation does not abrogate the risk. Based on the observed various frequencies of p53 alteration, it implied that p53 itself is not an adequate marker for patient selection in BE and EAC monitoring. Determination of p53 mutational status may ultimately be a component of a panel of biomarkers to risk-stratify patients with BE because of the complex apoptotic signaling transduction in the EAC carcinogenesis.

Apoptosis in EAC Chemotherapy Anticancer Drugs Activate the Apoptotic Pathway Anticancer Properties of Enhancing TRAIL-Induced Apoptosis Expression of TRAIL-R1 and TRAIL-R2 has been found in the majority of human EAC tissues. Younes et al. used agonist anti-TRAIL-R1 and TRAIL-R2 antibodies to induce apoptosis in human EAC cell lines and found that the antibodies induced apoptosis of EAC cells with expressing of these receptors in a time and dose dependent manner. The findings suggest that human EAC, with very poor prognosis and no accepted effective non-surgical treatment, may be good candidates for treatment with agonist antibodies targeted to TRAIL-R1 and TRAIL-R2 (Younes et al. 2006). Because TRAIL is a promising agent for development as a cancer-specific therapeutic agent, his-tagged protein and three versions of native recombinant human TRAIL protein have been isolated and purified from Escherichia coli (Kim et al. 2004). However, some reports have demonstrated a toxicity of some TRAIL preparations against primary normal human cells through a caspase-dependent mechanism that involves activation of the extrinsic death pathway (Jo et al. 2000). Kim et al. investigated the toxicity of certain TRAIL preparations against primary human esophageal epithelial cells, and found that primary cultured normal esophageal epithelial to be sensitive to all TRAIL preparations used in the study, including trimer TRAIL. However the TRAIL-induced death in esophageal epithelial cells can be prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure. This encouraging results suggest a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity, and the hyper-oligomerized TRAIL may be considered as an alternative agent for the treatment of EAC in clinical trials (Kim et al. 2004). Anticancer Properties of Fas/FasL Mediated Apoptosis Fas/FasL-induced apoptosis could participate in the mechanism of action of DNAdamaging anticancer drugs, but it has been disputed about this hypothesis, based largely on the inability of exogenously added anti-Fas/FasL reagents to attenuate drug-induced apoptosis. Poulaki et al. demonstrated that transfection of a construct encoding soluble (decoy) Fas protected SK-N-MC cells from doxorubicin induced call death. However, incubation with anti-Fas or anti-FasL neutralizing antibodies or exogenous addition of pre-synthesized recombinant soluble Fas decoy protein

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had no protective effect (Poulaki et al. 2001). It has been demonstrated that the expression of Fas on the cell surface by EAC is reduced. Wild-type Fas protein is found retaining in the cytoplasm in an EAC cell line. Therefore, the retention of wild-type Fas protein within the cytoplasm may represent not only a mechanism by which malignant cells evade Fas-mediated apoptosis but also chemoresistance (Hughes et al. 1997). This raises the possibility that the proposed Fas/FasL suicidal interaction may take place in an intracellular compartment and thus is not accessible to exogenously added reagents. Although Fas/FasL neutralizing reagents are available commercially, these reagents may not be a reliable indicator of the involvement of the Fas pathway in anticancer-drug-induced apoptosis in EAC. Therefore, the experiments using these agents in assistance of EAC chemotherapy, if any, should be carefully evaluated. Anticancer Properties of Bcl-2 Family Mediated Apoptosis Although members of the Bcl-2 family play an important role in the regulation of apoptosis, the role of bcl-2 family played in EAC resistance to chemotherapy is controversial. In total 48 EAC patients with received preoperative chemoradiotherapy followed by surgery, Raouf et al. studied bcl-2, bax and bcl-x protein expression. They found that preoperative chemoradiotherapy induces expression of bax and bcl-x protein, and bcl-2-positive patients had a significantly improved survival compared with bcl-2-negative tumors. Although expression of bcl-2 and related proteins did not predict response or resistance to neoadjuvant chemoradiotherapy, bcl-2 family was associated with improved survival suggesting that multiple regulators of apoptosis could determine the response of EAC to chemoradiotherapy (Raouf et al. 2003). Ogunwobi et al. demonstrated that statins, an agent clinically used to treat hypercholesterolemia, can induce apoptosis in EAC cells via inhibition of Ras farnesylation and inhibition of the ERK and Akt signaling pathways. They found that statins treatment increased messenger RNA and protein expression of the proapoptotic proteins Bax and Bad, but protein levels of the antiapoptotic proteins Bcl-2 and Bcl-XL were unchanged. This finding provided an evidence that the bcl-2 family could play an important role in the chemotherapeutic agent associated the induced apoptosis in EAC, and suggested that statins may have some potential as chemopreventative and adjuvant chemotherapeutic agents in EAC (Ogunwobi and Beales 2008). Anticancer Properties of p53 Protein Mediated Apoptosis It is generally accepted that p53 associated with prognosis of EAC and response to chemotherapy in EAC. Heeren et al. investigated immunohistochemically the expression of p53 in pre-treatment biopsy specimen of 30 patients, in phase II neoadjuvant studies for locally advanced EAC, who underwent surgery. They found that alteration in expression of p53-positivity in the pre-treatment specimens to p53negativity in the resection specimens was correlated with better chemotherapeutic response and survival. The results suggested that alteration of p53 expression rather

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than the initial expression seems to be related to chemotherapy response and overall survival in patients with EAC (Heeren et al. 2004). In contrast, Yang et al. also evaluated immunohistochemically p53 protein expression in 95 biopsy specimens from patients with EAC or with ESCC before chemoradiotherapy. p53 expression was correlated to the pathologic response identified in subsequent esophagectomy specimens. Overexpression of p53 protein is associated with decreased responsiveness to chemoradiotherapy in patients with EAC but that no such association exists in patients with ESCC (Yang et al. 1999). However, Duhaylongsod et al. found that p53 positivity correlated with residual cancerous tissue in the resection specimen but not with EAC-free survival. They concluded that although p53 protein overexpression is commonly observed in EAC, its prognostic value appears limited (Duhaylongsod et al. 1995). Nabeya et al. investigated the relationship of p53 mutation to drug sensitivity in EAC cells. They examined the EAC cell lines for p53 mutational status, chemosensitivity to 5-fluorouracil, mitomycin C, and cis-dichlorodiammineplatinum(II), alteration in p53 levels following exposure of cells to these drugs, and the mechanisms involved in regulating p53 levels. The results indicated that wild-type p53 protein levels increase after treatment with each of these drugs and that this increase in wildtype p53 appears to be required for effective chemotherapeutic growth inhibition of EAC cells. In contrast, EAC cells expressing either mutated p53 protein or no p53 protein are more resistant to the growth-inhibitory effects of these drugs, despite the fact that drug exposure can also increase mutant p53 levels by a translational mechanism (Nabeya et al. 1995). Schrump et al. investigated that flavopiridol, a synthetic flavone that induces apoptosis via p53-independent pathways, mediated growth inhibition via cell cycle arrest and apoptosis in the EAC cell lines expressed a mutant p53. They found that flavopiridol is efficacious in the prevention of cell growth in EAC cell lines. Although the mechanisms of flavopiridol-mediated multigene targeting cytotoxicity have not been fully elucidated, the harboring p53 mutations of these cell lines could have minimal resistance to the apoptotic effect of flavopiridol thereby make this an attractive agent for use in the treatment and possible prevention of EAC (Schrump et al. 1998). Apoptosis of Proteins (IAP) Family Mediated Apoptosis Resistence Survivin, a member of the inhibitor-of-apoptosis family, is reported not only to be overexpressed in ESCC, but also to be overexpressed increasingly along with the metaplastic/dysplastic sequence in EAC. Vallb¨ohmer et al. performed the survivin gene expression in normal squamous/columnar epithelium and in the various stages of development of Barrett’s adenocarcinoma. They concluded that survivin expression may be a biomarker in the development of Barrett’s adenocarcinoma that is able to distinguish between quiescent BE, BE with dysplasia and EAC (Vallbohmer et al. 2005). Survivin gene could also be used as a member of a panel of targeting genes to prevent BE patients progression to EAC. Ngan et al. provided further evidence of surviving targeting by the investigation of the effect of oxaliplatin on the EAC (TE7) cells. They found that TE7 cells died via mitotic catastrophe, and survivin was inhibited in TE7 cells compared with ESCC cells. Mechanistic studies

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showed that downregulation of survivin by oxaliplatin in TE7 cells was partially due to the proteasome-mediated protein degradation pathway and partially due to the downregulation of Sp1 transcription factor. Similar results were obtained for another gastric adenocarcinoma cell line, MKN45, in which survivin was previously shown to be inhibited by oxaliplatin. These data indicate that survivin may be a key target for oxaliplatin. The ability of oxaliplatin to induce different modes of cell death through surviving targeting may contribute to its efficacy in EAC chemotherapy (Ngan et al. 2008).

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Chapter 7

Molecular Targets in Gastric Cancer and Apoptosis Elizabeth K. Balcer-Kubiczek and Michael C. Garofalo

Abstract Gastric cancer remains one of the most common forms of cancer worldwide. With current diagnostic and therapeutic approaches the prognosis of gastric cancer is very poor. Similar to other solid cancers, gastric cancer is a complex disease resulting from combinatorial interactions among diverse factors including environmental, host-genetic and molecular mechanisms. Gastric cancers arise in part because of disruption cell death mechanisms including apoptosis contributing to cell expansion. This review provides an update (current up to April 6, 2008) on pathogenesis focusing on the specific abnormalities in gastric cancer genomes, conventional therapy approaches, genetic and epigenetic mechanisms of expression regulation, apoptotic pathways in gastric cancer and new emerging or potential therapeutic approaches focused around molecularly defined gastric cancers. Keywords Apoptosis · Cancer regulators · Gastric cancer · microRNA · Stem cells

Introduction The purpose of this chapter is to cover advances in gastric cancer research. The Medline database (www.ncbi.nlm.nih.gov/pubmed/) was searched using subject heading terms including “gastric cancer” or “stomach cancer”, in combination with the subheadings such as “risk factor”, “pathology”, “histology”, “Helicobacter pylori”, “Epstein-Barr virus”, “genetic marker”, “epigenetic marker”, “microsatellite instability”, “histone modification”, “DNA repair”, “cDNA microarray”, “tumor suppressor gene”, “oncogene”, microRNA”, “microRNA array”, “apoptosis”, and “cancer stem cell”. Ongoing trials were identified through the Physician Data Query database (www.cancer.gov/search trials). Gene nomenclature follows the Entrez Gene database (www.ncbi.nlm.nih.gov/sites/entrez). We provide an update

E.K. Balcer-Kubiczek Department of Radiation Oncology, University of Maryland School of Medicine, The Marlene and Stewart Greenebaum Cancer Center, Baltimore MD 21201, USA e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 7,

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(current up to April 6, 2008) on epidemiology and pathogenesis focusing attention on the specific abnormalities in gastric cancer genomes, conventional treatment approaches including chemo- and radiation therapy, genetic and epigenetic mechanisms of expression regulation, apoptotic pathways in gastric cancer and new emerging or potential therapeutic approaches focused around molecularly defined gastric cancers.

Epidemiology Gastric cancer remains one of the most common forms of cancer worldwide with 870,000 new cases and 650,000 deaths per year (Parkin 2001, Jemal et al. 2007). Approximately 23,000 patients per year are diagnosed in the United States alone, and about 11,000 patients are expected to die of the disease (Edwards et al. 2005, Hayat et al. 2007, Espey et al. 2007). In terms of incidence, gastric cancer is the fourth most common malignancy, only after lung (12.3%) and breast (10.4%) and colorectum (9.4%) (Parkin 2001). In terms of mortality, gastric cancer is the second most fatal malignancy (10.4%), only after lung (17.4%). In the United States, gastric cancers account for 2% of total cancer deaths. The incidence of gastric cancer shows intriguing geographic variations. The high incidence areas are in Southeast Asia, Japan, China, Korea, Eastern Europe and parts of South America, particularly Chile. The low incidence areas are in North America, India, most countries in Africa, some countries in Western Europe, New Zealand and Australia (Roder 2002) The high-to-low risk population ratio is about 10. According to the World Health Organization, the incidence rates range from approximately 18/100,000 in Finland and the United Kingdom to approximately 97/100,000 in China and South Korea (IARC 2000). The incidence of gastric cancer also shows gender variation. In every area of the globe, incidence rates in males are approximately two-fold higher than in females (IARC 2000, Roder 2002). For example, of approximately 23,000 new gastric cancer cases In the United States, there are approximately 14,000 new cases in males and 9,000 new cases females or a 1.5:1 ratio (Edwards et al. 2005, Hayat et al. 2007, Espey et al. 2007). Worldwide, male-to-female incidence ratios are in the 1.5 to 2.5 range (Roder 2002). Declining mortality and incidence of gastric cancer has been noted in both developing and developed countries (Verdecchia et al. 2003, Jemal et al. 2007). Over the past 30 years, the rates of decline approximated 70% in low-risk countries, including Western and Northern Europe (Finland, Norway, Sweden, Denmark, France, Belgium) and North America (the United States, Canada). Less dramatic but nevertheless significant rates of decline of approximately 40% have been observed in high-risk countries including those in Asia (Japan, China, Korea), South America (Columbia), Central Europe (Germany, Poland) and developing countries. The decline of overall incidence of gastric cancer has been attributed to a number of factors. These include a decrease in the intake of highly processed and chemically preserved foods, and increased consumption of fresh fruit and vegetables after the widespread introduction of refrigeration (Gonz`alez et al. 2006). Earlier detection of

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gastric tumors as well as improved sanitary conditions and wider availability and use of antibiotics with a resulting reduction of Helicobacter pylori (H. pylori) infections also play a part (El-Omar et al. 2001, Roder 2002). The IARC classified H. pylori as a group I, or definite carcinogen, in 1994 but discussion of the causal association between H. pylori infection and gastric cancer has been complicated by the African enigma, a high prevalence of H. pylori and low incidence of gastric cancer in Africa (Holcombe 1990, Segal et al. 2001, Wabinga 2005). Similar observations have been made about other populations including those in India, South America and Middle East (Bravo et al. 2002, Siavoshi et al. 2005). Much current research of H. pylori-associated gastric cancer has focused on a role of polymorphic genotypes of H. pylori and host factors such as individual variations of the inflammatory cytokine genes of the host. Recent evidence implicated the Epstein Barr virus as a risk factor (Kim et al. 2005a, Koriyama et al. 2007, Jung et al. 2007). In all, 5% to 15% of gastric carcinoma cases worldwide are associated with Epstein-Barr infection. The virus is proposed to play a causative role in Epstein-Barr virus-associated gastric cancer, because it is found in almost all tumor cells and the viral DNA shows monoclonality in the cancer (Takada 2000). Prior surgery for gastric ulcer and exposure to ionizing radiation also increases risk (Hall and Wuu 2003, Suit et al. 2007, Preston et al. 2007, Bahmanyar et al. 2007). Molecular aspects of H. pylori or Epstein-Barr infection and other environmental risk factors, including radiation, in relation to gastric carcinogenesis and apoptosis are discussed in Sections “Helicobacter PyloriInfection Associated Genes” and “Epstein-Barr Infection-Associated Genes”. For more general discussion of gastric carcinogenesis see Cohen (2003), Tahara (2004), Lynch et al. (2005), Correa and Schneider (2005) and Smith et al. (2006).

Pathological Classification It has been estimated that the majority (90%) of gastric cancers are sporadic and the remaining 10% of gastric cancers are hereditary (Park et al. 2000a). Possible predisposing factors to hereditary gastric cancer have been identified. They include germline mutations in CDH1 in approximately 30% of affected families (Guilford et al. 1998). The remaining 70% of affected families have not been genetically defined. However, gastric cancer may also be seen as part of other inherited cancer predisposition syndromes, such as hereditary nonpolyposis colorectal cancer syndrome, Li-Fraumani syndrome, Familial Adenomatous Polyposis, Cowden syndrome and Peutz-Jeghers syndrome (Lynch et al. 2005). Over 90% of gastric cancers are adenocarcinomas. Adenosquamous, squamous, and undifferentiated carcinomas also occur but are rare. There have been many attempts to classify gastric cancers on the basis of morphology or anatomical sites, and each has its own advantages and disadvantages. Classifications based on anatomy have identified carcinoma of the cardia as a possibly distinct disease from non-cardia carcinoma (Sidoni et al. 1989). The importance of cardia carcinoma is in

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steadily rising incidence, in contrast to a sharp decline in the incidence and mortality of gastric cancer (Powell and McConkey 1992). In terms of possible risk factors, Epstein-Barr infection rather than H. pylori infection associates more strongly with cardia than non-cardia carcinoma. Additionally, the division into the cardia and noncardia subtypes is therapeutically relevant because carcinoma of the cardia generally has worse prognosis compared to non-cardia carcinoma (Kim et al. 2005). The most widely used classification based on morphology and proposed by Lauren (1965) divided adenocarcinomas into diffuse and intestinal types. The Lauren classification corresponds to the Japanese classification of poorly differentiated (diffuse) and well-differentiated (intestinal) types (Tahara 2004). A small proportion of gastric cancer presents a hybrid type comprising a mixture of diffuse and intestinal types. The histological classification is useful because diffuse and intestinal gastric cancers show differences in epidemiology, demography, etiology, pathogenesis, biological behavior and prognosis. For example, the diffuse type occurs in younger patients and is often associated with genetic predisposition (Lynch et al. 2005). Diffuse adenocarcinoma has more invasive and metastasizing potential and poorer prognosis than intestinal adenocarcinoma. The intestinal type is more frequent than in men and older patients as well as among Japanese patients. The intestinal type is associated with exposure to various environmental factors, including H. pylori and a higher risk and poorer survival than the diffuse type. Carcinoma of the cardia is more frequently associated with the poor differentiation and the diffuse type of the Lauren classification compared to carcinoma of other parts of the stomach (Kim et al. 2005). Despite an increasing body of data, the overall view on molecular pathology of remains fragmentary. No consistent differences in the molecular pathology of gastric cancer subtypes to meet the Lauren classification have been established, but some authors suggest that genetic and epigenetic alterations involved in diffuse carcinoma may differ from those involved in intestinal cancer (Tahara 2004, Smith et al. 2006). Accordingly, altered cell-cell adhesion markers occur frequently in the diffuse-type gastric cancer, whereas proliferation markers occur frequently in the intestinal-type. Defective genes associated with predisposition to gastric cancers are similar to those found to be altered in sporadic gastric cancers (Aarnio et al. 1997, Lindor and Greene 1998, Varley et al. 2003, Cohen 2003, Lynch et al. 2005).

Conventional Treatment Options Conventional treatment options for patients with gastric cancers are driven by their stage of disease. The staging system utilized in the majority of the world is the American Joint Committee on Cancer/International Union against Cancer staging system, though the Japanese have also developed their own classification, which is based on anatomic involvement of the cancer, especially the lymph node stations (Roder et al. 1998, Japanese Research Society 1993). In either system, patient treatment and outcome is predicted by the initial stage of disease.

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Surgery Surgery is the primary treatment for medically fit patients with potentially resectable gastric cancers that have not metastasized to distant sites beyond the locoregional lymph nodes. The goal of surgery is to remove the tumor entirely with adequate (5 cm) margins. A curative resection with negative margins and no residual disease (R0) is possible in only approximately 50% of patients (Ajani et al. 1993). Surgery typically consists of a total versus subtotal gastrectomy and a lymphadenectomy. For distal gastric cancers, a subtotal gastrectomy is preferred as it is equally efficacious and associated with fewer side effects when compared with a total gastrectomy (Bozzetti et al. 1999). The extent of lymphadenectomy that should be performed is controversial and remains a subject of international debate. A D0 resection, by definition, is a partial dissection of the first echelon draining lymph nodes, whereas a D1, D2 or D3 resection indicate a full dissection of the first, second and third echelon draining lymph nodes, respectively. The Japanese have historically advocated for extensive (D2 or D3) lymphadenectomies, whereas Western researchers have been unable to demonstrate a survival advantage to D2 resections when compared with a less extensive (D1) lymphadenectomy. Several randomized trials have failed to demonstrate a survival advantage to performing a D2 lymphadenectomy compared with a D1 lymphadenectomy (Hartgrink et al. 2004, Cuschieri et al. 1999). Greater morbidity was seen in the D2 resection arms of these trials, however the Japanese argue that it is lack of experience that is the underlying cause of this increased morbidity and that a benefit would be seen to D2 resections if morbidity were otherwise equal. At this point in time, Western practitioners largely perform D0 or D1 resections, even when they are encouraged to perform extensive lymph node dissections (MacDonald et al. 2001).

Postoperative Therapy For patients with very early T stage node negative gastric cancer (stage IA), surgery alone is adequate and there is no advantage to postoperative treatment. For patients with advanced T stage disease or node positive disease, there is a benefit to postoperative therapy in the form of chemotherapy and radiation therapy (MacDonald et al. 2001). Studies examining the patterns of failure following surgery have suggested a potential role for postoperative treatment and have also been important with regard to radiation therapy planning (Gunderson and Sosin 1982, Smalley et al. 2002). The largest and most important trial evaluating the potential benefit of postoperative therapy was the Intergroup trial (INT-0116), which randomized patients with stage IB-IV gastric or gastroesophageal junction cancers to either no further therapy or chemoradiation (CRT) consisting of 5-fluorouracil (5-FU), leucovorin (LV) and 45 Gy of radiation therapy to the postoperative bed and draining lymphatics. The mature results of this study reported by MacDonald et al. demonstrated a survival advantage to postoperative CRT yielding an improvement in median survival from 27 months to 36 months ( p = 0.005). The 3-year overall

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survival for patients treated with postoperative CRT was 50% versus 41% for those who underwent surgery alone ( p = 0.005).

Preoperative Therapy Growing interest in neoadjuvant approaches to the treatment of gastric cancer led to a recent randomized phase III trial by the British Medical Research Council which demonstrated an advantage to preoperative and postoperative chemotherapy treatment when compared to surgery alone (Cunningham et al. 2006). In this study patients were randomized to undergo surgery alone, or to receive ECF (epirubicin, cisplatin and 5-fluorouracil) chemotherapy before and after surgery. Five-year survival rates were 36% for those patients randomized to perioperative ECF versus 23% for those who underwent surgery alone. When compared with the outcome of the INT-0116 trial, approximate 3-year overall survival rates in this trial were slightly inferior, however a direct comparison is hazardous. Nevertheless, ECF chemotherapy has become an attractive regimen as a result of this trial and has led to further investigation. Whether ECF chemotherapy is superior to 5-FU/LV when combined with radiation therapy in the postoperative setting is the subject of an ongoing phase III cooperative group trial (CALGB 80101).

Management of Unresectable Nonmetastatic Disease In patients with unresectable or medically inoperable gastric cancer that is not yet distantly metastatic, studies have demonstrated no survival advantage to radiation therapy as a single modality. However, when given concurrently with radiosensitizing chemotherapy radiation therapy can improve the median survival for these patients (Wieland and Hymmen 1970, Moertel et al. 1969). By combining 45-50.4Gy of external beam radiation therapy with radiosensitizing chemotherapy such as 5FU, median survival can be improved from 6 to 13 months and 5-year overall survival can be improved from 0% to 12% (Moertel et al. 1969). Therefore, for a patient of a performance status such that they can tolerate CRT, this is the preferred approach.

Chemotherapy for Metastatic Gastric Cancer Multiagent chemotherapy results in improved quality of life and overall survival when compared with best supportive care in patients with metastatic carcinoma (Pyrhonen et al. 1995, Murad et al. 1993, Glimelius et al. 1994, MacDonald et al. 1980). Combination chemotherapy results in improved response rates when compared with single agent chemotherapy and is the preferred approach at present (Cullinan et al. 1985). Modern first line regimens include DCF (docetaxel, cisplatin and 5-fluorouracil) and ECF (Van Cutsem et al. 2006, Ross et al. 2002). A summary of current treatment guidelines by stage can be found in Table 7.1 as well

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Table 7.1 Current treatment guidelines Resectable Patients: Suggested Current Management by Stage T1N0: Surgery alone T2-4N0-1M0: Surgery followed by chemoradiationa TxNxM1: Chemotherapyb or clinical trial versus best supportive care Unresectable Patients including Medically Inoperable: Suggested Management TxNxM0: Chemoradiationa TxNxM1: Chemotherapyb or clinical trial versus best supportive care a

Chemoradiation consisting of 45–50.4 Gy in 1.8 Gy daily fractions combined with concurrent radiosensitizing chemotherapy such as capecitabine or 5-Fluorouracil +/− leucovorin b Chemotherapy consisting of regimens such as DCF (docetaxel, cisplatin and 5-FU) or ECF (epirubicin, cisplatin, 5-FU). Alternative regimens include irinotecan doublets.

as suggested first and second line polychemotherapy regimens for patients with metastatic disease.

Molecular Aspects of Gastric Cancer The combined results of several decades of research lead to identification of a rather small number of features shared by most if not all types of human cancers. Hanahan and Weinberg (2000) list the following alterations:

r r r r r r

Self-sufficiency in growth signals, such as constitutive autocrine growth signaling via EGF or TFF1. Insensitivity to growth inhibitory signals, such as silencing of p16 by hypermethylation or loss of expression of TGFB1. Evasion of apoptosis, such as mutational inactivation of pro-apoptotic genes, BAK and BAX, mutational inactivation of p53 or loss of PTEN, downregulation of BIRC5 or upregulation of CDKN1A. Limitless replicative life span, such as constitutive expression of TERT. Sustained angiogenesis, such as expression of VEGF (vascular endothelial factor) or HIF1A (HIF-1α; hypoxia inducible factor-1α). Tissue invasion and metastasis such as inactivating mutations in CDH1/CTNNB1 or TFF1).

Multiple genes associated with the above phenotypes have been described in gastric cancers (Table 7.2). Their number is rapidly increasing with application of new technologies including cDNA array approaches and the serial analysis of gene expression (Lee et al. 2003, Oh et al. 2005, Kim et al. 2005). The frequent molecular abnormalities reported to play roles in some forms of gastric cancer include mutation and overexpression of oncogenes such as ERBB2, K-sam, MET, KRAS and CTNNB1, the inactivation of tumor suppressor genes such as p16, trefoil factors 1 and 3, p53, APC, DCC, RUNX3, alterations in DNA mismatch repair genes (MLH1 and MSH2), cell-cycle regulators (p16, CDH1) as well as effector genes related

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Oncogenes: MET (c-met; hepatocyte growth factor receptor) K-sam (KATO III cell-derived stomach cancer amplified) KIT (c-kit; v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) ERBB2 (HER-2, erbB-2; v-erb-b2 erythroblastic leukemia viral oncogene homolog 2) MYC (c-myc; v-myc avian myelocytomatosis viral oncogene homolog) MDM2 (mouse double minute 2 homolog) KRAS (Kirsten rat sarcoma viral oncogene homolog) Tumor suppressor genes: RUNX3 (runt-related transcription factor 3) P53 (tumor protein p53) P73 (tumor protein p73) TFF1 (pS2; gastrointestinal trefoil protein 1) TFF3 (ITF; gastrointestinal trefoil protein 3) CDH1 (E-cadherin) PTEN (phosphatase and tensin homolog) FHIT (fragile histidine triad) APC (adenomatous polyposis coli) DCC (deleted in colorectal cancer) MADDH3 (mothers against decapentaplegic (MAD) Drosophila homolog 3) MADDH4 (MAD homolog 4) MADDH7 (MAD homolog 7) Apoptosis-associated genes: Bcl-2 (B-cell lymphoma 2) BIM (Bcl-2 interacting mediator of cell death) BBC3 (PUMA; Bcl-2 binding component 3) BAX (Bcl-2-associated X protein) BAK1 (BAK; BCL-2 antagonist/killer) BIRC5 (survivin; baculoviral IAP repeat-containing 5) E2F1 (E2F transcription factor 1; retinoblastoma-associated protein 1) CASP3 (Caspase-3; apoptosis-related cysteine peptidase) CASP8 (Caspase-8; FLICE; apoptosis-related cysteine peptidase) CASP9 (Caspase-9; APAF3; apoptosis-related cysteine peptidase) CASP10 (Caspase-10; FLICE2; apoptosis-related cysteine peptidase) TRAIL (TNF-related apoptosis inducing ligand) DR5 (death receptor 5) SMAC (second mitochondria-derived activator of caspase) Cytochrome c Cell adhesion molecules: CDH1 (E-cadherin) CTNNB1 (β-catenin; cadherin-associated protein) MUC5AC (mucin 5AC; oligometric mucus/gel-forming) Cell-cycle regulators: CCNE1 (cyclin E) CCND2 (G1/S –specific cyclin D2) CDKN2A (p16; INK4A; cyclin-dependent kinase inhibitor 2A) CDKN2B (p15; INK4B; cyclin-dependent kinase inhibitor 2b) CDKN1B (p27; kip1; cyclin-dependent kinase inhibitor 1B) CDKN1A (p21; WAF1/CIP1; cyclin-dependent kinase inhibitor 1A) E2F1 (E2F transcription factor 1; retinoblastoma-associated protein)

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Table 7.2 (continued) Growth factors and cytokines: EGF (epidermal growth factor) FGF (fibroblast growth factor) TNFα (tumor necrosis factor α) TGFB1 (TGFβ; transforming growth factor beta 1) IL1A (interleukin 1, α) IL1F6 (interleukin 1 family member 6, ε) IL1F8 (interleukin 1 family member 8, ξ ) VEGF (vascular endothelial growth factor) PDGF (platelet-derived growth factor) PTGS2 (cyclooxygenase-2 ;COX2; prostaglandin endoperoxide synthase) DNA repair: MYH (MutY human homolog base excision repair) MLH1 (MutL human homolog DNA mismatch repair) MSH2 (MutS homolog 2) MSH6 (mismatch repair ATPase MutS family) TERT (telomerase reverse transcriptase) MicroRNA: (Upregulated miRNAs in non-tumor vs gastric carcinoma indicated by bold letters) miR-21 miR-223 miR-25 miR-17-5p miR-125b miR-181b miR-106a miR-107 miR-92 miR-103 miR-221 miR-83 miR-100 miR-106b miR-21 miR-214 miR-34 miR-16 miR-223 lMIRNLET7 (let7/miR-98; lethal 7) miR-136 miR-218 miR-212 miR-96 miR-339 miR-221

to evasion of apoptosis, cellular multiplication, mobility or adhesion, and matrix remodeling (reviewed by Tahara 2004, Lynch et al. 2005, Smith et al. 2006). Several altered gene-regulatory mechanisms have been demonstrated to play a role in the pathogenesis of gastric cancer. These include intragenic mutations, gene amplification, loss of heterozygosity, microsatellite instability, DNA methylation

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at CpG islands, and chemical modification of the chromatin (DNA and histones). In addition H. pylori-virulence associated genes, Epstein Barr viral genes, and microRNA genes, can influence downstream target gene expression or repression. Interplay between two or more mechanisms is frequently observed in various types of cancers, including gastric cancers. For example, inactivating of tumor suppressor genes requires at least two independent inactivating mechanisms, such as mutations and loss of heterozygosity (homologous deletion) or microsatellite instability and loss of heterozygosity. A novel tumor suppressor gene, RUNX3, is expressed at low levels in gastric cancers due to a combination of loss of heterozygosity and hypermethylation of the RUNX3 promoter region (Li et al. 2002).

Genomic Instability in Gastric Cancer Two major genomic instability pathways involved in the pathogenesis of gastric cancer are chromosomal instability and microsatellite instability. The chromosome instability phenotype is characterized by changes in chromosome copy number (aneuploidy) and alterations in chromosomal regions, including one or more non-random allele losses (loss of heterozygosity), gene deletion and/or amplification (Tahara et al. 1996). In gastric cancer, inactivation of proteins involved in regulation of chromosome segregation, cell-cell adhesion, cell proliferation, apoptosis and control of DNA damage is frequently associated with the chromosomal instability phenotype. Chromosomal instability involving several loci including the loss of 1p, 5q, 7q, 12q, 17p, and 18q has been reported in approximately 60% of gastric carcinomas. There have been attempts to correlate chromosomal instability at various loci with gastric cancer staging and prognosis (Kitayama et al. 2000). In fact, survival is reduced in patients with gastric cancers exhibiting a high level of chromosomal instability (Suzuki et al. 2003). Examples of gastric cancer genes regulated by chromosome instability are tumor suppressor genes, APC, p53 and CDH1. The APC gene is implicated with cell cycle control, chromosome stability and cell migration and adhesion. Loss of heterozygosity at chromosome 5q at the APC locus has been observed in 30% to 40% whereas APC mutations have been observed in 10% of gastric cancer. At the molecular level, APC controls the function of CTNNB1, which is involved in cell-cell adhesion by forming a complex with CDH1 as well as the activation of several signal transduction pathways (Giles et al. 2003). The p53 gene acts as a transcription factor that regulates downstream genes important in cell cycle arrest, DNA repair and apoptosis. Loss of p53 function due to loss of heterozygosity at chromosome 17p (60%) and/or mutations (30% to 50%) are the most frequent genetic defect observed in both diffuse and intestinal subtypes of gastric cancer. Loss of p53 leads to genomic instability, cell cycle deregulation, and inhibition of apoptosis. CDH1 is involved in contact inhibition of cell growth by inducing cell cycle arrest. Functional CDH1-dependent cell adhesion requires the formation of complexes between CDH1 with CTNNB1. The aberrant CDH1/CTNNB1 complex

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occurs in more than 30% of gastric cancers and correlates with loss of differentiation. Loss of heterozygosity at chromosome 16q and gene mutations was observed in 46% of all gastric cancers, but a higher frequency was reported in the diffuse subtype. CDH1 with CTNNB1 are possible markers for increased the risk for diffuse gastric cancer (Smith et al. 2006). Gastric cancer also exhibits genome-wide mutations in two to four repeat sequences that are referred to as microsatellite instability (Loeb 1994). The microsatellite mutator phenotype has been observed in 15% to 39% of all gastric carcinomas (Smith et al. 2006). This phenotype results from malfunction of the mismatch repair genes, caused by epigenetic inactivation of MLH1 (see below). Microsatellite instability of the D1S191 locus is considered a more frequent early event in intestinal gastric cancer than in the diffuse subtype (46% versus 26%, respectively; Smith et al. 2006). The target genes for microsatellite-driven gastric carcinogenesis include TGFB1, BAX, hMSH3 and hMSH6. DNA mismatch repair errors in these genes occur in coding repeats. Consequently frame shift mutations occur in coding sequences and genes produce aberrant proteins.

Epigenetic Control of Gene Expression There are two epigenetic mechanisms that can affect gene expression in gastric carcinogenesis: DNA methylation (Toyota et al. 1999) and histone modifications (Kondo et al. 2003, Meng et al. 2007). Recent advances highlight an intricate web of interaction between epigenetic processes in transcriptional repression of specific genes (Bird and Wolffe 1999). However, the genetic environment may influence transcriptional activity of silenced genes, as discussed next. DNA Methylation Methylation of cytosine residues in DNA has long been recognized as an epigenetic gene silencing mechanism in the gastric pathogenesis. Normal cells exhibit a global methylation with the exception of CpG sites that are generally hypomethylated. In contrast, cancer cells exhibit a global hypomethylation and CpG island hypermethylation. CpG sites are located within the promoters of about 60% of genes (Egger et al. 2004). The shift in the methylation pattern between normal versus cancer cells frequently results in heritable transcriptional silencing of genes, including several tumor suppressor genes, in cancer cells. Gastric cancer-related genes that are inactivated by DNA methylation include p16, p15, CDH1, APC, MLH1, TFF1, PTGS2 and a novel tumor suppressor gene RUNX3. Silencing of cyclin-dependent kinases p15 and p16 by hypermethylation can lead to disruption of the cell cycle whereas inactivating of the mismatch repair MLH1 gene is responsible for the microsatellite phenotype. TFF1, PTGS2 and RUNX3 are involved in several tumor progression mechanisms, including proliferation and apoptosis. Mutations in TFF1 and RUNX3 are rare (Park et al. 2000b, Ito et al. 2005) and thus their functions can potentially be restored in gastric cancer; for this reason, these genes are considered to be good targets for drug discovery.

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Reduction or loss of TFF1 and/or RUNX3 expression has been described in 40% to 65% of gastric cancer subtypes (Machado et al. 1996, Li et al. 2007). In contrast, PTGS2 is overexpressed in the majority (80%) of gastric cancers and its transcriptional silencing by hypermethylation is infrequent (Song et al. 2001). The positive PTGS2 methylation status is a favorable prognostic factor for overall survival of gastric carcinoma patients (de Maat et al. 2007). PTGS2 overexpression can be triggered or enhanced by H. pylori infection and TNFα-mediated nuclear factor (NF)-κ B. The TFF1 gene has an oncogenic potential and can be transcriptionally activated by GAST (gastrin; also called GAS) (Khan et al. 2003), a hormone whose production in enhanced by H-pylori infection. RUNX-3 is a downstream target of TGFB1 signaling (Li et al. 2002). Repression of RUNX3 leads to the reduced sensitivity to TGFB1 and a reduced rate of apoptosis. Similar to TFF1, RUNX3 also has oncogenic potential (Ito 2004). However RUNX3 expression is enhanced by Epstein-Barr virus infection (Ito 2008) via integration of the viral genome into cellular DNA. Histone Modification Post-translational modification of histone N-termini such as acetylation, phosphorylation and methylation play a fundamental role in chromatin structure and transcriptional regulation, and the list of covalent and non-covalent modifications to histone proteins is growing (Goldberg et al. 2007). These modifications are associated with both gene silencing and activation, depending on the nature of the modification and the specific amino acids involved (Egger et al. 2004, Bird and Wolffe 1999, Goldberg et al. 2007). For example, it has been established that acetylation of histone 3 (H3) at lysine 9 (K9), methylation of H3 at lysine 4 (K4) or methylation of H3 at arginine17 (R17) represent an activating step in gene transcription; TFF1/pS2 is an example of a gene that may be activated by R17-modification of H3 (Bauer et al. 2002). In contrast, methylation of H3 at lysine 9 (K9) represents an inactivating step in gene transcription. It has also been proposed that histone methylation (e.g., H3K9) is directed by DNA methylation at CpG islands (so called the “piggyback” model) (Bird and Wolffe 1999, Egger et al. 2004). However, the demonstration that DNA methylation is dependent on histone methylation or histone acetylation is the converse to what the piggyback model proposes. Nevertheless, there is a general agreement that the two epigenetic mechanisms of gene expression regulation, DNA methylation and histone modification, are interdependent. Methylation at lysine 9 of histone 3 is thought to be permanent, because it occurs in the promoter region and no demethylases are known to act on methylated lysines. In contrast, gene silencing by histone acetylation is a reversible process, which can be accomplished by the recruitment of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Histone deacetylation is associated with transcriptional repression and gene silencing, which has subsequent implication to both carcinogenesis and for the discovery of therapeutic agents (Moggs et al. 2004). In the context of gastric carcinogenesis, overexpression of HDAC was found to

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be inversely associated with overall survival of gastric cancer patients (Weichert et al. 2008). The effect of increasing histone acetylation by HDAC inhibitors (HDIs) on de-repression of specific genes is a subject of intense preclinical and clinical research such as Phase I/II studies for advanced solid tumors (Marks et al. 2004, Kelly et al. 2003). Gastric cancer-related genes inactivated by histone acetylation include TERT, CDKN1A and possibly RUNX3 (Richon et al. 2000, Takakura et al. 2001, Mitani et al. 2005). It remains controversial whether p53 function is affected by the histone acetylation status and whether reduced expression of CDKN1A due to histone acetylation is independent of the p53 pathway. As already mentioned, loss of RUNX3 activity is due mainly to homozygous deletion and DNA methylation (Li et al. 2002). More recently, an additional mechanism involving mislocation of the RUNX3 protein to the cytoplasm has been identified in an additional 20% of gastric cancer cases (Ito et al. 2005); thus the two mechanisms account for loss of RUNX3 function in more than 80% gastric cancer cases. The third regulatory mechanism proposed by Huang et al. (2007) may involve RUNX3 silencing by histone acetylation, because a histone deacetylase (HDAC) inhibitor reactivated RUNX3 expression and the protein function. However, it remains unclear how frequent is this phenotype in gastric cancer.

MicroRNA MicroRNAs (miRNAs) genes, unlike other genes involved in cancer, do not encode proteins. Instead, the products of miRNA genes are small noncoding single-strand RNA transcripts that regulate protein expression of specific messenger RNA (mRNA) by annealing to an mRNA containing a nucleotide sequence in the untranslated 3’ region (UTR) that complements the sequence of a miRNA transcript. miRNAs block protein translation when the match between miRNA and mRNA sequences is imperfect, or cause degradation of the mRNA when the match between the two sequences is perfect (Calin and Croce 2006, Esquela-Kerscher and Stack 2006). About 3% of human genes encode for miRNAs, and up to 30% of human protein coding genes may be regulated by miRNAs (Sassen et al. 2008). Expression profiling of miRNA genes has revealed that miRNAs can be upregulated or down-regulated in cancer versus corresponding normal cells. Upregulated miRNA genes can act as oncogenes by down-regulating tumor suppressor genes, whereas the down-regulated miRNA genes can act as tumor-suppressor genes by downregulating oncogenes. Moreover, the function of miRNA genes heavily depends on their gene targets in a specific tissue; thus a miRNA gene can be a tumor suppressor gene, if in a given cell type its critical target is an oncogene and it can be an oncogene if in a different cell type its target is a tumor suppressor gene (Esquela-Kerscher and Stack 2006). An example of miRNA acting as a tumor suppressor gene is provided by let-7/miR-98, which negatively regulates expression of Ras oncogenes (Johnson et al. 2005). A contrasting example of miRNA acting as

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an oncogene is upregulation of c-Myc by six miRNAs from the miRNA-17 family (O’Donnell et al. 2005). The miRNA-17 family is also involved in transcriptional regulation of transcription factor E2F1 (Koscianska et al. 2007). miRNA-106b, miRNA-93 and miRNA-25 regulate E2F1 expression via both positive and negative loop in gastric cancer (Petrocca et al. 2008). Since the discovery of the first miRNA gene (lin-4) in B-cell chronic lymphocytic leukemia (Calin et al. 2002), ∼ 450 miRNAs has been experimentally identified and more than 1,000 miRNAs are predicted by computational methods (Berezikov et al. 2006). Target genes of miRNAs have also been predicted computationally (using algorithms such as the PicTar algorithm) based on sequence analysis of UTRs or promoters of candidate target genes (Sood et al. 2006, Place et al. 2008). However, a direct interaction between the particular miRNA and mRNA of a specific gene has been demonstrated experimentally only in a few cases and in selected solid cancers. In the case of gastric cancer, Zhang et al. (2007) reported significant repression of let-7 (lethal-7) mi/R-98 in human tissue samples from gastric carcinoma patients. Let-7/miR-98 expression is reduced in other tumors (lung, colon and ovary) as well, potentially contributing to elevated activity of the Ras pathway and effects on growth control (Takamizawa et al. 2004, Akao et al. 2006, Yang et al. 2008). A further example of miRNAs deregulated in gastric cancer come from a recent study by Li et al. (2006) who reported that miR-155 targets PRL3 (protein tyrosine phosphatase of regenerating liver 3) whose high expression is associated with lymph node metastasis by enhancing in cell invasion and migration. The significance of the above findings could be that several common miRNAs participate in fundamental signaling participating in many types of tumors. Finally, Kim et al. (2007) reported incorporation of Epstein Barr virus miRNAs into human genome following viral infection, but further work is required to elucidate a role of viral miRNAs in gastric carcinogenesis. A large-scale microRNA microarray analysis of hundreds samples from different solid tumors (breast, colon, lung, pancreas, prostate and stomach) identified miRNA signatures associated with a particular type of cancer (Volinia et al. 2006, Schetter et al. 2008, Lowery et al. 2008). In a recent report, Petrocca et al. (2008) compared alteration in miRNAs in chronic gastritis and gastric adenocarcinoma and found a unique set of miRNAs associated with each condition (Table 7.2). Of special interest is miRNA-25, which regulates expression of the Bim whose protein product critically regulates apoptosis by activating proapoptotic molecules like BAX and BAD and antagonizing antiapoptotic molecules like Bcl-2. Notably, Bim is the most downstream apoptotic effector of the TGFB1 pathway and its downregulation abrogates TGFB1-dependent apoptosis. Other significantly up-regulated miRNAs and target genes involved in gastric carcinogenesis reported to date are: miRNA-21 and miRNA-214 target PTEN (Yang et al. 2008); miR-34 targets p53 (He et al. 2007); miR-1 and miR-16 target (suppress) Bcl-2 (Koscianska et al. 2007); miR-221 and miR-223 (suppress) target KIT. Also, the influence of miR-223 on CDH1 expression (Place et al. 2008), miR-9 on CDH1 expression (Dalmay and Edwards 2006), or miR-139 and miR-200 on CTNNB1 expression has been predicted (Dalmay and Edwards 2006).

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Helicobacter pylori-Infection Associated Genes Gastric adenocarcinoma develops through a multistep process from normal mucosa to chronic active gastritis, to gastric atrophy and intestinal metaplasia, and finally to dysplesia and neoplasia. It is thought that infection with H. pylori plays a causative role in the early phases of this malignant progression by inducing inflammatory, immune and genotoxic responses, including apoptosis, in the gastric mucosa (Siavoshi et al. 2005, Correa and Schneider 2005, Smith et al. 2006). The correlation between H. pylori infection and gastric carcinogenesis is demonstrated by the pathogen-induced activation of PTGS2 at mRNA and protein levels. This induction is a typical event in H. pylori infection with simultaneous production of other endogenous cytokines. Detailed discussion the biochemistry and molecular biology of this enzyme, as well as other upstream and downstream factors controlling prostaglandin synthesis and activity is beyond the scope of this chapter, but several relevant aspects are noteworthy. First, PTGS2 mRNA and protein expressions are higher in gastric cancer tissue than in normal stomach. Second, PTGS2 activation is connected with multiple aspects of carcinogenesis increased invasiveness and decreased adhesion, inhibition of apoptosis, stimulation of proliferation and angiogenesis, and increased DNA damage (by oxygen free radicals) (Hull 2005). Third, there is a growing body of experimental evidence as well as human data that genetic deletion or pharmacological inhibition of PTGS2 inhibits tumorigenesis. For example, in an H. pylori-associated gastric cancer model, long-term administration of the PTGS2 inhibitor (nimesulide) significantly delayed gastric tumorigenesis (Nam et al. 2004). The second aspect of H. pylori infection is the induction of oxidative stress. Oxidative stress is linked to carcinogenesis because it produces DNA-damaging reactive oxygen species (ROS). The most stable product of oxidative DNA, 8-oxo7,8-dihydro 2 deoxyguanosine (8-oxoG), tends to mispair with adenine residues, which leads to DNA mutations. Levels of 8-oxoG are significantly higher in Hpylori-positive gastric mucosa than in negative tissues. The cellular mechanism of protection against oxidative DNA damage is base excision repair (BER). Of three main components of BER, the adenine-specific BER gene MYH whose function is to remove adenine mispaired with 8-oxoG, was found to be defective in 2.1% gastric cancers associated with H. pylori infection (Kim et al. 2004a). Patients with inherited MYH mutations are at a high risk of the development of gastric cancer (Kim et al. 2004a). Several studies have identified TFF1 as a gene associated with H. pylori infection. TFF1 is expressed mainly in stomach mucosa (Balcer-Kubiczek et al. 2002 and Fig. 7.1) and H. pylori infection correlates with a decrease in the number of TFF1 positive cells in humans (Xing et al. 2005, Giraud et al. 2007). Normal functions attributed TFF1 include commitment to differentiation during embryonic development to give rise to functional secreting mucosa and to limit gland proliferation as well as protection against mucosal injury, stabilization of the mucous layer, and acceleration of repair of mucosal damage in the adult gastrointestinal tract.

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Fig. 7.1 Mouse TFF1 (mpS2) mRNA expression following whole body mouse X-irradiation (8 Gy on day 0) (Balcer-Kubiczek et al. 2003, unpublished). Approximately 67% of X ray-induced DNA damage is due to ROS generated by radiolysis of water. Normal high level of its expression in the stomach contributes to protection against mucosal injury by blocking apoptosis and limiting growth (Bossenmeyer-Pouri´e et al. 2002). A model of radiation-induced gastric injury proposes early death by apoptosis and onset of a rapid proliferation in irradiated gastric mucosa (Potten 1992). These events coincide dramatically with reduced expression of mpS2 on day 4, followed by a gradual increase, which coincides with a reduced rate of proliferation. Mouse pS2 or β-actin cDNA probes were generated using the published cDNA sequence data (Ribieras et al. 2001). Mice were sacrificed at time points shown. Lack of mpS2 mRNA expression in colon and intestine is consistent with the literature data (Ribieras et al. 2001). Upper panel: Ethidium iodine-stained membrane is shown to demonstrate RNA quality and loading

It has been proposed that H. pylori infection-induced repression of TFF1 protein is which essential to maintaining mucosal function, repair and proliferation is one of mechanism by which H. pylori might subvert gastric defense (Giraund et al. 2007). The association between TFF1and gastric cancers is derived from the observation that approximately 60% of gastric carcinomas show decreased levels of TFF1 mRNA and protein compared with normal gastric mucosa (Ribieras et al. 1998). The ability of TFF1 to block apoptosis, thereby permitting cells to survive even if they lose cell-cell adhesion (e.g. due to loss of E-cadherin expression) is likely to be beneficial to cancer cells. Mechanisms underlying the loss of TFF1expression include

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allelic loss at TFF1 locus (chromosome 21) in 28% to 50% cases and methylation of the TFF1 promoter. Inactivating mutations are rare and to date found only in Korean patients (Yio et al. 2006).

Epstein-Barr Infection-Associated Genes As discussed in Section “DNA Methylation”, a frequent mechanism of gene inactivation in gastric cancer is epigenetic silencing by DNA methylation, which occurs mainly in CpG dinucleotides by the action of methyltransferase enzymes. It has been shown that Epstein-Barr virus infections correlate with upregulation of DNMT1, which leads in turn to hypermethylation of the CpG islands within multiple genes (Etoh et al. 2004). According to Etoh et al. (2004), no DNMT1 activity was detected in non-cancerous tissue. Epstein-Barr virus-related DNMT1 activity and aberrant CpG methylation was found consistently within the MLH1 and CDH1 promoters (Etoh et al. 2004) as well as the p16 promoter (Chang et al. 2006). Kang et al. (2002) confirmed the high methylation frequency of p16, but also reported a change in the methylation status of genes coding for PTEN, RASSF1A (Ras association RalGDS/AF-6 domain family 1), GSTP1 (glutathione S-transferase π ), MGMT (O-6-methylguanine-DNA methyltransferase and MINT2 (Munc18-1interacting protein 2). Ushiku et al. (2007) examined whether an Epstein-Barr virus infection-associated change in the gene methylation profile is a global or genespecific phenomenon; these authors reported that a loss of p73 expression through aberrant methylation of the p73 promoter is specifically associated with EpsteinBarr virus-associated gastric carcinoma together with the global methylation of p14ARF and p16. Further literature examples of correlations among Epstein-Barr virus-positivity, gastric cancer and DNMT1 overexpression are represented by a loss of gene expression of p16, p53, MADDH4, MADDH7, MUC5AC, ERBB2 (Lee et al. 2004).

Apoptotic Pathways in Gastric Cancer Gastric cancer can be partially attributed to the defects in the regulation of apoptotic cell death. Failure to trigger the cellular suicide program not only predisposes to the development of gastric malignancy, but also increases resistance of cancer cells to anticancer drugs and irradiation. As indicated in the previous sections, numerous genes listed in Table 7.2 may directly or indirectly modulate the process of cell death by apoptosis. Apoptosis can occur in response to a broad range of stimuli such as loss of cell-cell adhesion, oxidative stress, or DNA damage, but ultimately this type of cell death response results in caspase activation, cleavage of caspase substrates, and finally cell demise. The two best-characterized molecular pathways for achieving caspase activation are termed extrinsic and intrinsic.

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Extrinsic Pathway In the extrinsic pathway, apoptosis is triggered by the interaction of death ligands of the tumor necrosis factor (TNF) family, such as Fas/CD95 ligand, and TNF-related apoptosis-inducing ligand TRAIL/ApoL, with their corresponding death receptors, Fas-associated death domain (FADD) (Fas/CD95, DR4 and DR5) and the activation initiator CASP8 and CASP10 (Ashkenazi and Dixit 1998). CASP8 cleaves and activates CASP3 and other downstream effector caspases, which gives rise to the cell death phenotype characterized by DNA fragmentation, chromatin condensation, cell shrinkage, and membrane blebbing (Kerr et al. 1972). In sporadic gastric cancer, CASP3, CASP8 and CASP10 are overexpressed in more than 90% of patients, and weakly expressed in normal gastric mucosa (Yoo et al. 2002), however, less frequent inactivation of CASP8 and CASP10 by inactivating mutations or loss of heterozygosity was also reported in 28% and 10% gastric patients, respectively (Park et al. 2000). In addition to participating in the extrinsic death pathway, TNF has a pro-survival function due to its ability to activate NFKB1, which plays a major role in protecting cells against apoptotic stimuli such as TNF and Fas/CD95. Important to TNF anti- or pro-survival functions is crosstalk between TNF and TGFB1 via MADDH7 signaling (Hong et al. 2007). MADDH7 mediates the inhibitory activity of TGFB1 on TNF-induced NFKB1 activation and the synergistic activity of TGFB1 on TNF-induced apoptosis. MADDH7 is expressed in approximately 33% of the analyzed gastric cancers and has been shown to correlate with poor prognosis (Kim et al. 2004).

Intrinsic Pathway The intrinsic pathway involves release of cytochrome c from the inter-membrane space of mitochondria into the cytoplasm, a process triggered by various stimuli, including elevations in the levels of pro-apoptotic Bcl-2 family proteins such as BAK or BAX, reductions of the levels of anti-apoptotic proteins such as NFKB1 or elevation of ROS and other death signals. In the cytoplasm, cytochrome c interacts with APAF1 (apoptotic protease-activating factor 1) and together with 2 deoxyadenosine 5 -triphosphatase (ATP) form a complex that recruits and activates CASP9, leading to the activation of CASP3 and other downstream caspases, and the death response. The original characterization and subsequent study of the Bcl-2 family members provided an early indication that their function is linked to cancer. The founding member, Bcl-2, was discovered as the defining oncogene in follicular lymphoma (Cleary and Sklar 1985). Subsequently, it has been shown that the Bcl-2 protein is overexpressed in many solid tumors that do not harbor the t(14;18) translocation, such as gastric cancers (Lauwers et al. 1995, Inada et al. 1998). The clinical importance of Bcl-2 is that this anti-apoptotic protein is associated with chemoand radio-resistant states because of its ability to block cell death induced by

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DNA-damaging agents, microtubule-modifying agents, and anti-metabolites. Cells that are doubly deficient in BAX and BAK fail to release cytochrome c and are resistant to all apoptotic stimuli that activate the intrinsic pathway. Chemoresistance and survival of gastric cancer patients have been linked to BAX expression (Deeks and Scott 2007). With further regard to aberrant expression of proteins involved in the intrinsic pathway, CASP9 was observed to be overexpressed in 90% of gastric cancers (Yoo et al. 2002). Yoo et al. (2004) reported that repression of CASP2, CASP6, and CASP7 is decreased compared to normal gastric mucosa in approximately 35% of sporadic gastric cancers. Interestingly, loss of the three caspases in a subset of gastric cancer appeared to occur in the early stage of gastric carcinogenesis, and thus their deficiency might be a candidate marker of gastric malignancy. With regard to other biomarkers, 50% of gastric cancers with the microsatellite instability phenotype carry mutations in the BAX coding region (Ionov et al. 2000). The Bcl-2 protein is expressed in up to 72% of sporadic gastric cancer cases (Lauwers et al. 1995). In sporadic gastric adenocarcinomas (Inada et al. 1998), H. pylori-positive human gastric carcinomas (Konturek et al. 1999, 2001), or Epstein-Barr virus-positive human gastric carcinomas (Kume et al. 1999), a low rate of apoptosis has been reported in Bcl-2 positive gastric cancers, compared to their Bcl-2 negative counterparts as well as BAX positive gastric cancers. Chronic infection with H. pylori is associated with an increase in the circulating concentrations of the gastric hormone GAST. Several experimental studies have concluded that an increased GAST level may contribute to apoptosis indirectly by the inhibition of anti-apoptotic NFKB1 and TFF1, and repressing the transcription of various anti-apoptotic genes (such as TFF1, BIRC5 and Bcl-2) (Khan et al. 2003, Cui et al. 2006).

Other Core Components of Apoptotic Pathways In addition to the proteins that are directly involved in cell death signaling there are many other gastric cancer proteins that contribute to the activation of apoptotic pathways. One important protein family that regulates apoptosis is the family of inhibitor of apoptosis proteins (IAP), which bind to and inhibit caspases and block apoptotic signaling. The best-characterized IAP is BIRC5, a bifunctional protein that regulates both mitosis and apoptosis (Li et al. 1998). The clinical relevance of BIRC5 is that BIRC5 is not present in most normal tissue but is overexpressed in more than 40% of gastric cancers (Meng et al. 2004). Differential expression of BIRC5 suggests a means by which these cells evade apoptosis during neoplastic progression and become resistant to chemotherapy and radiation treatments. Detection of survivin protein is considered a negative predictor of survival in gastric cancer patients (Meng et al. 2004). According to a recent model proposed by Altieri (2008), anti-apoptotic functions of survivin require physical interactions between survivin and other adaptor and cofactor molecules. One interaction involves BIRC4 (XIAP; X-linked inhibitor

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of apoptosis) and SMAC. SMAC (along with cytochrome c) is released from the mitochondria once the cell has received a death signal and binds to XIAP in the BIRC5-XIAP complex, which relieves the inhibitory effect of IAP on CASP3, CASP7 and CASP9 and thus promotes cell death Altieri (2008). The actual physiological relevance of the survivin-SMAC complex in vivo has not been fully established, and other models involving SMAC and IAP have been proposed. For example, Li et al. (2004) suggested that SMAC interacts with XIAP rather than with survivin. This and other studies demonstrated that SMAC-mimic compounds bind to XIAP and promote TRAIL- and TNFα-induced cell death (Li et al. 2004, Bockbrader et al. 2005) suggesting that IAP may partner with proteins controlling the external apoptotic pathway (Altieri 2008). MDM2 and p53 are also important regulators of apoptosis (Haupt et al. 1996). The critical role of p53 in gastric carcinogenesis is indicated by the large number of gastric tumors (∼60%) that harbor inactive p53. After DNA damage, wild-type p53 arrests the cell at G1 /S to allow DNA damage repair. If the damage is irreversible, cell death by apoptosis (or mitotic catastrophe) may be triggered. As a key regulator of the p53 pathway, MDM2 can directly bind to p53 protein and inhibit its activity and degradation (Haupt et al. 1996). On the other hand, p53 can transactivate MDM2 promoter and elevate the expression of MDM2 (Barak et al. 1993). In response to DNA stress, such as DNA damage, p53 is upregulated; however, over-expression of MDM2 may inhibit p53 function, which enables damaged cells to escape cell cycle arrest and become carcinogenic (Barak et al. 1993, Haupt et al. 1996). It has been shown that gastric cancers over-express MDM2 and, in some cases, MDM2 over-expression but not p53 mutation is observed (Yang et al. 2007). Whereas p53 loss of function has been observed in half of all cancers, RUNX3 loss of function appears to be more specifically linked to gastric cancer. Firstly, p53 knockout but RUNX3-positive mice do not develop gastric cancer and secondly, RUNX3 loss is linked to gastric cancer progression, with ∼ 60% of early stage but 90% of late stage gastric tumors exhibit reduced RUNX3 levels (Li et al. 2002). At the molecular level, RUNX3 activity is closely associated with TGFB1 signaling. For example, the gastric mucosa of Runx3 knockout mouse is less sensitive to TGFB1-induced apoptosis and growth arrest (Li et al. 2002). Indeed, loss of RUNX3 tumor suppressor function in cancer cells has a pro-survival effect by promoting proliferative and anti-apoptotic effects. TGFB1 causes growth inhibition by modulating the functions of p15, p27 as well CDKN1A. Chi et al. (2005) has shown that RUNX3 is an essential component of TGFB1-mediated CDKN1A induction. Interestingly, upregulation of miR-103 and miR-96, both of which frequently are overexpressed in gastric cancers, disrupts the TGFB1 pathway and represses the expression of CDKN1A (Petrocca et al. 2008). It has been shown by other investigators that in the absence of CDKN1A, DNA-damaged cells arrest in G2 , undergo additional DNA synthesis without intervening with normal mitosis, and die through an apoptosis-like mechanism (mitotic catastrophe) (Waldman et al. 1996). DNA-damaging or mitotic apparatus-damaging agents that can cause uncoupling of S-phase and mitosis have been pre-clinically and clinically tested for gastric cancer therapy (Balcer-Kubiczek et al. 2006, Deeks and Scott 2007).

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Apoptosis-Targeting in Gastric Cancer As indicated above, the process of apoptosis is controlled on multiple levels, each of which is influenced by different pro- and anti-apoptotic proteins and the system tilts towards cell survival in most of the cell types relevant to the major forms of solid cancer. Many of the proteins involved in apoptosis have redundant functions, and many apoptotic pathways may include signals that are involved in either or both the extrinsic and intrinsic pathways depending on the specifics of how apoptosis pathways are regulated in a given tumor. Thus, given the intricacy of multiple redundant pathways, each balanced against multiple countervailing measures, selective targeting of core components of the cell apoptotic machinery generally proved to be insufficient to induce significant in vivo effects even in genetically simple malignancies and display great efficacy in clinical trials. Fisher and Schultze-Osthoff (2005), Fesik (2005) and Reed (2006) among others reviewed advances in the field of apoptosis-based therapies. In those reviews, the authors also provided comprehensive lists of drugs targeting key players in apoptosis, such as TRAIL, TNF, caspases, IAP, p53, MDM2, NFKB1 and SMAC, with the focus on drugs that could progress from preclinical and early clinical studies to actual clinical application. Although most of these drugs showed anticancer effects in single agent trials, the introduction of a drug into a more general clinical setting proved to be more daunting. A good example of this challenge has been the attempt to nullify the anti-apoptotic effects of Bcl-2 (Reed et al. 1990). Promising phase I/II results of the antisense Bcl-2 molecule oblimersen (also called Genasense or G3139; Genta Inc, Berkeley Heights NJ USA) prompted multiple phase III clinical trials in chronic lymphocytic leukemia, melanoma, multiple myeloma and acute myelogenous leukemia. This trial demonstrated that oblimersen, as a single agent, did not improve survival in any randomized phase III trials (Genta, Inc 2007). In recently reported results, oblimersen in combination with cyclophosphamide and flurarabine (a purine analog, DNA synthesis inhibitor) improved complete response in chronic lymphocytic leukemia patients (16% response in the oblimersen arm versus 7% in the control arm) (O’Brien et al. 2007). In preclinical testing reported by Wacheck et al. (2001), minimally cytotoxic concentrations of oblimersen in combination with cisplatin, significantly enhanced the anti-tumor effect of cisplatin in a gastric cancer model. Taken together, the above studies suggest a novel anticancer therapeutic strategy, namely the use of to apoptosis targeting drugs as chemo- or radiation sensitizers rather than as a single anticancer agent. Indeed, this concept is supported by data from several recent studies that evaluated TRAIL (TNFα-related apoptosis inducing ligand)-based combination therapy for cancer-cell specific sensitization towards TRAIL. For more than a decade, TRAIL has been considered to be a promising anticancer agent due to its ability to induce apoptosis in a variety of tumor cell types while having negligible effects on normal cells (Sheridan et al. 1997). Administration of some truncated and other recombinant versions of TRAIL proved to be toxic to normal tissues in single agent cancer therapy. In addition, the resistance to TRAIL-induced apoptosis is frequent in gastric cancer. TRAIL induces apoptosis

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via the death receptor pathway using mechanism similar to that of TNF (Sheridan et al. 1997) but has also been shown to cross-link with the death receptors DR4 or DR5. Thus, TRAIL resistance may be in part due to low expression levels of DR4 and DR5 in gastric cancer. One emerging approach for TRAIL-therapy is to combine with cytostatic agents that target DR4 or DR5 receptors and are not toxic to normal tissues (Kim et al. 2008, Jung et al. 2006, Kim et al. 2006, Shiraishi et al. 2005) but it remains to be demonstrated whether this promising approach can be translated to clinical gastric cancer treatment. Recently, histone deacetylase inhibitors (discussed just below) have emerged as a potential strategy to sensitize cells to TRAIL-induced apoptosis (Fulda 2008). High expression of HDACs suggests decreased survival of gastric cancer patients (Weichert et al. 2008). Thus, treatment response to TRAIL– based combination therapy with HDIs could be significant in gastric cancer patients overexpressing HDACs. Histone deacetylase inhibitors (HDIs) have potential as new anticancer drugs due to their ability to reverse aberrant epigenetic changes and subsequently to induce differentiation, cell cycle arrest or apoptosis. A growing list of HDIs includes sodium butyrate, trapoxin, trichostatin A, apicidin, suberoylanilide hydroxamic acid (SAHA; vorinostat), depsipeptide and pyroxamide (reviewed by Kouraklis and Theocharis 2006). Several groups provided evidence that SAHA mediates apoptosis via a caspase-independent pathway characterized by the release of cytochrome c and the production of ROS (Ruefli et al. 2003, Henderson et al. 2003, Ungerstedt et al. 2005), but activation of both the intrinsic pathway, involving Bcl-2, BAX, APAF1, CASP3 and CASP6 as well the BNIP and GADD families, and the extrinsic pathway involving the transcription of TNF superfamily genes and genes involved in death-receptor signaling have also been reported (Peart et al. 2005, Fulda 2008). HDI treatment has been found to induce cell death of a broad spectrum of transformed cells, while relatively sparing normal cells (Ungerstedt et al. 2005, Gaymes et al. 2006). The mechanism by which HDI may cause differential effects in cancer versus normal is unclear. However, some results suggest that chromatin changes induced by HDIs can directly activate the DNA damage pathway (Bakkenist and Kastan 2003) or/and that HDIs might induce actual DNA damage that then trigger apoptosis (Gaymes et al. 2006). Gene expression profiling demonstrated that the treatment with HDIs (SAHA and depsipeptide) altered more than 40% of gene transcription within the human genome, which led to a conclusion that HDIs affect multiple molecular pathways as well as multiple genes within the same pathway (Peart et al. 2005). It is possible then that differential effects of HDIs on transformed or normal cells arise from the fact that cancer cells have multiple gene defects in redundant or cooperating pathways that affect survival, compared to normal cells. Overall, the literature data strongly supports the idea of developing clinically relevant combinations with ionizing radiation and other anti-cancer agents. Indeed, clinical trials of HDIs are being considered for gastric cancer (Weichert et al. 2008). As noted earlier, RUNX3 loss of function appears to be specifically linked to gastric cancer (Li et al. 2002, Ito et al. 2005, Chi et al. 2005). Huang et al. (2007) has demonstrated that SAHA induces re-expression of RUNX3 and apoptosis in human

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gastric cancer cell lines by activating proapoptotic Bim, the most downstream apoptotic effector of the TGFB1 pathway. Therefore, re-activation of RUNX3 by HDIs may have the therapeutic effect in gastric cancers characterized by the RUNX3 loss-of-function phenotype and treated with conventional cancer therapies. There is renewed interest in developing small-molecule inhibitors of DNA methyltransferases that can potentially be used as anticancer agents, because of their ability to reverse methylation-mediated silencing of critical genes in cancer (methylation does not change DNA sequence). The first generation of DNA methyltransferase inhibitors such as 5-azacytidine and 5-aza-deoxycytide (decitabine) has shown to be effective against leukemias. In contrast their effectiveness with solid tumors appears to be less promising due to their instability in vivo as well as the toxicity secondary to their excessive incorporation into DNA, which causes cell cycle arrest (Fenaux 2005). Drugs that can facilitate gene activation in cancer cells by enhancing DNMT1 degradation using anti-sense technology are under development (Fenaux 2005). Development of stable drugs that can facilitate gene expression in cancer cells by enhancing degradation of DNA methyltransferases without being incorporated into DNA would be ideal for chemotherapy. MicroRNAs have potential as both diagnostic markers and therapeutic targets in various types of cancers, including gastric cancer (Schetter et al. 2008, Lowery et al. 2008, Yang et al. 2008, Sassen et al. 2008, Petrocca et al. 2008). For example, microRNA microarray profiling of colon adenocarcinoma and paired normal tissues revealed that high expression of miR-21 is associated with poor survival and poor therapeutic outcome (Schetter et al. 2008). Overexpression of miRNA-21 acts as an anti-apoptotic factor in human gastric carcinoma (Petrocca et al. 2008). Thus, inhibiting miR-21 activity may help to inhibit growth of gastric cancers by increasing apoptosis. To date, experimental and clinical evidence supports a substantial role for five miRNAs (miR-21, let-2/miR-98, miR-16, miRNA-214 and miR-25) in suppressing apoptosis as well as in modulating sensitivity/resistance to anti-cancer drugs (Yang et al. 2008, Petrocca et al. 2008, Blower et al. 2008). The aberrant expression of all the five miRNAs was observed in human gastric cancers and suppressing the oncogenic microRNAs (miR-21, miR-16 mir-214 and miR-25) or re-expressing the tumor suppressor let-7/miR-98 could sensitize gastric cancer cells to conventional cancer therapies by overriding intrinsic resistance to apoptosis. Several methods for manipulating miRNA expression levels have been developed. Target oncogenic miRNA downregulation can be achieved by using miRNA inhibitors such as 2-O-methyl antisense single-strand oligonucleotides (Hutv´agner et al. 2004), locked nucleic acid (LNA) oligonucleotides (LNA-antimiRs) (Kr¨utzfeldt et al. 2005, Stenvang et al. 2008, Elm´en et al. 2008), synthetic small interfering RNAs (siRNAs) (John et al. 2007), or a modified version of synthetic siRNAs, interfering nanoparticles (iNOP) (Baigude et al. 2007). The utility of these synthetic molecules in reducing levels of oncogenic miRNAs has been demonstrated in vitro as well as rodent and non-human primate models (John et al. 2007, Elm´en et al. 2008). Conversely, the induction of target tumor suppressor miRNAs can be

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achieved by tissue-specific delivery of miRNA mimetic compounds into a tissue via endogenous routes (Vasudenvan et al. 2007, Yu et al. 2007). Overall results identify miRNA inhibitors (or inducers) as a new class of therapeutics for cancerassociated miRNAs.

Cancer Stem Cells, MicroRNAs and Potential Cancer Therapeutics Most basic cancer research has focused on the molecular analysis of cells comprising cancer tissues. This has led to formulating the traditional multistep carcinogenesis model in which most tumor cells have accumulated multiple genetic and epigenetic alterations, which enable them to proliferate extensively. However, in many tissues in which cancer arises, mature cells have a limited lifespan and thus limited opportunity to accumulate multiple genetic alternations required for transformation and tumor development. Consequently, the probability of a single cell accumulating the necessary alterations is small (Reya et al. 2001). An alternative model of carcinogenesis is the cancer stem cell model, which suggests that many cancers are maintained by rare, slowly dividing “cancer stem cells” (also called “tumor-initiating cells”). In the cancer stem cell model malignant tumors are considered a disease of deregulated self-renewal in which mutations convert normal stem-cell self-renewal pathways into aberrant stem cell self-renewal pathways driving neoplastic proliferation (reviewed by Reya et al. 2001, Pardal et al. 2003). In addition to being the source of the tumor, the cancer stem cells are responsible tumor progression, metastasis, resistance to chemo- and radiation therapy, and subsequent recurrence. The cancer stem cell hypothesis was first documented in human acute myeloid leukemia (Bonnet, Dick 1997). Malignant tumors represent a highly heterogeneous cell population that can be separated on the basis of cell surface marker antigen expression by flow cytometry. Using this experimental approach, cancer stem cells have been identified in human cancers of the breast (Al-Haji et al. 2003), brain (Singh et al. 2004, Galli et al. 2004), prostate (Patrawala et al. 2006), ovary (Szotek et al. 2006), colon (O’Brien et al. 2007, Ricci-Vitiani et al. 2007, Petrocca et al. 2008, and pancreas (Li et al. 2007)). Clearly, much greater insight into the precise role miRNAs play in the survival and maintenance of cancer is needed. Progress in gastric stem cell biology has been hampered by the lack of distinct surface markers and the associated necessity to use indirect approaches for characterization of gastric stem cells (Haraguchi et al. 2006, Qiao et al. 2007). These authors have collectively demonstrated that a rare subpopulation (0.6% to 2%) of gastric cells has selective attributes of cancer stem cells, such as the drug-resistant phenotype and enhanced proliferation. Houghton et al. (2004) showed that bone marrow-derived cells could migrate to sites of irradiation injury or inflammation, engraft into gastric epithelial cells and repopulate gastric mucosa. The demonstration of malignant progression of a bone marrow-derived progenitor cell in the setting of chronic inflammation or acute injury offers the basis for a new model of H. pylori infection-induced gastric cancer as well as radiation-induced gastric injury. The

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observation that bone marrow-derived cells could adopt the phenotype of recipient cells has been demonstrated in other tissues (Harris et al. 2004, Rizvi et al. 2006). The identification of cancer stem cells has potential implications for cancer therapy (Reya et al. 2001). Conventional cancer therapies including radiation and chemotherapy are designed to target primarily rapidly dividing cancer cells and transiently reduces the tumor bulk, while sparing slowly dividing cancer stem cells. Thus, cancer stem cell survival and subsequent regeneration of the tumor from a few surviving cancer stem cells may be one reason for conventional treatment failure (Reya et al. 2001). To date, resistance of cancer stem cells to chemotherapy agents (temozolomide, etoposide, taxol) and radiation therapy has been demonstrated in breast cancer and glioblastoma (Bao et al. 2006, Philips et al. 2006, Piccirillo et al. 2006, Liu et al. 2006, Yu et al. 2007). Cancer stem cells share similarities with normal stem cells, including the ability to self-renew and proliferate. These functions are controlled by concerted actions of extrinsic and intrinsic factors, but only a few factors have tentatively been identified to date. For example, the WNT (frizzled homolog), SHH (sonic hedgehog), Notch (neurogenic locus notch homolog), PTEN and BMI1 (polycomb group ring finger oncogene) have been shown to promote both the self-renewal of somatic stem cells and neoplastic proliferation in the same tissue, when deregulated (reviewed by Reya et al. 2001, Grinstein and Wernet 2007). Several studies demonstrated that normal stem cells are efficiently protected against apoptosis. If cancer stem cells maintain this property of normal stem cells, knowledge of stem cell apoptotic pathways could help to optimize therapies for killing these cells. The ability of a single miRNA to regulate many target genes, and the ability of many miRNAs to regulate a single target gene make miRNA attractive candidates for regulating stem cell renewal and their developmental fate (Hatfield and Ruohola-Baker 2008). Although no studies have been published, to date, describing miRNAs profiles of cancer stem cells versus normal stem cells, there is direct evidence that specific miRNAs such as let-7/miR-98 and miR-21 are important to the establishment and/or progression of various cancers (Yu et al. 2007, Yang et al. 2008, Schetter et al. 2008) stem cells will be required before stem cell/miRNA therapeutic possibility can be fully realized.

Conclusions As in the case of other solid cancers, human gastric cancer is a complex disease resulting from combinatorial interactions among bacterial, viral, environmental, host-genetic and molecular mechanisms. In particular, the remarkable progress has been made towards understanding gastric cancer as a genetic disease. Aberrant genes and protein products have been the basis for modern anticancer drug development. However, most key molecules in gastric cancer are beyond the targeting capabilities of established drug discovery technologies and thus are considered presently “undruggable”. Among the most chemically intractable targets are components of the apoptotic pathways, because their biological function is heavily dependent on protein-protein interactions and the formation of mutiprotein complexes.

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The resistance to apoptosis in cancer cells hinders the success of gastric cancer therapy. Overcoming therapeutic resistance represents a big challenge. Fortunately most regulatory and mediatory components of apoptosis are present in redundant forms. This redundancy has important implications for development novel types of anti-tumor therapy since gastric cancer cells that have lost pro-apoptotic components are likely to retain other components having similar functions. As the basic biology of apoptosis continues to be unraveled, more chemically amenable targets for the development of a new generation of therapeutics, such as microRNAs, are being defined. It is likely that further studies aimed at defining the molecular signature of cancer stem cells, together with the individualized molecular diagnosis will provide considerable treatment advances.

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Chapter 8

Apoptosis and the Tumor Microenvironment in Hematologic Malignancies Danielle N. Yarde and Jianguo Tao

Abstract Hematologic malignancies arise from defects in normal hematopoiesis and are often associated with aberrant expression of growth and survival factors, such as TGF-β, VEGF, bFGF, IL-6, and BAFF. These growth factors have also been shown to be involved in dysregulated apoptosis. Once a hematopoietic cell becomes malignant, these growth factors can be produced by both the tumor cell as well as by cells surrounding the tumor. These surrounding cells, such as bone marrow stromal cells, together with the extracellular matrix, cytokines and growth factors, and blood vessels, comprise the tumor microenvironment. This microenvironment provides a safe haven for the tumor cells to grow, and, following chemotherapeutic treatment, contributes the emergence of minimal residual disease (MRD), where a small number of drug resistant tumor cells survive cytotoxic stress. These drug resistant tumor cells, which often exhibit upregulation of anti-apoptotic pathways, are typically the cause of relapse of hematologic diseases. Thus, targeting these tumor cells along with the tumor microenvironment in which the tumor cells reside is vital in overcoming the devastating effects associated with hematologic malignancies. Keywords Drug resistance · Leukemia · Lymphoma · Multiple myeloma · Tumor microenvironment

Introduction The formation of new blood cells is termed hematopoiesis. Pluripotent hematopoietic stem cells (HSCs) can give rise to both lymphoid and myeloid cells. Lymphoid progenitors, in turn, can give rise to B and T cell lymphocytes, whereas myeloid and erythroid progenitors are responsible for the generation of macrophages,

J. Tao (B) Hematopathology and Laboratory Medicine, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida College of Medicine, 12901 Magnolia Drive, MCC 2071F, Tampa, FL 33612, USA e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 8,

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granulocytes and erythrocytes. Hematopoiesis is a dynamic process involving numerous growth factors, cytokines, and microenvironmental stimuli, and errors along the way involving any of these homeostatic regulators can have detrimental effects, causing malignancies of these different cell lineages. According to the National Cancer Institute (http://www.cancer.gov), incidence and mortality rates of leukemia, the most common hematopoietic malignancy, have shown no significant change over the course of the last 20 years. Furthermore, although advances have been made in the treatment of multiple myeloma and time to relapse has been extended, this disease still remains incurable. In the United States, an estimated five percent of cancers are lymphomas. Unfortunately, although the lymphoma mortality rate has declined due to better treatment options, curative treatment of many subtypes of lymphoma are still being sought. Although profound advances have been made in the treatment of hematologic malignancies, many of these cancers remain incurable due to the effects of minimal residual disease (MRD) and the emergence of drug resistance. Certain cancer cells can elicit mechanisms, such as the evasion of apoptosis, to avoid cell death and persist in the patient even after many rounds of chemotherapy. Unfortunately, many of these patients relapse and eventually succumb to disease due to these remaining, often initially undetectable, tumor cells. Some of these cancer cells are intrinsically resistant to chemotherapeutic agents, while others become resistant to drugs throughout the course of treatment. The evasion of apoptosis in hematologic cancer cells is influenced, if not enhanced, by factors, both soluble and physical, of the tumor microenvironment. The tumor microenvironment of a hematologic malignancy is a source of nutrition and survival factors for the tumor cell, and provides a sanctuary for the tumor cells to prosper, even following chemotherapeutic insult. This microenvironment is comprised of physical determinants, such as bone marrow stromal cells (BMSCs), blood vessels, and extracellular matrix (ECM) components, including fibronectin; and soluble factors, such as growth factors and cytokines (Li and Dalton, 2006). (See Fig. 8.1). The many ways by which the tumor microenvironment bolsters tumor cell survival are becoming increasingly understood, and much research is now dedicated to understanding these influences, and how to overcome them, as a means to combat what may otherwise be an incurable hematopoietic malignancy. This chapter is divided into three sections, which each discuss the intermingled roles of the tumor microenvironment and apoptosis in hematologic malignancies. In the first section, the role of the tumor microenvironment in hematologic tumorigenesis and cancer cell survival is explained. This section highlights one of the main mechanisms by which certain cells become malignant: via evasion of apoptosis. Next, drug resistance due to microenvironmental influences in hematologic malignancies are discussed. The emergence of drug resistance and the role that the tumor microenvironment and cell cycle proteins play in evasion of apoptosis are described. Finally, the last section of this chapter will summarize current treatments of hematologic malignancies aimed at overcoming drug resistance and completely eradicating the cancer, by targeting the tumor cell, the tumor microenvironment, and/or the interactions between the tumor cell and the tumor microenvironment. Each

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Fig. 8.1 Tumor microenvironment of a hematalogic malignancy. The microenvironment of hematopoietic tumor cells is composed of: (1) physical determinants, such as bone marrow stromal cells (BMSCs) and extracellular matrix (ECM) components, such as fibronectin; and (2) soluble determinants, such as growth factors and cytokines, including IL-6, BAFF, VEGF, bFGF, and TGF-β. Components of the micro environment differ depending on tumor type

section focuses on a different regulatory aspect of the tumor microenvironment, with an emphasis on microenvironmental regulations which aid the tumor cell in evading apoptosis.

The Role of the Tumor Microenvironment in Hematologic Tumorigenesis and Tumor Cell Survival In 2000, Hanahan and Weinberg identified six hallmarks of cancer, and suggested that these six alterations within the cell dictate the origination of a malignancy (Hanahan and Weinberg, 2000). These six characteristics are: “self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis” (Hanahan and Weinberg, 2000). Hematologic malignancies exhibit many, if not all, of these traits, and understanding the mechanisms of tumorigenesis is crucial to both diagnosing and finding a cure for these diseases. To this end, much effort has been focused on determining how hematopoietic cells evade apoptosis, a key component of cellular homeostasis, and eventually become malignant. This section focuses on the role of dysregulated apoptosis in the formation of hematologic malignancies by highlighting the involvement of the tumor microenvironment in this process. Numerous growth factors and cytokines, too many to describe in this chapter, are known to play a role in hematologic tumorigenesis, and the survival factors described below provide examples of how environmental cues can support the growth of the tumor cell.

Transforming Growth Factor-β An abundance of growth factors are known to regulate hematopoiesis and, consequently, aberrant growth factor expression, regulation, and signaling have been

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shown to contribute to hematopoietic tumorigenesis. The transforming growth factor-β (TGF-β) family of growth factors is known to play a vital, negativeregulatory role in hematopoiesis. The TGF-β signaling pathway has been shown to regulate the cellular processes of differentiation, motility and adhesion, proliferation and apoptosis (Glasgow and Mishra, 2008). Specifically, TGF-β, an inhibitor of the growth of hematopoietic cells, causes arrest at the G1 phase of the cell cycle (Alexandrow and Moses, 1995), which can in turn induce apoptosis (Dong and Blobe, 2006; MacDonald et al., 1996). Improper expression of TGF-β can, therefore, have detrimental outcomes due to uninhibited cell growth, and this growth factor has indeed been linked to hematologic tumorigenesis. For example, a study analyzing murine plasmacytomas found that these tumor cells exhibited inactivated TGF-β receptors, and so were refractory to the treatment effects of TGF-β when compared to non-transformed plasma cells (Amoroso et al., 1998). Studies of TGFβ in various other hematopoietic malignancies have shown similar results, where loss of TGF-β receptors or receptor mutations, and thus resistance to the growth inhibitory effects of TGF-β, has also been found in chronic lymphocytic leukemia (CLL) (DeCoteau et al., 1997; Lagneaux et al., 1997), non-Hodgkin lymphoma (NHL) (Capocasale et al., 1995), and multiple myeloma (Fernandez et al., 2002). Altered TGF-β signaling due to defects downstream of the TGF-β receptor have also been discovered in a number of hematologic malignancies (Dong and Blobe, 2006), including acute myeloid leukemia (AML) (Imai et al., 2001), acute lymphocytic leukemia (ALL) (Wolfraim et al., 2004), and essential thromobcythemia (Kuroda et al., 2004). There are three mammalian isoforms of TGF-β (TGF-β1, 2, and 3) (Ruscetti et al., 2005). Bone marrow stromal cells (BMSCs), a component of the tumor microenvironment, as well as multiple myeloma (MM) cells, have been shown to produce TGF-β1 (Urashima et al., 1996). This publication also reported that BMSCs from MM patients secrete significantly more TGF-β1 than do BMSCs from diseasefree subjects (Urashima et al., 1996), and BMSCs derived from ALL patients also show enhanced TGF-β1 expression when compared to their normal counterparts (Corazza et al., 2004). These results suggest that these tumor cells may be resistant to the inhibitory effects of TGF-β. Finally, it has been reported that TGF-β1 and –β2 can stimulate the production of such growth factors as IL-6 and VEGF (Hayashi et al., 2004; Kaminska et al., 2005), leading to enhanced tumor cell survival in a microenvironment where TGF-β is overexpressed.

Vascular Endothelial Growth Factor and Fibroblast Growth Factor Two angiogenic growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), have also been implicated in the growth of a number of hematologic malignancies. Although the importance of angiogenesis in the progression of hematopoietic malignancies is not fully understood, these growth factors have been shown to play a crucial role. Bellamy et al. analyzed a number of

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hematopoietic tumor cell lines and found that all of these cell lines expressed VEGF, bFGF, or both, and believe that this expression may be associated with tumor cell growth (Bellamy et al., 1999). Patients with CLL, as characterized by accumulation of B-lymphocytes, typically display elevated levels of bFGF, and these levels correlate with the disease stage and are associated with resistance to the apoptosis-inducing drug fludarabine (Menzel et al., 1996). Non-Hodgkin’s lymphoma (NHL) survival has also been shown to be decreased in patients with elevated VEGF and bFGF levels (Salven et al., 2000), and increased plasma levels of VEGF and bFGF when compared to normal controls have been found in myelodysplastic syndrome and certain leukemias (Zhou et al., 2005). Also, increased angiogenesis in multiple myeloma (MM) patients correlates with prognosis (Bisping et al., 2003). In fact, FGF and VEGF receptors are found to be expressed on MM patient cells, which implies an autocrine loop that may be important for MM cell survival (Podar et al., 2001; Zhou et al., 2005). Additionally, a frequent translocation seen in MM patients is the t(4:14), which involves the FGF receptor (FGFR) 3 locus. This activating translocation has been shown to occur during tumor progression, and to promote transformation by activating the MAP kinase pathway (Chesi et al., 2001). Finally, MM cell survival is further aided by enhanced secretion of IL-6 by the BMSCs, which has been shown to be regulated in part by VEGF (Dankbar et al., 2000). Fragoso et al. analyzed the effects of activation of a VEGF receptor, VEGFR1/FLT-1, on ALL survival (Fragoso et al., 2006). This study revealed that activation of this receptor on ALL cells in vitro resulted in cell proliferation as well as tumor cell migration. Moreover, in vivo neutralization of FLT-1 led to increased leukemic apoptosis (Fragoso et al., 2006), indicating that VEGF plays an important role in tumor cell survival. Both VEGF and bFGF have been shown to influence apoptotic pathways, leading to enhanced tumor cell survival. The Bcl-2/Bax ratio has been found to be increased following activation of VEGF, which leads to a decrease in apoptosis (Dias et al., 2002a). It has been reported that VEGF increases the expression of heat shock protein (Hsp) 90, which results in activation of the mitogen-acitvated protein kinase (MAPK) cascade, and increased expression of Bcl-2 (Dias et al., 2002b). Furthermore, this group infected VEGF receptor positive normal endothelial cells with VEGF and found that these cells displayed enhanced Hsp90 and Bcl-2 expression, which resulted downregulation of apoptosis following both serum starvation and treatment with the Hsp90 inhibitor geldanamycin (Dias et al., 2002b). The anti-apoptotic effects of these growth factors have also been studied in CLL cell lines. For example, bcl-2 mRNA and protein expression in B-CLL cell lines and patient specimens were analyzed following treatment with bFGF (Konig et al., 1997). It was determined that bFGF did indeed upregulate bcl-2 expression, and that this upregulation was likely causative for delayed fludarabine-induced apoptosis. In total, these analyses underscore the importance of two angiogenic factors, VEGF and FGF, in hematopoietic tumor cell survival and evasion of apoptosis. Although the criticality of angiogenesis and angiogenic factors has yet to be fully elucidated for the tumorigenesis and survival of hematologic tumors, it is becoming

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increasingly evident that these factors are vital and may be relevant targets for the treatment of such diseases.

Interleukin-6 Interleukin (IL)-6 is another growth factor that has also been implicated in hematologic carcinogenesis. IL-6 is a pleiotropic cytokine that has been shown to have roles in gene activation, differentiation and proliferation (Cheung and Van Ness, 2002). Increased levels of IL-6 have been found in the supernatants of pediatric ALL patient marrow samples when compared to control samples, and these levels decreased back to normal levels once these patients were in remission (Espinoza-Hernandez et al., 2001). This cytokine has also been shown to have growth inhibitory effects on B cells while promoting growth and survival of MM cells (Cheung and Van Ness, 2002). Moreover, one study found that autocrine production of IL-6 by MM patient mononuclear cells produced a “highly malignant phenotype”, and these IL-6- producing clones were also shown to be more resistant to spontaneous and dexamethasone-induced apoptosis (Frassanito et al., 2001), indicating that IL-6 promotes tumor cell survival via protection from apoptosis. Providing further support of the importance of this cytokine in MM cell survival, treatment of MM cells with anti-IL-6 monoclonal antibodies inhibited MM cell proliferation (Bataille et al., 1995). The cell survival effects of IL-6 have been vastly studied in MM. In the development of normal B cells, B lymphocytic maturation into antibody producing plasma cells is known to be induced by IL-6 (Chen-Kiang, 1995), and these short-lived cells die via apoptosis within a matter of weeks (Cheung and Van Ness, 2002). However, IL-6 acts as a growth factor in MM cell lines and patient specimens (Kawano et al., 1988; Klein et al., 1995), often through inhibition of apoptosis. IL-6 has been found to be produced by both the MM cell itself as well as by the BMSCs (Hata et al., 1993; Kawano et al., 1988; Klein et al., 1989), indicating a positive regulation of MM cell growth and survival by tumor microenvironmental influences. IL-6 secretion by BMSCs induces a number of survival pathways, including the nuclear factor (NF-K B), signal transducers and activators of transcription (STAT) and MAPK pathways. The STAT family of transcription factors, which contains seven members (Ferrajoli et al., 2006), have been shown to regulate the processes of cellular differentiation, proliferation and apoptosis (Bromberg and Darnell, 2000). Catlett-Falcone et al. reported constitutive activation of Stat3 in the IL-6 dependent U266 MM cell line, as well as in MM patient mononuclear cells (Catlett-Falcone et al., 1999). Further, it was determined that the U266 cells, which exhibit resistance to Fas-induced apoptosis, overexpressed anti-apoptotic Bcl-xL , and blocking IL-6 receptor signaling led to attenuated Bcl-xL expression and induction of apoptosis (Catlett-Falcone et al., 1999). This report indicates that IL-6 can prevent apoptosis of MM cells via activation of Stat3 and subsequent upregulation of Bcl-xL , which contributes to overall tumor cell survival. In a different report, using B16 melanoma cells, overexpression of a STAT3 dominant-negative variant was shown to induce

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both cell cycle arrest and apoptosis in the tumor cells (Niu et al., 2001). These reports indicate that tumor cell survival may be abrogated by targeting IL-6 secretion as well as by targeting the signaling pathways induced by this cytokine. In addition to activation of survival pathways, IL-6 is also known to influence the production of growth and survival factors. As mentioned above, TGF-β, VEGF, and bFGF have all been shown to induce IL-6 production and secretion. On the other hand, IL-6 has also been found to increase bFGF and VEGF production by MM cells (Bisping et al., 2006; Bisping et al., 2003; Dankbar et al., 2000). Additionally, MM cells can induce secretion of IL-6 by BMSCs following direct adhesion of the MM cells with the BMSCs (Chauhan et al., 1996). Thus, interactions of the tumor and the tumor microenvironment are crucial for MM cell survival, as communication between the two has a profound effect on the production of growth and survival factors and the subsequent upregulation of tumor cell survival pathways.

B Cell Activating-Factor of the Tumor Necrosis Factor Family B cell activating-factor of the tumor necrosis factor family (BAFF) protein is known to specifically target B lymphocytes, promote B cell proliferation, activation, and differentiation, enhance B lymphocyte survival, and thereby stimulate immunoglobulin production both in vitro and in vivo (Do et al., 2000). In normal mice receiving injections of BAFF, lymphoid compartments in the spleen undergo marked expansion, and plasma levels of immunoglobulins increase significantly (Parry et al., 2001). The role of BAFF in B cell homeostasis became evident from studies in BAFF null (Gross et al., 2001; Schiemann et al., 2001) and BAFF transgenic mice (Gross et al., 2000; Khare et al., 2000; Mackay et al., 1999). Mice lacking BAFF have extreme reductions of mature B cells in peripheral blood, whereas mice overexpressing BAFF have increased numbers of mature splenic and lymph node B cells, elevated immunoglobulin levels, and manifestations of autoimmune disease (Gross et al., 2000). The mechanism whereby BAFF exerts these effects is by improving survival of peripheral B cells (Mercurio et al., 1993; Naumann et al., 1993), which is thought to allow escape of B cells from cytotoxic apoptosis, leading to autoimmune diseases. Taken together, these findings have led to the proposal that BAFF plays a critical role in maintaining B cell homeostasis, with insufficient signaling by BAFF resulting in B cell deficiency and excessive signaling causing B cell disorders (Liou et al., 1992). For example, He et al. report that lymphoma B cells evade apoptosis through upregulation of BAFF (He et al., 2004). The role of BAFF in B lymphoma cell survival and drug resistance has been further substantiated by recent emerging reports on CLL and MM. Kern et al. demonstrated that addition of soluble BAFF or APRIL protected B-CLL cells against spontaneous and drug-induced apoptosis and conversely, addition of anti-BAFF and anti-APRIL antibodies enhanced B-CLL cell apoptosis (Kern et al., 2004). Most recently, Moreaux and colleagues provided evidence that BAFF was involved in the survival of primary MM cells and protected MM cells from dexamethasone-induced apoptosis (Moreaux et al., 2004). Taken together, these results indicate that B-CLL

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and MM cells can be rescued from apoptosis through a process involving BAFF and its receptors. The origin of BAFF remains undefined, even though an autocrine interaction can account for a subset of CLL. Although the majority of lymphoma cells exhibit characteristics consistent with prolonged cell survival in vivo, when cultured in vitro lymphoma cells often undergo spontaneous apoptosis. This observation suggests that autocrine regulation of BAFF is not sufficient to maintain hematopoietic cell survival. However, BAFF has been detected on BMSCs derived from MM patients, and secretion of BAFF was enhanced by adhesion of the tumor cell to the BMSC (Tai et al., 2006). This group also reported that addition of BAFF enhanced tumorBMSC adhesion, further implicating the involvement of this factor in mediating interactions between the tumor and its microenvironment. BAFF has been shown to activate the NF-K B signaling pathway and, consequently, NF-K B activation can lead to upregulation of BAFF (Tai et al., 2006). The transcription factor NF-κB is widely recognized as a critical mediator of immune and inflammatory responses. In most cell types, NF-κB is found in the cytoplasm where it is associated with an inhibitory protein known as IκB. After activation by a large number of inducers, the IκB proteins become phosphorylated, ubiquitylated and, subsequently, degraded by the proteasome. The degradation of IκB allows NFκB proteins to translocate to the nucleus and bind their cognate DNA binding sites to regulate the transcription of a large number of genes, including antimicrobial peptides, cytokines, chemokines, stress-response proteins and anti-apoptotic proteins. NF-κB has attracted attention because of both its unique activation pathways and its physiological importance as a key regulatory molecule in the immune response, cell proliferation and cell survival during stress. NF-κB protects cells from apoptosis by promoting expression of survival factors, such as members of the inhibitor of apoptosis (IAP) family (c-IAP-1, c-IAP-2, XIAP) and the Bcl-2 homologs (Bfl-1/A1 and Bcl-XL ). In a signaling cascade that involves activation of IKK, p100 and p105 can be phosphorylated and partially cleaved to yield the product proteins p52 and p50, respectively (Heissmeyer et al., 1999; Salmeron et al., 2001; Xiao et al., 2001). This allows nuclear translocation of p50 and p52 and binding to target DNA sequences. This pathway has been called the “alternative” or “non-canonical” NF-κB signaling pathway, with a bias towards differentiation, architecture and proliferation within the B cell compartment. BAFF are among few cytokines that are known induce non-canonical NF-K B activation (Hatada et al., 2003; Schiemann et al., 2001). As a consequence of BAFF-mediated NF-κB activation, antiapoptotic genes encoding Bcl-2, Bcl-xL and Bfl-1/A1 are activated, leading to enhanced cell survival (Do et al., 2000).

The Role of the Tumor Microenvironment in Drug Resistance The treatment of a hematologic malignancy often involves chemotherapy, radiation, or both. Also, many treatment regimens call for combination therapy, where more than one chemotherapeutic drugs are used as a means of trying to completely

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eradicate the cancer by targeting multiple mechanisms that contribute to tumor cell survival. Although patients with hematologic malignancies typically respond to initial therapy, many relapse and often cannot be cured of their disease. This relapse is mainly due to minimal residual disease (MRD), where a small population of cancer cells is able to withstand the chemotherapeutic insult. Tumor cells can be resistant to drug by means of intrinsic resistance, de novo resistance due to extrinsic factors, or acquired resistance. Many molecular changes are seen when a hematopoietic cell turns malignant, and some of these modifications and the affected signaling pathways are known to contribute not only to carcinogenesis but also to intrinsic drug resistance. For example, FLIP (Fas-associated death domain-like interleukin-1β-converting enzyme inhibitory protein) proteins prevent the effective activation of procaspase-8 and procaspase10 by forming complexes with these proteins (Reed and Pellecchia, 2005), and therefore inhibit apoptosis. FLIP overexpression has been shown to be imperative for tumor cell survival in a number of hematologic malignancies, including Burkitt’s lymphomas (Valnet-Rabier et al., 2005) and KSHV-associated lymphoma cells (Guasparri et al., 2004), and overexpression of FLIP has also been linked to intrinsic drug resistance to Fas and TRAIL (Reed and Pellecchia, 2005). Aside from being intrinsically resistant to chemotherapeutic drugs, tumor cells may also be able to survive initial cytotoxic insult by means of de novo resistance associated with a tumor cell’s interactions with its surrounding microenvironment. Once a cell survives initial chemotherapeutic treatment, it may then develop mutations that allow it to become resistant to the drug(s) being used, a phenomenon known as acquired drug resistance. For example, low-grade follicular lymphomas regress after treatment, but relapse due to MRD occurs with a drug resistant tumor at the same site (Horning, 1994), indicating that the microenvironment of the tumor acts as a sanctuary to promote tumor cell survival and drug resistance. The remainder of this section of the chapter will focus on the contribution of the tumor microenvironment to both de novo and acquired drug resistance, which typically occur via deregulation of apoptotic pathways.

De Novo Drug Resistance The microenvironment to which the tumor cells home provides a safe haven for tumor cells by supplying nutrients necessary for cell survival. This microenvironment, composed of both soluble factors, such as IL-6 and VEGF, and physical effectors, such as the extracellular matrix, bone marrow stromal cells, as well as other tumor cells, is also known to play a large role in MRD, by helping the cells to withstand the stress of cytotoxic insult. In 1972, Durand and Sutherland reported that single cells were less likely to survive damage due to radiation than were cells grown as a spheroid (Durand and Sutherland, 1972). They reported that cells that were grown in contact with one another were better able to repair radiation damage than those that were not grown as a spheroid, and repair capacity was also increased when these cells were irradiated

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while maintaining cellular contact (Durand and Sutherland, 1972). This work indicates that cells can impart essential signals to surrounding cells, signals that can enhance cell survival. In a later publication by Teicher et al. (1990), EMT-6 murine mammary tumors were made resistant to drugs in vivo, by treating the tumor-bearing mice with drugs commonly used as therapies these tumors. Interestingly, it was discovered that following six months treatment, high levels of drug resistance were seen in vivo, but these tumor cells were not resistant to the drugs in vitro. These results show that the environment in which these cells live is a crucial determinant of cellular survival and drug resistance. To address the involvement of the tumor microenvironment in tumor cell survival and drug resistance, much research is now focused not only on analyzing the tumor cell by itself, but also analyzing the tumor cell in the context of different components of what would be its in vivo environment. For example, Damiano et al. analyzed a multiple myeloma (MM) cell line, namely 8226, in the presence of the extracellular matrix component fibronectin (FN) (Damiano et al., 1999). This work demonstrated that MM cells adhered to FN via α4 β1 and α5 β1 integrins were more resistant to apoptosis following exposure to melphalan or doxorubicin than were cells grown in suspension. This group coined the term cell adhesion mediated drug resistance (CAM-DR), which emphasizes the fact that the cells must be physically adhered to the FN in order for the drug resistance phenotype to occur. Although it had previously been reported in that adhesion of B-CLL and Chinese hamster ovary cells to FN viaα4 β1 or α5 β1 integrins, respectively, supported cell survival through up-regulation of the anti-apoptotic bcl-2 protein (de la Fuente et al., 1999; Matter and Ruoslahti, 2001; Zhang et al., 1995), the mechanism of CAM-DR in the case of the 8226 cells was determined not to be due to altered expression of bcl-2 family proteins. It was later found that CAM-DR in the 8226 cell line was associated with increased levels of p27kip1 protein (Hazlehurst et al., 2000). This protein caused a G1 phase cell cycle arrest, and once cells were detached from FN, p27kip1 levels were rapidly reduced, cells entered S phase, and the CAM-DR phenotype was reversed. Furthermore, treatment of these cells with antisense p27kip1 attenuated drug resistance as measured by apoptosis, implicating this protein, a cell cycle regulatory protein, in the anti-apoptotic effects of CAM-DR (Hazlehurst et al., 2000). Lwin and colleagues have also shown that adhesion of non-Hodgkin’s lymphoma cell lines to bone marrow stromal cells (BMSCs), another component of the tumor microenvironment, resulted in a G1 cell cycle arrest that was associated with elevated p27Kip1 and p21 protein levels (Lwin et al., 2007a). And, as a final point, the clinical relevance of the CAM-DR phenotype has also been confirmed, in a report showing that adhesion of primary MM patient specimens to FN attenuated the percentage of apoptotic cells and thus conferred resistance to melphalan (Hazlehurst et al., 2003a). CAM-DR due to FN adhesion has also been found to be relevant in other hematopoietic tumors as well. Following adhesion of the histiocytic lymphoma U937 cell line to FN, enhanced resistance to the topoisomerase II inhibitor mitoxantrone was seen (Hazlehurst et al., 2001). Adhesion to FN also enhanced U937 cell survival following exposure to Fas, and inhibition of apoptosis in this case was

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determined to be due to a redistribution of c-FLIPL to the cytosol (Shain et al., 2002). Finally, the chronic myelogenous leukemia K562 cell line was also found to be resistant to melphalan and BCR-ABL inhibitors upon adhesion to FN (Damiano et al., 2001). A subsequent paper reported that adhesion of K562 cells to FN via β1 integrins enhanced proteasomal degradation of Bim, a pro-apoptotic bcl-2 family member, and reduction of Bim levels contributed to de novo drug resistance in these cells (Hazlehurst et al., 2007). Taken together, these works indicate that components of the tumor microenvironment augment tumor cell survival and drug resistance by enhancing anti-apoptotic signaling, and thus should be taken into consideration when studying and attempting to overcome drug resistance. Furthermore, the mechanisms of de novo resistance are likely dependent on the cancer type, as mechanisms found to be causative for CAM-DR in one tumor type are not necessarily causative for resistance in others. The FN model of CAM-DR has been widely used to elucidate mechanisms of de novo drug resistance. However, this model does not take into account the effects of soluble factors produced in the microenvironment by cells surrounding the tumor cells. To this end, the bone marrow stromal model is now commonly used to study mechanisms of CAM-DR, or, more broadly, environment mediated drug resistance (EM-DR), as bone marrow stromal cells (BMSCs) influence tumor cell survival not only through BMSC/tumor cell adhesion but also through the production of soluble factors. Tumor cells are co-cultured with BMSCs, and the effects that the combination of both soluble factors and cell-cell contact have on microenvironmentmediated drug resistance can be analyzed. As a means to decipher mechanisms involved in EM-DR, the transwell system was created; this system allows for analysis of the role of soluble factors, adhesion, or both, in this de novo resistance. In this system, the BMSCs are plated and the tumor cells can be adhered to these cells as a means to study the tumor microenvironmental effects as a whole, taking into consideration influences due to both adhesion and soluble factors. Alternatively, the tumor cells can be separated from the BMSCs by a thin, microporous membrane that allows for the free-flow of soluble factors, produced by both the BMSCs and the tumor cells, throughout the well, as a means to study only the impact of soluble factors on tumor cell survival without the added component of cellular adhesion. (See Fig. 8.2). The BMSC system has been utilized to study the impact that these cells have on the survival of a variety of hematological cancer types. For instance, chronic lymphocytic leukemia (CLL) cells were shown to be protected from spontaneous apoptosis via contact with normal BMSCs, a protection that was not extended to normal B cells under the same conditions (Lagneaux et al., 1998). The anti-apoptotic phenotype observed in the CLL cells was associated with bcl-2 expession. Additionally, using both the transwell system and stromal cell conditioned media, it was observed that direct contact with the BMSCs was necessary to attenuate spontaneous apoptosis (Lagneaux et al., 1998). Survival following chemotherapy of acute lymphoblastic leukemia (ALL) cells in the presence of BMSCs has been analyzed in acute lymphoblastic leukemia cells (Mudry et al., 2000). ALL cells alone or cocultured with BMSCs were treated with

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Fig. 8.2 Research models utilized to study tumor microenvironmental effects. (A) Fibronectin (FN) model. Tumor cells are adhered to plates coated with FN. (B) Co-culture model. Tumor cells are co-cultured with bone marrow stromal cells (BMSCs), and the concurrent effects of soluble factors and direct contact between the two cells are studied. (C) Transwell model. BMSCs and tumor cells are separated by a microporous membrane to study of the effect of soluble factor production on the tumor cells, without the component of direct adhesion

cytarabine (Ara-C) or etoposide (VP-16). Following drug treatment, a reduction in apoptosis was observed in leukemic cells cocultured with BMSCs compared to cells exposed in the absence of BMSCs. Furthermore, it was reported that adhesion to the stroma was necessary for protection from apoptosis, as leukemic cells treated with conditioned media, which contains soluble factors provided by the BMSCs, did not exhibit attenuated apoptosis (Mudry et al., 2000). A subsequent study by this group revealed that the BMSCs protected the ALL cells from apoptosis induced by Ara-C and VP-16 by regulating the caspase-3 activity of the leukemia cells (Fortney et al., 2001). In another study, this time analyzing a panel of acute myeloid leukemia (AML) patient specimens, it was also observed that direct adhesion of the tumor cells to BMSCs inhibited chemotherapy-induced apoptosis of the AML cells. Interestingly, bcl-2 protein expression in the AML cells was not consistently linked to the anti-apoptotic effect observed (Garrido et al., 2001), suggesting that the mechanisms of CAM-DR may vary from patient to patient. Finally, using the transwell system, this group also found that the mere presence of BMSCs could inhibit leukemic cell apoptosis as well, but to a much lesser degree than conferred by direct contact with the BMSCs (Garrido et al., 2001). Analysis of bone marrow-tumor cell interactions has also revealed that the tumor cells themselves can affect the microenvironment in which they reside, often leading to a microenvironment that is even more conducive to tumor cell survival. Viega et al. reported that ALL cells can stimulate bone marrow endothelium and promote angiogenesis in the bone marrow surrounding the leukemic cells (Veiga et al., 2006). In turn the bone marrow endothelium then supported tumor cell survival via regulation of anti-apoptotic bcl-2. In addition to bcl-2, Notch signaling has also been implicated in hematologic tumor cell survival and de novo resistance associated with BMSCs. Notch family

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members and their associated targets have been found to be overexpressed in various hematologic malignancies (Hubmann et al., 2002; Jundt et al., 2002; Tohda and Nara, 2001), including in T cell acute lymphoblastic leukemia, where NOTCH1 activating mutations were found in more than half of the cells analyzed (Weng et al., 2004). Notch1 is a transmembrane receptor expressed on hematopoietic cells. The intracellular domain of Notch1 is release upon ligand binding, and this intracellular region of Notch1 (IRN1) then translocates to the nucleus and regulates gene transcription (Gery and Koeffler, 2007). Notch signaling in MM cells was analyzed by Nefedova et al. (2004). This group found that adhesion to BMSCs activated Notch signaling. Furthermore, activation of Notch-1 due to BMSC adhesion was found to be involved in protection of the MM cells from apoptosis induced by melphalan and mitoxantrone, a protection that was also associated with enhanced regulation of the cell cycle protein p21WAF/Cip (Nefedova et al., 2004). As a final example of how the tumor microenvironment influences de novo drug resistance, it was recently reported that adhesion of the SUDH-4 and SUDH-10 cell lines, two B-cell lymphoma cell lines, to BMSCs protected the lymphoma cells from mitoxantrone-induced apoptosis, and this protection was associated with activation of the NF-K B (RelB/p52) pathway (Lwin et al., 2007b). The anti-apoptotic molecules XIAP, cIAP1 and cIAP2, which are known to be regulated by NFK B, were also found to be up-regulated following adhesion to BMSCs (Lwin et al., 2007b). In total, this section of de novo drug resistance has provided examples of some of the many mechanisms associated with this form of drug resistance. Many different models can be used to study the effects of the tumor microenvironment on tumor cell survival and drug resistance. Although these models are not equivalent to the true microenvironment of a tumor cell in a cancer patient, studies such as these are likely more accurate in recapitulating signaling pathways than looking at just the cancer cell by itself. Hopefully, these models provide enough insight into mechanisms actually used by the tumor cell in a patient with a hematopoietic malignancy to provide more targeted therapy and more effective treatments.

Acquired Drug Resistance If a tumor cell is able to survive the initial stress induced by chemotherapeutic drugs, by means of de novo drug resistance via tumor microenvironmental interactions, it may eventually develop acquired resistance to the drug. This acquired drug resistance is often the cause of treatment failure in hematologic malignancies, as these cancer cells can no longer be eliminated by way of standard therapy. To study acquired drug resistance mechanisms, unicellular models are commonly utilized, where a tumor cell line is treated with a particular drug over a period of time until the emergence of drug resistance. The cancer cell type and the selective pressure used both influence the mechanism of acquired drug resistance (Dalton, 2003). Known mechanisms of acquired drug resistance include: modifying the target of the drug, through overexpression of the target or point mutations; reducing drug

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concentrations within the cell by decreasing drug uptake or increasing drug efflux; enhancing the efficiency of drug metabolism; enhancing DNA repair; and inhibiting apoptosis by activating anti-apoptotic pathways while decreasing pro-apoptotic factors (Hazlehurst et al., 2003b; Li and Dalton, 2006). Furthermore, acquisition of drug resistance by a tumor cell may involve more than one of these mechanisms to avoid being killed by a cytotoxic agent. The 8226/MR4 MM cell line, for instance, was selected for resistance to mitoxantrone, and acquired resistance in this cell line was associated with decreased intracellular drug levels via an efflux pump, as well as redistribution and reduction of enzymatic activity of topoisomerase II (the target of mitoxantrone) (Dalton, 2003; Hazlehurst et al., 1999). In an effort to compare mechanisms associated with acquired drug resistance to those associated with de novo resistance, Hazlehurst et al. analyzed melphalan resistance in the 8226 MM cell line (Hazlehurst et al., 2003a). In this study, the characteristics of 8226 cells adhered to FN were compared to those of the 8226/LR5 cell line. The 8226/LR5 cell line was selected for resistance to melphalan by continued exposure of these cells to this drug for a period of 47 weeks, and thus represents a tumor cell line with acquired melphalan resistance (Bellamy et al., 1991). Cells adhered to FN and 8226/LR5 cells both display reduced levels of apoptosis following exposure to melphalan when compared to the parental 8226 line in suspension. To compare de novo resistance to acquired resistance, oligonucleotide microarray analysis was performed, comparing the parental 8226 cell line to the 8226/LR5 cells or to 8226 cells adhered to FN. Changes in 1479 genes were observed in the 8226/LR5 cell line when compared to the parental 8226 line; 69 changes were found when comparing the parent to FN-adhered cells; and only 21 of the gene changes observed in both comparisons overlapped (Hazlehurst et al., 2003a). These results suggest that the mechanisms of de novo and acquired drug resistance are diverse, and therefore should both be taken into account when attempting to determine resistance mechanisms operative in vivo. Although the work described above defined mechanisms of both de novo and acquired drug resistance, the influence of the tumor microenvronment in the acquisition of drug resistance was not addressed in this study. However, in another study, the role that the tumor microenvironment plays in acquired drug resistance was analyzed (Hazlehurst et al., 2006). In this study, U937 histiocytic lymphoma cell lines were selected for resistance to the topoisomerase II inhibitor mitoxantrone. These cells were selected for resistance both unicellularly (U937 cells in suspension) and while adhered to FN. Thus, the influence of CAM-DR on acquisition of drug resistance was studied. Interestingly, this group found that following acquisition of drug resistance, the FN-adhered cells displayed greater than 2-fold mitoxantrone resistance levels when compared to their suspension counterpart. To measure levels of drug resistance, MTT assays were performed to determine the mean IC50 for each cell line. Importantly, the drug resistance levels were determined by analyzing cells in suspension, so the cells that were selected for resistance on FN were first detached then analyzed. Therefore, the 2- to 3- fold difference seen in resistance between the suspension-selected and FN-selected cells was due to acquired drug resistance, without including the effects of de novo resistance; in other words, adhesion itself

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did not cause drug resistance, but FN-adhesion during selection of cells caused an increase in drug resistance. These results underscore the importance of considering the tumor microenvironment when studying drug resistance in hematologic malignancies. Hazlehurst et al. next wanted to determine the mechanism(s) by which these tumor cells, selected for resistance to mitoxantrone alone or in the presence of the extracellular matrix component FN, acquired drug resistance. It was reported that although drug resistance in both cases was associated with attenuated topoisomerase II activity and a decrease in DNA damage induced by drug, cells selected for resistance in suspension and cells selected while adhered to FN regulated this activity and damage utilizing different mechanisms (Hazlehurst et al., 2006). Specificlly, it was determined that the mitoxantrone resistant U937 cells selected for resistance while in suspension exhibited decreased topoisomerase IIβ RNA and protein expression, which was linked with reduced expression of a transcription factor, NF-YA, known to regulate topoisomerase IIβ expression. On the other hand, when the CAM-DR model was used to select for selection, decreases in topoisomerase IIβ protein levels were associated with resistance, but no change in topoisomerase IIβ RNA expression or NF-YA levels was observed (Hazlehurst et al., 2006). Taken together, this work and the work of others who have studied the role of the tumor microenvironment in drug resistance emphasizes the importance of considering tumor microenvironmental interactions when studying methods of enhanced cell survival. Taking into account the influences of the tumor microenvironment when studying mechanisms of cell survival and resistance to chemotherapeutics will be of benefit by more realistically recapitulating the setting of the tumor cell in the cancer patient. Hopefully, better understanding of the mechanisms associated with drug resistance will ultimately lead to better treatment options through targeted therapy, and even cures of hematopoietic malignancies.

Overcoming Drug Resistance in Hematologic Malignancies The influence that the tumor microenvironment has on tumor cell survival, as well as the changes in the microenvironment that can be induced by the tumor, should now be evident from the first two sections of this chapter. Researchers and clinicians alike now understand that it in order to successfully treat most hematologic malignancies, it is crucial to target not only the tumor cell, but also the microenvironment in which the tumor resides, by targeting both soluble factor production as well as direct interactions between the tumor cell and components of its microenvironment. This next section focuses on chemotherapeutics aimed at eradicating hematopoietic malignancies. Certain drug in use today directly target the tumor cell by altering pathways activated in the tumor cell by the microenvironment, while others have been shown to induce cytotoxic effects by influencing interactions between the tumor and the microenvironment. Another example of chemotherapy being used today involves targeting the microenvironment itself as a means to decrease tumor cell survival. As an example of why targeting the microenvironment itself may be important,

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Fig. 8.3 Chemotherapy targeting the tumor cell and its microenvironment. The treatments discussed in this chapter and their known targets are summarized. Chemotherapeutic treatment of hematologic malignancies can target both the tumor cell itself, soluble factors found in the tumor microenvironment, and/or tumor microenvironmental interactions

Moshaver and colleagues found that treatment of BMSCs with the chemotherapeutic agent cytarabine greatly reduced the ability of these cells to protect AML cells from spontaneous and cytarabine-induced cell death (Moshaver et al., 2008). Importantly, many chemotherapeutic regimens in effect today for the treatment of hematopoietic malignancies often involve combinations of drugs, as a means to target various networks of pathways that are deregulated and lead to tumor cell survival. For example, using a combination of two drugs, one of which induces apoptosis and the other which does not allow for cellular adhesion to tumor microenvironmental components, may ultimately be more efficacious in eliminating MRD and thus eradicating the cancer than treating a patient with only a single agent. The next few examples provide sound evidence for targeting deregulated pathways in the tumor cells as well as tumor microenvironmental influences as a means to overcome drug resistance and eliminate the tumor cells. (See Fig. 8.3).

Chemotherapy Targeting Bcl-2 As explained in the first two sections above, a hematopoietic tumor cell is formed and able to survive chemotherapeutic insult largely in part due to deregulation of apoptotic pathways. The bcl-2 family members are commonly aberrantly expressed in hematologic malignancies. For instance, Bcl-2 protein is commonly overexpressed in diffuse large B-cell lymphoma, CLL, MM and acute leukemias (Klener et al., 2006; Monni et al., 1997). Furthermore, the ratio of bax to bcl-2 has been

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shown to predict clinical outcome of AML patients (Del Poeta et al., 2003), and bcl-2 expression is upregulated in MM cells following treatment with various cytotoxics (Tu et al., 1996). Also, elevated expression of the pro-apoptotic bcl-2 family member bax in newly diagnosed AML patients has been found to be a good prognostic indicator (Ong et al., 2000). These studies, taken together with those above that implicate the apoptotic pathways in enhanced tumor cell survival, provide reason to target bcl-2 as a means to eliminate hematologic tumors. Antisense oligonucleotides targeting bcl-2 mRNA are currently undergoing laboratory as well as clinical studies. For example, oblimesen sodium (G3139, Genasense), which binds to the first six codons of Bcl-2 mRNA (Cotter et al., 1994), has been widely studied. Oblimesen has been shown to enhance the cytotoxic effects of a variety of chemotherapeutic agents (Kim et al., 2004), and this enhancement has been shown pre-clinically in hematologic tumor types such as NHL and EBV-associated lymphoproliferative disease (Guinness et al., 2000; Klasa et al., 2000). Also, Phase III clinical trials are currently underway using oblimersen for the treatment of CLL, which has shown promising results in combination with other chemotherapeutic agents (O’Brien et al., 2007). In addition to bcl-2 antisense oligonucleotides, other agents have also been shown to target bcl-2 activity and therefore induce apoptosis. For example, histone deacetylases (HDACs) inhibitors have been reported to attenuate BCL2 expression in leukemia cell lines as well as in primary myeloma and leukemia specimens (Khan et al., 2004; Mori et al., 2004a; Reed and Pellecchia, 2005; Rosato et al., 2003). Another inhibitor of bcl-2 and bcl-2 family proteins is the small molecule inhibitor ABT-737. This molecule binds to bcl-2 and bcl-xL, and has been shown to induce apoptosis in MM cell lines (Kline et al., 2007). Furthermore, this group also reported that addition of VEGF and IL-6 could not overcome the apoptotic effects of ABT737 (Kline et al., 2007), suggesting that this molecule may also be effective in overcoming tumor microenvironmental influences.

Targeting Soluble Factors and Adhesion Molecules Soluble factors such as TGF-β, VEGF, bFGF, IL-6 and BAFF, are known to influence and enhance hematologic tumorigenesis and tumor cell survival. These factors, which can be produced by the tumor cell or other cellular components of the tumor cell’s microenvironment, are thus rational chemotherapeutic targets. VEGF monoclonal antibodies and VEGF receptor tyrosine kinase inhibitors are currently being studied in both the lab and the clinic. Bevacizumab, a recombinant humanized monoclonal anti-VEGF antibody, has shown favorable results in AML patients resistant to traditional chemotherapy (Karp et al., 2004). Furthermore, Gabrilove postulates that since antiangiogenic therapies, such as inhibitors of VEGF and bFGF, do not directly target the tumor cell, drug-resistant tumor cells will not emerge (Gabrilove, 2001). Aside from neutralization of VEGF and bFGF, targeting IL-6 secretion may also be important in overcoming tumor cell survival. For example, Bisping and

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colleagues reported that IL-6 secretion by BMSCs was abrogated by treatment with a receptor tyrosine kinase inhibitor, and this inhibitor also induced apoptosis in a subset of MM patient specimens (Bisping et al., 2006). IL-6, as well as VEGF, production by stromal cells was also reported to be inhibited by treatment with an HDAC inhibitor (Golay et al., 2007). Additionally, neutralization of BAFF led to increased apoptosis in B-cell lymphoma cell lines (Klener et al., 2006). Antibody therapy may be effective not only in attenuating the effects of survival factors, but also in decreasing the positive influences that direct contact with the tumor microenvironment has on tumor cell survival. Monoclonal antibodies targeting tumor cell integrin α4 β1 or VCAM-1 on BMSCs, which are involved in tumor cell adhesion to the stromal cells, led to leukemic cell apoptosis. Also, treatment with an anti-α4 integrin antibody was found to suppress MM development in a mouse model (Mori et al., 2004b).

Immunomodulatory Drugs The immunomodulatory drug (IMiD) thalidomide and its second-generation derivatives, including lenalidomide, are now widely used in the treament of hematologic malignancies. These drugs are known to have anti-angiogenic and anti-inflammatory effects (Pangalis et al., 2006), and thalidomide has been reported to inhibit VEGF secretion by BMSCs (Gupta et al., 2001). Aside from inhibition of angiogenesis, these drugs have also been shown to induce apoptosis in MM cells (Mitsiades et al., 2002). IMiDs enhance apoptosis through inhibition of NF-kB activity and decreasing the expression of FLIP (Gupta et al., 2001). Furthermore, these drugs have been shown to affect tumor cell interactions with the surrounding environment. IMiDs have been shown to downregulate expression of cell adhesion molecules (Geitz et al., 1996; Settles et al., 2001), and also inhibit adhesion of MM cells to BMSCs (Anderson, 2003). Thus, treatment of hematologic malignancies with IMiDs will likely continue to prove beneficial, as these drugs target both the tumor and its microenvironment.

Bortezomib Bortezomib (PS-341, Velcade) is a reversible 26S proteasome inhibitor (Adams and Kauffman, 2004). The proteasome function is to degrade most intracellular proteins, such as apoptotic, cell cycle regulatory, and cell growth proteins (Zhou et al., 2005). Bortezomib, which has been approved for the treatment of MM (Jagannath et al., 2004), has shown encouraging results for the treatment of MM in combination with melphalan (Berenson et al., 2006), and is also being studied for the treatment of other hematopoietic tumors. This drug has been shown to enhance melphalan activity in MM cells by inhibiting NF-K B activity (Mitsiades et al., 2003). Furthermore, Hideshima et al. reported that bortezomib decreases IL-6 production in BMSCs, as well as decreases adhesion of MM cells to BMSCs (Hideshima et al., 2001). Therefore, like the IMiDs, bortezomib’s mechanisms of action include targeting the tumor cell as well as the tumor cell’s microenvironment.

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Conclusions Although today provides better treatment options for the treatment of many hematologic malignancies, the problem of minimal residual disease and drug resistance is still a major obstacle for overcoming these devastating diseases. The tumor microenvironment has been shown to be involved in hematologic tumorigenesis by providing growth and survival factors to the tumor, as well as enhancing antiapoptotic pathways in the tumor. This microenvironment, whose composition is different depending on the type of malignancy, has also been shown to contribute to both de novo and acquired drug resistance. Targeting the apoptotic pathways is a promising approach for the treatment of many of these devastating diseases, especially combination therapy to enhance both the intrinsic and extrinsic apoptotic pathways. It is also critical to target soluble factors produced in the tumor microenvironment as well as to directly target interactions between the tumor and its microenvironment, as this type of treatment has shown promising results to date and will likely prove most effective in eliminating minimal residual disease and drug resistance, ultimately leading to cures for hematologic malignancies.

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Chapter 9

Bcl-2 Family Members in Hepatocellular Carcinoma (HCC) – Mechanisms and Therapeutic Potentials Shihong Ma, George G. Chen and Paul B.S. Lai

Abstract Bcl-2 family members can be functionally divided into anti-apoptotic and pro-apoptotic groups. The balance between these two groups may determine the fate of tumor cells. In HCC, this balance is often tilted towards the anti-apoptotic members in tumor cells, leading to resistance to cell death and rapid proliferation. Recently, various strategies have been identified to correct this imbalance and to re-sensitize tumor cells to anti-tumor treatments. This chapter will summarize the recent development in our understanding of Bcl-2 family members in hepatocarcinogenesis and the therapeutic utilization of these molecules to increase the effectiveness in HCC treatment. Keywords Apoptosis · Bcl-2 family · BH3 domain only members · Hepatocellular carcinoma

Introduction Apoptosis is widely accepted as a prominent tumor-suppression mechanism. Bcl-2 family has emerged as a dominant regulator of apoptosis in cancer cells. Bcl-2 family proteins are structurally defined by their Bcl-2 homology domains (BH domains) into multi-domains and BH3-only, and functionally categorized into anti-apoptotic and pro-apoptotic. Bcl-2 family members fall into three subgroups. The first group includes Bcl-2, Bcl-xL, Mcl-1, Bcl-w, Bcl-B/Nrh/NR13 and Bfl-1/Bcl-2A1/GRS. These molecules contain multi-BH domains and function to inhibit apoptosis. The second group of Bcl-2 proteins also contains multi-BH domains but the proteins in this group function to promote apoptosis. These proteins include Bax, Bak, Bok/Mtd and Bcl-xS. The third group contains only a BH3 domain and it includes Bid, BAD, Bik/Nbk, Bim/Bod, PUMA, Noxa, Hrk/DP5, Bmf, Spike and BNip proteins. The

G.G. Chen (B) Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 9,

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Table 9.1 Expression of Bcl-2 family members in liver tissues of HCC and the normal subjects

Function

Structure

Antiapoptotic Multi-domain

Proapoptotic Multi-domain

Proapoptotic BH3 domain only

Bcl-2 family members

Expression in normal Expression in subjects HCC tissues

Bcl-2

−

−/+

Bcl-xL

+

↑

Mcl-1 Bcl-w Bcl-B/Nrh/NR13 Bfl-1/Bcl-2A1/GRS Bax

+ + + − +

↑ +/ND ND ND ↓/↑

Bak Bcl-xS Bok/Mtd Bid

+ + + +

↓/ND ↓ ND ↓

BAD

+

Phospho−/+

Bim/Bod Bik/Nbk PUMA Noxa Hrk/DP5 Bmf Spike BNip proteins

Low level ND ND ND −/low level + ND −/+

↑ −/ND ND ND ND ND ND ND

References Yoon et al., 1998 Chiu et al., 2003 Hussein, 2004 Pizem et al., 2001 Takehara et al., 2001 Chiu et al., 2003 Sieghart et al., 2006 O’Reilly et al., 2001

Guo et al., 2002 Luo et al., 1999 Liu et al., 2003 Chiu et al., 2003 Chen et al., 2001b Yoo et al., 2006 Chang et al., 2007 Miao et al., 2007 Zhao et al., 2007

ND = not determined.

BH3-only proteins bind and regulate the pro-survival Bcl-2 family members to promote apoptosis. Hepatocellular carcinoma (HCC) is considered highly resistant to chemotherapy (Avila et al., 2006). Defects in apoptosis signaling contribute to tumorgenesis and chemotherapy resistance of HCC cells. In HCC, there is an imbalance between the pro- and anti-apoptotic of Bcl-2 family members (Mott and Gores, 2007). As shown in Table 9.1, the expression of anti-apoptotic Bcl-xL and Mcl-1 is increased in HCC, whereas the expression of pro-apoptotic Bid and Bak protein is decreased (Chiu et al., 2003; Sieghart et al., 2006; Chen et al., 2001b; Fiorentino et al., 1999).

Physiological Role of Bcl-2 Family Members in Liver Anti-Apoptotic Multi-Domain Members of the Bcl-2 Family Bcl-2 family members have a number of essential roles in liver homeostasis. Although Bcl-2 is not generally expressed in human hepatocytes (Charlotte et al., 1994), ectopic Bcl-2 expression delays hepatocytes cell cycle progression (Vail

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et al., 2002). While Bcl-xL-/- mice die during embryogenesis with extensive apoptosis in liver (Motoyama et al., 1995). Over-expression of either Bcl-2 or Bcl-xL in mouse livers protects hepatocytes from Fas-induced apoptosis and liver destruction in a dose-dependent manner. The protective effect of Bcl-2 is not associated with the levels of Bcl-xL, Bax, Bad, Bak and p53 (Rodriguez et al., 1996). Bcl-xL was protective without any change in the level of Bax and inhibits hepatic caspase-3-like activity (de la Coste et al., 1999a). Excess Bcl-xL can inhibit p53induced oligomerization of Bak and the release of cytochrome c when being added to purified mouse liver mitochondria (Mihara et al., 2003). Mcl-1 is expressed in human liver (Krajewski et al., 1995) and plays a major role in mediating the anti-apoptotic effects of hepatocyte growth factor in primary human hepatocytes (Schulze-Bergkamen et al., 2004). Rapid up-regulation of other anti-apoptotic Bcl-2 family members such as Bfl-1 may also contribute to hepatocyte survival, although Bfl-1 gene is not detected by northern blot in human liver (Schoemaker et al., 2003). Bcl-B and Bcl-w are expressed in human liver (Ke et al., 2001; Gibson et al., 1996). However, their physiological roles in liver are unknown. A functional study showed that Bcl-B suppressed apoptosis by binding Bcl-2, Bcl-xL and Bax but not Bak (Luciano et al., 2007). Bcl-w is functionally similar to Bcl-2 since it enhances cell survival. Bcl-w and Bfl-1 protect against apoptosis induced by over-expression of Bax or Bad but not that induced by Bak or Bik (Holmgreen et al., 1999).

Pro-Apoptotic Multi-Domain Members of the Bcl-2 Family A high level of Bax and a moderate level of Bak are expressed in human liver (Krajewski et al., 1994 and 1996). No histological and functional abnormalities (determined by standard blood chemistries) have been observed in the liver of either Bak-/- or Bax-/-Bak-/- animals (Lindsten et al., 2000). Bok exhibits a higher level in fetal liver than in adult liver (Lee et al., 2001) but its physiological role in liver is unknown. Bok is the only member of the Bcl-2 family having a leucine-rich sequence indicative of a nuclear export signal within its BH3 domain (Bartholomeusz et al., 2006). In the yeast two-hybrid system, Bok interacts strongly with Mcl-1, Bfl-1 and BNip3 but not Bcl-2, Bcl-xL, and Bcl-w (Gao et al., 2005).

Pro-Apoptotic BH3 Domain Only Members of the Bcl-2 Family Bid is expressed predominantly in the cytosolic fraction of hepatocytes (Gross et al., 1999). Bid-/- mice apparently would develop normally and have a normal liver. Bid connects the death receptor apoptosis pathway to the mitochondrial apoptosis pathway (Ding et al., 2004). The cytosolic truncated Bid (tBid) targets mouse liver mitochondria while Bid does not. Anti-Fas antibody results in the appearance

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of tBid in the cytosol of hepatocytes and tBid translocates to the mitochondria where it stimulates the releases of cytochrome c. Bid is indispensable in Fas-induced hepatocytes apoptosis and liver injury (Li et al., 2002; Yin et al., 1999). However, the deletion of Bid only delays but does not block TNFα -mediated apoptosis of hepatocytes and liver injury (Ding et al., 2004; Zhao et al., 2001, 2003). Hepatocytes require a Bid-dependent mitochondrial amplification loop that releases cytochrome c, oligomerizing Apaf-1 and caspase-9 to activate sufficient effector caspases to execute apoptosis. In both cultured cells and animal models of TNFα-induced injury, Bid-independent mitochondrial activation could be demonstrated at later time points (Chen et al., 2007). Gene knockout mice model indicated that Bid functions upstream of either Bax or Bak to initiate mitochondrial dysfunction and cell death (Wei et al., 2001). Following Fas activation, Bid is singularly required to oligomerize Bak and release cytochrome c (Wei et al., 2000). Researchers also found that Bid-dependent generation of oxygen radicals promotes death receptor activation-induced apoptosis in murine primary hepatocytes (Zhao et al., 2001). Transient expression of anti-death Bcl-2 or Bcl-xL reduces the apoptosis of Bid-deficient hepatocytes treated with TNFα, suggesting that the Bcl-2 family proteins could interact with the Bid-independent mitochondrial activation mechanism. Consistent with this finding, treatment of hepatocytes in vitro with TNFα also cleaves of Mcl-1 on the mitochondria, independent of the status of Bid. The roles of Bad, Bmf, Bim, Hrk, Spike and BNip in the liver are less clear than Bid. Liver has abundant Bmf, detectable Bad, and very low or undetectable level of Bim and Hrk mRNA in liver (Inohara et al., 1997; Kitada et al., 1998; Miyashita et al., 2001). In the yeast two-hybrid system, Bmf can interact with Mcl-1, pro-survival Bcl-2, Bcl-xL, Bcl-w but not with the pro-apoptotic Bax, Bid and Bad (Puthalakath et al., 2001). Spike has been shown to regulate the interaction between Bcl-xL and Bap31, which is an adapter protein for procaspase-8 and Bcl-xL. No co-immunoprecipitation of Spike with all tested Bcl-2 family members such as Bax, Bcl-xL or Bcl-2 has been observed (Mund et al., 2003). The BNip proteins include BNip1, BNip2, BNip3, BNIPL-1, BNIPL-2 and Nix. They are pro-apoptotic members of the Bcl-2 family. The expression of BNip1 and its variants have been observed in liver. Bnip2, a putative pro-apoptotic protein, contains two isoforms BNIP-Sα and BNIP-Sβ. BNIP-Sα but not BNIP-Sβ is expressed in liver. Bnip3 induces necrosis rather than apoptosis. Several human homologues of BNip3 have been reported, all of which are pro-apoptotic molecules: BNip3L, BNip3h and Nix (Zhang et al., 2003). Nix homo-dimerizes similarly to Bnip3 and can overcome the suppressors Bcl-2 and Bcl-xL (Chen et al., 1999). The expression of BNip3 can be suppressed by nitric oxide (NO), and such suppression has been proposed to be a mechanism for NO-induced apoptosis in hepatocytes (Zamora et al., 2001). BNIPL-1 and BNIPL-2 are homologous to human Bnip2, can interact with Bcl-2, Cdc42GAP and induce apoptosis (Qin et al., 2003). BNIPL-2 may play its role in apoptosis through regulating the expression of genes associated with cell apoptosis, growth inhibition and cell proliferation (Xie et al., 2004).

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Bcl-2 Family Members and Hepatocarcinogenesis Given the importance of Bcl-2 family members in normal liver, the relevance of Bcl-2 family members in HCC has also been extensively studied.

Anti-Apoptotic Multi-Domain Members of the Bcl-2 Family Among this subgroup, Bcl-2 and Bcl-xL have been well studied in hepatocytes. In HCC, Bcl-2 is usually absent while Bcl-xL is predominately expressed. Using Northern blot, researchers reported that Bcl-2 and Bcl-xL may play important roles in regulating the apoptosis of normal liver and HCC (Guo et al., 2002). Upregulation of Bcl-xL is associated with tumorgenesis and resistance to chemotherapeutic treatment, thus becoming a promising target for cancer therapy (Takehara et al., 2001). The expression and the function of Bcl-w, Bcl-B and Bfl-1 are unknown in HCC. Some reports showed that the increased level of Bcl-2 RNA was frequently present in HCC (Fiorentino et al., 1999). However, its protein product is either absent (Yoon et al., 1998; Skopelitou et al., 1996) or present only in a very small proportion of tumor cells in HCC tissues (Soini et al., 1996; Hamazaki et al., 1995). The findings suggest a post-translational mechanism of Bcl-2 protein degradation, indicating that Bcl-2 may not play an important role in hepatocarcinogenesis (Ravazoula et al., 2002; Nakopoulou et al., 1999). In contrast, other studies have shown the increased level of Bcl-2 protein in HCC (Hussein, 2004; Pizem et al., 2001) and it may be involved in the development of HCC (Ali et al., 2004). This concept is supported by a study showing that the over-expression of Smad3, a major TGF-β signaling transducer, reduces the susceptibility to hepatocarcinogenesis in a mouse model by reducing the level of Bcl-2 to sensitize hepatocytes to apoptosis (Yang et al., 2006). This observation is in agreement with data demonstrating that excess Bcl-2 expression in TGFα/Bcl-2 double transgenic mice delays the development of liver tumors induced by the growth factor (Vail et al., 2001) and that Bcl-2 inhibits c-myc-induced liver carcinogenesis (de La Coste et al., 1999b). Furthermore, in vivo electroporetic transfer of Bcl-2 antisense oligonucleotide (ASO) into liver can inhibit the development of HCC in rats (Baba et al., 2000). The differential expression of Bcl-2 may be related to the status of p53 since Bcl-2 is remarkably up-regulated in p53-positive HCC tissues, but down-regulated in p53negative ones (Chiu et al., 2003). Alterations of both p53 and Bcl-2 proteins have been observed during hepatocarcinogensis (Hussein, 2004), and the over-expression of p53 correlates with a high level of proliferation cell nuclear antigen (PCNA), HCC de-differentiation and advanced HCC stages (Hu et al., 2007). Bcl-x can alternatively produce two distinct proteins, anti-apoptotic Bcl-xL and pro-apoptotic Bcl-xS (Yang et al., 2004). Bcl-xL is expressed at high levels in HepG2, Hep3B, and Huh7 cell lines and in a great percentage of murine and human HCC tissues. Bcl-xL can be up-regulated in HCC regardless of p53 status

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(Chiu et al., 2003). Bcl-xL is located not only in the cytoplasm but also in the nuclei of some HCC cells, suggesting that it is involved in the progression of HCC cells in vivo (Watanabe et al., 2002). Increased expression of Bcl-xL in human HCC cells inhibits apoptosis produced by serum starvation and p53 activation (Takehara et al., 2001). The function of Bcl-xL can be modulated through deamidation at asparagine residues in a post-translational manner, and human HCC cells may acquire resistance to apoptosis and a survival advantage by suppressing the deamidation (Takehara and Takahashi, 2003). Mcl-1 interacts with tBid and, thereby, inhibits intrinsic as well as extrinsic apoptotic signaling (Clohessy et al., 2006). Mcl-1 protein is considerably over-expressed in Huh7, Hep3B, HepG2 and human HCC tissues, and it correlates with Bcl-xL expression in HCC tissues (Sieghart et al., 2006). Enhancing cell survival Bcl-w is also expressed in HepG2 (O’Reilly et al., 2001). The expression of Bcl-2 fails to correlate with the natural prognosis or survival (Garcia et al., 2002). However, Bcl-xL is a significant prognostic factor for disease progression and poorer survival in human HCC (Watanabe et al., 2004). There is no correlation between the levels of Bcl-2 and Bcl-xL and age, gender, differentiation or stage of tumor in HCC patients (Guo et al., 2002). However, the induction of anti-apoptotic Bcl-2 related proteins at the early stage of differentiation is important for the maintenance of tumor cell differentiation by antagonizing pro-apoptotic molecules such as Bax (Wakabayashi et al., 2000). Although Bfl-1 gene may be involved in cancer progression by promoting cell survival and its level is overexpressed in other digestive cancers, Bfl-1 gene is not detectable in human hepatic tumors (Choi et al., 1995).

Pro-Apoptotic Multi-Domain Members of the Bcl-2 Family Bax, Bak, Bcl-xS and Bok function either by inactivating pro-apoptotic Bcl-2 family members or by forming pores in the mitochondria. Most studies indicated that the expression of these pro-apoptotic proteins is decreased in HCC. For example, Bax was down-regulated in HCC tissues (Guo et al., 2002) and the decreased Bax was with over-expressed non-functional p53 protein in HCC samples (Beerheide et al., 2000). The level of Bak is down-regulated in human hepatoma cell line BEL-7402 (Liu et al., 2003). Bcl-xS is remarkably down-regulated in p53 positive HCC (Chiu et al., 2003). However, there are some reports showing that the level of Bax was not reduced in human HCC QGY-7703 cells and that its level was even up-regulated in HCC regardless of the p53 status (Luo et al., 1999). The reason for the controversial results is unknown. The survival rate in the patients with Bax-expressing HCC appears to be better (Osada et al., 2004; Garcia et al., 2002). Immunostaining intensity of Bax tends to be positively correlated with overall survival (Garcia et al., 2002). The level of Bax does not correlate with either age or gender in HCC patients (Guo et al., 2002). Bax may be used to identify the patient prognosis of the lower grade histological cases but another study fails to show such a relationship (Guo

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et al., 2002; Osada et al., 2004). Although the ratio of Bcl-2 to Bax is generally regarded as a critical index for determining whether a cell is likely to undergone apoptosis, this ratio correlates with neither apoptosis nor clinical markers in HCC (Ikeguchi et al., 2002).

Pro-Apoptotic BH3 Domain Only Members of the Bcl-2 Family BH3 domain only members of the Bcl-2 family promote apoptosis by binding antiapoptotic Bcl-2 family members, leading to inhibit their activity. It can also interact with Bax or Bak to regulate cell death. Bid and BAD are found to be down-regulated or inactive in HCC. However, surprisingly the level of Bim is increased. The expression and function of other recently discovered members of this subgroup in HCC are unknown. The expression and the regulation of Bid may play crucial roles not only in liver tumorgenesis but also in the chemotherapeutic treatment of HCC. The level of Bid was shown to be decreased in HCC except in poorly differentiated HCC in which cells may undergo a process of apoptosis or necrosis (Miao et al., 2004, 2006; Chen et al., 2001b). The decreased Bid may be related to hepatitis B virus X protein or hepatitis C virus polyprotein (Chen et al., 2001a; Disson et al., 2004). Bid can block the inhibitory effect of Bcl-2 on Fas-mediated apoptosis of HCC cell line BEL-7404 cells through oligomerising Bak to release cytochrome c (Chang and Xu, 2000). In SK-Hep1 cells TNF-related apoptosis-inducing ligand (TRAIL) is known to induce the translocation of Bax, which subsequently leads to the cleavage of Bid (Kim et al., 2002a). BAD is a pro-apoptotic Bcl-2 family protein that regulates the intrinsic apoptosis pathway. Phosphorylation of BAD inhibits apoptosis whereas de-phosphorylation of BAD promotes it. In FaO hepatoma cells, TGF-β1 can induce cleavage of BAD at its N terminus to generate a 15-kDa truncated protein, leading to apoptosis (Kim et al., 2002b). A mutant BAD can prevent caspase 3 from cleavage and thus block TGF-β1-induced apoptosis (Kim et al., 2002b). The loss of phospho-BAD expression, but not BAD gene mutation has been shown to be a feature of HCC and the loss of phospho-BAD expression may play a role in hepatocarcinogenesis (Yoo et al., 2006). However, this seems not to be the case in all HCC, since the phospho-BAD has been observed in HBV-infected HCC (Chang et al., 2007). Bim mRNA and protein are strongly expressed in HCC (Miao et al., 2007). Bim EL, L, S, a1, a2, a3, b2, b4 and b6 are abundant isoforms according to their mRNA levels. However, only Bim EL, L and S proteins could be clearly detected in HCC. Although the pathological role of such an increase in HCC is unclear, over-expression of Bim EL, L, S and all alpha isoforms (containing BH3 domain in their translation products) appears to have a role in the regulation of apoptosis in HCC, which may contribute to not only the growth of tumor cells but also the sensitivity of HCC cells to 5-FU. Further study is necessary to identify whether the phosphorylation of Bim EL in Hep3B contributes to the sensitivity or resistance of HCC to chemotherapy (Miao et al., 2007).

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Unlike other BH3-only proteins, Bik is the first BH3-only protein to be identified as an endoplasmic reticulum-resident, from which it induces apoptosis (Mathai et al., 2005; Boyd et al., 1995; Zou et al., 2002). Bik could apparently induce apoptosis in Hep3B cells through activating caspase-12-dependent signal transduction pathway. Bik activates caspase-9 and depolarization of mitochondrial membrane potential, which is decreased concomitantly with caspase-12 silenced (Zhao et al., 2007). Bik is not normally detected in Hep3B and HepG2 cells, however after treatment with azacytidine or butyrate an increase of Bik mRNA expression can be observed (Wang et al., 1998), suggesting that the pro-apoptotic role of this molecule is secondary to other stimuli. Hrk/DP5 activates apoptosis and interacts selectively with Bcl-2 and Bcl-xL but not Bcl-xS (Boyd et al., 1995). Methylation of Hrk silences the gene expression. However, the methylation around the transcription start site was not detected in HCC cell lines and in 20 HCC primary tumors (Obata et al., 2003). PUMA and Noxa are BH3-only proteins that are involved in p53-dependent apoptosis (Nakano and Vousden, 2001; Hijikata et al., 1990). Previous studies reported that Noxa induces apoptosis in various cell lines (Oda et al., 2000). Bnip3 and its homologue Nix are hypoxically regulated in many tumor types (Sowter et al., 2001). Unfortunately, the roles of these BH3-only proteins are still unknown in HCC.

Bcl-2 Family Members in HCC Treatment Bcl-2 Family Members as Targets for Anti-HCC Therapies Over-expression of Bcl-2 protein protects several human HCC cells from various apoptotic stimuli such as TRAIL (Guo and Xu, 2001), FAS (Takahashi et al., 1999) and TGF-β (Huang and Chou, 1998). Therefore, a high level of Bcl-2 may raise the apoptotic threshold and cause HCC cells more likely to survive. In contrast, a decrease in the expression of Bcl-2 protein promotes cell death in HCC cells, which is the mechanism responsible for apoptosis of HCC cells induced by TNFα and TGF-β (Li et al., 2001). Bcl-2 and Bcl-xL play important roles in the resistance to chemical therapy in HCC. Bcl-xL inhibits staurosporine-induced apoptosis in human HCC. Primary cultured HCC cells, resistant to paclitaxel, are known to express high levels of the anti-apoptotic Bcl-2 and Bcl-xL proteins. The level of Bcl-xL can be further induced upon paclitaxel treatment (Chun and Lee, 2004). Constitutive expression of Bcl-2 can render HCC QGY-7703 cells more resistant to Taxol and doxorubicin. Contrarily, a decreased level of Bcl-2 renders the tumor cells to be more sensitive to the drugs (Luo et al., 1999). ASO strategy has been used to target Mcl-1 to down-regulated its level, leading to an increase in apoptosis or a decrease in cell viability in HCC cells. In combination with cisplatin or doxorubicin, Mcl-1 ASO has an additive effect (Sieghart et al., 2006; Fleischer et al., 2006). Similar to ASO strategy, siRNA can mediate the

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inhibition of Mcl-1, resulting in significantly enhanced susceptibility of HCC cells to chemotherapy or TRAIL-mediated apoptosis. The regulation of Mcl-1 involves in phosphatidylinositol 3-kinase (PI3K) since the inhibition of PI3K reduces expression of Mcl-1 in Hep3B cells (Fleischer et al., 2006). Therefore, treatment of HCC cells with PI3K inhibitors and chemotherapeutics in combination can greatly enhance the apoptosis induced. The important role of Mcl-1 in HCC treatment is further demonstrated by the Mcl-1 over-expression experiment, in which the overexpression of Mcl-1 reduces apoptosis of Huh-7 cells induced by chemotherapeutic agents or TRAIL (Wirth et al., 2005). Therefore, Mcl-1-expressing HCC cells are resistant to apoptosis and usually show low sensitivity to chemotherapeutic drugs. Up-regulation of Bax expression either by p21/ceramide or by the introduction of high level p53 in Hep3B cells induces apoptosis. JNK- and p38 kinase-mediated phosphorylation of Bax leads to apoptosis of human HepG2 cells (Kim et al., 2006). These results demonstrated Bax may participate in inducing p53-dependent apoptosis in HCC (Kang et al., 1999; Lai et al., 2007). Over-expression of Bax sensitizes HCC-9204 cells to adriamycin-induced apoptosis (Zheng et al., 2005). Also, an inverse relationship of Bax expression with doxorubicin resistance has been shown in HCC (Hu et al., 2004). The over-expression of Bak leads to apoptosis in HCC-9204 cells and sensitizes the cells to apoptosis induced by doxorubicin (Li et al., 2000). Transfer of Bcl-xS plasmid is effective in preventing and inhibiting rat HCC induced by N-nitrosomorpholine (Baba et al., 2001). Over-expression of BNIPL-1 suppresses Hep3B cell growth probably though cell cycle arrest or apoptotic cell death pathway (Xie et al., 2005).

Strategies to Target Bcl-2 Expression in HCC In HCC, resistance to currently used chemotherapeutic drugs is partly mediated by over-expression of the cell survival proteins. Interactions between anti-apoptotic and pro-apoptotic members of the Bcl-2 family should determine the fate of the cells. Strategies which aim to down-regulate anti-apoptotic Bcl-2 proteins or interfere with its anti-apoptotic function are likely to sensitize cancer cells to chemotherapeutic agents as well as lower the dose of therapy required to kill the cancer cells. Therapeutic strategies currently in use or potentially useful to modulate apoptosis in HCC are summarized in Fig. 9.1. ASO and siRNA present a genetic approach to inhibit the anti-apoptotic function of Bcl-2 family members. Results from human phase I clinical trials of Genasense (G3139), ASO of Bcl-2, have demonstrated good efficacy with low toxicity in nonHodgkins lymphoma patients (Webb et al., 1997; Waters et al., 2000). However a recently report of a phase I-II study of G3139 in combination with Doxorubicin in 21 patients with advanced HCC fails to have a positive result and the failure of the treatment is likely due to the low level of Bcl-2 in the patients studied (Knox et al., 2008). Taken together, it appears that anti-Bcl-2 treatment is not suitable for all HCC and HCC with a high level of Bcl-2 likely responds well to the treatment. Therefore, it may be critical to determine the level of Bcl-2 before the treatment.

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p53 transcription activation

Noxa Puma

Dephosphorylation Bad Hrk

Cytokine deprivation

Dissoclation from dynein LC

Bim

Loss of cell attachment (anoikis)

unleash Bmf

Bmf

BH3 mimetic

Bcl-2 Bcl-xL Mcl-1 Bfl f -1 Bfl-1 Bcl-w

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Mitochondria Bax Bak Bok

Cytochrome C

ER Bik

ER stress Hypoxia etc.

Caspase 12

Adenovirus carrying pro-apoptotic gene

Apaf-1 Caspase 9

Bnip proteins Effector caspase

Death receptor activation

Caspase- 8 Bid activation

tBid

Death

Fig. 9.1 Signaling of Bcl-2 family members in hepatocytes and potential therapeutic windows for treatments of HCC

Adenoviral gene therapy is an effective mode of gene delivery that has been demonstrated to be safe in clinical trials of gene therapy. Alpha-fetoprotein (AFP) promoter-mediated tBid (Ad/AFPtBid) have been shown to significantly and specifically kill AFP-producing Hep3B cells in vitro and in mice subcutaneously implanted with HCC cells (Miao et al., 2006). Adenovirus that carries a lethal gene PUMA under the control of a beta-catenin/Tcf-responsive promoter (AdTOP-PUMA) has also been demonstrated to efficiently kill HepG2 HCC cells (Dvory-Sobol et al., 2006). The binding pocket formed by the BH1, 2 and 3 domains of Bcl-2 and related survival molecules has great potential as a target for small molecule inhibitors. Antimycin A has emerged as a Bcl-2 groove binder upon screening mitochondrial respiration inhibitors for pro-apoptotic activity in hepatocytes with graded expression of Bcl-xL (Kim et al., 2001; Tzung et al., 2001). Antimycin A interacts with the BH3-binding hydrophobic groove of Bcl-xL and can also compete with a Bak BH3 peptide for binding to recombinant Bcl-2. BH3 mimetics ABT-737 is a small molecule that binds within the hydrophobic cleft of Bcl-2, Bcl-xL, and Bcl-w and kills cells in a fashion dependent on Bax and Bak. A combination of sorafenib, a multi-kinase inhibitor and ABT-737 has been shown to be effective in the treatment of HCC (Lin et al., 2007). Likewise, sorafenib has a beneficial effect in the treatment of HCC (Abou-Alfa et al., 2006), likely in part by down-regulating Mcl-1. Finally, cyclooxygenase-2 (COX-2) can act in HCC cells to increase Mcl-1 protein expression (Kern et al., 2006), and thus the inhibition of COX-2 can decrease Mcl-1 protein expression and in turn sensitize tumor cells to apoptosis.

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Conclusions Physiological apoptosis is associated with liver homeostasis. In contrast, deficient apoptosis contributes to the development of HCC. Members of the Bcl-2 family serve as critical regulators of apoptosis, acting to either inhibit or promote cell death. Aberrant expression patterns of some members of the Bcl-2 family have been reported in HCC. However the roles of some Bcl-2 proteins such as Bcl-B, Bfl-1, Bok, Hrk and Bmf in HCC are largely unknown. A proper balance between anti-apoptotic and pro-apoptotic molecules or signals is required for normal hepatocytes, and the imbalance caused by either an increase in the anti-apoptotic Bcl-2 members or a decrease in pro-apoptotic members may contribute to hepatocarcinogenesis as well as to the sensitivity of HCC to anti-tumor treatments. Therefore, the balance between pro-apoptotic and anti-apoptotic Bcl-2 family members determines the outcomes of HCC in terms of resistance or susceptibility to triggers for apoptosis induction. Our understanding of Bcl-2 family members has widened the horizon in the research of novel and promising treatments for HCC. Acknowledgments This work was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region (No: CUHK 4534/06M).

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Chapter 10

Apoptosis in the Development and Treatment of Laryngeal Cancer: Role of p53, Bcl-2 and Clusterin ´ Maximino Redondo, Rafael Funez and Francisco Esteban

Abstract In cancer, the balance between proliferation and programmed cell death is disturbed, and defects in apoptotic pathways allow cells with genetic abnormalities to survive. Alterations in apoptotic pathways may result in resistance to drugs and radiation. Such alterations might serve as predictors of chemotherapyand radiotherapy-sensitivity and, most importantly, as new treatment targets. The proteins p53, bcl-2 and clusterin influence the apoptotic activity of the cells. In this report we describe the distribution and possible functional significance of these proteins in squamous cell carcinoma of the larynx. Their implications in carcinogenesis and progression make these genes worthy of investigation. Keywords Laryngeal cancer · p53 · bcl-2 · Clusterin

Introduction Laryngeal squamous cell carcinoma occur most frecuently in the sixth and seventh decades and they are more common in men (Cataruzza et al., 1996) though the male:female ratio is decreasing in some countries because of increased prevalence of women smoking over the last two decades (De Rienzo et al., 1991). Squamous cell carcinoma comprises about 95% of laringeal malignancies and there are geographic variations. The incidence in men is high in southern and central Europe, southern Brazil, Uruguay and Argentina and among Blacks in the United States. The lowest rates are recorded in Souht East Asia and Central Africa. An estimated 140.000 new cases ocurred worldwide in 1990, 86% of these patients were men (Parkin et al., 1999; Parkin et al., 2003). The incidence of laryngeal cancer is increasing in much of the worl, both in men and in women. This increase is related to changes in tobacco and alcohool consumption (Cataruzza et al., 1996).

M. Redondo (B) Department of Biochemistry, Hospital Costa del Sol, Carretera de C´adiz Km 187, 29600, Marbella, University of Malaga, Marbella, M´alaga, Spain e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 10,

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There are also geographic differences in the topographic distribution of the laryngeal squamous cell carcinoma. In France, Spain, Italy, Finland and the Netherlands supraglotics carcinomas predominates, while in the United States, Canada, England and Sweden glottic carcinomas is more common (Barnes and Gnepp, 2000). Apoptosis, or programmed cell death, is a major control mechanism by which cells die if DNA damage is not repaired (Lowe and Lin, 2000). Apoptosis is also important in controlling cell number and proliferation as part of normal development. Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation, cellular senescence and cell death. Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities. Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms. Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death. The intimate link between the cell cycle, cellular senescence, apoptosis regulation, cancer development and tumor responses to cancer treatment has become eminently apparent (Villard et al., 2001). Morphologically, in cells undergoing apoptosis there is ruffling, blebbing, and condensation of the plasma and nuclear membranes, and, subsequently, aggregation of nuclear chromatin. Mitochondria and ribosomes retain their gross structure and at least partial function. There is disruption of the cytoskeletal architecture; the cell shrinks and then fragments into a cluster of membrane-enclosed “apoptotic bodies” that are rapidly ingested by adjacent macrophages or other neighbouring phagocytic cells. The fact that apoptosis is a genetically defined pathways has lead to the expectation that new therapies based on apoptosis will be superior to current anticancer treatments. The most studied genes related to apoptosis are the tumour suppressor gene p53 and the anti-apoptotic gene bcl-2. p53 functions as a transcription factor regulating downstream genes important in cell cycle arrest, DNA repair, and apoptosis. The critical role that p53 plays is evident by the large number of tumors that bear a mutation in this gene. Normal wild type p53 can limit cell proliferation after DNA damage by two mechanisms: arresting the cell cycle or activating apoptosis (Wallace-Brodeur et al., 1999). If cellular damage is considered reparable, p53induced cell cycle arrest allows time for DNA repair. With more extensive damage, to prevent the cell with an impaired DNA sequence from proliferating as a defective or malignant clone, p53 moves the cell into the apoptotic pathway. p53 protein, normally, is present in the cytosol in low concentration. It is negatively regulated by another transcription factor, MDM2 (murine double minute 2), which downregulates p53 transcription, and binds to p53 protein, decreasing its activity and accelerating its degradation. In the event of DNA damage, p53 gene induction is accompanied by increased synthesis and phosphorylation of p53. Phosphorylation has profound consequences—it renders the protein more active and reduces its binding and inactivation by MDM2, thereby doubling its half-life. As a result, p53 protein activity may increase a hundredfold. p53 promotes cell cycle arrest in late G1 at a restriction point guarded by the retinoblastoma (Rb) protein. Phosphorylation of Rb permits the cell to pass through

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this cell-cycle transition point—the cell now irreversibly committed to exit G1 and proceed unhindered into S phase. p53 exerts control of the cell cycle through upregulation of p21, an inhibitor of the cyclin-dependent kinases (CDKs) responsible for moving the cell through G1 . The cyclinD/CDK4 complex normally promotes phosphorylation of Rb. Hypophosphorylated Rb binds E2F, a transcription factor required for passage through the G1 restriction point; upon Rb phosphorylation E2F is released, translocates to the nucleus, and induces transcription of a number of proteins, prompting the cell to move into S phase. Cell injury results in increased expression of p53 followed by p53-regulated induction of p21, and consequently, by the inhibition of cyclinD/CDK4 phosphorylation of Rb. Maintenance of Rb in its active hypophosphorylated state holds the cell in G1 , allowing time for repair. In the event that DNA damage is more severe and non-reparable, p53 performs its alternate role of moving the cell into apoptosis through the Bax/Bcl-2 pathway (Israels and Israels, 1999) Tumors with mutated p53 for example can be more anaplastic, have a higher rate of proliferation, and have a more aggressive phenotype than similar tumors with wild-type p53, thereby giving rise to a worse prognosis. However, p53 has a dual and complex role in chemosensitivity; it can either increase apoptosis or arrest growth and thereby increase drug resistance. The importance of p53 for chemosensitivity, however, is supported by the fact that, currently, the most curable cancers are among the minority of tumours in which p53 is not mutated , that is, some haematopoietic and germ cell tumours. The Bcl-2 family of proteins stands among the most crucial regulators of apoptosis and performs vital functions in deciding whether a cell will live or die after cancer chemotherapy and irradiation. In addition, several studies have revealed that members of the Bcl-2 family also interface with the cell cycle, DNA repair/ recombination and cellular senescence, effects that are generally distinct from their function in apoptosis (Zhan et al., 1999). At present, the bcl-2 family of proteins together with related members is known to include more than 30 proteins with either pro-apoptotic or antiapoptotic functions, suggesting that they might also play different roles in carcinogenesis (Cory et al., 2003). The bcl-2 gene is overexpressed in most follicular B-cell lymphoma, CLL, and about 25% of B- large cell NHL (Schattner, 2002). The Bcl-2 protein is overexpressed in many solid organ malignancies that do not harbor the t(14;18) translocation such as prostate cancer, breast cancer, nonsmall cell lung cancer, small cell lung cancer, and melanoma (Selzer et al., 1998). When bcl-2 was expressed in cells in tissue culture, it not only protected them from apoptosis due to removal of growth factors, it also prevented apoptosis following treatment with a diverse range of drugs and toxins, giving cells a multidrug resistance phenotype (Strasser et al., 1991; Tsujimoto, 1989). This suggested that apoptosis inhibitory genes such as bcl-2 might not only play a role in the development of malignancy, but also determine the response to therapy. Thus, Bcl-2 also plays a critical role in regulating response to chemotherapy, hormonal therapy, and irradiation (Miyashita and Reed, 1992). However, in vivo, bcl-2 expression has been associated with a more favourable prognosis in some malignant diseases. Indeed, in breast cancer, tumours positive for bcl-2 often have

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oestrogen receptors and a more favourable prognosis. Oestrogen has been shown to be a positive regulator of bcl-2 gene expression in breast cancer cell lines (Teixeira et al., 1995). Indeed both proliferation and apoptotic cell death are very complex process that involves the participation of many genes. Thus, another gene as clusterin (CLU) has been implicated in anti-apoptotic fuctions. Because it was studied independently by a number of laboratories and in different systems, it was given various names, such as SGP-2, clusterin, apolypoprotein J, SP 40-40, complement lysis inhibitor, gp80, glycoprotein III and T64 (Jordan-Stark et al., 1992). The human homologue of CLU is comprised of 449 amino acids with two 40 kDa subunits (alpha and beta) joined by a unique five disulfide bond motif (Kirszbaum et al., 1992). The protein precursor is encoded on a single 2 Kb mRNA that is transcribed from a single copy gene located on chromosome 8 (8p21) (Fink et al., 1993). The high degree of sequence conservation across species, the widespread tissue distribution and the high circulating concentration suggest that CLU play an important biologic role. It is well known that the glycoprotein CLU has two known isoforms generated in human cells (Pucci et al., 2004). A nuclear form of CLU protein (nCLU) is pro-apoptotic, while a secretory form (sCLU) is pro-survival. Both forms are implicated in various cell functions, including DNA repair, cell cycle regulation, and apoptotic cell death (Chen et al., 2004). CLU expression has been associated with tumorigenesis and the progression of various malignancies. One of the most effective ways to combat different types of cancer is through early diagnosis and administration of effective treatment, followed by efficient monitoring that will allow physicians to detect relapsing disease and treat it at the earliest possible time. Dysregulation of programmed cell death mechanisms plays an important role in the pathogenesis and progression of cancer as well as in the responses of tumours to therapeutic interventions. Clusterin and many members of the bcl-2 family of apoptosis-related genes have been found to be differentially expressed in various malignancies. the clinical importance of these antiapoptotic proteins has stimulated interest in using antisense therapy to modulate their expression (Chi and Gleave, 2004). Agents that can affect the Bcl-2 protein include antisense oligonucleotides such as G3,139 (Genasense [Genta, Inc., Berkeley Heights, NJ], oblimersen sodium), small molecules that recognize the surface pocket of Bcl-2 or Bcl-xL , and antisense Bcl-xL , which is in preclinical development. Concerning clusterin, because of its important role, the inhibition of CLU using sequence-specific antisense oligonucleotides has been shown in both in vitro and in vivo models to enhance chemotherapy effects in hormone refractory prostate cancer (Gleave and Jansen, 2003) and breast cancer (Redondo et al., 2007).

Role of p53 and bcl-2 in Layngeal Intraepithelial Neoplasia Squamous cell carcinoma of larynx (SCCL), usually develops in a multistep process: normal mucosa→ dysplasia → (laryngeal intraepithelial neoplasia, LIN) → SCC in situ → invasive SCC. Although the molecular events which induce this

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process are still unknown, they seem to depend on the disruption of the genomic stability (Vogelstein, 1990; Harris, 1990).The latter reflects an interaction among tumor suppressor genes (p53) and oncogenes (bcl-2). Several studies have proposed close interactions between p53 and bcl-2 proteins during carcinogenesis (Rudoltz et al., 1993; Skuse 1996; Kastan et al., 1991). This proposition is supported by the inhibitory effect of p53 on bcl-2 through its regulatory domain in the bcl-2 gene (Kastan et al., 1992). One study demonstrated that the development of LIN is associated with upregulation of p53 and bcl-2 protein expression (Hussein 2005).

P53 and Laryngeal Cancer Identification of a prognostic indicator that is both reliable and an easily assayed marker of cancer recurrence remains one of the goals of translational research. Obviously, this would be of greatest interest to treating physicians because it would enable more effective treatment and the early detection of recurring tumors. One gene that has received the greatest amount of attention is the tumor suppressor p53 gene. The p53 gene is one of the most commonly mutated genes identified in head and neck carcinomas (Fig 10.1A and B), being associated with a large number of tumor sites (Watling et al., 1992; Awwad et al., 1996). In laryngeal carcinomas overexpression of the p53 product has been detected in approximately 50% to 60% of the cases (Nadal and Cardesa, 2003). Mutations in the p53 gene are not the primary lesions responsible for tumorogenesis, but probably occur as an intermediate step in tumor progression (Skuse, 1996). Although p53 overexpression is frequent in head and neck squamous cell carcinomas, controversy remains regarding the prognostic significance of that overexpression. It has been proposed that P53 overexpression is an independent prognostic factor parameter (Narayana et al. 1998), but the claim has not been substantiated by others (Friedman et al. 2001, Kokoska et al. 1996). Another study of Narayana et al. showed that p53 is an important prognostic factor and is independent of histologic grade in patients with early glottic carcinoma treated with radiotherapy alone (Narayana et al., 2000). This raises one question: does p53 mutation mediate changes in radiosensitivity? There are conflicting data, with some studies suggesting increased sensitivity (Kastan et al., 1991; Kastan et al., 1992), some increased resistance (Brachman et al., 1993; Leith, 1994; Alsner et al., 2001; Lee et al., 2006), and others no changes (Awwad et al., 1996; Field et al., 1993; Pai et al., 1998). It is tempting to speculate that p53 mutation resulted in radioresistance and eventual local recurrence. Indeed, Raybaud-Diogene et al. reported a statistically significant increased risk of local recurrence in 101 patients treated for head and neck carcinoma by radical radiotherapy (Raybaud-Diogene et al., 1997). Koch et al. examined the relation between p53 mutation and local failure in patients with head and neck squamous cell carcinoma treated with primary or adjuvant radiotherapy (Koch et al., 1996). Both the incidence of local recurrence and time to local

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Fig. 10.1 (A) Laryngeal carcinoma (LC) with strong immunoreactivity of p53 next to laryngeal mucosa (LM) without expression (×100). (B) p53 staining is seen in a poorly-differentiated laryngeal squamous cell carcinoma (×200). (C) Immunoreactivity of bcl-2 protein in a poorlydifferentiated laryngeal squamous cell carcinoma. The positively staining lymphocytes in stroma serve as an internal control (×200). (D) In situ hybridization of clusterin mRNA in most cells of a moderately-differentiated laryngeal squamous cell carcinoma (×400)

recurrence significantly were worse for patients whose tumors contained mutant p53 genes. Without comparison with surgical series, it is difficult to determine whether it is due to radioresistance or intrinsic biologic behavior. In this sense, however, Alsner et al. found a strong relationship between p53 mutations and poor prognosis after radiotherapy, but not after surgery (Alsner et al., 2001). Because p53 is only a component of the apoptotic pathway, DNA repair process, and G1 cell cycle arrest, it is unlikely that one event by itself would result in radioresistance.

Bcl-2 and Laryngeal Cancer The expression of bcl-2 has been measured in a variety of human neoplastic tissues including some works on laryngeal carcinomas (Yuen et al., 2001; Fracchiolla et al., 1999; Jackel et al., 1999). However, the prognostic significance of bcl-2 protein expression in tumor pathology is contradictory. Some authors have suggested a

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favorable prognostic role of bcl-2 positivity in breast and non-small cell carcinomas (Villar et al., 2001; Gasparini et al., 1995; Pezzella et al., 1993) while others have found a direct relationship between bcl-2 protein expression and worse prognosis in prostate carcinomas and meningiomas (Krajewska et al., 1996; Karamitopoulou et al., 1998). However, the prognosis of bcl-2 expression has been reported in laryngeal carcinoma though with variable results (Fracchiolla et al., 1999; Jackel et al., 1999; Hirvikoski et al., 1999). Our incidence of positive bcl-2 expression (25%) (Redondo et al., 2006) is similar to most reports on larynx cancer (Pezzella et al., 1993; Trask et al., 2002; Whisler et al., 1998). Bcl-2 expression was found to correlate with tumor grade with a significant higher incidence of bcl-2 expression in poorly-differentiated tumors compared with well-differentiated tumors (Fig 10.1C). The same finding has been reported by others (Pezzella et al., 1993; Krajewska et al., 1996; Karamitopoulou et al., 1998; Whisler et al., 1998). Nodal metastasis was also found to be associated with the expression of bcl-2 which has also been previously reported (Pezzella et al., 1993; Karamitopoulou et al., 1998), although in one series the opposite result was found (Krajewska et al., 1996). Our results also showed a higher incidence of bcl-2 expression in supraglotic tumors compared to other location. Similar findings have been described previously (Karamitopoulou et al., 1998; Hirvikoski et al., 1999). This association may suggest that tumours originating from different sites in larynx may have different tumor biology. By uni- and multivariate analysis bcl-2 was shown to be an independent predictor of good prognosis in these tumors Our data were in agreement with several reports that found an association between high expression of bcl-2 and increased survival in larynx cancer (Xie et al., 1999; Wilson et al., 1996; Homma et al., 2001). However, other reports did not find any relationship between bcl-2 and prognosis (Hirvikoski et al., 1999; Friedman et al., 2001; Kuropkat et al., 2002). A smaller number of patients included in these latter studies may have influence this negative result. It can not be discerned whether bcl-2 is involved directly in contributing to this more indolent phenotype or is simply an epiphenomenon that is a marker for another molecular or biologic process. As bcl-2 does not promote cell proliferation, in the absence of additional genetic alterations bcl-2 positive tumors tend to be relatively non-aggressive. Therefore, bcl-2 expression can be a useful prognostic marker in laryngeal carcinomas and might be helpful in distinguish which tumors with pathological aggressive characteristic (poorly differentiated or with node metastasis) might present a better outcome. Concerning radioresistance, few works have analyzed the relationship between bcl-2 expression and response to radiotherapy. A study reported no changes on bcl-2 expression in recurrent laryngeal cancer after radiotherapy (Lee et al., 2006). However, other works have shown a positive association between expression of bcl-2 and radioresistant laryngeal cancer which suggests a potential mechanism by which cancer cells avoid the destructive effect of radiotherapy (Condon et al., 2002; Nix et al., 2005). Finally, Gallo et al. reported a cumulative prognostic value of simultaneous detection of bcl-2 overexpression and p53 gene aberration in head-and-neck cancer treated by radiotherapy (Gallo et al., 1999).

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Clusterin and Laryngeal Cancer Clusterin has been also shown to present important roles in various physiopathological processes, including tissue remodeling, reproduction, lipid transport and complement regulation (Rosemberg and Silkensen, 1995) and has been found to be altered in various human carcinomas (Trougakos and Gonos, 2002). Its implication in carcinogenesis and progression of some carcinomas (Steinberg et al., 1997, Redondo et al., 2000, Chen et al., 2003) designate CLU as an interesting gene to be explored in other types of carcinomas. We studied a large series of laryngeal carcinomas (Redondo et al., 2006). CLU expression in normal laryngeal tissue is limited to the mucosecretory gland and lymphocyte infiltrate in some cases. Squamous epithelium is negative for CLU expression. However, CLU is up-regulated in a low proportion of laryngeal tumors compared with tumors of glandular origin as prostate or breast (Steinberg et al., 1997; Redondo et al., 2000) (Fig. 10.1D). In these tumors its up-regulation is associated with carcinoma progression. Although the clusterin gene is involved in the carcinogenic pathways in some types of tumors, in laryngeal carcinomas it seems not to exert a significant role in carcinogenesis because its mRNA and proteins are detected in few specimens. However, the low proportion of CLU positive tumors seems to present an aggressive tumor behavior since their expression were associated with local invasiveness. It should be clarified the underlying mechanisms of differential clusterin gene regulation in different tissues. Interestingly, we found a statistical significant association between the expression of CLU and bcl-2 proteins. In a report (Trougakos et al., 2004) CLU knockdown by siRNA induced down-regulation of bcl-2 in two sarcoma cell lines. The exact mechanism of this association should be elucidated. We also tried to correlate the expression of CLU mRNA by in situ hybridization with its protein expression by immunohistochemistry. One study reported differences between both methods in astrocytes and neurons (Pasinetti et al., 1994). In our series of laryngeal carcinomas only one tumor presented RNA expression in cytoplasm without CLU protein expression, which is in accordance with previous studies in other localization (Redondo et al., 2000). Differences in antibody affinity according to cell types or cell state may explain this opposite results. The CLU protein is an inhibitor of apoptosis with a cytoprotective function (Zhang et al., 2005) and thus represents a promising target for molecular intervention strategies such as antisense therapy designed to inhibit its expression, (Olie and Zangemeister-Wittke, 2001). The over-expression of exogenous CLU has been shown to result in resistance to paclitaxel (Miyake et al., 2000), doxorubicin (Cervellera et al., 2000), cisplatin (Chung et al., 2004) and radiation therapy (Zellweger et al., 2001). In contrast, decreased CLU expression by antisense or siRNA expression enhances the chemosensitivities of various cell lines (Gleave et al., 2005; Trougakos et al., 2004; Klokov et al., 2004; So et al., 2005; Redondo et al., 2007), suggesting that CLU expression is a prominent resistance factor in cancer cells.

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Conclusions Identification of prognostic markers of cancer recurrence remains one of the goals of translational research. The proteins p53, bcl-2 and clusterin influence the apoptotic activity and therefore, their alterations may change the prognosis and resistance to chemo- and radiotherapy. Overexpression of bcl-2 and p53 is frequent events in squamous cell carcinoma of the larynx, however controversy remains regarding the prognostic significance of that overexpression. In addition, there are also conflicting data concerning radiosensitivity. We should consider that other components of the apoptotic pathway might play an important role. It is unlikely that only one event by itself would result in radioresistance. In fact, results have made it clear that a number of coordinating alterations in apoptosis-related genes must occur to inhibit apoptosis and provoke carcinogenesis in a wide variety of cancers. Concerning clusterin, although is detected in a low proportion of laryngeal carcinomas, it seems to exert a significant role in local invasiveness. More research is required to increase our understanding of the extent to which and the mechanisms by which they are involved in cancer development, providing the basis for earlier and more accurate cancer diagnosis, prognosis and therapeutic intervention that targets the apoptosis pathways. Acknowledgments This research was supported by a grant from Fondo de Investigaciones Sanitarias (06/1062), Spain.

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Chapter 11

Cyclooxygenase 2 and its Metabolites: Implications for Lung Cancer Therapy Kin Chung Leung and George G. Chen

Abstract The cyclooxygenase 2 (COX-2) and its downstream metabolite prostaglandins play an important role in lung cancer development, progression and metastasis. COX-2 and some of its metabolites have been regarded as markers for diagnosis and prognosis for lung cancer prevention and therapy. There is increasing evidence indicating that the inhibition of the expression or/and the activity of COX-2 can sensitize tumor cells to anti-tumor treatments by promoting apoptosis. Thus the development of selective COX-2 inhibitors has become a hot area in anti-cancer treatment. This chapter will summarize the current development in COX-2 and its metabolites in lung cancer and will particularly focus on the therapeutic value of COX-2 inhibition in lung cancer treatment. Keywords Cyclooxygenase 2 · Prostaglandins · Lung cancer · Apoptosis

Introduction Lung cancer is the leading cause of cancer cell death in the world (Vineis et al., 2004). In western countries, both men and women suffered from this type of cancer with the highest cancer morbidity and mortality. Based on the histological feature, lung cancer can be divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) (Li et al., 2005). According to the data of American Cancer Society, it is predicted there will be around 162,460 new cases of NSCLC in 2007 and the mortality rate is exceeding the sum of colorectal, breast and prostate cancer in US. In Japan, there are annually more than 50,000 people died in lung cancer. 80% of lung cancer is characterized into NSCLC and the remaining belongs to SCLC. NSCLC are subdivided into squamous, adenocarcinoma and large cell carcinoma and it is the prevalent form of cancer found in adults of western countries (Toussaint et al., 1993). SCLC is characterized by its fast dissemination and clinically more G.G. Chen (B) Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 11,

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aggressive than NSCLC. Lung cancer, particularly NSCLC, possesses the high metastatic ability and it is mostly diagnosed after the disease has been developed into an advanced stage. SCLC shows the highest apoptotic index when compared to NSCLC and other types of cancer (Soini et al., 1998). It is not entirely understood what makes such a difference between SCLC and NSCLC. However, the variation may explain their difference in the susceptibility to anti-cancer drugs. The formation of lung cancer is the consequence of multiple factors including environmental, molecular, genetic and epigenetic changes. It has been suggested the lung is the major target for inhaled toxicants and 85–90% of lung cancer is caused by smoking (Jemal et al., 2003). There are 4000 components in cigarette smoking, in which more than 100 of them have been identified as carcinogens. Among them, two of the tobacco components 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and nicotine enhance lung cancer cell proliferation and survival by activation of nuclear factor kappa B (NF-κB) (Tsurutani et al., 2005). NF-κB, a transcriptional factor, may control cell angiogenesis, carcinogenesis, inflammation and apoptosis via regulating several signaling pathways including cycclooxygenase-2 (COX-2) which is a key enzyme in the production of prostaglandins. Cigarette smoking condensate (CSC) is known to activate NF-κB by increasing the p50 and p65 subunits, phosphorylating and degrading IκBα and IκB kinase (IKK) (Fig. 11.1). All these alterations contribute to CSC-induced p65 physophorylation and translocation in NSCLC cells (Shishodia et al., 2004). The expression of COX-2 and its corresponding luciferase activity are also elevated when cells are treated with CSC, suggesting that CSC-mediated carcinogenesis could be caused by the alteration of various prostaglandins in lung cancer cells. Another environmental factor is the exposure to asbestos which contribute to 5–7% lung cancer cases. It has been pointed out that this carcinogen could damage chromosome and DNA and the underlining mechanisms are involved in MAPK and NF-κB signaling pathways (Nymark et al., 2008; Ruosaari et al., 2008). Therefore it is no doubt that NF-κB plays an essential role in lung tumorgenesis. Nicotine derivate NNK is the most potent carcinogen in lung tumorgenesis, which has been tested in a variety of animal models. A couple of molecular modifications, such as single-strand DNA breaks and DNA adduct formation, p53 and RAS mutations are highly associated with NNK (Cloutier et al., 2001; Schuller, 2002). Accumulation of DNA adduct is one of the major causes of cancer formation. NNK is able to regulate Bcl-2, Akt, ERK1/2 and PKCalpha, c-myc, all of which are antiapoptotic molecules, and they play important roles in lung cell proliferation, tumor development and chemoresistance (Jin et al., 2004; West et al., 2003) (Fig. 11.1). Information related to NNK and prostaglandins is limited. There is a study indicating that NNK is able to promote DNA synthesis, which could be the consequence of increase arachidonic acid (AA) production through its binding to β1 and β2adrenergic receptors (Schuller et al., 1999). NNK is suggested to be bioactivated by COX-2 while COX-2 inhibitor NS-398 reduces prostaglandin E2 synthesis in NNK-treated human U937 cells (Rioux and Castonguay, 2000). The above results suggest that increased prostaglandins synthesis may be one of the mechanisms of NNK-induced carcinogenesis.

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Cyclooxygenase 2 and its Metabolites EGFR

NNK

CSC

NF-˧ B

NF-IL6 (C/EBPbeta) NNK

253

MAPK

c-myc

Bcl-2

COX-2 Akt

STAT6

ERK 1/2 PKC alpha

Damage DNA/chromosome PGH2

Cell proliferation

Chemoresistance Apoptosis

Prostaglandin F2˞ Prostaglandin D2 Thromboxane A2 Prostaglandin E2

Prostacyclin

Fig. 11.1 Putative mechanisms by which COX-2 metabolites, CSC and NNK affect lung cancer cell proliferation, apoptosis and chemosensitivities

A study concerning both of the genetic and environmental factors in lung cancer estimated that genetic factor involvement was about 14% (Hemminki et al., 2001). A great quantity of research has also been working on the genetic alterations in lung cancer which has been focusing on markers such as p53, Fas, retinoblastoma (Rb), TNF-related apoptosis inducing ligand (TRAIL) and NF-κB pathways (Motadi et al., 2007). For example, one copy of the chromosomal region 17p13 encodes p53 is frequently mutated in both SCLC and NSCLC. The tumor suppressor protein Rb, one of the G1 cell cycle regulating proteins, is founded to be inactivated in 90% SCLC and 15% SCLC (Motadi et al., 2007). Generally, lung cancer treatments with chemotherapy, radiation and surgery have not yet achieved a satisfactory result. The dismal less than 15% average 5-year survival rate and the disappointing low curing rate has prompted us to discover new therapies for lung cancer. Before the development of novel treatments, it is necessary to better understand the lung carcinogenesis and the molecular pathway responsible for lung cancer chemoresistance.

Apoptosis in Lung Cancer Apoptosis, which is a defined genetically controlled process, is the central mechanism to control cell death in order to maintain cellular homeostasis by balancing cell death and cell proliferation (Shivapurkar et al., 2003). Cells undergo apoptosis

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is characterized by their plasma membrane blebbing, formation of apoptotic bodies, cytoplasm contraction, chromatin condensation and DNA fragmentation that do not generate inflammatory response. An increasing amount of studies in oncology has demonstrated the importance of apoptosis in cancer, as defect of apoptosis leads to cancer development and resistance to treatment with anticancer agents. DNA is the major target of carcinogens. Once DNA adduct is formed, normal cells have the ability of DNA repair by turning on the tumor suppressor genes to facilitate cell cycle arrest or start apoptosis machinery. Whereas cells with oncogene overexpression or defects in tumor suppressor genes, which is frequently observed in tumor, are unable to eliminate mutated or transformed cells by apoptosis thus favor cancer development. Therefore apoptosis induction is a promising approach in cancer therapy. Since the turn-on and -off of pro-apoptotic and anti-apoptotic factors depends on the type of stimulation and cell type, it is of particularly important for clinicians and scientists to understand the molecular events that contribute to drug-induced apoptosis and by what mechanism cancer cells escape from apoptotic cell death. Certain pro-apoptotic and anti-apoptotic molecules that are highly associated with lung cancer are summarized here.

Cysteine Aspases (Caspase) Pathway Apoptosis is mainly executed by a group of cysteine proteases called caspases. Caspases are cysteine-aspartic-acid-proteases that cleave protein with an aspartic acid residue in the P1 position. Normally caspases are maintained at their inactivated forms in order to keep the apoptotic program under control. Caspases are involved in both apoptotic pathways, namely the extrinsic death receptor and intrinsic mitochondria pathway. The extrinsic pathway depends on the ligand binding to the cell membrane receptors to initiate apoptosis. Ligands that are responsible to trigger extrinsic pathways and their respective receptors are TRAIL (DR4 and DR5), tumor necrosis factor TNF (TNFR1) and FasL (FAS-R). Once the ligands bind to their receptors, they recruit adaptor molecules Fas-associated death domain (FADD) and TNFR-associated death domain (TRADD) leading to the activation of two initiator molecules caspase 8 and caspase 10. This will promote the cleavage of Bid to its truncated form, enables the BAX/BAK pro-apoptotic molecules to insert into the mitochondrial membrane and release cytochrome c into the cytosol. Loss of caspase 8 expression is frequently observed in 8 of 10 SCLC cell lines (Joseph et al., 1999) and homozygous deletion at chromosome 2q33 encoding capase 8 has been noted in SCLC (Shivapurkar et al., 2002). The latter experiment also indicates that neither the expression of caspase 8 protein nor its activity is present in SCLC cells, reflecting the caspase 8 is one of the tumor suppressor proteins in SCLC. The second type of apoptosis which depends on the release of cytochrome c from the mitochondrial intermembrane to the cytosol, is called the intrinsic pathway. After releasing to the cytosol, cytochrome c will be in complex with apoptosis protease activating factor-1 (Apaf-1), ATP and pro-caspase 9. They will then form

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the apoptosome to enable the cleavage of caspase 9. Caspase 9 is an initiation protein which will in turn activate several executioner proteins such as caspase 3, caspase 6 and caspase 7 (Adrain and Martin, 2001). The activated executioner proteins will cleave protein substrates within the cells, resulting in apoptosis (Huerta et al., 2007). The effectiveness of several chemotherapeutic drugs depends on the activation of caspase in lung cancer treatment. For example, bortezomib induces the mitochondrial dependent apoptosis in NSCLC H460 cells by activating caspase 9 and caspase 3 (Voortman et al., 2007a).

Bcl-2 Family Bcl-2 is overexpressed in 50% NSCLC and 69–90% SCLC (Mortenson et al., 2005; Ohmura et al., 2000). At least 20 Bcl-2 members have been identified and they divide into 2 groups as pro- and anti-apoptotic Bcl-2 members. The pro-apoptotic (Bax, Bak, Bid, Bik) members share a short BH3 region. The ratio of pro- and anti-apoptotic Bcl-2 family members determines the permeability of cytochrome c to pass through the mitochondrial membrane. Thus an increase in the relative ratio of pro- to anti-apototic molecules enables the leakage of cytochrome c into the cytosol, which leads to apoptosome formation. Overepression of Bax has been demonstrated to promote apoptosis in lung cancer cell lines but not in normal cell lines with the activation of caspase (Kaliberov et al., 2002). The anti-apoptotic members (Bcl-2, Bcl-xL, Bcl-M) prevent apoptosis through stabilizing the mitochondrial membrane modifications, preventing cytochrome c translocation and apoptosome formation. The anti-apoptotic Bcl-2 members may protect lung cancer cells from cell death induced by various agents. Such apoptosis protective effect has been proved in several models; such as overexpression of Bcl-2 reduced ∼50% apoptosis in Bortezomib-treated H460 cells (Voortman et al., 2007a). In addition, the application of platinum-based therapeutic agents on SCLC and NSCLC improves the quality of life and survival rate. Cisplatin (cis-diamminedichloroplatinum) is a chemotherapeutic agent commonly used in many cancer treatments. The expression of Bcl-2 contributes to the resistance to cisplatin in many cancer cell types (Wang et al., 2008; Wu et al., 2005; Yang et al., 2004), partly due to the upregulation of Bcl-2. An in vitro study revealed a synergistic enhancement of apoptosis in NSCLC A549 cells treated with cisplatin and the knockdown of Bcl-2 in combination (Losert et al., 2007). The reduction of Bcl-2 expression and activity is important for bortezomib-induced apoptosis in SCLC cells (Mortenson et al., 2005).

NF-κB NF-κB is a group of transcriptional factors (p50/p105, p52/p100, RelA, c-Rel, Rel-B) that normally present in an inactive form in the cytosol binding with its regulator IκB. The crucial role of NF-κB in tumoregenesis and their chemoresistance has been broadly described in different publications (Baldwin, 2001; Karin

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and Lin, 2002). IκB controls the activation of NF-κB by degradation and phosphorylation. In response to injury, infection, inflammation and other stress conditions, IKK phosphorylates IκB that enables its degradation and detach from NF-κB/IκB complex, allowing the two NF-κB subunits (p50/p65) to translocate into the nucleus. Activated NF-κB binds to the responsive promoter region to increase the target gene level. Numerous target genes of NF-κB have been identified, which are correlated with cell proliferation, metastasis, apoptosis and inflammation. They included Bcl-2, Bcl-xL, inhibitors of apoptosis (IAP), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) (Bharti and Aggarwal, 2002; Karin and Lin, 2002; Zhang et al., 2007). NF-κB has been related to lung cancer tumoregensis and inhibition of NFκB is effective to facilitate apoptosis. It is well documented about the involvement of chemoresistance by NF-κB, especially in TRAIL-induced apoptosis (Luo et al., 2005; Papa et al., 2006; Voortman et al., 2007b). Early NF-κB activation is only observed in ethyl carbamate (urethane)-induced lung tumor (Stathopoulos et al., 2007). Blockage of NF-κB by its dominant inhibitor has been shown to reduce Bcl-2 expression and enhance apoptosis leading to decreasing carcinogenesis by more than 50% in a transgeneic mice model. The prognostic value of RelA (an indicator for NF-κB activity) and IκBα has been evaluated in 116 patients with stage I and II NSCLC (Zhang et al., 2007). Immunohistochemisty demonstrated that more positive RelA/NF-κB and phosphorylated IκBα staining was detected in tumor tissues than adjacent normal tissues, suggesting that RelA/ NF-κB and IκBα phosphorylation may be used as a prognostic marker in lung cancer patients. Activation of NF-κB leads to resistance to apoptosis induced by celecoxib and aspirin in lung cancer cell line, possibly due to increasing its downstream anti-apoptotic genes, such as Bcl-2, Bcl-xL and survivin (Gradilone et al., 2007).

COX-2 in Lung Cancer AA is a 20 carbon unsaturated fatty acid and it mainly situates in the lipid bilayer of the cell. Once it is liberated by phospholipase, it can be metabolized by three pathways namely the COX pathway, the lipoxgenase pathway and the cytochrome P-450 monooxygnease pathway (Williams et al., 1999). COX is the rate limiting enzyme responsible for converting AA to prostaglandin H2 (PGH2 ). The intermediate product will then be metabolized to several types of prostaglandins (prostacyclin, thromboxane A2 , prostaglandin D2 , prostaglandin E2 and prostaglandin F2α ) by prostaglandin synthase (PGES) and thromboxane synthase (THXS) (Fig. 11.1). The end products of COX are often called eicosanoids. The local signaling eicosanoids in turn exert their activities through binding to their corresponding receptors (EP1, EP2, EP3, EP4, FP and thromboxane receptor (TXR)). At least two isoforms of COX, COX-1 and COX-2, have been identified. COX1 is constitutively found in most tissues while COX-2 is an inducible enzyme. COX-1 and COX-2 are encoded by different genes (Herschman, 1996). Each of them have unique properties. Both of the isoforms can synthesize prostaglandins.

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Prostaglandins metabolized by COX-1 are mainly responsible for the protection of stomach lining whereas those COX-2 producing prostaglandins are associated with pain and inflammations. Therefore, COX inhibitors that specifically act on COX2 but not COX-1 can reduce the toxicity to the gastro-intestinal tract to prevent gastric ulceration (Sarkar et al., 2007). The expression of COX-2 could be regulated by several factors such as interleukin (IL-1), phorbol ester, tumor necrosis factor α (TNFα), platelet-derived growth factor, serum, peroxisome proliferatoractivated receptor-gamma (PPARγ), Bombesin (BN) or gastrin-releasing peptide (GRP), epidermal growth factor, lipopolysaccharide, retinolic acid, tumor promoters and forskolin (Bazan et al., 1994; Bren-Mattison et al., 2008; Brown and DuBois, 2004; DeWitt and Meade, 1993; Hohla et al., 2007; Kujubu et al., 1991; Kujubu and Herschman, 1992). A recent in vitro experiment suggests that COX-2 is the target of epidermal growth factor receptor (EGFR) in lung cancer cell because EGFR kinase inhibitor decreases COX-2 protein level (Chen et al., 2008a) and it may be probably mediated by inhibition of NF-κB (Fig. 11.1). The role of COX-2 in cancer research has been extensively studied. Recent studies suggest that this enzyme and its metabolites play an active role in tumorgenesis through promoting tumor angiogenesis, suppressing apoptosis, stimulating cell growth (Antonakopoulos and Karamanolis, 2007; Liao et al., 2007; Sarkar et al., 2007). Because of the role of COX-2 and its metabolites in tumorgenesis, COX-2 has been used as a diagnostic marker and a therapeutic target in lung cancer (Campa et al., 2004; Shaik et al., 2004). Similar to many other cancers, the majority of lung cancers show the overexpression of COX-2 (Krysan et al., 2006). It has been reported that there are 70–90% NSCLC overexpressed COX-2 (Csiki et al., 2005). In one of the investigations in human lung adenocarcinomas, the overexpression of COX-2 is found in all of the tumor specimens. Several downstream enzymes under COX-2 pathway, such as THXS, prostaglandin D2 synthase and prostaglandin E2 synthase, could also be stained in adenocarcinomas (Ermert et al., 2003). This study has also demonstrated the high levels of COX-2 and THXS immunostaining in the endothelial cells inside the lung tumor area, suggesting that a possible role of COX-2 and THXS in lung tumor angiogenesis. Therefore, COX-2 together with its downstream enzymes and corresponding prostaglandins are believed to participate in NSCLC pathogenesis and development. It is also worth noting that COX-2 overexpression is observed in NSCLC but not in SCLC. This is evident by the protein expression comparison between NSCLC cell line NCI-H2126 and SCLC cell line DMS-79, which shows that COX-2 is present in NSCLC but absent in SCLC (Alam et al., 2007). The result is in line with the study performed in lung tumor specimens (Petkova et al., 2004) and in other culture experiments (Tsubouchi et al., 2000). However, some COX-2 inhibitors can still exert its pro-apoptotic function in several SCLC cell lines, implying the anti-cancer (pro-apoptotic) effect of COX-2 inhibitors is possibly due to the unspecific COX-2 inhibition in SCLC. The chemoprevention effect of COX-2 inhibitors and non-steriodal antiinflammatory drugs (NSAID) has been well reported. Blockage of COX-2 by daily intake of NSAID (aspirin and ibuprofen) for 1 or 2 years reduces 60–68% of relative

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risk of lung cancer (Harris et al., 2007; Harris et al., 2002). The result prompts to develop a new therapeutic tool in the lung cancer treatment by using COX inhibitors. It is now proved that COX-2 inhibitors can successfully inhibit tumor cell growth in animal models of lung cancer. Table 11.1 shows currently available COX-2 inhibitors. Table 11.1 List of COX-2 inhibitors Name

Aspirin

Structure

Market name

Reference

Aspirin

(Wennogle et al., 1995)

NS-398

(Yamamoto and NozakiTaguchi, 1996)

Etoricoxib

Arcoxia

(Wilton, 2004)

Celebex

Celebrex

(Wilton, 2004)

rofecoxib

Vioxx, Ceoxx and Ceeoxx.

(Wilton, 2004)

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Prostaglandins and its Enzymes/Inhibitors Physiological functions of prostaglandins include the regulation of immunity, kidney development and reproductive biology (Williams et al., 1999). Dysregulation of these molecules has been proved to take part in the fundamental role in carcinogenesis, tumor progression, metastasis, the production of proangiogenic factors and drug resistance (Steele et al., 1999; Tsujii et al., 1997; Tsujii et al., 1998). The levels of prostaglandins are frequently increased in tumor cells. For example the endogenous level of PGE2 was much lower in premalignant bronchial epithelial cell lines BEAS-2B and 1198 (0.2 and 0.04ng/106 cells respectively) compared to 2.7ng/ 106 cells in cancer cell line A549 (Schroeder et al., 2007). Since the increased level of PGE2 is closely related to the enhanced expression of COX-2 in tumors and COX2 is the rate-limiting step for the catalysis of prostaglandin synthesis (Dohadwala et al., 2002; Hubbard et al., 1989; Sharma et al., 2003), it is believed that COX-2 inhibitors can effectively arrest cancer cell growth, metastasis, invasion and angiogenesis through the reduction of prostaglandin production. The major prostaglandin found in NSCLC is PGE2 which is synthesized by microsomal PGE2 synthase. The amount of PGE2 is reduced when COX-2 expression is suppressed by PPARγ in NSCLC cells (Bren-Mattison et al., 2008) or by celecoxib in a hamster model of lung cancer (Vegeler et al., 2007). Mounting researches have been taken to examine the apoptosis induced by COX2 inhibitors. NS-398, one of the selective COX-2 inhibitors, can effectively induce apoptosis in NSCLC A549 cells, characterized by the formation of DNA fragmentation and the release of cytochrome c from the mitochondria (Fang et al., 2004) (Fig. 11.2). Moreover, it can also alter the expression of PEG2 receptors (EP1, EP2, EP3, EP4) and PGF2α receptors (FP) in the mitochondria, suggesting that the modulation of cytochrome c may be mediated through the change in prostaglandins. In the A/J mouse model of NNK-induced lung cancer, NS-398 inhibits lung tumorgenesis, promotes the apoptosis and stimulates the expression of FasL and BAD (Yao et al., 2000). The suppression of lung cancer growth by NS-398 has also been associated with the increase of p21 (Han and Roman, 2006). In contrast to the proapoptotic response shown above, NS-398 promoted cell growth in A549 cell (Duan and Zhang, 2006). Another study showed that NS-398 induced G1 cell cycle arrest rather than apoptosis in A549 cell lines and H229 lung cancer cells are resistance to apoptosis triggered by NS-398(Chang and Weng, 2001). This indicated that that NS-398 may possess both cell growth and cell inhibition effect on lung cancer cells and its effect on lung cancer treatment should be further elucidated. In addition to NS-398, other COX-2 inhibitors have also been shown to inhibit the growth of lung cancer both in vitro and in vivo. For example, it is reported that Nimesulide is able to inhibit both NSCLC and SCLC cell growth by promoting the apoptosis (Hida et al., 2000). The pro-apoptotic effect of Nimesulide seems not to related to the p53 status, suggesting that Nimesulide-mediated anti-tumor effect may be p53-independent. Celecoxib, the first approved NSAID to treat arthritis and adenomatous polyposis (Everts et al., 2000), can also significantly increase apoptosis

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↑ FasL, Bad NS-398 ↑ caspase 3

↑ pERK ↑ pERK ↑ p21

↑ PGE 2

↑ p21

↑ PPARγ

EP1, EP2, EP3, EP4, FP Cytochrome C CSC

Celecoxib

↑ caspase 8 Cleavage

↑ caspase 3 ↑ PARP

↓ Mcl-1

↓ cFLIP

NF-˧ B

↑ DR5

Fig. 11.2 Anti-lung tumor signal pathways of COX-2 inhibitors Celecoxib, Nimesulide and NS-398

of lung cancer cells (Schroeder et al., 2007). In Female nu/nu mice model, apoptosis was dose-dependently triggered by Nimesulide without affecting the COX-2 expression level, indicating that the apoptosis elicited by Nimesulide is COX-2 independent. The overexpression of PPAR-gamma and reduction of PGE2 is suggested to take part in the Nimesulide-induced apoptosis (Shaik et al., 2004) (Fig. 11.2). The apoptotic pathway mediated by Celecoxib is likely through the extrinsic death receptor pathway (Liu et al., 2004). There are three pieces of evidence to support the extrinsic death receptor pathway mediated by Celecoxib (Fig. 11.2). First, Celecoxib induces the cleavage caspase 3, caspase 8 and PARP. The caspase 8 may be particularly critical in Celecoxib-mediated cell death since the Celecoxib-induced apoptosis can be inhibited by caspase 8 siRNA. Second, Celecoxib stimulates the transcriptional level of DR5. Third, Bcl-2 does not seem to play a role in the Celecoxib-induced apoptosis in this study, suggesting that the action of Celecoxib is not through the intrinsic mitochondrial-mediated apoptotic pathway. The anti-tumor effect of Celecoxib may also be associated with NF-κB since it can abrogate the CSC-induced NF-κB by modulating the phosphorylation status of NF-κB and IκBα (Shishodia and Koul, 2004). However, Celecoxib fails to exert an anti-tumor effect on lung tumor hamster model induced by N-nitroso-bis(2oxopropyl)amine (BOP) (Vegeler et al., 2007). Therefore, there appears to be some differences between human lung cancer and the hamster lung tumor model, which requires further study to clarify the difference. Natural products of COX-2 inhibitors have also shown to exert anti-proliferation or pro-apoptotic effects in lung cancer. For example, deguelin a natural product

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isolated from Mundulea sericea Willd blocks the COX-2 protein expression in squamous human bronchial epithelia cells (HBE), induces apoptosis in H322 non-small cell lung carcinoma and HBE and upregulates BAX (Lee et al., 2004). There is a study indicating that the suppression of lung tumor growth as well as the increased p21 by COX-2 inhibitor NS-398 may be independent of COX-2 inhibition since both indexes are not affected by COX-2 siRNA (Han and Roman, 2006). However, this point of view is not supported by other studies using either NS-398 or celecoxib as COX-2 inhibitors. NS-398 augments the effects of radiation in COX-2-overexpressing cells but such an effect is not found to be true in COX-2 nonexpressing cells (Pyo et al., 2001), suggesting that the effect of NS-398 relies on the expression of COX-2. Similarly, Celecoxib’s radiation-enhancing effect is observed in COX-2-expressing A549 and NCI-H460 cells but not in the COX-2 nonexpressing MCF-7 and HCT-116 cells (Shin et al., 2005). Furthermore, the Celecoxib’s radiation-enhancing effect in A549 cells disappears after COX-2 is knocked down, whereas the HCT-116 cells are radiosensitized by celecoxib after being transfected with COX-2 expression vector. The findings strongly support that the anti-tumor effect of Celecoxib is COX-2 dependent in lung cancer cells. The concept of COX2-dependent tumor suppression by COX-2 inhibitors is also supported by COX-2 sense and anti-sense study, showing that COX-2 sense DNA significantly heightens the resistance of NSCLC cells to radiation- and drug-induced apoptosis whereas COX-2 antisense DNA sensitizes NSCLC cells to apoptosis induction (Krysan et al., 2004).

Prostaglandins in Lung cancer Thromboxane Thromboxane is one of the metabolites produced in the AA pathway in which the CYP superfamily member THXS converts the PGH2 into thromboxane A2 (THXA2 ) (Miyata et al., 1994; Shen and Tai, 1986a). This enzyme is located in the long arm of chromosome 7 band q33-q34 (Miyata et al., 1994) and it has been purified from human platelets (Haurand and Ullrich, 1985) and porcine lungs (Shen and Tai, 1986b). Its metabolite will exert it function by binding to its specific receptors called TXR, which belongs to the heptahelical superfamily of G-protein-coupled receptors. The level of thromboxane B2 (THXB2 ), a stable product of THXA2 is increased in various types of lung cancers, including lung squamous cell carcinoma, adenocarcinoma, small cell carcinoma, mixed cell carcinoma, bronchioloalveolar cell carcinoma, large cell undifferentiated carcinoma, bronchial carcinoid and metastatic tumors (McLemore et al., 1988); (Chen et al., 2006). The findings suggest that thromboxane may play a positive role in lung tumor development, progression and metastasis. To confirm the role of thromboxane in lung cancer, a number of THXS inhibitors or thromboxane antagonists have been tested in several models of lung tumors. Thromboxane antagonists (S-1452) and (ONO-NT-126) enhance the cisplatin-induced apoptosis in human lung adenocarcinoma cells by increasing caspase 3 protein expression (Fujimura et al., 1999). The role of caspase 3

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in the apoptosis enhanced by thromboxane antagonists is verified in an inhibitory experiment, in which the apoptosis is blocked by caspase 3 inhibitor (Ac-DEVDCHO) but not by caspase 1 inhibitor (Ac-YVAD-CHO). In another study, the THXS inhibitor Ketoconazole is used to confirm the role of thromboxane against metastatic lung tumors. Ketoconazole significantly prevents melanoma cell metastasis to lung in mice receiving subcutaneous injection of tumor cells (Nardone et al., 1988). Our current unpublished data also show that thromboxane synthase inhibitor 1Benzylimidazole (1-BI) leads to either cell cycle arrest or apoptosis in NSCLC and SCLC cells and that p53 is crucial for 1-BI induced apoptosis. The mechanism responsible for anti-lung tumor effect by THXS inhibitors or thromboxane antagonists is not clear. However, the above studies appear to suggest that it may involve a multiple factors including caspase 3, p53 and cell cycle. It may also be related to NF-κB and COX-2 since the activation of TXR alpha can result in the increase of NF-κB activity and the induction of COX-2 expression in A549 human lung adenocarcinoma cells (Wei et al., 2007). As discussed in previous sections, both NF-κB and COX-2 play a positive role in lung cancer cell growth and the inhibition of COX-2 has been a well-known strategy to prevent or suppress the lung cancer development. It is possible that the reduction of thromboxane by THXS inhibitors may leave TXR in a rest status, leading to the inhibition of NF-κB activity and COX-2 expression in lung cancer.

Prostaglandin E2 Microsomal prostaglandin E synthase (mPGES) has been shown to be overexpressed in lung cancer (Yoshimatsu et al., 2001). An anti-tumor pigment Shikonin has been shown to reduce the expression of COX-2 and the level of PGE2 in a concentration-dependent manner and the inhibition results in the increased cell death of A549 lung tumor cells via an apoptotic pathway, in which caspase 3, caspase 9, and poly-ADP ribose polymerase (PARP) are activated; the sub-G1 population is increased and the ratio of Bax to Bcl-2 is elevated (Lim et al., 2007). In addition to Shikonin, several other chemicals also show to inhibit the growth of lung cancers by suppressing the production of PGE2 . For example, Wikyungtang, a herbal chemical, exerts its apoptotic effect in A549 cells by activating caspase 3 and caspase 9, cleaving PARP, suppressing Bcl-xL and COX-2 (Park et al., 2004). Bee venom (BV), a chemical used in rheumatoid arthritis treatment, can trigger apoptosis in H1299 lung cancer cells via a similar mechanism to Shikonin and Wikyungtang (Jang et al., 2003). Collectively, Bcl-2 family and caspase activation appear to be the major molecules participated in apoptosis induced by the reduction of PGE2 .

Prostacyclin I2 Prostacyclin synthase (PGIS) converts the PGH2 to either prostacyclin I2 (PGI2 ) or PGE2 . The role of PGI2 in cancer tumorgenesis is different from other prostaglandins. It has been hypothesized that this metabolite negatively regulates lung cancer cell

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growth. The production of PGI2 is abundant in normal lung but is significantly lowered in lung cancer adenocarcinoma cells (Hubbard et al., 1988; Keith et al., 2004), suggesting that there may be some protective function exerted by PGI2 in lung cancer development and that the lack of PGI2 may be important in lung tumorgenesis. The negative role of PGI2 in lung cancer development may also be supported by the immunohistochemical staining result, which shows that the THXS, prostaglandin D2 synthase, and prostaglandin E2 synthase are all positive in adenocarcinomas and squamous cell carcinomas but negative for PGIS (Ermert et al., 2003). The level of PGI2 mRNA and protein is down-regulated in lung tumor tissues compared to their surrounding normal lung tissues (Nana-Sinkam et al., 2004). Further studies show that NSCLC cells lack PGIS and thus do not synthesize appreciable amounts of PGI2, and that the epigenetic mechanism contributes to the down-regulated PGIS expression (Kreultzen et al. 2007; Stearman et al. 2007). In addition to the inhibition of primary tumors in lung, PGI2 may also effectively attenuate lung metastasis (Cuneo et al., 2007). Although an increasing number of studies supports that PGI2 is against carcinogen- and tobacco smoke-induced lung tumor formation and thus favors chemoprevention to lung cancer, the information on the molecular pathway leading to inhibitory function of PGI2 is limited. However, there is a study showing that PGI2 may exert its inhibitory effect on lung cancer cells through a cell surface G protein-coupled receptor (PGI2 -binding receptor) and also by interacting with a nuclear hormone receptor, peroxisome proliferator-activated receptor delta (PPARdelta) (Fukumoto et al., 2005). There is also a study indicating that PGI2 may not have a role in the inhibition of lung cancer. Neilan et al. (2006) show that lung tumor growth inhibition caused by doxorubicin (DOX) is not affected in the presence of a synthetic analogue of PGI2 , iloprost, in the model of C57BL/6 mice implanted with Lewis lung carcinoma.

COX-2 Inhibitors and Chemotherapy Most of lung cancers are known to express a high level of COX-2 and the increased COX-2 expression is associated with a worse prognosis (Edelman et al., 2008). Further it appears that the higher level of COX-2, the more resistant to anti-tumor treatments in lung cancer. COX-2 and its metabolites are believed to confer cells resistant to targeted therapy (Krysan et al., 2006; Reckamp et al., 2006). Therefore, the level of COX-2 has been regarded as a chemoresistant marker in lung cancers (Mathieu et al., 2004). The subcellular location of COX-2 may also contribute to the chemoresistance of tumors since it shows that tumor cells with the increased COX-2 in the mitochondria are highly resistant to apoptosis (Liou et al., 2005). The above data have clearly pointed out that the inhibition of COX-2 may potentially enhance chemotherapy by removing the COX-2-mediated chemoresistance. Accordingly, the application of COX-2 inhibitors to enhance the chemosensitivites of anti-tumor drugs has been become a feasible approach in the treatment of lung cancer.

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Lung cancer, especially advanced tumor, is often resistant to gemcitabine, a chemotherapeutic drug. However, in the presence of NS-398, lung adenocarcinoma becomes susceptible to apoptosis induced by gemcitabine (Chen et al., 2008b). In another in vitro study using Nimesulide as an adjunct agent in a panel of lung cancer cell lines, the IC50 of SM-5887-13-OH, SN-38, Taxotere, VP-16 and CDDP can be decreased to various levels with supra-addictive effect shown in some cases (Hida and Kozaki , 2000). A simply additive effect has also been noted in the same study with the co-treatment with radiation and Nimesulide. JTE-522 is another COX-2 inhibitor that can reduce the cell proliferation as well as PGE2 production in lung cancer cells. Combined treatment with Nimesulide and Doxorubicin can enhance apoptosis and caspase 3 activity in A549 cells (Haynes et al., 2003). The significant inhibition of human lung cancer cell growth both in vitro and in vivo has been achieved by the use of JTE-522 and conventional anticancer agents in combination (Hida et al., 2002). However, chemosensitivity enhancement by COX-2 inhibitors is not guaranteed in some situations. For example, combination therapy using Celecoxib and NF-κB inhibitor (LC-1) fails to show statistically significant in the reduction of both the tumor size and the tumor number in a hamster lung tumor model (Vegeler et al., 2007) and the authors explain that the failure may result from the insufficient inhibition of NF-κB. The elevated apoptosis by Celecoxib and other anti-cancer agents in combination is correlated to the decreased level of PGE2 . Docetaxel, a cytotoxic antimicrotubule agent, is clinically approved to treat breast cancer, ovarian cancer and NSCLC. However this drug generates the side effect of phosphorylating Bcl-2 and prevents cancer cells to undergo apoptosis. As a result, the combination of Celecoxib and Docetaxel may be a promising approach in lung cancer treatment. This strategy has been demonstrated to be very effective in mice implanted with A549 tumors (Shaik et al., 2006). Celecoxib and Docetaxel in combination can greatly suppress the growth of the tumor with a marked reduction of mPEGS-generating PGE2 levels but a significant increase in apoptosis. A similar result was also observed in Nu/Nu mice implanted with A549 cells. It revealed that apoptosis was detected by co-treatment with Docetaxel and Celecoxib, which elevated Fas and caspase 3 level and decreased PGE2 (Fulzele et al., 2006). Indomethacin, one of the COX-1 and COX-2 inhibitors, can also reinforce the chemosensitivity of anti-cancer agents by promoting apoptosis. In A549 lung adenocarcinoma cells the cancer killing effect of peroxisome proliferator-activated receptor-delta (PPARδ) agonist L-165041 can be enhanced by indomethacin and the cell death is believed to be related to the increased levels of pro-apoptotic Bax and p53 and the decreased level of anti-apoptotic Bcl-xL (Fukumoto et al., 2005). The inhibitory effect of indomethacin and L-165041 in combination may associate with the reduction of polyamine, a molecule that plays a role in maintaining cell proliferation and apoptosis (Seiler and Raul, 2005). Indomethacin and L-165041 in combination can stimulate the activity of spermidine/spermine N1-acetyltransferase (SSAT) (Fukumoto et al., 2005). The increased SSAT reduces the level of the polyamine and subsequently induces apoptosis of lung cancer cells. The molecular mechanism responsible for the anti-tumor effect of COX-2 inhibitors such as Celecoxib has been evaluated. In one of the studies, celecoxib was

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used together with epidermal growth factor receptor kinase inhibitor (EGFR-KI) ZD 1839 to treat lung cancer cells and it is found that apoptosis is synergistically increased in three of the lung cancer cell lines (A549, GLC82 and SW 1573), with a greater extend of the downregulation of cell proliferative signal transduction kinases p-AKT, p-ERK, the reduction of the nuclear NF-κB and the increased level of cytosolic NF-κB (Chen et al., 2008a). Celecoxib can also increase the apoptotic response when cells are co-treated with TRAIL, leading to the great activation of caspase and the increased cleavage PARP (Liu et al., 2004). Both COX-2 and 5-lipoxygenase (5-LOX) are categorized as the AA metabolizing enzymes and implicated in lung tumorgenesis. It is interesting that blocking one of the AA pathways (COX-2 and 5-LOX) can favor the others due to the re-directing the breakdown of their common substrate AA. Based on this concept, the dual inhibition of COX-2 and 5-LOX may therefore generate a synergistic effect on lung cancer cell growth. This has been proven by an in vitro study, which demonstrates that the treatment of lung cancer cells with Celecoxib, MKK886 (5-LOX–activating protein inhibitor) and REV5901 (5-LOX inhibitor) dramatically increases cell death (74.4% apoptotic and 23.7% necrotic) compared to the treatment with a single agent (Schroeder et al., 2007). Moreover, the exogenous addition of PGE2 and 5-HETE partially rescues the cell death induced, suggesting that the reduction of these two metabolites is one of the factors involved in the synergistic effect by MKK886, REV5901 and Celecoxib. Collectively, it is encouraging to use COX-2 inhibitors as an adjunct agent with anti-cancer drugs in order to benefit from reducing their side effects and to potentiate their cancer killing abilities.

Conclusions Growing incidence of lung cancer has been observed in both developed and developing countries and the cancer is with the less than 15% 5 year survival rates. This malignancy is often highly resistance to chemotherapies and the current treatments have changed little on this statistics. Examination of COX-2 and prostaglandins levels is one of the molecular approaches not only to enable us to make early diagnosis but also to provide evidence to target COX-2 or its metabolites in lung cancer. The modulation of COX-2 and prostaglandin levels, along with other anti-cancer agents, has shown promising results in increasing chemosensitivity, and reducing side-effects in treatment of lung cancer. Acknowledgments The study was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region (Project No. CUHK4390/03M).

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Chapter 12

Roles of Negative and Positive Growth Regulators in Nasopharyngeal Carcinoma Mong-Hong Lee, Huiling Yang, Ruiying Zhao and Sai-Ching J. Yeung

Abstract Cell cycle dysregulation plays an important role of tumorigenesis. Nasopharyngeal carcinoma (NPC) cells are affected by numerous cell cycle regulators either act as negative or positive regulators, and their aberrations result in proliferative advantage for NPC cancer cells. Many cell cycle regulators are deregulated in NPC providing the growth advantage. The molecular regulators associated with cell cycle growth are of particular interest because they are potential therapeutic targets for NPC. We focus on recent advances in regulators of the cell cycle and discuss their potential use as therapeutic targets for NPC. Keywords Cell cycle · Nasopharyngeal carcinoma · Signaling transduction

Introduction Nasopharyngeal carcinoma (NPC), a malignancy arising from the epithelium lining of the posterior nasopharynx, is endemic in Southern China and Southeast Asia, with a characteristic of remarkable racial and geographic distribution, and has caused very serious health problem in these areas (Yu and Yuan 2002). Etiological studies suggested that Epstein-Barr virus (EBV) infection, dietary exposure to carcinogens (Yu 1990), and genetic susceptibility are associated with NPC (Hildesheim and Levine 1993). Studies showed that the tumorigenesis of NPC could be caused by Epstein-Barr virus infection and accumulation of epigenetic and genetic deregulation. Radiotherapy and chemotherapy are the most common treatment modalities for NPC. However, the patient outcome is not ideal. Therefore, there is an urgent need to improve NPC treatment. To develop better treatment approaches, it is important to understand the molecular basis of the development and progression of NPC. Like many cancers, NPC is also a proliferative disease in which the cell cycle regulatory machinery becomes deregulated during tumorigenesis. There is an obvious M.-H. Lee (B) Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 12,

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unbalance between negative and positive regulators of the cell cycle machinery in NPC. Many NPC researchers have determined how oncogenic signals and compromised tumor suppressor activity are involved in regulating cell cycle during tumorigenesis. Understanding the roles of these oncogenic mediators and tumor suppressors in normal and cancerous cells will help us exploit the pathways involved in their regulations to rationally develop novel cancer therapy. In this review, we focus on various molecular targets dysregulated in NPC, discussing their potentials as therapeutic targets for NPC. Negative growth regulators (Table 12.1) and positive growth regulators (Table 12.2) in NPC can be strategically manipulated for targeted therapy.

Negative Growth Regulators of NPC p16 The p16 Ink4a protein, a CDK4 interacting protein that inhibits CDK4 kinase activity (Serrano et al. 1993), is located on chromosome 9p21 (Kamb et al. 1994). 9p21 is the hot spot of genomic alterations in cancers, and has been frequently deleted in NPC (61% of primary tumors) (Lo et al. 1995; Makitie et al. 2003). Numerous studies have demonstrated a high frequency of p16 deletion in other malignancies (Cairns et al. 1995; Kamb 1995). The frequent inactivation of p16 in cancers is consistent with its function as a tumor suppressor, and p16’s tumor suppressive activity can be attributed to its ability to bind both CDK4 and 6 and to inhibit the catalytic activity of the cyclin D/CDK enzyme complex, which is required for phosphorylation of the retinoblastoma protein (pRb) for cell cycle progression. The inactivation of p16 in NPC can occur by three mechanisms: homozygous deletion, promoter hypermethylation, and point mutation. Methylation of 5’ CpG islands is an important mechanism of transcriptional repression, and evidence suggests that methylation of p16 promoter can play an important role in NPC tumorigenesis (Kwong et al. 2002). Treatment of with 5-deoxyazacytidine, a DNA methylaion inhibitor, results in a significant decrease in p16 promoter methylation, leading to reexpression of p16 and G1 cell cycle arrest in cell culture. Given p16’s role in NPC development, restoration of p16 expression have been investigated as gene therapy for NPC (Lee et al. 2003) (Wang et al. 1999). It has been shown that adenoviralmediated p16 gene transfer is highly effective in inhibiting growth of NPC that lack functional p16. Thus therapeutic strategies aiming at restoring p16 is a biologically rational approach, and gene replacement of p16 can offer a therapeutic strategy for NPC.

ARF Ink4a/arf locus encodes two different proteins derived from alternative splicing: p16 (Ink4a) and p14 (ARF, alternative-reading frame protein)) (Lloyd 2000; Quelle

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Table 12.1 Selected negative growth regulators for NPC Protein

Function/characteristics

P16

a CDK4 interacting protein that inhibits CDK4 associated kinase activity; located on chromosome 9p21; 9p21 loss, where p16 is located, has been frequently observed in NPC (61% of primary tumors) Ink4a/arf locus encodes two different proteins derived from alternative splicing: p16 (Ink4a) and p14 (ARF); p14ARF (ARF stands for alternative-reading frame protein) modulate MDM2-mediated degradation of p53; Homozygous deletion and promoter hypermethylation of p14ARF are found in high percentage of primary NPC tumors. p27Kip1 (p27), a CDK inhibitor involved in inhibiting G1 cyclin-CDK activities, causes G1 cell cycle arrest. p27 functioned as a new class of tumor suppressor and is haplo-insufficient in tumor suppression; There are studies showing that a low level of p27 expression was observed in high percentage of NPC patient by immunohistochemical studies. In addition, a low level of p27 expression significantly correlates with loco-regional recurrence. The 14-3-3 σ(sigma) gene product, upregulated by p53 in response to DNA damage; 14-3-3 sigma can function as a CDK inhibitor, akt antagonist, p53 positive regulator. NPC, a tumor of epithelial origin, has reduced expression in 14-3-3 sigma (stratifin) level; 14-3-3 sigma overexpression in NPC reduced the tumor volume in nude mice. BRD7 is a novel bromodomain containing protein and is downexpressed in NPC; over-expression of BRD7 leads to inhibition of NPC cell growth and causing cell cycle arrest. NGX6, a putative tumor suppressor gene, is located at 9p21-22 and is downregulated in NPC; the function of NGX6 was characterized to cause cell cycle arrest and influence the expression of adhesion molecules; NGX6 expression can block NPC cell migration and compromise invasive ability as well as inhibit tumorigenicity. TSLC1 (tumor suppressor in lung cancer) is characterized as a tumor suppressor located on 11q23; deletion of 11q is one of the major genetic changes in NPC; frequent epigenetic inactivation of TSLC1 gene correlated with tumor progression of NPC; expression of TSLC1 can inhibit cell proliferation and tumorigenesis in NPC. RASSF1A is characterized as a tumor suppressor located on chromosome 3p; LOH of 3p21.3 is frequently observed in NPC; missense, frameshift mutations, and promoter methylation of this gene has been identified in primary NPC; Expression of RASSF1A can inhibits colony formation and tumorigenesis in NPC. Promyelcytic leukemia (PML) tumor suppressor is implicated in acute myeloid leukemia and other types of cancer; For NPC, PML expression in Subtype-III was very low or undetectable in NPC, suggesting that decreased PML expression correlated with more aggressive tumors. THY1, located close to 11q22-2 3, is a tumor suppressor gene candidate of NPC. THY1 is a surface glycoprotein, and is expressed on the cytoplasmic membrane of cells; THY1 is downregulated in high percentage of NPC and lymph node metastatic NPC. Studies have shown that THY1 gene can be inactivated by promoter hypermethylation in tumor cells; Expression of THY1 gene can inhibits NPC cell colony formation, suggesting inhibition of NPC transformation phenotype.

ARF

p27Kip1

14-3-3 σ

BRD7

NGX6

TSLC1

RASSF1A

PML

THY1

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Protein c-Myc

Function/characteristics

The c-Myc protooncogene has been found to be involved in the progression of a wide range of neoplasia; It is known that oncogenic signals of c-Myc have profound effects on cell cycle components including cyclin D, CDK4, INK4 and CIP/KIP families, thus providing important growth advantage; c-Myc overexpression was observed in NPC patient and that c-Myc protein expression levels in NPC are significantly higher when compared with normal nasopharyngreal epithelial cells. Importantly, and its expression correlates with early recurrence of NPC. Survivin Survivin, a novel member of inhibitor of apoptosis (IAP) protein family, appears to be involved in regulation of apoptosis as well as cell division; There is a positive correlation between survivin expression and poor prognosis of the NPC. It has been demonstrated that overexpression of survivin and was significantly associated with late stage of NPC. MAD2 Mitotic arrest deficient 2 (MAD2), is a protein involved in spindle checkpoint; MAD2 regulates mitotic checkpoint and the activity of anaphase-promoting complex (APC), an ubiquitin ligase complex; expression levels of MAD2B correlate with cellular resistance to DNA damaging agent in NPC. CD44 CD44, a glycoprotein molecule on cell surface, plays important roles in proliferation, differentiation, migration and survival of cells; A study showed that that expression levels of CD44 correlate with cellular NPC cell growth. When CD44 expression is decreased, it reduces the malignant activities of NPC such as cell growth, enhanced e-cadherin expression, and inhibiting tumorigenicity in NPC xenograft model. LMP1 LMP1, the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV), plays an important role in nasopharyngeal carcinoma (NPC); Study has indicated that expression of LMP1 by RT-PCR can be found in nasopharyngeal swabs in over 90% of NPC patients; Overexpression of LMP1 leads to anchorage-independent growth and invasive phenotypes in NPC cells. On the other hand, when LMP1 expression is silenced, it causes cell cycle arrest by reducing protein expression of CDK4 and cyclin D1 and increasing p27. Akt Akt oncogene is a crucial regulator of a variety of cellular processes, including cell survival and proliferation; For NPC, Akt signaling can be enhanced by LMP1, thus providing cell growth and cell survival advantage. It has been shown that phosphorylation of Akt and its downstream targets IkB, FKHR was inhibited by LMP1 depletion. Twist Twist, a basic helix-loop-helix transcription factor, is involved the development and progression of human cancer. It was shown that Twist is responsible for the development of resistance to taxol in NPC. When Twist expression is inactivated through small RNA interference, cells have increased sensitivity to taxol-induced cell death. Cyclin D1 D-type cyclins associate with CDK4 or CDK6 and function at the early G1 phase; For NPC, there are studies show that cyclin D1 is overexpressed in high percentage of NPC; Also, cyclin D1 is a target oncogene at 11q13 in NPC. VEGFR/ Nasopharyngeal carcinomas also have the abnormal regulation in endothelial growth EGFR factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) pathway; ZD6474, a tyrosine kinase inhibitor, is shown to inhibit both VEGFR and EGFR signaling; ZD6474 causes cell cycle arrest by causing downregulation of CDK4, CDK6 and CDK2 and upregulation of p21 and p27 in NPC. Id1 Id1 (inhibitor of differentiation/DNA binding-1), a basic helix-loop-helix transcription factor, is involved the development and progression of human cancer; LMP1 upregulates Id1 expression in NPC; Also, the expression of Id-1 was present in NPC cells but absent in normal tissues.

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et al. 1995; Sharpless and DePinho 1999). As described above p16 (Ink4a) is a G1 cell cycle inhibitor (Serrano et al. 1993), whereas p14ARF modulates MDM2mediated degradation of p53 (Sherr and Weber 2000; Tao and Levine 1999; Weber et al. 1999; Zhang et al. 1998). Ink4a/arf locus is one of the hot spots of genomic alterations in many cancers. As for NPC, homozygous deletion and promoter hypermethylation of p14ARF are found in a high percentage of primary NPC tumors (Lo and Huang 2002). ARF plays an important role in regulating p53-dependent apoptosis. ARF can be induced by oncogenes such as Myc, adenovirus E1A, and activated Ras (Sherr and Weber 2000). Importantly, E1A protein and c-Myc enhanced p53 protein stability and p53 transcriptional activity, thereby promoting apoptosis (de Stanchina et al. 1998; Zindy et al. 1998). ARF is the mediator that stabilizes p53 by antagonizing MDM2 because ARF-null cells became resistant to E1A and cMyc-induced apoptosis (de Stanchina et al. 1998; Zindy et al. 1998). Deficiency of ARF or p53 results in elimination of this tumor surveillance mechanism, allowing oncogenes to promote uncontrolled growth (Kamijo et al. 1999). Thus, we could imagine that loss of p14ARF through inactivation or promoter hypermethylation in NPC will compromise p53’s important function, thereby allowing unlimited growth of the cancer cells. It has been shown that adenovirus-mediated p14ARF expression greatly increases the sensitivity of breast cancer cells to the DNA-damaging chemotherapeutic drug Cisplatin (Deng et al. 2002). Gene therapy aiming to restore ARF expression to inhibit carcinogenesis or to enhance conventional chemotherapy remains to be explored.

p27 p27Kip1 (p27), a CDK inhibitor involved in inhibiting G1 cyclin-CDK activities, causes G1 cell cycle arrest (Lee and Yang 2001). p27 functions as a new class of tumor suppressor, and is haplo-insufficient in tumor suppression (Fero et al. 1998). Reduced expression of p27 is frequently detected in many types of human cancers, such as breast (Catzavelos et al. 1997; Porter et al. 1997), prostate (Cordon-Cardo et al. 1998), gastric (Mori et al. 1997), lung (Esposito et al. 1997), skin (Florenes et al. 1998), colon (Loda et al. 1997), and ovarian (Masciullo et al. 1999) cancers. Importantly, decreased expression of the p27 protein correlates with cancer development and poor survival (Catzavelos et al. 1997; Porter et al. 1997). A high percentage of NPC cases have been shown to have a low level of p27 expression by immunohistochemistry (Baba et al. 2001; Hwang et al. 2003). In addition, low p27 expression significantly correlates with loco-regional recurrence (Hwang et al. 2003). In animal models, p27 has been demonstrated to have tumor suppressor activity in lung cancer (Park et al. 2001) and HER2-overexpressing xenograft cancer models (Yang et al. 2001). Given that reduced expression of p27 protein is correlated with NPC cancer development, manipulating the expression of p27 can be an important strategy for NPC cancer treatment for future investigation.

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14-3-3 Sigma The 14-3-3 sigma (σ) gene product (stratifin), upregulated by p53 in response to DNA damage, is involved in cell-cycle checkpoint control and is a human cancer epithelial marker down-regulated in various tumors (Hermeking 2003; Lee and Lozano 2006). Several lines of evidence indicate that loss of 14-3-3 sigma function contributes to malignant transformation. First, 14-3-3 sigma suppresses the anchorage-independent growth of several breast cancer cell lines (Laronga et al. 2000). Second, 14-3-3 sigma expression levels are diminished in v-Ha-rastransformed mammary epithelial cells (Prasad et al. 1992), mammary carcinoma cells, SV40-transformed human keratinocytes (Dellambra et al. 1995), head and neck squamous cell carcinoma lines (Vellucci et al. 1995), primary bladder tumors (Ostergaard et al. 1997), and colonic polyp specimens (Ferguson et al. 2000). Third, transcriptional silencing of the 14-3-3 sigma gene is frequently observed in breast cancer (Ferguson et al. 2000), gastric cancer, and hepatocellular carcinoma (Iwata et al. 2000). Finally, 14-3-3 sigma can function as a CDK inhibitor (Laronga et al. 2000), an Akt antagonist (Yang et al. 2006a, b), and a positive regulator of p53 (Yang et al. 2003; Yang et al. 2007). These characteristics together have made it an important target for cancer therapeutic design. Recently it was found that NPC also has reduced expression of 14-3-3 sigma (Wang et al. 2008). Moreover, failure to upregulate 14-3-3 σ in response to DNA damage has been observed in two NPC cell lines (Yang et al. 2006c). Given that 14-3-3 σ interacts with p53 in response to DNA damage and stabilizes the expression of p53, antagonizes Akt-mediated cell survival, and inhibits CDK kinase activity, these coordinated antineoplastic acitivity of 14-3-3 sigma can be harnessed for potential cancer treatment. Indeed, 14-3-3 sigma overexpression in both NPC cell lines reduced the tumor volume in nude mice (Yang et al. 2006c), thus modulating 14-3-3 sigma activity should be further explored in the treatment of NPC.

BRD7 BRD7 is a novel bromodomain-containing protein and is downregulated in NPC (Zhou et al. 2004). It has been shown that over-expression of BRD7 leads to inhibition of NPC cell growth and causing cell cycle arrest (Peng et al. 2007a). Especially cyclin D1 is significantly decreased after BRD7 transfection (Peng et al. 2007a). This is particularly interesting, because cyclin D1 associates with CDK4 to mediate the phosphorylation of the retinoblastoma (Rb) family protein. This event inactivates the ability of pRb–E2F complexes to negatively regulate the transcription of genes required for S phase entry. Also, it has been shown that cancer cells usually have elevated levels of cyclin D1 (Lee and Yang 2001). Thus BRD7 loss will lead to CyclinD1 accumulation and abnormal cell proliferation. The molecular mechanism behind BRD7-mediated inhibition of NPC cells has been investigated by cDNA microarray transcriptome profiling to examine difference

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in gene expression induced by BRD7 (Peng et al. 2007a). Important molecules involved in Ras/MEK/ERK and Rb/E2F pathways are regulated by the expression of BRD7 (Zhou et al. 2004). BRD7 can serve as an marker for NPC and a candidate for cancer gene therapy.

NGX6 NGX6, a putative tumor suppressor gene located at 9p21-22, is downregulated in NPC. Importantly, the loss of heterozygosity (LOH) on 9p is one of the most frequent genetic alterations in many types of cancers. Indeed, LOH at 9p21-22 has been observed in up to 60% of NPC. The NGX6 gene encodes a putative protein (338 amino acids) that has two transmembrane regions. It has an extracellular region containing one EGF (epidermal growth factor)-like domain and three potential N-glycosylation sites, and it inhibits the EGFR-MAPK signaling pathway (Wang et al. 2005). The function of NGX6 was characterized to cause cell cycle arrest and influence the expression of cell adhesion molecules (Ma et al. 2005). Also, it can associates with ezrin, which is a linkage between the cell membrane and the cytoskeleton (Peng et al. 2007b). Importantly, NGX6 expression can block NPC cell migration, inhibit invasion, improve cell adhesion, and increase gap junctional intercellular communication. NGX6 has demonstrated growth inhibitory effect in a xenograft NPC mouse model (Ma et al. 2005), suggesting its function in controlling NPC tumorigenicity.

TSLC1 TSLC1 (tumor suppressor in lung cancer) is characterized as a tumor suppressor located on 11q23 (Lung et al. 2004). The expression of TSLC1 is dysregulated in many types of human cancers, including lung (Fukami et al. 2003; Kuramochi et al. 2001; Murakami 2002), prostate (Fukuhara et al. 2002), gastric (Honda et al. 2002), pancreatic (Jansen et al. 2002), and breast cancers (Allinen et al. 2002; Heller et al. 2007). For NPC, deletion of 11q is one of the major genetic changes (Lo et al. 2000). Tissue microarray and immunohistochemical staining showed downregulation or loss of TSLC1 in a high percentage of metastatic lymph node NPC (Lung et al. 2006). Thus TSLC1 could be one of the NPC-associated tumor suppressor genes. Studies have shown that TSLC1 gene can be inactivated by promoter hypermethylation in tumor cells (Heller et al. 2006), and the promoter of TSLC1 in some NPC cells is hypermethylated (Zhou et al. 2005) as expression of TSLC1 can be restored after treatment with a demethylating agent 5-aza-deoxycytidine. Indeed, promoter hypermethylation of the TSLC1 gene has been observed in a high percentage of primary NPC tumor samples (Hui et al. 2003). Frequent epigenetic inactivation of TSLC1 gene correlated with tumor progression of NPC. TSLC1 is a transmembrane glycoprotein containing an extracellular domain with

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three immunoglobulin-like C2 domains, a transmembrane domain and a cytoplasmic domain. This protein structure indicates that it may function as a cell adhesion molecule and may be involved in tumourigenesis by controlling cell–cell interactions during growth. Expression of TSLC1 can inhibit cell proliferation, induce apoptosis, and inhibit tumorigenesis in lung cancer cells (Mao et al. 2004). Its tumor suppressive activity is also documented in NPC (Lung et al. 2006). Therefore, TSLC1 can also serve as a marker for NPC and a candidate for cancer gene therapy.

RASSF1A RASSF1A, a Ras-association domain family of protein, is characterized as a tumor suppressor located on chromosome 3p (Chow et al. 2006) (Lo et al. 2004). The gene is frequently deleted in many types of cancer including breast and lung cancers (Kwong et al. 2002). Also, epigenetic inactivation of RASSF1A gene has been found in small cell lung cancer, non-small cell lung cancer, breast cancer, and renal cell carcinoma (Lo et al. 2004). For NPC, LOH of 3p21.3 is frequently observed (Chow et al. 2004; Lo et al. 2000), suggesting the presence of a tumor suppressor gene at that locus. RASSF1A is the critical tumor suppressor on 3p21.3 involved in NPC. Missense and frameshift mutations of this gene has been identified in primary NPC (Pan et al. 2005). Also, promoter of RASSF1A in NPC cells is hypermethylated (Lo et al. 2001; Zhou et al. 2005), suggesting yet another mechanism to inactivate RASSF1. Expression of RASSF1A can inhibit colony formation in soft agar and tumorigenesis in NPC. Thus, RASSF1A is an important marker for NPC and its tumor suppressive activity can be employed for NPC gene therapy.

PML Promyelocytic leukemia (PML) tumor suppressor is implicated in a subtype of acute myeloid leukemia and other types of malignancies (Salomoni and Pandolfi 2002). PML is concentrated in subcellular structures termed as PML-nuclear bodies (PMLNBs). Strikingly, all the proteins involved in post-translational modification of p53 (de Stanchina et al. 2004), including ARF, HAISP, CBP, hSir, PIAS, and Mdm2, are found to be accumulated in PML-NBs . It is possible that PML-NBs form a scaffold to bring roteins together for efficient post-translational modification of p53 (de Stanchina et al. 2004; Gottifredi and Prives 2001). Recently, PML has been shown to directly interact with MDM2 (Bernardi et al. 2004), and PML can sequester MDM2 to the nucleolus after DNA damage, thereby enhancing p53 stability. In the absence of PML, the sequestration of MDM2 to the nucleolus is impaired. Downregulation of PML has been characterized in small cell lung carcinoma (Zhang et al. 2000) and various carcinomas (Gurrieri et al. 2004). For NPC, PML expression in Subtype-III was very low or undetectable (Chan et al. 2002), suggesting that decreased PML expression correlates with increased aggressiveness of NPC. Thereofre, PML may

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be an important prognostic marker for NPC, but so far the studies are still limited. Given its tumor suppressive role in regulating p53, future exploration of PML expression as NPC gene therapy is warranted.

THY1 THY1, located close to 11q22-23, is a tumor suppressor gene candidate in NPC (Lung et al. 2005). THY1 is a surface glycoprotein expressed on the plasma membrane of cells, eliciting a variety of cellular functions such as proliferation, lymphokine release, differentiation, and apoptosis. THY1 has been associated with tumour suppressive activity in human ovarian cancer (Abeysinghe et al. 2003; Abeysinghe et al. 2004), but its molecular mechanism remains undefined. THY1 is downregulated in a high percentage of primary NPC and lymph node metastatic NPC. Studies have shown that THY1 gene can be inactivated by promoter hypermethylation in tumor cells (Lung et al. 2005). Expression of THY1 gene can inhibits NPC cell colony formation, suggesting inhibition of NPC transformation phenotype (Lung et al. 2005). It will be interesting to continue exploring its tumor suppressive activity in NPC.

Positive Growth Regulators of NPC c-Myc The c-Myc protooncogene has been found to be involved in the progression of a wide range of neoplasia (Adhikary and Eilers 2005). The c-Myc protein is a transcription factor in conjunction with its transcriptional activation partner Max. Miz1, a Myc-interacting zinc-finger protein 1, was found to upregulate the expression of p15, a CDK4 inhibitor by binding the initiator element of the p15 promoter; however, this process can be antagonized by Myc (Staller et al. 2001). c-Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit Miz-1-mediated transcriptional activation of p15 (Staller et al. 2001). It is known that oncogenic signals of Myc have profound effects on cell cycle components including cyclin D, CDK4, INK4 and CIP/KIP families (Lee and Yang 2001), thus overall providing an important growth advantage. Also, c-Myc contributes to tumorigenesis as an antagonist of the DNA damage-sensing mechanism (Karlsson et al. 2003). Modest overexpression of c-Myc is sufficient to disrupt the DNA repair check point control, impede the repair of DNA double-strand breaks and cause genomic instability, thereby accelerating tumor progression. c-Myc overexpression is observed in NPC compared with normal nasopharyngreal epithelial cells (Fan et al. 2000). Importantly, its overexpression correlates with early recurrence of NPC (Fan et al. 2000) as well as a poor prognosis. Thus far, the studies on c-Myc network in NPC are still limited. Given the important role of c-Myc’role in carcinogenesis, c-Myc regulation and signaling in NPC certainly deserve further study.

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Survivin Survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, appears to be involved in regulation of apoptosis (Altieri 2008) as well as cell division (Delacour-Larose et al. 2007). It inhibits apoptosis in vitro and in vivo. Overexpression of survivin inhibits cell death initiated via the extrinsic or intrinsic apoptotic pathways. Also, survivin is mainly expressed during mitosis. Survivin associates with various components of the mitotic apparatus, including centrosomes, the mitotic spindle, and midbodies. Survivin forms a complex with molecules regulating cytokinesis (Jeyaprakash et al. 2007), including Aurora B kinase and INCENP on kinetochores and the anaphase central spindle, enhancing the activity of Aurora B kinase. Compared with the adjacent normal tissues, expression of survivin is enhanced in many types of human cancers including esophageal, ovarian, laryngeal, colorectal, and breast cancers as well as lymphomas (Altieri 2008). There is a positive correlation between survivin expression and poor prognosis of NPC (Xiang et al. 2006). Overexpression of survivin is significantly associated with late stages of NPC (Li et al. 2008), and the 5-year survival rate of NPC patients with survivin overexpression is significantly lower than that of patients with low-expression. Lmp1, the latent membrane protein of EBV (see below), can increase the expression and activity of survivin, thus promoting cell proliferation and antagonizing apoptosis (Ai et al. 2005; Faqing et al. 2005). Various strategies have been explored to downregulate survivin expression or function to reduce tumor growth, increase apoptosis and sensitize tumor cells to chemotherapeutic drugs in tumor models (Pennati et al. 2008). These strategies can certainly be applied to NPC too. Thus, survivin is an important marker and target for therapy to be further investigated in NPC.

MAD2 Mitotic arrest deficient 2 (MAD2), a protein involved in spindle checkpoint (Li et al. 1993), is important to arrest cells in mitosis when chromosomes are not attached to the mitotic spindle. MAD2 regulates mitotic checkpoint and the activity of anaphase-promoting complex (also known as cyclosome, abbreviated as APC/C), a ubiquitin ligase complex (Wassmann and Benezra 1998). The APC/C controls critical transitions in mitosis by degrading securin and cyclins (Shah and Cleveland 2000). MAD2 is an inhibitor of APC/C, whereas CDC20 and CDH1 are activators (Reddy et al. 2007). MAD2 can bind to and inhibit ubiquitin ligase CDC20-APC/C and CDH1-APC/C, thus controlling chromosome segregation and mitotic exit. Its role in mitotic checkpoint control also makes MAD2 important in response to DNA damage. MAD2 plays an important role in chromosomal segregation as loss of the MAD2 leads to embryonic lethality due to chromosome missegregation (Dobles et al. 2000; Michel et al. 2001). MAD2B, a homolog of the MAD2 protein, was identified based on its sequence similarity to MAD2. Functionally, MAD2B is also an inhibitor of APC/C. Recent studies have shown that

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expression levels of MAD2B correlate with cellular resistance to DNA damaging agent in NPC (Cheung et al. 2006). When MAD2B expression is decreased by RNA interference, sensitivity to DNA-damaging anti-cancer agents such as cisplatin and γ-IR is restored (Cheung et al. 2006). The increased drug sensitivity is due to MAD2B-mediated mitotic arrest defect, thus causing reduced frequencies of spontaneous and drug-induced mutations and elevated phosphorylation of histone H2AX. This finding suggests a novel strategy for sensitizing NPC cells to DNA-damaging anticancer drugs through inactivation of the MAD2B gene.

CD44 CD44, a glycoprotein molecule on cell surface, plays important roles in proliferation, differentiation, migration and survival of cells (Goodison et al. 1999; Naor et al. 2002). The principal ligand of CD44 is hyaluronic acid, which is an integral component of the extracellular matrix. CD44 interacts with ligands such as osteopontin, serglycin, collagens, fibronectin, and laminin (Goodison et al. 1999). CD44 is usually upregulated in neoplasia (Goodison et al. 1999; Sy et al. 1997). CD44 can mediate growth factor presentation to facilitate metastasis formation (Ponta et al. 1998). Due to CD44’s tumor-promoting activity, therapeutic agents specifically targeting CD44 have been developed. Experiments have shown that targeting of CD44 by antibodies or antisense DNA can dramatically reduce the malignant behavior of various neoplasms, confirming the potential of CD44 as a cancer therapeutic target (Naor et al. 2002). Another study has shown that the expression levels of CD44 correlate with NPC cell growth (Shi et al. 2007). When CD44 expression is decreased, the NPC cells are inhibited in terms of cell growth, enhanced e-cadherin expression, and tumorigenicity in NPC xenograft model (Shi et al. 2007). These findings suggest that inhibiting CD44 expression may be an important strategy for NPC therapy.

LMP1 LMP1, the latent membrane protein 1 of EBV, plays an important role in NPC (Liebowitz 1994). Infection of EBV is one of the major etiological factors in NPC. Expression of LMP1 can be detected by RT-PCR in nasopharyngeal swabs in over 90% of NPC patients (Tsao et al. 2002). LMP1 encoded by EBV is a classic oncogene, and it stimulates at least four signaling pathways: NFkB, c-Jun N terminal kinase, p38MAPK, and JAK/STAT, thus resulting in the highly invasive malignant phenotype of NPC tumors (Tsao et al. 2002). LMP1 can also induce the expression of Id1 to facilitate cellular immortalization and stimulates cell proliferation (Li et al. 2004). Recently, it has been shown that LMP1 expression can suppress the transcriptional activity of the RASSF1A core promoter, adding another important mechanism to drive the tumorigenesis of NPC (Man et al. 2007). Overexpression of LMP1 leads to anchorage-independent growth and invasive phenotype in NPC cells. On the other hand, silencing LMP1 expression causes cell cycle arrest by reducing

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protein expression of CDK4 and cyclin D1 and increasing p27 (Mei et al. 2007). Overall, these studies suggest that inhibiting LMP1 expression may be a rational strategy for NPC therapy.

Akt Protein kinase B (also called Akt) is the cellular homologue of the oncogene of the AKT8 oncovirus (v-Akt). Akt is activated when particular extracellular signals activate receptor tyrosine kinases to enhance phosphatidylinositol 3-kinase (PI3K) activity on phospholipids (Vivanco and Sawyers 2002). The oncogene is a crucial regulator of a variety of cellular processes, including cell survival and proliferation (Manning and Cantley 2007; Sun et al. 2001). Importantly, Akt activity is elevated in several types of human malignancy, including ovarian, breast, lung, and thyroid cancers (Altomare and Testa 2005). The kinase activity of Akt is constitutively activated in human cancer as a result of dysregulation of its regulators, e.g., the tumor suppressor PTEN and 14-3-3 sigma (Yang et al. 2006a, b), and amplification of the catalytic subunit of PI3K (Altomare and Testa 2005). Akt also has an impact on p27 expression and its subcellular localization. For example, in breast cancer cells, Akt-mediated p27 phosphorylation at Thr(157) (within the nuclear localization signal sequence of p27, amino acids 153–166) impairs the nuclear import of p27 (Liang et al. 2002; Shin et al. 2002). In addition, Akt phosphorylates p27 at Thr(198) (Fujita et al. 2002), which results in the nuclear export and degradation of p27. For NPC, Akt signaling can be enhanced by LMP1, thus providing cell growth and survival advantages, and LMP1 depletion inhibits phosphorylation of Akt and its downstream targets IkB, FKHR (Mei et al. 2007). Thus, the LMP1-Akt axis may be an important target for strategic design of novel NPC therapy.

Twist Twist, a basic helix-loop-helix transcription factor, is involved the development and progression of human cancer. Twist, originally identified as an important regulator of morphogenesis in the embryo, is implicated in the onset of invasive behavior during tumor progression and plays an essential role in metastasis (Yang et al. 2004). Overexpression of Twist leads to loss of E-cadherin-mediated cell-cell adhesion, activation of mesenchymal markers, and induction of cell motility (Yang et al. 2004). These changes contribute to Twist-mediated metastasis by promoting an epithelial-mesenchymal transition (EMT) (Kang and Massague 2004). Twist expression positively correlates with lymph-node metastasis and distant metastasis (Song et al. 2006). Twist may also be responsible for the development of resistance to paclitaxel in NPC (Wang et al. 2004) because overexpression of Twist blocks paclitaxel-induced apoptosis by decreasing Bak and Bax, increasing Bcl-2, and inhibiting PARP and caspase cleavage. When Twist expression is inactivated

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through small RNA interference, cells have increased sensitivity to paclitaxelinduced cell death (Zhang et al. 2007). The Twist-mediated paclitaxel resistance is probably mediated by the Akt signaling pathway (Zhang et al. 2007). Together these findings suggest a method to sensitize NPC cells to paclitaxel through regulating the expression of the Twist gene.

Cyclin D1 The G1 phase of the cell cycle integrates many extracellular growth regulatory signals. When quiescent cells are stimulated by growth factors or mitogenic signals, the D-type cyclins are the first to be activated. D-type cyclins associate with CDK4 or CDK6 and function at the early G1 phase (Lee and Yang 2003a). CDK4 associates with the D-type cyclins to mediate the phosphorylation of the retinoblastoma (Rb) family protein. This event inactivates the ability of pRb–E2F complexes to negatively regulate the transcription of genes required for S phase entry. Given its role in promoting cell cycle progress, cyclin D is usually dysregulated in cancers. For example, up to 50% of breast cancer cases have elevated levels of cyclin D1 (Lee and Yang 2003b). Cyclin D1 is also overexpressed in a high percentage of NPC (Hui et al. 2005; Lai et al. 2002). Cyclin D1 is a target oncogene at 11q13 in NPC (Hui et al. 2005). It has been shown that several transcription factors including beta-catenin, Msx1, Notchic can positively regulate cyclin D1 expression (Lee and Yang 2003b). Mutations in the tumour suppressor gene adenomatous polyposis coli (APC) play important role in human cancers (Bienz and Hamada 2004). Loss of functional APC protein results in the accumulation of beta-catenin. Betacatenin interacts with transcription factors of the TCF/LEF family to activate target genes (Tetsu and McCormick 1999). Beta-catenin can activate transcription from the cyclin D1 promoter, which contains consensus TCF/LEF-binding sites (Shtutman et al. 1999). Mutant forms of beta-catenin have been discovered in many human cancers, suggesting that abnormal levels of beta-catenin may lead to neoplastic transformation through induction of cyclin D1. So far, it is not clear whether the APC-Beta-catenin axis is involved in Cyclin D1 dysregulation in NPC. This is an area that needs to be further explored. Interestingly, knockdown of cyclin D1 in NPC results in significant decrease in cell proliferation (Hui et al. 2005), suggesting that cyclin D1 is an important therapeutic target and antagonizing cyclin D1 expression can be an important strategy for NPC therapy.

VEGFR/EGFR Epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) pathways are associated with tumorigenesis. VEGF is a major mitogen for endothelial cells, enhancing vascular permeability (Glade-Bender et al. 2003). Enhanced VEGF expression in human cancers correlates with increased tumor neovascularization. EGFR pathway plays an important role in cancer cell growth (Johnston

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et al. 2006). Therapeutic approaches targeting VEGF, EGF receptor (EGFR) or VEGF receptor (VEGFR) signaling have been designed for cancer treatment (Drevs et al. 2003). Nasopharyngeal carcinomas also have the abnormal regulation in the VEGFR and EGFR pathways. ZD6474 (Vandetanib, ZACTIMA) (Ryan and Wedge 2005), a tyrosine kinase inhibitor, is shown to inhibit both VEGFR and EGFR signaling. ZD6474 causes cell cycle arrest through downregulation of CDK4, CDK6 and CDK2 and upregulation of p21 and p27 in NPC. Furthermore, it can induce apoptosis in NPC by reducing Bcl-2 and/or Bcl-XL, inducing Bak and/or Bax, and activating caspases (Xiao et al. 2007). Significantly, ZD6474 inhibits NPC tumorigenesis in a xenograft model. Therefore, inhibiting the VEGFR and EGFR pathways is an effective treatment strategy for NPC.

Id1 Id1 (inhibitor of differentiation/DNA binding-1), a basic helix-loop-helix transcription factor, is involved the development and progression of human cancer (Perk et al. 2005; Ruzinova and Benezra 2003). Id1 can regulate gene transcription by heterodimerizing with other basic helix-loop-helix transcription factors, therefore inhibiting their DNA binding and transactivation of their target genes (Perk et al. 2005). It causes the inactivation of the retinoblastoma (Rb)/p16 pathway through downregulation of p16 and increasing phosphorylation of Rb (Ling et al. 2006). Elevated expression of Id1 is found in many types of solid cancers (Ling et al. 2006; Swarbrick et al. 2008). It has been shown that LMP1 upregulates Id1 expression in NPC (Li et al. 2004). Also, Id-1 is differentially expressed in NPC cells but not expressed in normal tissues (Wang et al. 2002). Ectopic expression of Id1 in NPC cells leads to increases in serum-independent cell growth, the population of cells in S phase of the cell cycle and phosphorylation of Rb, suggesting that Id-1 plays an important role in cancer cell proliferation (Wang et al. 2002). Also, overexpression of Id1 results in resistance to an anticancer drug, paclitaxel, by suppressing the apoptosis signal. These studies suggest that Id1 is an important therapy target in NPC and that inactivation of Id1 can be a treatment strategy target to potentiate paclitaxel-induced apoptosis.

Conclusion In this review, we reviewed some of the representative growth regulators deregulated in NPC for the potential strategy for the treatment of NPC. These targets are candidates for rational cancer therapeutic approaches. With the explosive discoveries in understanding the abnormal proliferation of malignant NPC, we expect that deregulation of numerous cell cycle components, tumor suppressors, or signal molecules involved in NPC carcinogenesis will continue to be characterized and that new therapeutic approaches will continue to be developed. The important goal of the NPC cancer researchers is to study carcinogenesis cancer biology in sufficient molecular details to design rational interventions and cure the disease. The common disease management modalities for NPC, such as chemotherapy and radiotherapy,

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can potentially be combined in novel treatment regimes with cancer gene therapy to kill NPC cells with improved efficacy. With the newly increased mechanistic insights into the NPC tumorigenesis, the potential for future applications of therapeutic approaches designed based on the molecular targets discussed in this chapter is rather optimistic.

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Chapter 13

Cellular Signaling Mechanisms in Pancreatic Apoptosis Nawab Ali, Stewart MacLeod, R. Jean Hine and Parimal Chowdhury

Abstract It is evident that various types of tumor cells use different mechanisms to inhibit apoptosis. Recent increased understanding of the many factors involved in the apoptotic process has identified potential targets for the restoration of the apoptotic response in pancreatic tumor cells. The ultimate goal is to develop effective therapeutic strategies to control this devastating disease. The aim of this chapter is to review signaling pathways involved in apoptosis by providing an account of the signaling molecules involved. This chapter reviews the literature on traditional apoptotic signaling pathways with special emphasis on pancreatic cancer. Involvement of G-protein coupled receptors and inositol phosphates in pancreatic apoptosis is also reviewed. Finally we have reviewed the literature on nutritional impacts on pancreatic apoptosis as an example of an environmental risk factor for pancreatic cancer. Knowledge about diverse effects on signaling molecules may serve as a basis for pancreatic cancer chemotherapeutic applications focused on apoptotic mechanisms. Keywords Apoptosis · Inositol phosphates · Nutrition · Pancreatic carcinogenesis · Cellular signaling mechanisms

Introduction The biology of the pancreatic cancer and the understanding of molecular pathogenesis of pancreatic adenocarcinoma through alterations of biochemical signaling pathways have been extensively reviewed (Bardeesy, 2005). It is important to dissect the molecular pathways in order to develop a targeted approach to effective drug development to combat this deadly disease. Pancreatic adenocarcinoma (cancer) is one of the most severe forms of human cancer, with the lowest survival rate of any human tumor. Pancreatic cancer is a relatively rare tumor with an incidence of P. Chowdhury (B) Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 13,

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approximately 10 cases per 100,000 persons and the lifetime risk of dying from pancreatic cancer is between 1 and 2% in the general population (Parker et al., 1997). More than 30,000 Americans and 60,000 Europeans are diagnosed annually with pancreatic cancer rating it as the fourth leading cause of cancer deaths (Ghaneh et al., 2000). Patients diagnosed with pancreatic cancer typically have a poor prognosis due to lack of early diagnostic symptoms, leading to metastasis at time of diagnosis. Median survival from diagnosis to death is approximately 3–6 months; However, pancreatic tumors are extremely aggressive, with a 5-year survival rate of 1–2% (Coppola, 2000) and a mortality rate of nearly 100%. Symptoms of pancreatic cancer are not apparent until late in the development of the disease. At the point of diagnosis, conventional forms of chemotherapy have little impact on the progression of the malignancy since pancreatic cancer cells are resistant to chemotherapy, radiotherapy (Beger et al., 1994; Neoptolemos et al., 2004) and immunotherapy (von Bernstorff et al., 2001).The cause of this resistance is probably the end result of a combination of many factors, including cancer cell insensitivity to normal cell growth restraints, drug resistance acquired during therapy, and lack of response to apoptotic stimuli, which may be an underlying cause of resistance in all of these mechanisms (Korsmeyer, 1992; Miyashita and Reed, 1993). Various types of tumor cells use different mechanisms to inhibit apoptosis (Hager and Hanahan, 1999). Recent increased understanding of the many factors involved in the apoptotic process has led to the identification of potential targets that may lead to the restoration of the apoptotic response in pancreatic tumor cells, with the goal of developing an effective therapeutic strategy to control this devastating disease. The aim of this chapter is to review signaling pathways involved in pancreatic apoptosis by providing an account of the signaling molecules involved. These signaling molecules may serve as a basis for pancreatic cancer chemotherapeutic applications by way of inducing apoptosis to prevent cellular proliferation.

Apoptosis The word apoptosis is derived from Greek apo – from, ptosis – falling. It is a naturally occurring programmed cell death for destruction of living cells that may represent a threat to the integrity of an organism. Cell division and programmed cell death are physiological processes that occur simultaneously on a continuous basis in almost all living tissues. It is required to maintain a balance between cell division and programmed cell death to ensure the integrity of the organs. In programmed cell death, a series of orderly patterns of biochemical events bring about cellular changes such as cell shrinkage, changes in mitochondrial morphology, chromatin condensation, membrane perforation, activation of caspases and inhibition of survival pathways such as Akt/PKB (protein kinase B, a serine/threonine kinase), all resulting in DNA fragmentation and formation of apoptotic bodies that in turn lead to cell death (Fig. 13.1). Phagocytes safely dispose of the apoptotic bodies and cell corpses without invoking inflammatory reactions.

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Fig. 13.1 Apoptosis: The Programmed Cell Death. Process of apoptosis showing major events in the apoptotic pathway

Cancerous cells represent abnormal cellular proliferation due to genetic malfunctioning and therefore represent a threat to normal cell physiology. Cellular proliferation leads to tumor formation. Researchers have developed certain chemical treatment procedures to induce apoptosis specifically in cancerous cells to prevent tumor formation.

Position for Figure Apoptosis is a highly regulated and efficient cell death program requiring involvement of many factors. Apoptotic signaling pathways are present in an inactive state in viable cells and are turned on only after induction of death stimuli. There can be various types of apoptotic signals both from outside (extrinsic) and inside (intrinsic) of the cell that eventually lead to the same fate of programmed cell death. Cellular homeostasis is maintained in normal tissue by a balance between cellular growth and programmed cell death or apoptosis. Defective cells are also eliminated by apoptosis, however tumor cells develop mechanisms that allow them to avoid apoptosis, in spite of the accumulation of multiple defects in these cells (Igney and Krammer, 2002). Tumor cells use many different pathways to evade apoptosis (Hager and Hanahan, 1999; Okada and Mak, 2004).

Signaling Pathways in Pancreatic Apoptosis Apoptosis can be initiated through two well-characterized pathways. The first involves ligand-specific binding of cell surface death receptors (Ashkenazi and Dixit, 1999) that are related to the tumor necrosis factor (TNF) superfamily (Itoh et al., 1991; Suda et al., 1993). This mechanism is referred to as the extrinsic

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pathway (Vucic and Fairbrother, 2007), since the death ligand originates outside the cell. In extrinsic apoptosis or when death-inducing stimuli are from outside the cell, ligands bind with and ligate death receptors such as Fas, TNFR1, DR5, etc. These death receptors recruit adaptor molecules (FADD, TRADD) through their cytoplasmic death domains (DD) (Sartorius et al., 2001). Death effecter domains (DED) of these adaptors in turn recruit procaspase-8, forming a death-inducing signaling complex (DISC). Active caspase-8 is then formed by autoproteolytic cleavage of procaspase 8, which in turn cleaves and activates other caspases (caspase-3, -6, -7) in the cascade (Denault et al., 2002). In intrinsic apoptosis, death-inducing signals are from within the cell; signals induce the release of cytochrome c (cyto c) from mitochondria into cytosol (Salvesen and Renatus, 2002). Cytosolic cyc c binds with apoptotic protease activating factor-1 (Apaf-1) inducing the binding with procaspase -9 that results in proteolytic processing and activation of procaspase-9 (Denault et al., 2002). This activation of procaspase-9 leads to proteolytic processing and activation of procaspase-3, -6, -7 resulting in activation of the caspase cascade and cell death (Earnshaw et al., 1999). Current pancreatic cancer therapies such as radio- and chemotherapy indirectly promote apoptosis in cancerous cells. These treatments induce apoptosis by causing DNA damage. In doing so, they stimulate apoptosis through the intrinsic pathways. Both mechanisms are mediated by activation of various members of a family of serine proteases called cysteine-dependent aspartyl-specific proteases or caspases. These enzymes are part of a cascade that includes initiator caspases that are activated by recruitment of signaling complexes, such as caspases 8 and 9 (Fuentes and Salvesen, 2004). Activation of these caspases links the signaling caspases to the so called effector or executioner caspases that directly cleave multiple cellular components including DNA (Timmer and Salvesen, 2007), leading to cell death. Apoptosis can be initiated in some cell types by ligand binding to death receptors and activation of caspase-8, which can then activate caspase-3, directly triggering the apoptotic process. It has been shown that in pancreatic cancer cells, the release of mitochondrial products is always involved in the apoptotic mechanism, regardless of whether the initial triggering mechanism was by activation of the TNF related death receptors or by environmental stress (Adrian, 2007). Apoptosis can also be induced by apoptotic inducing factors (AIF), which unlike other pathways do not use caspases. AIF protein is located in the inner space of mitochondrial membrane. Upon induction of death signal, AIF migrates into the nucleus and binds with DNA. AIF binding with DNA triggers DNA destruction and thus results in cell death.

Death Receptor Mediated Signaling Pathways The four cell surface receptors and one circulating receptor for this pathway belong to the tumor necrosis factor TNF super family and include TNF-α, Fas and TNFrelated apoptosis-inducing ligand or TRAIL. These receptors share an internal

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domain called the death domain (Itoh et al., 1991; Suda et al., 1993). These receptors are activated by binding of their ligands, TNF-α, FasL and TRAIL. Binding of the ligand to the receptor results in receptor trimerization, then recruitment of Fasassociated death domain protein or FADD and procaspase-8 to the complex, which has been termed the death-inducing signaling complex or DISC (Sprick et al., 2000). Assembly of this complex results in the cleavage of procaspase-8 to its active proteolytic form. In some cell types, the activation of caspase-8 is sufficient to then activate caspase-3, the main effector caspase, thus instituting the apoptotic program, ending in cell death. In other cell types including pancreatic tumor cells, the signaling resulting from the release of mitochondrial products is necessary to institute apoptosis (Scaffidi et al., 1998). In pancreatic cancer cells, death receptor activation is signaled to the mitochondria by caspase-8 mediated cleavage of Bid, a member of the bcl family. Cleavage of Bid produces the truncated form, tBid, which is translocated to the mitochondria (Scaffidi et al., 1998), and becomes integrated into the mitochondrial outer membrane. This results in the permeabilization of the mitochondria and the release of cytochrome c and other apoptotic factors into the cytoplasm (Martinou and Green, 2001; Zamzami and Kroemer, 2001). Upon its release into the cytoplasm, cytochrome c binds with Apaf-1(apoptotic protease-activating factor), ATP and procaspase-9 to form a complex known as the apoptosome (Pop et al., 2006). Assembly of the apoptosome results in cleavage of procaspase-9 to its active form, and allows it to function as an initiator caspase by activating the executioner caspases, primarily caspase-3, -6 and -7. These activated caspases are responsible for the DNA fragmentation and cleavage of cytoskeletal proteins that result in cell death (Rathmell and Thompson, 1999). Since caspase-3 has been recognized as the main effector caspase in the apoptotic cascade, activation of this enzyme is an attractive target to restore apoptotic response in resistant cells. The inactive precursor, procaspase-3, is expressed at high levels in pancreatic cancer cells and this expression is related to the invasiveness of these tumors (Satoh et al., 2000). Procaspase-3 is maintained in an inactive state by a so-called safety catch, consisting of a triplet of aspartic acid residues in the proenzyme sequence that blocks access to the procaspase-3 proteolytic activation site (Roy et al., 2001). A small molecule called procaspase-activating compound1 (PAC-1) has been discovered that increases hydrolysis of a peptidic caspase-3 substrate and activates caspase-3. PAC-1 has been shown to induce apoptosis in a number of tumor cell lines and to inhibit tumor growth in murine models of human cancer (Putt et al., 2006).

Apoptotic Factors in Pancreatic Cancer Elnemr et al. (2001) determined that despite high expression of Fas ligand by a number of pancreatic cell lines, exposure of these cells to Fas agonist induced only minimal amounts of apoptotic cell death. Further investigation demonstrated that

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these cells expressed high levels of Fas decoy receptors (DcR3), Fas-associated phosphatase-1 (FAP-19) and cellular FLICE-like inhibitory protein c-FLIP (Elnemr et al., 2001a). All of these proteins have the ability to abrogate Fas function at different levels of the Fas signaling cascade, thus rendering pancreatic cancer cells insensitive to apoptosis. Other groups have reported down-regulation of Fas expression in pancreatic cancer cells (Bernstorff et al., 2002), as well as increased expression (Kornmann et al., 2000), however , all of these reports conclude that pancreatic cancer cells are insensitive to Fas mediated apoptosis. Pancreatic cells are also resistant to TRAIL induced apoptosis, and the mechanism of this resistance is not related to an absence of TRAIL receptors. Unlike most other tumor cell types, pancreatic carcinoma cells express high levels of TRAIL receptors on the cell surface (Hinz et al., 2000). Resistance to TRAIL mediated apoptosis in pancreatic tumor cells was related to increased expression of anti-apoptotic proteins Bcl-XL (Hinz et al., 2000), X-linked inhibitor of apoptosis protein (XIAP) (Vogler et al., 2007), and FADD-like ICE inhibitory proteins (FLIPs) (Matsuzaki et al., 2001). Inhibition of production of these proteins by treatment of the cells with actinomycin-D rendered some pancreatic cancer cell lines sensitive to TRAIL mediated apoptosis (Matsuzaki et al., 2001). The ability of TRAIL itself to induce apoptosis in tumor cells while having no effect in normal cells has attracted interest in the use of recombinant TRAIL as a therapeutic agent. However, Trauzold et al. (2006) demonstrated increased distant metastasis of pancreatic tumors in TRAIL treated severe combined immunodeficiency mice transplanted with human pancreatic ductal adenocarcinoma cells. They emphasize that the in vivo effects of such treatment must be thoroughly evaluated in order to develop therapeutic regimens that enhance apoptosis but also prevent metastasis. Another mechanism for resistance to apoptosis in pancreatic cancer cells is overexpression of FAP-1 (Fas-associated phosphatase-1). Stable expression of FAP-1 in Capan-1, a pancreatic cell line which is highly sensitive to Fas mediated apoptosis and lacks FAP-1 expression, rendered the cells more resistant to apoptosis (Ungefroren et al., 2001). In addition, FAP-1 is highly expressed in pancreatic carcinoma tissue (Ungefroren et al., 2001). Down-regulation of FAP-1 expression or inhibiting its enzymatic activity may be a therapeutic strategy for sensitizing human pancreatic cancer to apoptosis-inducing therapy.

Mitochondrial Signaling Pathways in Pancreatic Apoptosis The Bcl family of apoptosis regulator proteins comprises at least 16 members, some of which have anti-apoptotic (Bcl-2, Mcl-1, Bcl-XL) or pro-apoptotic (Bax, Bak and Bad) activity (Adams and Cory, 1998). The Bcl family is responsible for regulating the mitochondrial apoptotic response, which is necessary for activation of the apoptotic program in pancreatic cancer cells (Scaffidi et al., 1998; Hinz et al., 2000). Bcl proteins interact with other proteins through an α-helical domain, called the BH-3

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domain, which is thought to be important in the balance between cellular apoptosis and survival (Kelekar and Thompson, 1998). Pro-apoptotic Bcl proteins function by increasing the permeability of the mitochondrial outer membrane, releasing cytochrome-c into the cytoplasm. Bcl-2 is expressed on the outer surface of the outer mitochondrial membrane, on the endoplamic reticulum membrane and nuclear membrane and may act as sensor for cellular damage to these compartments (Green and Reed, 1998; Reed and Green, 2002). Although high expression levels of Bcl-2 are found in many human tumors, Bcl-2 levels are normal or decreased in pancreatic tumors (Campani et al., 2001; Evans et al., 2001). Bcl-x may be more important than Bcl-2 in protecting pancreatic cancer cells from Fas and TRAIL induced apoptosis (Hinz et al., 2000). Bcl-x exists in two molecular forms, one longer and anti-apoptotic, called Bcl-xL , and one shorter, Bcl-xS that functions to promote apoptosis. Bcl-xL is highly expressed in pancreatic tumor cells and not only prevents release of cytochrome-c from mitochondria, but it also binds to Apaf-1, thereby preventing the activation of caspase-9 (Hu et al., 1998). For these reasons, Bcl-xL is an attractive target for pancreatic cancer therapy. Recently Masui et al. (Masui et al., 2006) delivered Bcl-xL antisense oligonucleotides into the AsPC-1 pancreatic tumor cell line, as well as in mice harboring AsPC-1 xenografts. They found that antisense treatment reduced Bcl-xL expression by 90% in cell lines and increased the apoptotic index 48 hours after irradiation, In addition, tumor wet weight was significantly reduced in antisense treated mice (Masui et al., 2006). Reducing the expression of Bcl- xL may be an effective strategy to sensitize pancreatic tumors to radiotherapy or chemotherapy. Xu et al. (2002), demonstrated in pancreatic tumor cell lines that transfection and overexpression of Bax resulted in increased sensitivity to gemcitabine and 5-Fu (Xu et al., 2002). Schniewind et al. confirmed these results and showed that restoration of the mitochondria mediated apoptotic signaling pathway by overexpression of Bax demonstrated resulted in increased sensitivity of pancreatic tumor cell lines to gemcitabine treatment (Schniewind et al., 2004). It appears that the restoration of pro-apoptotic proteins in pancreatic cancer cells may be effective in increasing the sensitivity to chemotherapeutic agents, and a number of research groups are developing strategies to move this research into the clinic (Baell and Huang, 2002) .

Biomarkers of Apoptosis Caspases are intracellular proteases (cysteine-dependent aspartate-specific proteases) that function as both initiators (caspase-2, -8, -9, -10) as well as effectors (caspase-3, -6, -7) of programmed cell death and are widely used as markers for apoptosis. These are synthesized as inactive precursors called procaspases, which upon cleavage become active. Cleaved caspases are taken as indicative of active forms of caspases. A variety of antibodies available for both cleaved and un-cleaved caspases are used in Western blot techniques to asses their involvement upon induction of apoptosis by a given treatment agent. These proteases cleave their substrate

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after aspartic acid residues. Catalytic activity of caspases depends on a critical cysteine-residue within a highly conserved active-site pentapeptide QACRG (Yuan et al., 1993; Thornberry and Lazebnik, 1998). Phosphatidylinositol 3 Kinase (PI3K) and Akt/PKB are another set of markers widely used to study apoptosis. Akt/PKB is a serine/threonine protein kinase that provides resistance against apoptotic stimuli. Akt/PKB signaling is the major pathway that promotes cell survival. Activation of Akt is necessary for cell survival as it regulates the phosphorylation of other signaling proteins. These signaling proteins are implicated in cell signaling pathways that affect apoptosis (Franke et al., 1997; Kennedy et al., 1997). Akt acts as an apoptosis suppressor by providing Ras (V12)-mediated resistance to cyto c-induced proteolytic processing and activation of procaspase-9. Procaspase-9 is negatively regulated by Akt/PKB kinase and promotes survival through inactivation of caspase-9 (Cardone et al., 1998). Akt/ PKB is one of the best-characterized PI3K downstream targets (Downward, 1998) that interact with PIP3 or PIP2 through its PH (pleckstrin homology) domain. The PI 3-kinase family of enzymes phosphorylates PIP2 into PIP3 . PIP2 is also the substrate for phospholipase C that produces the key calcium regulator via Ins(1,4,5)P3 . In cancerous cells, the copy number of the PI3KCα gene increases, which encodes for p110α catalytic subunit of PI3K (Shayesteh et al., 1999). Inappropriate activation of the PI3K/Akt pathway leads to phosphorylation of several signaling proteins resulting in cellular proliferation (Burgering and Coffer, 1995). Activation of caspases and Akt is being routinely used by many investigators to study the effect of InsPs as an anticancer agent. For example, InsP6 inhibits growth of tumors that accompanies activation of caspase-3 in pancreatic (Somasundar et al., 2005), prostate (Sharma et al., 2003; Singh et al., 2003; Diallo et al., 2006), cutaneous melanoma (Rizvi et al., 2006), and colon cancer (Weglarz et al., 2006). Further, InsP6 also blocks cervical tumor cell growth by inhibiting the Akt mediated pathway. InsP6 causes a dose and time dependent degradation and cleavage of caspase-3, and caspase-9 in Hela cells. (Ferry et al., 2002). InsP6 also increases PKC δ activity and decreases Erk1/2 and Akt activity. InsP6 -induced inhibition of the Akt pathway appears to be dependent on PKC δ (Vucenik et al., 2005) in prostate and pancreatic cancer cells (Agarwal et al., 2004).

Apoptosis Mediated by Caspase Inhibitors It has been demonstrated that pancreatic cancer cell lines that are resistant to apoptosis have normal expression levels of cell surface receptors for death receptors such as TRAIL-R1-4 and CD95 (Trauzold et al., 2003). This information leads researchers to investigate other factors in the apoptotic cascade, such as the many natural inhibitors of caspase activity in order to determine the mechanism of apoptotic resistance in pancreatic cancer. Major natural inhibitors of caspase activity include the FLIP (FADD-like ICE inhibitory proteins) family and the inhibitor of apoptosis proteins(IAP) family. FLIP is expressed as two splice variants, a long

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form, FLIPL and a short form, FLIPS (Krueger et al., 2001). Expression of FLIPS confers resistance to apoptosis to pancreatic cancer cell lines (Mori et al., 2005). FLIPL lacks caspase activity, and competes with caspase-8 for binding to FADD at the DISC, thereby preventing the activation of caspase-8 and inhibiting apoptosis. Therefore increased expression of either FLIP variant, as observed in pancreatic cancer cells and pancreatic tumors that are resistant to Fas mediated apoptosis, may be a target to increase the sensitivity of these cells to apoptotic stimuli. Mori et al. (Mori et al., 2005) found that the resistance to TRAIL induced apoptosis in human pancreatic cancer cell lines was a result of elevated anti-apoptitic FLIPS expression. Cycloheximide treatment to inhibit protein synthesis reduced FLIP levels and combined treatment with cycloheximide and TRAIL restored the apoptotic response in resistant cells. In more recent experiments, Mori et al. (Mori et al., 2007) treated TRAIL-resistant cells with FLIP antisense, thereby reducing FLIP expression and restoring TRAIL-induced apoptosis. Wang et al. (2007) also determined that insensitivity to TRAIL- induced apoptosis in pancreatic cancer cells was related to high expression levels of FLIP variants. In an alternate strategy for restoring apoptotic response, knockdown of both FLIP variants was accomplished by a short hairpin RNA (shRNA), leading to cleavage of caspase-8 and activation of the mitochondrial apoptotic pathway. A synergistic effect was observed when these cells were also treated with chemotherapeutic agents such as camptothecin, celecoxib or cisplatin, suggesting that reducing FLIP levels in conjunction with chemotherapy may be effective therapy for inducing apoptosis in pancreatic tumors.

Apoptosis Mediated by the IAP Family Members of the inhibitor of apoptosis, or IAP family are proteins which contain a baculoviral IAP repeat domain (Birnbaum et al., 1994), which enables them to interact with caspases. This family includes cIAP, XIAP and survivin. Elevated expression levels of these proteins in many types of tumors have been shown to suppresses apoptosis (Deveraux and Reed, 1999; Deveraux et al., 2001). Protein overexpression of IAP family members cIAP-2, survivin, livin, and XIAP have been detected in pancreatic cancer (Lopes et al., 2007). Survivin expression increases through the development of pancreatic intraepithelial neoplasia, which is a precursor of pancreatic cancer (Bhanot et al., 2006). Survivin has a different structure from other members of the IAP family, containing only one BIR repeat and lacking the carboxy terminal RING finger domain common to other IAP family members. Survivin is expressed in a regulated manner during the G2 /M phase of the cell cycle (Li et al., 1998) and binds directly to the executioner caspases 3 and 7, thereby inhibiting their action and apoptosis (Tamm et al., 1998). Although the expression of survivin has not been detected in normal tissue (Ambrosini et al., 1997) it has been detected in a wide array of tumor types including pancreatic cancer (Satoh et al., 2001; Sarela et al., 2002). In one study, survivin protein expression was detected in 68% of pancreatic cancer tissues, while

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no expression was detected in normal pancreatic tissue (Kami et al., 2004). A significant association was found between survivin expression and patient survival, with patients whose tumors were positive for survivin expression having shorter survival time compared to those without survivin expression. Survivin expression has been identified as an inducible radioresistance factor in pancreatic cancer cells (Asanuma et al., 2000, 2002). A number of different strategies have been attempted in order to decrease survivin expression and render pancreatic cancer cells more susceptible to apoptosis. The introduction of a survivin-specific small inhibitory RNA (siRNA) into pancreatic cancer cell lines resulted in a 90% reduction in survivin mRNA and protein, reduced cell growth and increased apoptosis (Tsuji et al., 2005). Liang et al. constructed a DNAzyme to target and cleave survivin mRNA, which resulted in reduced survivin mRNA and significantly increased apoptosis after transfection into the human pancreatic cancer cell line PANC-1 (Liang et al., 2005). A survivin DNA vaccine has been effective in a murine pancreatic cancer model in slowing tumor growth and increasing survival (Zhu et al., 2007). Another group used a chemical CDK4 inhibitor or RNA interference to arrest pancreatic cancer cells in G1 phase of the cell cycle. They found that G1 arrested cells had increased sensitivity to TRAILinduced apoptosis due to transcriptional down-regulation of survivin (Retzer-Lidl et al., 2007). In another series of experiments, survivin RNAi expression vectors were transfected into the PANC-1 pancreatic tumor cell line resulting in a 70% decrease in survival levels and an increased apoptotic index (He et al., 2006). As survivin is expressed in tumor cells but rarely by normal cells, it has been used as a target for immunotherapy for pancreatic cancer patient suffering from pancreatic cancer resistant to chemotherapy where gemcitabine was given with survivin-based peptide vaccinations (Wobser et al., 2006). The patient had a partial remission of a liver metastasis for 8 months that progressed to a complete remission of 6 months duration. Unfortunately, the patient developed recurrent disease after he was weaned from vaccination. However, this result demonstrates the promise of targeting factors that suppress apoptosis as therapy for pancreatic carcinoma. SMAC/DIABLO (second mitochondria-derived activatior of caspase/directIAP binding protein with low pI) is a protein inhibitor of IAPs which are highly expressed in some tumors and block apoptosis by inhibiting caspases. Overexpression of SMAC/Diablo in neuroblastoma, glioblastome and pancreatic cancer cells has been shown to enhance gamma-irradiation induced apoptosis (Giagkousiklidis et al., 2005) and reduce clonogenic survival (Vogler et al., 2005).

Apoptosis Mediated by G-Protein Coupled Mechanisms Most literature reviews described relate to major apoptotic pathways mediated by intrinsic or extrinsic mechanisms, however it is not well known whether heterotrimeric G-proteins or its signal transduction pathways are involved in the regulation of apoptosis. There have been a number of studies that clearly demonstrate that heterotrimeric G-protein mediated signal transduction pathways are also involved in

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apoptotic regulation of cell survival in a number of cell types including pancreatic cells (Turner et al., 2000; Laychock et al., 2006). Heterotrimeric G-proteins exist as alpha, beta and gamma subunits which upon activation by activated G-protein coupled transmembrane receptors form a trimer complex. This complex in turn activates the effector enzymes that lead to formation of second messengers (Ali and Agrawal, 1994). Gs and Gi alpha subunits couple receptors to induce stimulation or inhibition of adenylyl cyclase that generates cAMP. Gq alpha subunit couples phospholipase C to production of Ins(1,4,5)P3 and diacyglycerol (DAG) that cause a rise in intracellular calcium and activation of protein kinase C respectively. First evidence that the signal transduction pathway controlling the apoptotic process in pancreatic beta-cells regulated by heterotrimeric G-proteins originated from studies employing the global G-protein activator, fluoride, that showed that this agent induced apoptosis in clonal RINm5F pancreatic beta-cells and also in the cells of normal rat islets of Langerhans (Loweth et al., 1996). Later on, it was suggested that cGMP and protein kinase G were involved in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15 (Loweth et al., 1997). The role of G-proteins was then confirmed in an osteoblast cell line where parathyroid hormone (PTH)/ parathyroid hormone related peptide(PTHrP) induced apoptosis via activation of the phospholipase C/ Ca++ signaling pathway, although signaling appeared to be converged with the tumor necrosis factor (TNFalpha) pathway (Turner et al., 2000). Recently, an unique bioactive phospholipid, sphingomysine 1-phosphate (S1P) that couples to S1P G-protein coupled receptor via activation of a Gq protein has been shown to alter cytokine-induced apoptosis in pancreatic islet beta cells (Laychock et al., 2006). Involvement of G-proteins in regulation of apoptosis in pancreatic cells (RINm5F) is also evident from experiments where tyrosine kinase inhibitors affected sodium fluoride and purtusis toxin induced cell death (Elliott et al., 2001). Further evidence regarding the role of G-protein mediated signal transduction pathways in the regulation of apoptosis comes from involvement of protein kinase C and its isoforms (Basu et al., 2001; Refsnes et al., 2002; Okhrimenko et al., 2005).

Apoptosis Mediated by Inositol Polyphosphates Exogenous InsPs have been shown to inhibit cellular growth by inducing apoptosis in certain cancer cell lines (Ozaki et al., 2000) thus displaying properties of anticancer agents (Janeb and Thompson, 2000; Vucenik et al., 2003). Most notably, InsP6 treatment at millimolar concentration range (1 mM InsP6 for 72 h) has shown significant reduction in cell proliferation and induction of apoptosis in pancreatic MIAPACA and PAN1 cancerous cell lines (Somasundar et al., 2005). This effect appeared to be mediated due to induction of apoptosis. InsP6 at these concentration ranges also inhibit cellular proliferation in prostate carcinoma (Sharma et al., 2003), DU145 (Agarwal et al., 2003; Singh et al., 2004) and LNCaP cells (Agarwal et al., 2004). Both pharmacological (2mM) and physiological (100 μM) doses of InsP6 were inhibitory to cell proliferation in breast cancer MCF7 (Vucenik et al., 2005) and

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Hep-2 laryngeal cancer cells (Dorsey et al., 2005). InsP4 and InsP5 were also tested for their anticancer activities in several cancer cell lines (Piccolo et al., 2004; Maffucci et al., 2005). InsP4 and InsP5 showed anti-tumor activity at relatively lower concentrations as compared to InsP6 (Razzini et al., 2000). InsP3 5/6 kinase that phosphorylates InsP3 into InsP4 , also phosphorylates cJun and IκBα involved in TNFα signaling; the overexpression of which protects TNFα-induced apoptosis (Wilson et al., 2001). The increased InsP3 5/6-kinase activity also results in a concomitant increase of InsP5 and InsP6 providing a possible mechanism for increased levels of InsP6 in protecting cells from TNFα and Fas induced apoptosis (Verbsky and Majerus, 2007). InsP5 / InsP6 promotes cell death by inhibiting PI3-K/AKT signaling pathway in lung, ovarian, and pancreatic cancer cell lines thus implicating a role in cancer prevention (Piccolo et al., 2004). Comparative effect of Ins(1,4,5,6)P4 , Ins(3,4,5,6)P4 , Ins(1,3,4,5,6)P5 , and InsP6 showed that InsP5 was more potent than InsP4 and InsP6 in these studies. PI3-kinsae, which phosphorylates PIP2 to PIP3 (Stephens et al., 1993) is shown to be active in ovarian cancer cells (Klippel et al., 1998). Further, Ins(1,3,4,5,6)P5 , Ins(1,4,5,6)P4 , and Ins(3,4,5,6)P4 are shown to inhibit cell growth of SCLC cells by competing with PIP3 binding to the PH domain of Akt/PKB (Jimenez et al., 1998; Razzini et al., 2000) suggesting their role in cancer prevention. Further, it was reported that InsP5 treatment shows antiangiogenic and antitumor effects in human umbilical vein endothelial cells by inhibiting PI3K/AKT signaling pathway and was more effective in causing this effect than InsP4 and InsP6 (Maffucci et al., 2005). Higher InsPs (InsP7 ) are also known to compete for PIP3 PH domain binding with Akt (Luo et al., 2003).

Changes in Cellular Levels of Inositol Polyphosphates During Apoptosis During receptor stimulation of phospholipase C, cellular levels of both InsP3 and InsP4 increase. Recently, inositol hexakisphosphate kinase-2 (Morrison et al., 2001) that coverts cellular InsP6 into InsP7 was shown to mediate apoptosis as a result of increased cellular levels of InsP7 (Nagata et al., 2005). Endogenous accumulation of InsP7 can also be achieved by treatment with NaF due to inhibition of inositol polyphosphate pyrophosphatases (Menniti et al., 1993) or diphosphoinositol polyphosphate phosphohydrolases, the phosphatases involved in hydrolysis of [PP]2 -InsP4 / InsP8 and PP-InsP5 / InsP7 (Shears et al., 1995; Safrany and Shears, 1998). Incubation of cells with millimolar or even lower concentrations increases cellular levels of PP-InsP5 / InsP7 and [PP]2 -InsP4 / InsP8 . (Safrany and Shears, 1998); higher NaF concentrations may cause G-protein coupled activation of phosphatidylinositol specific phospholipase C (PI-PLC) and thus accumulation of lower InsPs. NaF is also known to induce apoptosis in a number of cell lines. It is likely that sodium fluoride-induced apoptosis is mediated by accumulation of higher inositol polyphosphates. Recently, it has been shown that synthesis of InsP8 is activated during apoptosis due to hyper osmotic stress by sorbitol as a consequence

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of activation of ERK1/2 and p38MAPα/β kinases (Pesesse et al., 2004). Nagata et al., 2005, showed that enhanced InsP6 kinase activity during apoptosis leads to increased cellular levels of InsP7 suggesting that InsP7 is involved in cell death. Multiple inositol polyphosphate phosphatase (MINPP) located in endoplasmic reticulum (Ali et al., 1993; Craxton et al., 1995, 1997) hydrolyzes InsPs in vitro. However, there is no evidence that shows its activity in vivo. In vitro studies revealed that the most abundant InsPs, InsP5 and InsP6 are high affinity substrates. MINPP also hydrolyzes InsP7 and InsP8 . MINPP gene deletion leads to elevation in InsP5 and InsP6 levels, which can be reversed by genetic re-introduction of the MINPP gene (Chi et al., 2000). Further studies on MINPP revealed that there are other activities involving the role of MINPP in inositol polyphosphate metabolism and thus in regulation of apoptosis and carcinogenesis. For example, mutations in the MINPP gene involving amino-acid residues, which are remote from the phosphatase active site, result in tumorigenesis of malignant follicular thyroid carcinomas (Gimm et al., 2001). This indicates that the MINPP gene is important in maintaining the normal cell signaling in relation to tumorigenesis. PTEN (phosphatase and tensin homolog deleted on chromosome 10), a tumor suppressor, is another phosphatase the that also hydrolyzes InsPs. Its chromosomal location is interesting in relation to MINPP location; they both are located in close proximity to each other on human chromosome 10q23. Survival signaling by AKT is inhibited by PTEN antagonizing the action of PI3K. PI3K and AKT are overexpressed in a variety of cancers whereas PTEN is deleted in advanced tumors. PTEN deletion may cause a rise in cellular InsPs and hence induction of apoptosis.

Treatment Agents That Induce Apoptosis in Pancreatic Cells The major challenge in pancreatic carcinogenesis lies in developing effective chemical treatment strategies to inhibit the growth of adenocarcinoma of the pancreas. Pancreatic tumors display strong resistance to almost all classes of conventional chemotherapeutic agents that are known to be cytotoxic (see for a review, Adrian, 2007). This resistance is inherent to pancreatic cancer rather than acquired during the course of drug administration. The success of effective treatment depends on specifically targeting those agents to inhibit proliferative and induce antiproliferative (apoptotic) signaling pathways. Recent advances in understanding the molecular events involved in signaling pathways for cancer initiation, progression and metastasis have led to multiple strategies that induce cell death and apoptosis (Mimeault et al., 2005; Cascinu et al., 2006; Yeh and Der, 2007). Inhibition of phosphatidylinositol 3-kinase/AKT /BKB (PI3K/ AKT) cell survival pathway that leads to induction of apoptosis is the commonly exploited target. Indeed, the most active agent, gemcitabine, a DNA chain terminator, used alone or in combination with wortmanin or the epidermal growth factor receptor inhibitor OSI-774, Tarceva, enhances apoptosis by this mechanism (Ng et al., 2000, 2002). However, gemcitabine, when used with C225, also an epidermal growth factor receptor inhibitor agent, results in regression of pancreatic carcinoma by antiangiogenic mechanisms (Bruns et al., 2000).

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Lipooxygenase inhibitors that directly affect arachidonic acid metabolism involving phospholipase A2 have been shown to induce apoptosis through the mitochondrial pathway (Tong et al., 2002). More recently, the same group of investigators has shown that leukotriene B4 receptor antagonist, LY293111, inhibits proliferation and induces apoptosis in human pancreatic cancer cells both in vitro and in vivo, perhaps by a cell cycle arrest mechanism (Tong et al., 2007). A number of naturally occurring agents such as polyphenols, retinoic acid and phytic acid have also been studied for their effectiveness in inducing apoptosis. A list of chemical treatment agents that are known to induce apoptosis in certain pancreatic cancer cell lines or model systems and their apoptotic signaling pathways involved are given in the Table 13.1. Epigallocatechin-3-gallate (EGCG) from green tea extract exerts growth-suppressive effect on human pancreatic cancer cells in vitro and induces apoptosis by nuclear condensation, caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage that accompanies growth arrest at an earlier phase of the cell cycle (Qanungo et al., 2005). Retinoids are potent growth inhibitory and differentiating agents in a variety of cancer cell types. Recently, in pancreatic cancer cells, retinoids have been shown to induce growth arrest regardless of their differentiation status perhaps by inducing expression of TGF-β, a growth inhibitory factor, in a p53-independent manner (Pettersson et al., 2002; Singh et al., 2007). Phytic acid, inositol hexkisphosphates, on the other hand mediate induction of apoptosis in a number of cancer cells including pancreatic cancer cell lines MIAPACA and PANC1 (Somasundar et al., 2005) and in vivo models by inhibiting phosphatidylinositol 3-kinase/ AKT/PKB cell survival pathways, perhaps due to interference of phosphate groups in the pleckstrin homology (PH) domain of the phosphatidylinositol 3-kinase.

Table 13.1 List of chemical treatment agents that are known to induce apoptosis in pancreatic cancer cell lines or models and the target site and/or apoptotic signaling pathways affected Treatment Cancer cell Signaling agents lines/model pathways/target sites References Gemcitabine

Human pancreatic adenocarcinoma

Ng et al., 2000, 2002; DNA chain terminator, Bruns et al., 2000 inhibition of phosphatidylinositide 3-kinase and epidermal growth factor, antiangiogenic Inhibition of cell proliferation, Tong et al. 2007 cell cycle arrest

Leukotriene B4 Human pancreatic cancer cells, receptor antagonist, LY293111 Lipoxygenase Human pancreatic Induction of apoptosis through Tong et al. 2002 inhibitors cancer xenografts the mitochondrial pathway Qanungo et al. 2005 Epigallocatechin- Human pancreatic Nuclear condensation, 3-gallate cancer cells caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage, earlier phase cell cycle arrest

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Table 13.1 (continued) Cancer cell Treatment agents lines/model Retinoids

Human pancreatic cancer cells

Signaling pathways/tar get sites

Activation of RAR-gamma and expression of Bcl-2/Bax, expression of TGF-β in a p53-independent pathway Pancreatic cell lines Inhibition of Phytic acid phosphatidylinositol MIAPACA and (inositol hexa3-kinase/ AKT/PKB cell PANC1 hosphates) survival pathways Oil A/ Red oil Human pancreatic Activation of caspase cascade, A5 cancer cells modification of cell cycle-regulating proteins P21 and P27 and GADD expression Arsenic trioxide Human pancreatic Activation of caspase cascade cancer cells and GADD expression Sphingosine Rat pancreatic islet Cytokine-induced apoptosis 1-phosphate beta-cells Soy isoflavone Human pancreatic Inactivation of nuclear factor (genistein) and cancer cells kappaB flavonoid (apigenein) Resveratrol Human pancreatic Induction of apoptosis via a (phytoalexin) cancer cells mitochondrial pathway Higher baseline Human smokers in Exposure to higher insulin blood glucose (alpha tocopherol, concentration and insulin and insulin beta carotene) resistance predicts exocrine concentrations clinical trial pancreatic cancer risk Gingerol Human pancreatic Induction of cell cycle arrest (polyphenol) cancer cells and cell death of mutant p53-expressing cells Vitamin D Human smokers in Subjects with higher serum Vitamin D levels had higher (alpha tocopherol, incidence beta carotene) clinical trial Oleandrin PANC-1 human Inhibition of cell proliferation (Lipid-soluble pancreatic cancer associated with induction of cardiac cell line G(2)/M cell cycle arrest glycosides) Erlotinib Metastatic Inhibition of epidermal growth (Tarceva) adenocarcinoma factor receptor type of the pancreas 1/epidermal growth factor receptor (HER1/EGFR) tyrosine kinase Nicotine and/or Rat pancreatic tumor Increased proliferation via hydrogen cell line (AR42J activation of ERK 1/2 peroxide cells) Nicotine Isolated rat Increased proliferation via pancreatic acinar activation of ERK 1/2 cells

References Pettersson et al., 2002; Singh et al. 2007

Somasundar et al., 2005

Dong et al., 2003, 2004

Li et al., 2003 Laychock et al., 2006 Li et al., 2005

Sun et al., 2008 Stolzenberg-Solomon et al., 2005

Park et al., 2008

Stolzenberg-Solomon et al., 2006

Newman et al., 2007

Senderowicz et al., 2007

Bose et al., 2005 Chowdhury and Walker, 2008 Chowdhury et al., 2007

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Position for Table Other treatment agents that have been recently studied include oil A/ Red oil A5 that induce apoptosis of pancreatic cancer cells via activating caspase cascade, modifying cell cycle progress and changing cell cycle-regulating proteins P21 and P27 and GADD expression (Dong et al., 2003, 2004). These findings showed that oils or their extracts could potentially be used as anti-proliferative agents on human pancreatic cancer cells to induce apoptosis. In another study, arsenic trioxide has been shown to induce apoptosis in pancreatic cancer cells via a similar mechanism of activation of caspase cascade and GADD expression (Li et al., 2003).

Diagnostic Markers for Pancreatic Cancer The prognosis of pancreatic cancer remains poor primarily because of its aggressive metastasis and lack of early stage clinical diagnostic tests. This hampers the application of appropriate therapies and hence results in low survival rates. Patients with early stages of pancreatic cancer development usually do not display any specific symptoms. Surgical removal of the localized cancer tissues is still the best treatment option. Therefore, there is further demand in clinical pancreatology to find more specific molecular markers that are sensitive enough to detect early stages of pancreatic cancer. A number of serum-based markers such as CA 19-9, CA 125 and M2-PK and tissue-based markers such as K-ras (Moskaluk et al., 1997), tumor suppressor genes p16, p53 (Lu et al., 2002), mucins, telomerase activity, growth factors and DNA methylation in combination with global gene profiling can be used to develop tests. However, none of these markers is currently developed to be useful for early detection. Patients with pancreatic adenocarcinoma are generally tested by CT scan followed by MRI to assess any morphological abnormalities. Further examination by endoscopic ultrasound (EUS) to produce images of the pancreatic ducts (Salek et al., 2007) followed by a second examination called endoscopic retrograde cholangiopancreatography (ERCP) using a contrast dye injection before x-ray often detects irregularities in pancreatic ducts. Currently, CA 19-9 is the only serum marker used for pancreatic cancer diagnosis. Carcinoembryonic antigen (CEA) is also used to detect advanced pancreatic cancer in some patients but it is not sensitive enough to diagnose at an early stage and hence it is not recommended as a screening test. Recently, RON, a phosphotyrosine kinase receptor, associated with the tumor suppressor gene Smad4/DPC4, a key transcription factor in transforming growth factor-beta (TGF-β) signaling cascades, has been suggested as a key factor contributing to pancreatic cancer progression (Zhao et al., 2008). Normaly, pancreas has very low levels of RON, but the expression of RON increases with increased metastasis. To identify biomarkers for pancreatic carcinogenesis, high-throughput proteomic and genomic approaches (Koopmann et al., 2006; Yip and Lomas, 2002) in combination with bioanalytical methods have identified potential genes and proteins such

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as clusterin, MUC4, survivin and mesothelin that could have roles in pancreatic carcinogenesis. Clusterin (apolipoprotein J), a glycoprotein that also serves as a heat shock protein is overexpressed in pancreatic cancer. MUC4, a transmembrane apomucin has been reported to be overexpressed in more than 75% of pancreatic adenocarcinomas but not in chronic pancreatitis. Thus, expression of MUC4 could be used as a prognostic indicator for pancreatic adenocarcinoma. Survivin is another candidate protein that inhibits apoptosis. Overexpression of survivin in human pancreatic tissues is considered an early molecular event that leads to cellular proliferation during carcinogensis and thus can serve as a diagnostic biomarker for pancreatic cancer. Mesothelin, a differentiation antigen, has been shown to be expressed in more than 85–100 % pancreatic cancer cells. Except for mesothelin, none of these candidate proteins have been translated to diagnostic markers.

Therapeutic Approaches to Identify Diagnostic Biomarkers for Pancreatic Cancer Human pancreatic cancer cells produce a number of growth factors, contributing to the unregulated growth potential of these tumors. Depriving tumor cells of autocrine growth factors induces apoptosis and this mechanism has been explored as a strategy for targeted therapies. Important growth factor pathways for pancreatic cancer cells include the phosphatidylinositol 3-kinase {PI3K}/protein kinase B (AKT) survival pathway, and the epidermal growth factor receptor (EGFR), nuclear factor κB (NF κB), the p53 tumor suppressor gene and the lipoxygenase/cyclooxygenase pathways. In addition, approximately 90% of human pancreatic tumors have activating mutations in the k-ras gene (Hruban et al., 1993). These mutations occur early in pancreatic tumorigenesis and result in constitutively activated ras signaling, giving affected cells a growth advantage early in their development. Both EGF and its receptor EGFR are over-expressed in the majority of pancreatic tumor cells, along with other EGFR ligands including transforming growth factor α {TGF- α}, and heparin binding EGF (Korc et al., 1992). Binding of these ligands to EGFR initiates a signal cascade that involves mutated k-ras and leads to stimulation of tumor cell growth. Buchsbaum et al. (Buchsbaum et al., 2002) treated pancreatic cancer cell lines and murine xenografts with the anti-EGFR antibody Erbitux (IMC-C225), resulting in inhibition of tumor growth and, in combination with gemcitabine and radiation, 100% complete regression of tumor xenografts. Activation of the PI3K/AKT pathway occurs through binding of growth factor receptors to their specific ligands, signaling cellular survival and growth (Datta et al., 1999). AKT activation generates cell survival signals by activating or inducing the expression of anti-apoptotic members of the Bcl family as well as MDM2 and NF κB. AKT activation also inactivates pro-apoptotic proteins, including Bad, forkhead, caspase-9 and glycogen synthase kinase 3 (GSK3) (Jones et al., 2000, 2005). PI3K activity and survival signaling through AKT is counteracted by PTEN (phosphatase and tensin homolog), which is often deleted in advanced tumors (Podsypanina et al., 1999). Pancreatic cancer cells exhibit increased expression of PI3K, AKT

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and numerous growth factors, while PTEN is often down-regulated (Adrian, 2007). Activation of PI3K/AKT leads to a constitutively active survival signaling pathway that contributes to insensitivity to apoptotic signals in tumor cells (Westphal and Kalthoff, 2003). For this reason, this pathway is an important target for resensitizing pancreatic cancer cells to apoptotic signals. Treatment of pancreatic cancer cells with wortmannin, a fungal metabolite that inhibits PI3K (Boehle et al., 2002), results in decreased proliferation, increased apoptosis and increased sensitivity to the chemotherapeutic agent gemcitabine (Ng et al., 2000, 2001). Another PI3K inhibitor, GN963 with activity against AKT, platelet-derived growth factor and Src kinases, was effective in reducing tumor volume and increasing the effectiveness of gemcitabine, while completely inhibiting liver metastasis in model of human pancreatic cancer cells grown orthtopically in nude mice (Baker et al., 2006). It is known that nicotine is a potential risk factor for induction of pancreatic cancer in smokers. In recent studies, it has been shown that pancreatic tumor cells derived from rat or pancreatic acinar cells isolated from rat that are exposed to nicotine show increased proliferation via activation of mitogen activated ERK 1/2 siganling pathways (Bose et al., 2005; Chowdhury et al., 2007, Chowdhury and Walker, 2008). UO126, an specific inhibitor of MAPK signals abolished the proliferative ability by nicotine suggesting that drug induced proliferation of acinar cells is MAP kinase dependent. A number of strategies have been devised to normalize the activity of k-ras in human pancreatic cancer cells. It has been shown that protein kinase c {PKC} activation by phorbol esters resulted in growth arrest in pancreatic cancer cell lines (Salabat et al., 2006). A possible mechanism for this inhibition of growth may be the phosphorylation of Ser181 of k-ras, which causes the translocation of k-ras from the plasma membrane to the mitochondrial membrane, where it initiates apoptosis by interacting with Bcl-X (Bivona et al., 2006). These results suggest that agents that stimulate the phosphorylation of Ser181 of k-ras may be candidates for therapeutic agents for ras dependent tumors such as pancreatic cancer.

Diet and Risk Factors for Pancreatic Cancer Epidemiological studies have shown the importance of genetic and environmental risk factors in colon and breast cancer (Shrubsole et al., 2004; Reszka et al., 2006). Similar interactions may play prominent roles in pancreatic cancer, although studies examining risk factors have yet to provide consistent strategies for disease prevention (Hine et al., 2003). Several other studies suggested positive associations between pancreatic cancer and diabetes and chronic pancreatitis (Ghadirian et al., 2003; Jee et al., 2005; Giovannucci and Michaud, 2007; Lowenfel and Maisonneuve, 2005). In 2007, American Institute for Cancer Research/World Cancer Research Fund (AICR/WCRF) review panel found a relationship between “body fatness”, as assessed by BMI, and cancer risk factors (Food Nutrition (AICR/WCRFP Publication,

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2007); Food Food Nutrition et al., 2007). Additionally, the panel found a 70% rise in risk and significant interaction between BMI and caloric intake in subjects who are above the median for both BMI and caloric intake. An increased risk for pancreatic cancer among people with longstanding diabetes has been observed. This association was independent of insulin use (Silverman, 2001). Among those who had diabetes, however, BMI was not related to pancreatic cancer risk. A multi-center case-control study of subjects from five European countries demonstrated that the highest intake of carbohydrates resulted in a 2.57-fold risk in pancreatic cancer. When dietary data from the prospective cohort Nurses’ Health Study (NHS), n=88,802, were examined for the relationship to pancreatic cancer risk, results indicated that high carbohydrate, sucrose and fructose intakes were not significantly correlated with pancreas cancer risk, (Michaud et al., 2002). These findings support the hypothesis that impaired glucose metabolism plays a role in the pathogenesis of pancreatic cancer and that diet high in glycemic load may increase the risk of pancreatic cancer among individuals who have an underlying degree of insulin resistance or hyperglycemia (Nothlings et al., 2007; Patel et al., 2007). Analysis of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study indicated a significant inverse association between pancreatic cancer and overall carbohydrate intake indicating that food macronutrients have a positive impact on pancreatic cancer prevention (Stolzenberg-Solomon et al., 2008). Pancreatic tumor cells are characterized by a specific high-glucose utilizing phenotype (Boros et al., 2005). Investigation of the metabolomic profiles of tumor cells may be crucial in the study of the effects of energy and dietary macronutrients on pancreatic cancer. Evidence indicates that intermediary metabolic enzymes, such as those involved in substrate flux through metabolic pathways and the contribution of substrates to macromolecule synthesis, are associated with the regulation of growth-signaling pathways that may promote malignant cell transformation (Boros et al., 2005). This is a critical area of research because differing levels of metabolites can be closely linked to normal and aberrant phenotypes. Potential breakthroughs in early detection and treatment of pancreatic cancer are benefits that may emerge through employing stable isotope based metabolic monitoring using GC-mass spectrometry (Boros et al., 2002).

Role of Oxidative Stress in Pancreatic Cancer and Prevention Inflammation and oxidative stress have also been implicated in pancreatic cancer etiology. Inflammatory cytokines, reactive oxygen species (ROS), and mediators of inflammatory pathways, such as cyclooxygenase-2 (COX-2) and Nuclear Factor kappa B (NFkB), are associated with oncogene expression, silencing of tumor suppressor genes, and affect the cell cycle, all of which may facilitate pancreatic carcinogensis (Giovannucci and Michaud, 2007; Kaneto et al., 2001). Mediators of the inflammatory response may also induce genetic damage, cell proliferation, and inhibition of apoptosis in the pancreas. Because ROS contribute to the

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inflammatory process, evaluating the potential cancer protective effects of dietary antioxidants is a logical step in this area of research (Srinivasan et al., 2007; Song et al., 2005). NFkB, when activated, increases secretion of inflammatory cytokines. Antioxidants inhibit both NFkB activation and secretion of IL-6 and IL-8 in CAPAN-2 cells, but not in CAPAN-1 cells, suggesting that cytokine expression in these cells may be more complex and could involve mechanisms other than NFkB activation. Phospholipase A2 is overexpressed in pancreatic cancer, which cleaves arachidonic acid (AA) from membrane phospholipids, with a concomitant overexpression of AA-metabolizing enzymes of the COX and lipoxygenase (LOX) families. For example, COX-2 is overexpressed in the atrophic acinar cells, hyperplastic ductal cells, and islets cells in most patients with chronic pancreatitis, but not in normal pancreatic tissues (Cuendet and Pezzuto, 2000). Agents that block COX enzymes have been shown to significantly inhibit pancreatic cancer cell growth and to induce apoptosis (Yip-Schneider et al., 2007). Other data suggests that a selective COX-2 inhibitor may slow down the growth of human pancreatic cancer through changes in gene expression that favor cell cycle arrest (Xu et al., 2005). Furthermore, exposure to reducing sugars such as glucose and fructose may increase oxidative stress in pancreatic cells. Fructose increases cellular peroxide levels and lipid peroxidation in hamster islet tumor (HIT) cells (Suzuki et al., 2000). Fructose suppresses the expression of glutathione peroxidase (GPx) mRNA and causes inactivation of GPx in HIT cells. The impact on GPx suggests a mechanism by which fructose induces oxidative stress. The role of fructose and its interactions with nitric oxide, which also increases intracellular peroxide levels in HIT cells, may represent an important mechanism affecting apoptosis in proliferating pancreatic tumors.

Nutritional Impact on Pancreatic Apoptosis Besides use of chemical treatment agents to induce apoptosis, in an effort to prevent pancreatic cancer, a number of studies suggest that bioactive nutritional components such as soy proteins and other phytochemicals have the ability to significantly alter pancreatic apoptosis. For example, the soy protein genistein and the dietary flavonoid apigenein augment the cytotoxic action of gemcitabine in pancreatic tumor cells via down regulation of transcription factor-NF-kappaB and anti-apoptotic Akt expression (Li et al., 2005). Resveratrol, a phytoalexin found in grape skin (Mouria et al., 2002) displays a protective effect by inhibition of tumor cell proliferation triggering changes in mitochondrial cytochrome C, which affect apoptosis (Sun et al., 2008). Gingerol, a major phenolic compound found in ginger mediates in vitro anti-tumor activity in pancreatic cancer cells (Park et al., 2006) by inhibiting p53/ AKT cell survival signaling pathway. Thus, there are multiple ways in which diverse environmental variables may influence development and progression of pancreatic malignancies.

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Conclusions Experimental models indicate that NF-kB, a proinflammatory transcription factor plays an important role in the regulation of apoptosis, as well as tumor angiogenesis, proliferation, invasion and metastasis. More recently it has been shown that suppression of NF-kB inhibits the oncogenic potential of transformed cells thus making it as an interesting therapeutic target for cancer. This approach has recently been reviewed extensively by Sethi et al. (2008). Thus therapeutic targeting of NF-kB in cancer drug design could be considered to be a very potential promising area. The growth and development of pancreatic tumors, as well as their resistance to most chemotherapeutic agents and radiation, have a basis in the resistance of these cells to apoptotic stimuli. The therapeutic strategies outlined in this chapter that aim to re-sensitize pancreatic tumor cells to apoptotic stimuli show great promise in reducing tumor growth and increasing response to radiotherapy and chemotherapy. It is hoped that some of these strategies can be translated to the clinic in order to increase survival of patients with this devastating disease.

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Chapter 14

Strategies to Circumvent Resistance to Apoptosis in Prostate Cancer Cells by Targeted Necrosis Richard D. Dinnen, Daniel P. Petrylak and Robert L. Fine

Abstract Cancer cells escape apoptosis by intrinsic or acquired mechanisms of drug resistance. One alternative strategy to circumvent resistance to apoptosis could be through re-direction into other death pathways, such as necrosis. However, necrosis is a non-specific, non-targeted process resulting in cell lysis and inflammation of both cancer and normal cells and therefore, not a viable alternative. However, if the necrosis could be targeted only to cancer cells which are undergoing an aborted apoptosis, then it may be possible to achieve a targeted and successful cell death. We termed this process “targeted necrosis”. We reported that a C-terminal peptide of p53, called p53p-Ant, induced high levels of targeted necrosis only in multiple mutant p53 human prostate cancer cells and not normal cells, because the mechanism of cytotoxicity by p53p-Ant is dependent on the presence of high levels of mutant p53. Other non-prostate cancer lines (breast, lung, colon, mesothelioma, glioma, bladder) were induced by p53p-Ant into apoptosis and not into targeted necrosis as the prostate cancer cell lines. Topotecan and paclitaxel resistant prostate cancer lines were as sensitive to p53p-Ant-induced targeted necrosis as parental lines. Intracellular generation of reactive oxygen species was involved in the mechanism of targeted necrosis, which was inhibited by O2 −. scavengers. We hypothesized that targeted necrosis by p53p-Ant is dependent on mutant p53, mediated by O2 −. and can circumvent chemotherapy resistance to apoptosis. Targeted necrosis, as an alternative pathway for selective killing of cancer cells, may overcome the problems of non-specificity in utilizing the necrotic pathway. Keywords Apoptosis · Necrosis · Drug resistance · p53 · Reactive oxygen species

R.L. Fine (B) Experimental Therapeutics, Division of Medical Oncology, College of Physicians and Surgeons, Columbia University, 650 West 168th Street, BB 20-05, New York, NY 10032, USA e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 14,

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Introduction The currently accepted paradigm for the action of most anticancer agents, is that at clinically achievable concentrations, they act by inducing cell death in cancer cells via pathways of apoptosis. Therefore, research efforts have primarily focused on developing anticancer agents to maximize caspase-dependent or caspaseindependent apoptosis. Unfortunately, the great majority of cancer cells eventually develop multiple, specific apoptotic resistance mechanisms. These include, for example, changes in bcl-2 (Raffo et al., 1995), p53 (Effert et al., 1992, Isaacs et al., 1991), p21 (Wang et al., 2001), and a myriad of other changes (Chen et al., 2002; Miyake et al., 2003). Therefore, even if one disable one form or one pathway of drug resistance, the cancer cell may still remain resistant, or quickly develop resistance because of the multiple, anti-apoptotic mechanisms which exist concomitantly within cancer cells (Howell, 2000). Therefore, an alternative strategy for cell death induction could be to circumvent resistance to apoptosis through re-direction into other death pathways, such as necrosis. A switch to the necrotic pathway is a promising possibility in that it has fewer mechanisms of resistance compared to apoptosis. But, because necrosis is a non-specific process resulting in cell lysis and inflammation of cancer and normal cells, necrosis has not generally been considered a viable alternative to apoptosis. However, it maybe possible to exploit necrosis, specifically against cancer cells, by induction of what we termed “targeted necrosis” (Dinnen et al., 2007). Targeted necrosis has potential clinical utility, since its cell death mechanism retains the specificity gained from the initiation of apoptosis but bypasses apoptotic resistance by re-direction into necrosis after the cell death process of apoptosis has begun. A method that seeks to bypass apoptosis resistance by initially inducing apoptosis in cancer cells and switching them to a necrotic death pathway may hold promise in the continuing effort to seek cell death specificity in prostate cancer cells without the development of cell death resistance. The molecular mechanisms of necrosis have not been well defined, but it can occur as a result of an incomplete execution of apoptosis (Formigli et al., 2000). Necrosis and apoptosis share some common pathway elements (Leist et al., 1997), and often the same stimulus can direct a cell into either mode of death (Bonfoco et al., 1995). Apoptosis requires energy through ATP, while necrosis is an ATP-independent mechanism of cell death. Therefore one primary factor which determines whether a cell follows an apoptotic or necrotic pathway is the intracellular concentration of ATP (Eguchi et al., 1997; Formigli et al., 2000). The absence of sufficient energy reserves can switch cells from an apoptotic to a necrotic form of death. Poly(ADP-ribose) polymerase (PARP) has been proposed as one of the switch points which determines whether a cell undergoes apoptosis (when PARP is cleaved and inactivated, not depleting ATP pools), or necrosis (when PARP is not cleaved and remains active, causing or contributing to low ATP pools) (Ha and Snyder, 1999). PARP is normally cleaved by caspases 3 and/or 7. Lack of PARP cleavage through inhibition of caspases, such as caspase 3 (Prabhakaran et al., 2004)

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or activation of PARP can ultimately cause necrosis by depletion of ADP-ribose leading to decreased ADP pools to form ATP (Ha and Snyder, 1999). In a recent study, we reported that a C-terminal peptide of p53, called p53pAnt, induced targeted necrosis only in multiple mutant p53 human prostate cancer cells and not normal cells, because the mechanism of cytotoxicity by p53p-Ant was dependent on the presence of high levels of mutant p53. p53p-Ant is a 22aa peptide from the COOH terminus of p53 (p53p, aa 361–382), linked to a truncated 17aa peptide from the Drosophila antennapedia homeobox domain (Ant) to facilitate cellular uptake. We have found that this peptide induced classical apoptosis in several colon, breast carcinoma and Burkitt’s lymphoma lines with mutant p53 (Kim et al., 1999; Selivanova et al., 1997; Selivanova et al., 1999). The mechanism of apoptosis by p53p-Ant was through a non-transcriptional/ non-translational, Fas/caspase 3 and 8-dependent pathway with cleavage of PARP (Kim et al., 1999). Our lab and others have shown that the apoptosis in tumor cells was directly correlated with levels of mutant p53 while non-toxic to non-malignant or normal cells with normal levels of wild type (wt) p53 (Kim et al., 1999; Selivanova et al., 1997). p53p-Ant-induced classical apoptosis in multiple human carcinoma cell lines expressing mutant p53, but not in null p53 tumor lines. Nonmalignant human colon and breast cell lines expressing low levels of wt p53, and normal human peripheral blood CD34 positive stem cell progenitors for CFU-GEMM (Senatus et al., 2006), were unaffected by the p53p-Ant peptide; but pre-malignant, mutant p53 colon and breast cell lines underwent Fas-mediated apoptosis (Kim et al., 1999; Li et al., 2005). Thus, this p53-derived peptide’s ability for induction of apoptosis was directly correlated to levels of endogenous p53. The binding target site of the peptide on whole p53, wild type or mutant, was at the tetramerization domain of p53 (aa 320– 360) (unpublished data). Further studies in our lab with p53p-Ant and purified wt and mutant p53 in surface plasmon resonance (Biacore) studies, revealed potent dissociation constants (Kd ∼ 10−12 M) for mutant p53, while the Kd for wt p53 was 3–4 fold weaker. This may explain why p53p-Ant was not toxic to null p53 cells, normal cells and CD34+ pluripotent marrow stem cells, which have low levels of wt p53 (Li et al., 2005). Our initial studies in prostate cancer cell lines concluded that p53p-Ant is equally transduced across plasma and nuclear membranes in both a sensitive prostate cancer cell line (DU-145, mutant p53) as well as a resistant cell line (PC-3, null p53) as determined by western blotting. Immunoblotting of p53p-Ant alone without cells produced a band of 4.4 kDa. A 4.4 kDa band was detected in DU-145 lysates. Similar results were found for PC-3 cells (Fig. 14.1A). The 4.4 kDa band was not observed in DU-145 cells incubated with p53p alone without Ant (Fig. 14.1B). To determine subcellular localization of p53p-Ant, live cells were incubated with p53p-Ant-RhoB and its fluorescence was monitored in DU-145 cells by confocal microscopy over 10 min. p53p-Ant rapidly crossed the plasma membrane with cytoplasmic, nuclear and nucleolar localization within 1 min in both DU-145 and PC-3 cells (Fig 14.1C). Morphological examination, observed by phase contrast microscopy, indicated rapid and distinct changes in the sensitive DU-145 line in

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Fig. 14.1 p53p-Ant uptake. (A) DU-145 or PC-3 cells exposed to 30 μM p53p-Ant for up to 10 min. Anti-p53 pAb421 antibody (epitope 371–380 aa) detects peptide. Blots were stripped and re-probed with anti-p53 DO-1 antibody (epitope 11–25 aa) to confirm endogenous p53 status. (B) DU-145 cells were untreated (CONT) or incubated with 30 μM p53p or p53p-Ant for 10 min. Cell lysates were analyzed as above. (C) DU-145 cells incubated with 30 μM p53p-Ant-RhoB and fluorescence monitored by confocal microscopy. The same group of cells was followed for up to 10 min. Untreated cells showed no staining. Low intensity fluorescence is represented by gray (background color), medium intensity by white and high intensity by black

response to p53p-Ant (Fig 14.2A). Loss of the typical cobblestone-like appearance was observed, with cells rounding and detaching from the plate at less than 1 h post treatment. Both plasma and nuclear membranes became more distinct, associated with subsequent swelling. In contrast, PC-3 cells (null p53) showed an initial rounding and plasma membrane ruffling by 1 h, but then the majority of PC-3 cells regained the appearance of untreated cells by 6 h with approximately 10% rounded and detached cells observed (Fig 14.2A). In contrast, the majority of PC-3 cells regained the appearance of untreated cells by 6 h. In addition, p53p-Ant, but not the controls Ant or p53p alone, was significantly more inhibitory to DU-145 than PC-3 cells at all time-points tested by MTT assay (Fig 14.2B, C). In addition, cell death increased 7–14 fold in all prostate cell lines with mutant p53 (DU-145, 22rv1 and VCaP) and approximately 1.6 fold in LNCap (wt p53), and p69 cells (immortalized, non-malignant prostate cell line, wt p53). To further confirm that p53p-Ant-induced cell death was dependent on the presence of mutant p53, cell death in stably transfected temperature sensitive PC-3 cells expressing mutant p53 −143 at 37◦ and wild type p53 at 32◦ was determined. Cells were approximately 1.6 fold more sensitive to p53p-Ant at the mutant p53 temperature (37◦ ) than at the wt p53 temperature (32◦ ) (Fig 14.2D). This is consistent with

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BIACORE data showing that p53p-Ant binds wt p53 with 3-4 fold lower affinity than mutant p53. We previously had reported that in p53 mutant breast cancer cell lines, p53pAnt induced apoptosis without necrosis (Kim et al., 1999). Therefore we examined apoptotic markers in p53p-Ant-induced cytotoxicity in prostate cancer cell lines.

Fig. 14.2 Morphological changes and growth inhibition induced by p53p-Ant treatment. (A) DU145 or PC-3 cells were untreated (CONT) or exposed to 30 μM p53p-Ant. Cells were photographed under phase contrast microscopy for up to 6 h., DU-145 (B) or PC-3 (C) cells were untreated (CONT) or exposed to 30 μM Ant, p53p or p53p-Ant for 3–24 h, or to 10, 20 or 30 μM p53p-Ant for 6 h. For (B) and (C) cell viability was determined by MTT assay. Results represent mean absorbance ± SD, n=3. (D) Trypan blue cell death in PC-3 cells or PC-3 stably transfected cells expressing a temperature sensitive p53–143 at 37◦ (mutant p53) or 32◦ (wt p53) for 3h. At least 200 cells were counted in triplicate

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Fig. 14.2 (continued)

We found some signs of apoptosis in prostate cancer death including a concentration and time-dependent sub-G1 peak (typically 30–50%), which was detected by flow cytometry of PI-stained cells, but not with the controls Ant or p53p alone (Fig 14.3A). We also found an increase in nucleosomal DNA fragments (mono- and oligosomes) in cytoplasmic fractions and an increase in Anx V+/PI- cells (apoptotic), within 0.5 min of peptide exposure (Fig 14.3B). However, in contrast to the breast cancer cell lines, the p53p-Ant-treated prostate cancer cell lines became Anx V+/PI+ cells (necrotic), after 5 min. (Fig 14.3C). This indicated that p53p-Ant first induced apoptosis within 0.5 min of peptide exposure which was later switched to necrosis after 5 min of peptide exposure. However, the initial apoptosis was incomplete since we did not detect caspase 3 or 8 activation and the pan caspase inhibitor,

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Fig. 14.3 Apoptotic markers in p53p-Ant-treated cells. (A) DU-145 or PC-3 cells ± 30 μM Ant, p53p or p53p-Ant for 6 h. Cells were analyzed for PI-stained DNA content by flow cytometry. The percentage of cells with sub-G1 DNA content are indicated. (B), Nucleosomes in cytoplasmic fractions of DU-145 and PC-3 lysates treated with Ant, p53p, or p53p-Ant peptides determined using an ELISA. Results represent mean absorbance ± SD, n = 3. (C) Anx V and PI staining time course. DU-145 cells were exposed to 30 μM p53p-Ant for up to 1 h, and analyzed by flow cytometry. Dot plots indicate PI positive (necrotic) cells (FL-3) and Anx V positive cells (FL-1). The percentage of cells in each quadrant are indicated

BOC-Asp-FMK failed to block the cell death as determined by accumulation of subG1 PI-stained particles. We further determined that there was no change in the basal levels of Bax, Bcl-2, Bak, or PUMA from p53p-Ant exposure in prostate cancer cells. In addition, treatment with p53p-Ant for 1h did not increase Fas membrane expression by FACS analysis (Fig 14.4A). However, the same treatment increased Fas expression 3.3 fold in mutant p53 MDA-468 human breast cancer cells consistent with our previous findings in breast cancer cells exposed to p53p-Ant (Kim et al., 1999). The PARP cleavage product was also not observed in DU-145 cells under conditions of maximal cell death by peptide. However, DU-145 cells treated with 50 nM paclitaxel (PAC) induced classical apoptosis (Haldar et al., 1996) with PARP cleavage (Fig 14.4B, C). This demonstrated that under conditions which induced apoptosis (PAC treatment), PARP cleavage does occur in DU-145 cells. Also, PAC, but not p53p-Ant, increased caspase-3 and -8 activities in DU-145 cells. In addition, pre-treatment of DU-145 cells with the pan-caspase inhibitor, BOC-Asp-FMK, significantly blocked the apoptotic effects of PAC (sub G1 fraction), but not the p53p-Ant-induced sub-G1 fraction (Fig 14.4B) and nucleosomes (Fig. 14.4C). To determine whether the p53p-Ant-induced cell death in DU-145 cells was dependent on p53 as it was in breast cancer cells, DU-145 cells were treated with

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Fig. 14.4 Apoptosis in p53p-Ant-induced DU-145 cell death. (A) Fas expression changes from p53p-Ant in DU-145 cells or MD-468 breast cancer cells ± 30 or 50 μM p53p-Ant for 1 h. Results expressed as fluorescent intensity of 5000 cells. (B) DU-145 cells were untreated (−) or preincubated (+) with the pan caspase inhibitor, BOC-Asp-FMK (BAF) (50 μM for 20 min) and then untreated or exposed to 30 μM p53p-Ant for 6 h. Cells were analyzed for PI-stained DNA. Values indicate percentage of cells with sub-G1 DNA content. (C) DU-145 cells were treated as in (B). Nucleosomes in cytoplasmic fractions of cell lysates were determined using an ELISA. Results represent mean absorbance ± SD, n = 3. Paclitaxel (PAC) treatment (50 nM, 48 h). (D) p53 Western and (E) Annexin V staining of pAd/U6/p53-SiRNA (p53 siRNA) vs pAd/U6/shuffled-p53-SiRNA (Cont) after 6 h treatment with 30 μM p53p-Ant

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Fig. 14.4 (continued)

adenovirus-containing shRNA against p53 or an adenovirus containing the same nucleotides in a shuffled sequence. After treatment with the sh-RNA containing adenovirus, p53 expression was reduced by >90% as determined by western blot (Fig 14.4D). p53-shRNA adenovirus-infected cells showed a >50% increase in resistance to p53p-Ant as compared to cells treated with the shuffled sequence (Fig 14.4E). Thus, p53p-Ant-induced death in DU-145 cells did not involve the extrinsic (FAS, caspase-8) or intrinsic pathways (Bcl-2, Bax, Bak, PUMA, or caspase-3), yet it was partially p53-dependent. Since p53p-Ant-induced cell death in DU-145 cells was associated with incomplete features of apoptosis, we investigated the necrotic aspects of p53p-Antinduced prostate cancer cell death in more detail. We found that extracellular LDH release doubled after a 10 min exposure to p53pAnt, and continued to increase over time (Fig 14.5A). Fluorescence microscopy showed an increase in the percentage of ethidium homodimer-stained cells after 10 min, indicative of a porous or damaged plasma membrane. These results were quantitated, showing a similar slope pattern analogous to the curve for LDH release (Fig. 14.5B). Intracellular energy levels of ATP decreased 25% from initial baseline levels after 5 min and 63% after 30 min

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Fig. 14.5 Necrosis in p53p-Ant-induced DU-145 cell death. (A) LDH Release time course. ±30 μM p53p-Ant. Results are expressed as a percentage of the maximum LDH release and represent mean absorbance ± SD, n=3. (B) Cell fluorescence time course. Cells cultured on poly-D -lysine-coated coverslips ± 30 μM p53p-Ant, stained with Calcein AM and ethidium homodimer-1. Green fluorescent cells (living) and red fluorescent cells (with damaged plasma membranes) were quantitated and expressed as a percentage. Results represent mean number of ethidium homodimer positive cells ± SD, n = 3, 300 cells/point. (C) Intracellular ATP time course. Cells ± 30 μM p53p-Ant. ATP levels were determined on a TD 20/20 luminometer at 560 nm. Results are expressed as a percentage of untreated controls and represent mean luminosity ± SD, n = 3. (D) ATP analysis as in (C). Cells were pre-incubated with 3-AB (10 mM) for 30 min. followed by incubation with 30 μM p53p-Ant ± 3-AB for 10 min. Results are expressed as a percentage of untreated controls and represent mean luminosity ± SD, n = 3

exposure to peptide (Fig 14.5C). The ATP decline was not affected by the PARP inhibitor, 3-aminobenzamide (3-AB) (Fig. 14.5D). Electron microscopy showed a mixture of necrotic and apoptotic cells, with a higher proportion of necrotic cells at 30 min, characterized by swollen cellular size and lower cytoplasmic density

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Fig. 14.6 Electron microscopy of p53p-Ant-treated DU-145 cells. (A) DU-145 cells were untreated or exposed to 30 μM p53p-Ant for the times indicated. Representative photomicrographs are shown for each time point. Bars = 5 μm. (B) Higher magnifications of the plasma membrane of DU-145 cells after p53p-Ant treatment for the indicated times. Arrows indicate breaks in the plasma membrane. Bars = 0.5 μm

(Fig 14.6A) with definite broken plasma membranes, consistent with a necrotic form of cell death (Fig 14.6B). At 3 and 6 h after peptide exposure, some cells showed apoptotic features. Together, the Annexin/PI studies, rapid LDH release, rapid decrease of intracellular ATP, ethidium homodimer fluorescence and electron microscopic studies suggested that cell death in DU-145 by p53p-Ant begins as apoptotic within 0.5 min, which is converted to necrosis after approximately 5– 10 min. However, not all cells were converted to necrosis and a few still developed an apoptotic morphology after 3 h. The drop in ATP levels suggested that mitochondrial disruption may be an important event in the early stages of death induced by p53p-Ant in mutant p53 prostate cancer cells. We found an increase in ROS production of 5.4–9 fold from p53p-Ant treatment as assessed using the fluorescent probes DCFDA and DHE (primarily detect H2 O2 and O2 −. , respectively) (Fig 14.7A). The increase in DHE fluorescence was peptide-dose-dependent (Fig. 14.7B), and not seen in PC-3 cells which have a null p53 status (Fig. 14.7C). The O2 −. scavenger, Tiron and the SOD mimetic MnTMPyP partially reversed the p53p-Ant-induced DHE fluorescence (Fig. 14.7D), as well as p53p-Ant-induced cell death (Fig 14.7E). In an attempt to elucidate why the prostate cancer cells underwent necrosis and the breast cancer cells undergo apoptosis from p53p-Ant exposure we assessed O2 − levels.

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Fig. 14.7 O2 − levels in p53p-Ant-induced DU-145 cells. (A) DU-145 cells ± increasing concentrations of p53p-Ant for 1 h. DHE was added to the cell culture in the last 30 min of incubation. Values indicate relative percentage of cells demonstrating DHE fluorescence (superoxide O2 −. accumulation). (B) Plot of data in (A). (C) DU-145 or PC-3 cells were treated with or without 60 μM p53p-Ant for 1 h and DHE (O2 −. levels) fluorescence determined. (D) Cells were preincubated for 30 min with either Tiron (5 mM) or MnTMPyP (30 μM) prior to p53p-Ant (60 μM) for 1h and DHE fluorescence was analyzed as in (A). (E) Trypan blue analysis of DU-145 cells treated as in (D). At least 600 cells were counted per sample. All experiments were repeated. (F) DHE fluorescence of DU-145 vs MD-468 after 5 min incubation with p53p-Ant under conditions that induce necrosis and apoptosis, respectively. (G) DHE fluorescence and Anx V+ time course in DU-145 cells. (H) Time course of apoptotic cells (Ann V+/PI-) and necrotic cells (Ann V+/PI+) and TUNEL positive cells. (I) Ann V+ and DHE fluorescence after 5 min ± p53p-Ant ± 5 mM KCN

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Fig. 14.7 (continued)

Breast MD-468 cells showed a 2.3 fold increase in O2 − levels compared to 4.7 fold in prostate DU-145 cells under conditions which induce apoptosis and necrosis, respectively (Fig 14.7F). Further examination of O2 − accumulation over time indicated that O2 − increased as early as 1 min after exposure to p53p-Ant, and corresponded closely with increased AnnV+/PI- cells until after a 5 min exposure to p53p-Ant (Fig 14.7G). In addition, TUNEL+ cells increased after the rise in AnnV+/PI- (apoptotic) cells and after 5 min, AnnV+/PI+ (necrotic) cells began to accumulate with fewer Ann V+/PI- (apoptotic) cells observed (Fig 14.7H). We found that O2 − accumulation and Anx V+ cells were blocked by pre-treatment with 5 mM KCN, suggesting that mitochondria were the source of O2 − accumulation in response to p53p-Ant (Fig. 14.7I). KCN is a potent inhibitor of mitochondrial cytochrome c oxidase, the fourth complex of the electron transport chain. We investigated whether the p53p-Ant-mediated necrosis in DU-145 cells may circumvent resistance to chemotherapy-induced apoptosis. The DURC-1 cell line, derived from parental DU-145 cells were made resistant to topotecan and camptothecin. The DU-TaxR cell line are parental DU-145 cells made resistant to PAC by continuous exposure in our lab. By TUNEL analysis, DU-145 parental cells were sensitive, and DURC-1 and DU-TaxR cells were resistant to topotecan (1.0 μM, Fig 14.8A) and PAC (10 nM, Fig. 14.8B), respectively. However, DURC-1 and DU-TaxR cells were just as sensitive as DU-145 parental cells to p53p-Ant (Fig. 14.8C). Similar results were obtained for these lines in PI and trypan blue assays (data not shown). DURC-1 and DU-TaxR cells, exposed to p53p-Ant,

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showed characteristics of necrosis as indicated by rapid LDH release (1 h) and rapid decline in intracellular ATP (1 h; Fig. 14.8D,E). There are different explanations why p53p-Ant induced targeted necrosis in multiple mutant p53 prostate cancer cells and classic apoptosis in breast, colon and lung cancer cell lines. One explanation for necrosis-induction may be an incomplete execution of the mechanisms of apoptosis as a consequence of a limited supply of intracellular ATP in all of the mutant p53 prostate cancer lines. This is under investigation in the lab. The aborted mechanisms of apoptosis may explain why

Fig. 14.8 Effect of p53p-Ant in DU-145 drug resistant cells. (A–C) TUNEL analysis in DU-145, DURC-1 or DU-TaxR cells exposed for 48 h to indicated concentrations of topotecan (A) Paclitaxel (PAC) (B) 30 μM p53p-Ant (3 h) (C). The percentage of particles gated is shown. (D) LDH release in DU-145, DURC-1 or DU-TaxR cells exposed to 30 μM p53p-Ant for 1 h (see Fig. 14.5A). (E) Intracellular ATP levels in DU-145, DURC-1 or DU-TaxR cells exposed to 30 μM p53p-Ant for 1 h (see Fig. 14.5C). Experiments were performed 3 times and mean ± SD are shown

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Fig. 14.8 (continued)

some features of apoptosis were detected without signs of classical apoptosis in the intrinsic or extrinsic pathways. We found that upon p53p-Ant treatment, PARP was not inactivated in DU-145 cells. However, incubation with the PARP-inhibitor, 3-aminobenzamide (3-AB) did not block the ATP decline in peptide-treated DU-145 cells, suggesting that continued PARP activity was not responsible for the decline in ATP. Another explanation for necrosis is that the ATP loss occurred from plasma membrane rupture. p53p-Ant was found to induce rapid membrane ruffling in both DU-145 and PC-3 cells , and it has been predicted that Antennapedia is internalized by a penetration mechanism (Derossi et al., 1998). Thus, necrosis could be from entry of the peptide through the plasma membrane. However, contrary to this idea, both prostate cancer lines (DU-145 and PC-3) displayed rapid and equal penetration of Ant-p53p-RhoD into all cellular compartments, yet only the DU-145 cells underwent cell death, while the PC-3 cells recovered and showed only 8–10% cytotoxicity with no decrease in ATP levels (Figs. 14.1A, 14.2A, 14.5C and 14.5D). In addition, Ant alone, and the control peptide p53-ANTCONT, which penetrates membranes, did not produce significant ATP or LDH loss or cytotoxicity. We have found in other studies that Ant with p53p produced a hydrophobic structure with a high density of positive charges from the multiple basic, positively charged aa residues that, when passing through the plasma membrane caused a transient loss of integrity and non-apoptotic death in a minority of cells (<10%) (Li et al., 2002). However, Tiron, an O2 −. -scavenger, and the SOD mimetic MnTMPyP partially abrogated p53p-Ant-induced cell death. Since these inhibitors are unlikely to inhibit penetration of p53p-Ant, this result argues that O2 −. accumulation may be important for the mechanism of the necrotic death pathway induced by p53p-Ant. Necrosis was found to occur at the peak of O2 −. accumulation (10 min) in our study. Other studies have proposed that low concentrations of ROS trigger apoptosis, but higher concentrations of ROS (O2 −. ) trigger necrosis (Nakano et al., 2006). These results suggested to us that the mechanism of p53p-Ant cell death in mutant p53 DU-145, as well as other mutant p53 prostate cancer lines, may be

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mediated by mitochondrial ROS in the form of O2 −. , and p53p-Ant initially produced apoptosis which was converted to necrosis when O2 − accumulation reached a critical level. ROS can induce a caspase-independent form of apoptosis, possibly because caspases can be inhibited by a high oxidizing environment. We found that p53p-Ant increased ROS (O2 −. and H2 O2 ) in DU-145 cells, but not in PC-3 cells. NAC, which is not a direct O2 −. scavenger (Aruoma et al., 1989), had no effect on p53p-Ant-induced cell death but the O2 −. scavenger Tiron and SOD mimetic MnTMPyP partially abrogated cell death, suggesting that O2 −. was responsible for inducing caspase-independent apoptosis in mutant p53 DU-145 cancer cells. However, we can not yet exclude the role of novel, yet undiscovered caspases, which were not inhibited by BOC-Asp-FMK or ROS and that are independent of the Fas and Bcl-2 family pathways in the mechanism of p53p-Ant-mediated apoptosis in mutant prostate cancer lines including DU-145 cells (Sperandio et al., 2000). Alternatively, expression of inhibitory protein(s) at the apex of the caspase activation cascade which prevents activation of caspase –8 may occur. An example of this is the action of over-expressed c-FLIP, which inhibits caspase-8 activation. However, we found no evidence of c-FLIP over-expression in DU-145 cells by western blot. Some studies have found ROS to be downstream mediators of wt-p53-dependent apoptosis (Johnson et al., 1996), suggesting that functional p53 can regulate the intracellular redox state for induction of apoptosis. This regulatory function of p53 may explain our previous findings where p53p-Ant restored partial functional activity to mutant p53 (Kim et al., 1999), and the involvement of O2 −. in p53pAnt-induced necrosis. We hypothesize that p53p-Ant may be restoring partial wt p53 activity to mutant p53 prostate cancer cell lines, including DU-145 cells. This partially functional, mutant p53 may induce O2 −. formation, and subsequent cell death via aponecrosis or targeted cell death. The finding that ROS is not formed in null p53 PC-3 and that this cell death is caspase-independent are consistent with this model. ROS has been found to induce a form of cell death lacking features of classical caspase-dependent apoptosis (Maianski et al., 2003; Shih et al., 2004). However, ROS has also been found to trigger classical apoptosis (Mates and Sanchez-Jimenez, 2000; Raha and Robinson, 2001) or necrosis, dependent upon its intracellular levels. We have found that p53p-Ant also induced a lower level of ROS in mutant p53 breast cancer cells (MDA-468), which undergo only extrinsic (Fas-mediated) apoptosis from p53p-Ant (data not shown). However, the level of induction of ROS may not totally explain why DU-145 cells and other mutant p53 prostate cancer lines are directed more into necrosis and breast cancer cells are directed more into apoptosis from p53p-Ant. As we found, p53p-Ant may induce different levels of ROS in different cell types. In addition, different cell types may be more or less sensitive to different levels of ROS due to intrinsic differences in levels of free-radical scavengers. In addition, the basal level of ATP or recovery of ATP levels may be an important determinant for apoptosis versus necrosis. In fact, in preliminary experiments p53p-Ant exposure in MB-468 breast cancer cells produced a 42% decline in intracellular ATP from basal ATP levels, but fully recovered to basal ATP levels after 4 h; while p53p-Ant exposure to DU-145 cells produced a

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67% decline, which did not recover above 50% of basal levels after 4 h (unpublished observation). This may also explain the mixture of necrotic and apoptotic DU-145 cells that were observed. Within the DU-145 cell population there may be cells that have a higher or lower sensitivity to ROS levels due to differences in free radical scavengers and/or basal ATP levels, driving some toward apoptosis and some toward necrosis. However, the major difference between DU-145 and PC-3 cells is the null p53 status; thus the target for p53p-Ant is missing and explains the lack of cell death, ATP decline, and ROS accumulation in PC-3 cells. The peptide may be mechanistically working through partial restoration of p53 function which generates critical levels of O2 −. ; which causes a decline in ATP levels and switches the death pathway from initial apoptosis to targeted necrosis, that kills the cells efficiently even if they are resistant to classical apoptosis. Importantly, this work is the first to show that peptide-mediated restoration of wt function to mutant p53 in cells can cause necrosis, suggesting that wt p53 may induce necrosis, as well as apoptosis, but each pathway is causally related to differential levels of ROS (low = apoptosis; high = necrosis). Thus, the induction of targeted necrosis in our model can bypass apoptotic resistance. This suggests that p53p-Ant has the potential for circumventing resistance to classical apoptosis by eliciting “targeted necrosis”. In addition, targeted necrosis induced by p53p-Ant is specific to prostate cancer cells expressing mutant p53, which may obviate the major problem in utilizing the necrotic death pathway in cancer therapy, namely its inflammatory reaction and lack of specificity for cancer and normal cells. This targeted necrosis did not occur in any of the human or rodent tumor types of adenocarcinoma cell lines except mutant p53 prostate cancer cell lines. Mutant p53 occurs in ≥75% of metastatic hormone refractory prostate cancer cases. Mutant p53 does occur in earlier prostate cancer and is detectable in approximately 33% of PIN III/IV cases. Targeted necrosis by p53 peptide can potentially retain the specificity of apoptosis for mutant p53 prostate tumor cells and bypass resistance mechanisms for apoptosis. The determinant for whether a cell undergoes apoptosis or targeted necrosis may depend upon its ability to generate critical levels of ROS, especially O2 −. , and to recover ATP levels before there is an irreversible commitment to necrosis. Thus, this work adds to the previous report by S. Snyder’s lab which demonstrated that the “switch” point for turning apoptosis into necrosis was through inhibition of PARP cleavage which lowered ATP pools (Ha and Snyder, 1999). In our findings, the increased ROS O2 −. levels induced mitochondrial damage causing a rapid and precipitous drop in ATP pools inducing the switch from apoptosis to necrosis. Thus, inhibition of caspases 3 and 7, which normally cleave PARP, would lead to continual PARP activity and O2 −. can lead to lower ATP pools, inducing the “switch”. This switch is dependent upon the levels of ATP- high levels of ATP lead to apoptosis and low levels of ATP (<50% of basal levels) lead to necrosis which bypasses the mechanisms of resistance in many cancers to apoptosis. The actual critical threshold of ATP levels, which would commit the cancer to targeted necrosis would probably be different for each histologic tumor type.

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Conclusion p53p-Ant induced significant cell death only in multiple mutant p53, human prostate cancer cell lines, in a p53-dependent and caspase-independent manner via ROSassociated loss of mitochondrial ATP. The form of cell death may be dependent upon the cell type, and suggests the possibility of an unknown pathway leading to activation of targeted necrosis (Fig. 14.9). Because p53p-Ant can induce multiple

Fig. 14.9 Pathways of non-specific necrosis, targeted necrosis and apoptosis. Wild type p53, through the action of ROS, Fas or Bcl-2 family of proteins, activates caspases, resulting in apoptosis. Necrosis is activated by injury or perfusion defects, leading to generation of ROS. Alternatively, p53p-Ant preferentially binds to mutant p53 and alters its conformation resulting in partially functional p53. The functional mutant p53 may mediate generation of ROS leading to DNA nicks, nucleosomal degradation and mitochondrial membrane damage but without caspase-3 activation. This leads to targeted necrosis because it will only be activated in mutant p53 cancer cells, which express sufficient threshold p53 target for the peptide. Alternatively, partially functional p53 by p53p-Ant may act via the Fas/FADD pathway leading to apoptosis in non-prostate cancer cell lines

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mechanisms of cell death dependent on cell type, it may provide an interesting tool to investigate the mechanisms of targeted necrosis and the “switch” from apoptosis to necrosis. If the mechanisms within the switch can be definitively elucidated, then it may become possible to induce targeted necrosis in prostate cancer cells resistant to apoptosis, and perhaps other types of cancers as well. One possible way to regulate this decision point is by generating high levels of ROS (O2 −. ) and modulating ATP pools or their recovery so that the cell is committed to necrosis after exposure to an inducer of apoptosis.

References Aruoma OI, Halliwell B, Hoey BM, Butler J (1989) The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Rad Biol Med 6:593–597 Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA (1995) Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci USA 92:7162–7166 Chen MW, Vacherot F, De La Taille A, Gil-Diez-De-Medina S, Shen R, Friedman RA, Burchardt M, Chopin DK, Buttyan R (2002) The emergence of protocadherin-PC expression during the acquisition of apoptosis-resistance by prostate cancer cells. Oncogene 21:7861–7871 Derossi D, Chassaing G, Prochiantz A (1998) Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol 8:84–87 Dinnen RD, Drew L, Petrylak DP, Mao Y, Cassai N, Szmulewicz J, Brandt-Rauf P, Fine RL (2007) Activation of targeted necrosis by a p53 peptide: a novel death pathway that circumvents apoptotic resistance. J Biol Chem 282:26675–26686 Effert PJ, Neubauer A, Walther PJ, Liu ET (1992) Alterations of the P53 gene are associated with the progression of a human prostate carcinoma. J Urol 147:789–793 Eguchi Y, Shimizu S, Tsujimoto Y (1997) Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 57:1835–1840 Formigli L, Papucci L, Tani A, Schiavone N, Tempestini A, Orlandini GE, Capaccioli S, Orlandini SZ (2000) Aponecrosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis. J Cell Physiol 182:41–49 Ha HC, Snyder SH (1999) Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc Natl Acad Sci USA 96:13978–13982 Haldar S, Chintapalli J, Croce CM (1996) Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 56:1253–1255 Howell SB (2000) Resistance to apoptosis in prostate cancer cells. Mol Urol 4:225–229; discussion 231 Isaacs WB, Carter BS, Ewing CM (1991) Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles. Cancer Res 51:4716–4720 Johnson TM, Yu ZX, Ferrans VJ, Lowenstein RA, Finkel T (1996) Reactive oxygen species are downstream mediators of p53-dependent apoptosis. Proc Natl Acad Sci USA 93:11848–11852 Kim AL, Raffo AJ, Brandt-Rauf PW, Pincus MR, Monaco R, Abarzua P, Fine RL (1999) Conformational and molecular basis for induction of apoptosis by a p53 C-terminal peptide in human cancer cells. J Biol Chem 274:34924–34931 Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P (1997) Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med 185:1481–1486 Li Y, Rosal RV, Brandt-Rauf PW, Fine RL (2002) Correlation between hydrophobic properties and efficiency of carrier-mediated membrane transduction and apoptosis of a p53 C-terminal peptide. Biochem Biophys Res Commun 298:439–449

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Li Y, Mao Y, Rosal RV, Dinnen RD, Williams AC, Brandt-Rauf PW, Fine RL (2005) Selective induction of apoptosis through the FADD/caspase-8 pathway by a p53 c-terminal peptide in human pre-malignant and malignant cells. Int J Cancer 115:55–64 Maianski NA, Roos D, Kuijpers TW (2003) Tumor necrosis factor alpha induces a caspaseindependent death pathway in human neutrophils. Blood 101:1987–1995 Mates JM, Sanchez-Jimenez FM (2000) Role of reactive oxygen species in apoptosis: implications for cancer therapy. Int J Biochem Cell Biol 32:157–170 Miyake H, Hara I, Kamidono S, Gleave ME, Eto H (2003) Resistance to cytotoxic chemotherapyinduced apoptosis in human prostate cancer cells is associated with intracellular clusterin expression. Oncol Rep 10:469–473 Nakano H, Nakajima A, Sakon-Komazawa S, Piao JH, Xue X, Okumura K (2006) Reactive oxygen species mediate crosstalk between NF-kappaB and JNK. Cell Death Differ 13:730–737 Prabhakaran K, Li L, Borowitz JL, Isom GE (2004) Caspase inhibition switches the mode of cell death induced by cyanide by enhancing reactive oxygen species generation and PARP-1 activation. Toxicol Appl Pharmacol 195:194–202 Raffo AJ, Perlman H, Chen MW, Day ML, Streitman JS, Buttyan R (1995) Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res 55:4438–4445 Raha S, Robinson BH (2001) Mitochondria, oxygen free radicals, and apoptosis. Am J Med Genet 106:62–70 Selivanova G, Iotsova V, Okan I, Fritsche M, Strom M, Groner B, Grafstrom RC, Wiman KG (1997) Restoration of the growth suppression function of mutant p53 by a synthetic peptide derived from the p53 C-terminal domain. Nat Med 3:632–638 Selivanova G, Ryabchenko L, Jansson E, Iotsova V, Wiman KG (1999) Reactivation of mutant p53 through interaction of a C-terminal peptide with the core domain. Mol Cell Biol 19:3395–3402 Senatus PB, Li Y, Mandigo C, Nichols G, Moise G, Mao Y, Brown MD, Anderson RC, Parsa AT, Brandt-Rauf PW, Bruce JN, Fine RL (2006) Restoration of p53 function for selective Fasmediated apoptosis in human and rat glioma cells in vitro and in vivo by a p53 COOH-terminal peptide. Mol Cancer Ther 5:20–28 Shih CM, Ko WC, Wu JS, Wei YH, Wang LF, Chang EE, Lo TY, Cheng HH, Chen CT (2004) Mediating of caspase-independent apoptosis by cadmium through the mitochondria-ROS pathway in MRC-5 fibroblasts. J Cell Biochem 91:384–397 Sperandio S, de Belle I, Bredesen DE (2000) An alternative, nonapoptotic form of programmed cell death. Proc Natl Acad Sci USA 97:14376–14381 Wang LG, Ossowski L, Ferrari AC (2001) Overexpressed androgen receptor linked to p21WAF1 silencing may be responsible for androgen independence and resistance to apoptosis of a prostate cancer cell line. Cancer Res 61:7544–7551

Chapter 15

Carcinogenesis and Therapeutic Strategies for Thyroid Cancer Zhi-Min Liu and George G. Chen

Abstract Thyroid carcinogenesis is complex and several etiological factors such as deficit of iodine and selenium, genetic predisposition, female sex hormone, irradiation, chemical carcinogenic agents, are involved in the development of thyroid cancer. These factors can cause molecular abnormalities of signaling pathways involved in proliferation, differentiation, and apoptosis, which eventually lead to the development and progression of various thyroid neoplasmas. In this chapter we will discuss these molecular abnormalities and emerging anti-cancer strategies to correct the abnormalities and thus to prevent or treat thyroid cancer. Keywords Thyroid · Carcinogenesis · Anti-cancer therapy

Introduction Thyroid cancer (TC) is the most common endocrine neoplasm in humans, affecting approximately 1% of the population, with increasing incidence around the globe (Sheils, 2005). Except for medullary thyroid cancer (MTC) derived from parafollicular C cells, thyroid lymphoma, and the very rare case of intrathyroidal sarcoma, roughly 95% of all thyroid tumors are of thyroid follicular epithelial cell origin, and include papillary thyroid carcinoma (PTC), follicular variant of PTC (FVPTC), follicular carcinoma (FC), its oncocytic variant, the Hurthle cell carcinoma (HC), and anaplastic thyroid carcinoma (ATC). Table 15.1 provides a simple classification for thyroid cancers. The subclassification of thyroid cancers is clinically significant. Papillary thyroid carcinomas metastasize via lymphatics to local lymph nodes in an estimated 50% of cases but have the most favorable prognosis, with a 98% 10-year survival rate. Follicular and tall cell variants of papillary thyroid carcinoma are associated with poorer prognosis. Follicular thyroid carcinomas are more prevalent in areas of Z.-M. Liu (B) Department of Biochemistry and Molecular Biology, The School of Basic Medical Sciences, Chongqing Medical University, Chongqing, China e-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-1-4020-9597-9 15,

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Z.-M. Liu and G.G. Chen Table 15.1 Classification of thyroid cancer Cell type

Type of cancer

Variants

Follicular cell

Papillary cancer

Mixed papillary follicular Follicular variant Tall cell type Columnar cell type

Follicular cell

Follicular cancer

Hurthle cell type Clear cell

Follicular cell

Anaplastic cancer

Parafollicular

Medullary cancer

MEN 2a MEN 2b

Lymphoid

Lymphoma

Mucosa associated lymphoid tissue lymphoma MALT-L B cell lymphoma

Connective tissue

Sarcoma

Nonthyroidal cancers

Metastases

Melanoma Breast Lung Gastric Renal

dietary iodine deficiency, metastasize hematogenously, and are less likely than papillary thyroid carcinomas to take up radioactive iodide for imaging and therapeutic ablation. However, the 10-year survival rate for follicular thyroid carcinomas is still high at 92%. Anaplastic thyroid carcinomas are almost invariably fatal as a result of rapid invasion of critical structures in the neck, distant metastases, and a failure to take up radioactive iodide. In many cases, anaplastic thyroid carcinomas arise from dedifferentiation of follicular follicular or papillary carcinomas or in patients with a history of multinodular goiter (Cotran et al., 1999). Medullary thyroid carcinomas, also commonly known as C-cell carcinomas, arise from calcitonin secreting C cells and are associated with inherited syndromes, such as multiple endocrine neoplasia (MEN 2a and 2b), in approximately 20–25% of cases (Sherman, 2003; Prades et al., 2002). Medullary carcinomas frequently metastasize via the bloodstream, in addition to lymphatic spread, and are treated with surgical resection and/or external beam radiation. In contrast to follicular origin thyroid tumors, C cells and tumors arising from them do not have the ability to take up radioactive iodide. The 5-year survival rate for medullary thyroid carcinomas is approximately 50%. Primary lymphoma can arise in the thyroid (Thieblemont et al., 2002). There are rare primary sarcomas of the thyroid. Metastases to the thyroid are uncommon as the only evidence of spread of disease from other organs. They have been described in patients with melanoma, breast, lung, gastric, and renal cancers (Nakhjavani et al., 1997). The pathogenesis of thyroid cancer is complex and some factors such as deficit of iodine and selenium, genetic predisposition, female sex hormone, irradiate in

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childhood, chemical carcinogenic agents, are involved in the development and progression of thyroid cancer. These etiological factors can cause genetic events and molecular changes, which result in signaling pathways involved in proliferation, differentiation and apoptosis, to be dysregulated (as summarized in Table 15.2). In this chapter we will discuss specific carcinogen-induced genetic events and molecular Table 15.2 Pathways and/or molecular events in thyroid cancer Pathways and/or molecular events

Type of thyroid cancer

TSHR-Gsp pathway Increased TSH Activating mutations of TSHR Activating mutations of gsp

Thyroid nodules, differentiated TC Thyroid nodules, differentiated TC Thyroid nodules, differentiated TC

RTK-Ras-Raf pathway Activating mutations of Ras Ret/PTC rearrangements Activating mutations of Ret Activating mutations of Raf NTRK1 rearrangements Amplification of Met

Thyroid nodules, adenoma, FC, PTC, ATC PTC, MTC MTC PTC, tall cell variant of PTC PTC, ATC PTC, ATC, FC, MTC

PI3K-AKT pathway Inactivation of PTEN Enhanced AKT activation

FA, FC, PTC, ATC PTC, FC, ATC, associated with progression of TC

Angiogenesis in thyroid cancer High level of VEGF in the serum Overexpression of EGFR

Metastases, worse prognosis Metastases, worse prognosis

Several functional proteins of TC Overexpression of TPO Expression of CP and LF Expression of DAP4 Expression of HMGI

Benign thyroid lesions and thyroid malignancy ATC, PTC, FC ATC, PTC, FC ATC, PTC, FC

Modulators of differentiattion Pax-8/PPAR-γ rearrangement Reduced TTF1

FC, ATC FC, ATC

Adhesion molecules Reduced E-cadherin Increased Gal3

FC, ATC PTC, FC, ATC

Estrogen binding proteins ERα GPR30

FC, ATC ?

Inactivating mutations of p53

Poorly differentiated and undifferentiated TC

Apoptosis associated molecules Expression of hTERT Overexpression of Bcl-2 and BAG3 Overexpression of FLIP

PTC, FC, ATC, associated with progression of TC PTC, FC, ATC PTC, FC, MTC, ATC

Selenium deficiency Loss of NIS

TC Poorly differentiated and undifferentiated TC

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changes during the development and progression of the thyroid cancer and how the emerging anti-cancer strategy can be built by the utilization of the specific carcinogen-related apoptotic pathway.

Activation of TSHR-Gsp Pathway Is Involved in Benign Thyroid Lesions Iodine Deficiency Leads to Compensatory Increases in TSH Subthreshold concentrations of iodine intake in geographic areas of endemic goiter are associated with a variety of alterations in thyroid-function indicators. These include reduced circulating inorganic iodine concentrations, increased thyroid iodine uptake, an increased triiodothyronine/thyroxine (T3/T4) secretion ratio, increased serum thyroid stimulating hormone (TSH) concentrations, and a tendency to goiter which may develop into thyroid cancer (Fisher, 1996). Iodine supplementation has a beneficial effect on reduced incidence of thyroid cancer in areas of preexisting iodine deficiency (Braverman and Utiger, 2000). The effect of dietary iodine on histological subtype is more evident, with a higher incidence of FC in endemic goiter areas. With the introduction of iodine supplementation, the proportion of PTC to FC is reversed (Bakiri et al., 1998). Data on additional dietary factors, particularly goitrogens, are even less clear cut, but anything leading to compensatory increased TSH will increase the risk of thyroid neoplasia, given the central role of TSH in thyrocyte proliferation (Roger and Dumont, 1984).

TSHR Mutations TSH transduces its signal through TSH receptor (TSHR) and results in stimulation of the adenyl cyclase (AC)/cyclic AMP (cAMP) and phospholipase C (PLC) pathways. TSHR is a member of the G-protein associated 7-transmembrane-segment receptors and is considered a growth factor receptor (Vassart and Dumont, 1992). Aberrant stimulation of TSHR is associated with benign overactive thyroid nodules such as multinodular goiter or Graves’ disease, and hyperfunctioning thyroid adenomas. This can occur through mutations of TSHR that cause its constitutive activation, except for TSH binding (Parma et al., 1993). Specifically, mutations of the third intracellular loop signal transduction region of the TSHR have been associated with transformation of thyrocytes (Vassart and Dumont, 1992). Less evidence exists regarding a possible association between TSHR mutations and thyroid malignancy. Although a major factor in thyrocyte malignant degeneration seems to be TSHR pathway activation independent of ligand binding (Spambalg et al., 1996), fewer mutations of TSHR have been described in thyroid carcinomas compared to benign tumors. The level of TSHR mRNA is similar for benign thyroid tumors compared to normal thyroid tissues (Brabant et al., 1991; Shi et al., 1993a),

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but it declines with decreasing differentiation (Shi et al., 1993b). Loss of TSHR expression has been documented in thyroid carcinomas (Br¨onnegard et al., 1994; Cai et al., 1992), and it is likely to be a late event in the tumor progression (Shi et al., 1993b).

Gsp Mutation Given the evidence that the TSHR pathway plays a key role in thyrocyte proliferation and oncogenesis, it follows that proteins found along the TSHR dependent signaling pathway might also be potential thyroid oncogenes. One such protein is G protein subunit alpha (gsp), the alpha subunit of the heterotrimeric G protein family of GTP-binding proteins that facilitate TSHR ligand binding and activation of AC, resulting in increase of cAMP (Bourne et al., 1991). Like mutations of TSHR, gsp mutations are found mostly in hyperfunctioning adenomas (Russo et al., 1995).

Receptor Tyrosine Kinase-Ras-Raf Pathway is Constitutively Activated in Approximately 70% of all Thyroid Cancers and is an Important Potential Therapeutic Target for Thyroid Cancer Ras Mutation Constitutive activation of Ras, by mutations or by activation of one of its upstream regulators, and activation of its downstream effector Raf, are the most common oncogenic events in human cancers, including thyroid cancer. In thyroid carcinoma, activating mutations of Ras (N-, K-, or H-Ras) can be found in as many as 30% of cases (Mizukami et al., 1988). Although activated Ras is an oncogene in vitro, activating mutations of Ras also occur in benign follicular adenomas, suggesting that additional genetic hits are required for thyroid malignancy (Namba et al., 1990a,b).

Rearrangement, Mutation and Amplification of Ret, NTRK1 and Met It is well-known that papillary thyroid carcinomas can occur secondary to ionizing radiation exposure, particularly in children (Boice, 2005; Sherman, 2003). After the Chernobyl nuclear reactor accident in 1986, the incidence of thyroid carcinomas in children in affected areas of Belarus increased from less than 1 per million to more than 90 per million (Cardis et al., 2006). The primary known molecular mechanism of radiation-induced papillary carcinoma development is through the Ret/PTC group of oncogenes. Ret proto-oncogene is a receptor tyrosine kinase (RTK) normally involved in the glial-derived neurotropic factor-signaling pathway in neuroendocrine and neural cells. In thyroid follicular cells, the alignment of chromosomes

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during interphase places the Ret proto-oncogene in close proximity to several other constitutively expressed genes with which it can recombine during repair of ionizing radiation–induced double-stranded DNA breaks (Nikiforov, 2002; Nikiforova et al., 2000) These Ret/PTC rearrangements allow for unregulated expression of chimeric oncoproteins with constitutive tyrosine kinase activity. Ret/PTC expression is found in roughly one third of all papillary carcinomas, but in the majority of all radiation-induced papillary carcinomas. In medullary carcinoma, Ret proto-oncogene can also undergo rearrangements as in papillary carcinoma. In addition, the full-length Ret proto-oncogene has gainof-function point mutations involving one of several possible codons (Cranston and Ponder, 2003; Michiels et al., 1997; Quayle and Moley, 2005) As a result, Ret is constitutively active in the neuroendocrine tissues normally expressing this protein, such as thyroid C cells and neuroendocrine cells of the adrenal medulla. All of the known hereditary forms of medullary carcinoma are autosomal-dominant and involve Ret mutations. While sporadic medullary carcinomas comprise more than 75% of cases, little is known about specific genetic mutations initiating this form of thyroid cancer. However, somatic Ret point mutations have been identified in up to 50% of sporadic medullary carcinomas (Machens et al., 1999). NTRK1 is a RTK normally involved in nerve growth factor signaling (Tallini, 2002). Like Ret/PTC rearrangements, NTRK1 can recombine with the 59 end of other heterologous genes and form a constitutively active oncogene, such as TRK-T1, leading to papillary carcinomas. However, NTRK1 rearrangements are less frequent than Ret/PTC rearrangements and are not associated with radiation exposure. Met, a 190 kDa RTK important in thyroid growth (Eccles et al., 1996), is activated through associating with its ligand, hepatocyte growth factor/scatter factor (HGF/SF). Amplification of Met has been described in approximately 70% of PTC and ATC and in 25% of FC, with little expression in benign thyroid disease and normal thyroid tissues (Di Renzo et al., 1992; Di Renzo et al., 1991). Some have postulated that activation of Met may occur through a paracrine mechanism, as parafollicular C cells secrete HGF/SF (Ivan et al., 1997). Met expression has been positively associated with aggressive and metastatic behavior of thyroid tumors.

Raf Mutation Sporadic papillary thyroid carcinomas unrelated to radiation exposure make up more than two thirds of all cases, and several genetic events have been identified as important in their tumorigenesis (Nikiforov, 2004). A form of the B-type Raf kinase, or BRAF, with a point mutation resulting in V600E has been identified in approximately 45% of sporadic papillary carcinomas, particularly the tall-cell variant (Xing, 2005). BRAF expression has been associated with dedifferentiation and disease progression.

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Therapeutic Strategies to Target Receptor Tyrosine Kinase-Ras-Raf Pathway Taken together, activation of receptor tyrosine kinase-Ras-Raf pathway through oncogenic events occurs in approximately 70% of all thyroid cancers; therefore, this pathway represents an important potential therapeutic target for thyroid cancer (Kondo et al., 2006). Some emerging therapeutic strategies for thyroid cancer have been built by the utilization of the specific carcinogen-related apoptotic pathway. Inhibition or reduction of molecules along this pathway should be effective for treatment of thyroid cancer, including antisense compounds for Ras and Raf, specific inhibitors for Raf and MEK kinases, antagonists for RTK, and corrective gene therapy by expression of a dominant negative Ret mutant. In addition, translocation of activated Ras to the cytoplasmic membrane is a key step of its activation. For this step, several posttranslational modifications must occur to allow membrane localization. The initial modification step involves farnesylation (i.e. addition of a farnesyl moiety to a cysteine residue) of Ras by the enzyme farnesyl transferase. Inhibition of farnesyl transferase has been shown to inhibit membrane accumulation of Ras in vitro and therefore reduce Ras signal transduction (Gibbs et al., 1993).

PI3K-AKT Pathway is Overactive in Development and Progression of Thyroid Cancer and is also a Novel Therapeutic Target for Thyroid Cancer PTEN Inactivation AKT, also known as protein kinase (PKB), is a central signaling molecule in the PI3K pathway. Phosphatase and tensin homolog (PTEN) is a dual-function lipid phosphatase acting on the phosphatidylinositol 3-kinase (PI3K) and the Akt/PKB pathways (Stambolic et al., 1998). PTEN dephosphorylates Akt (Akt-P), causing apoptosis and/or G1 cell-cycle arrest. Consequently, dysfunctional PTEN leads to high levels of Akt-P, which inhibits apoptosis (Maehama and Dixon, 1998; Gustin et al., 2001). A role for AKT signaling in thyroid tumorigenesis was first recognized when loss of PTEN expression was identified to be the genetic cause of Cowden’s syndrome, an autosomal dominant multiple hamartoma syndrome in which more than 50% of patients develop thyroid neoplasia (Liaw et al., 1997). Although somatic PTEN mutations are rare in primary epithelial thyroid tumors, hemizygous deletion occurs in thyroid adenomas and carcinomas. Expression and genetic analysis of benign and malignant thyroid tumors showed that FA, FC, and PTC all have a 20–30% frequency of hemizygous deletion, while 60% of ATC have hemizygous PTEN deletions associated with decreased PTEN expression. Epigenetic silencing of PTEN and perhaps inappropriate subcellular compartmentalization are two novel

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mechanisms of PTEN inactivation that appear pertinent to thyroid carcinogenesis (Gimm et al., 2000; Zysman et al., 2002; Soria et al., 2002).

Enhanced AKT Activity Sporadic FTCs are characterized by increased expression of AKT 1 and AKT 2 and have increased levels of total AKT activity in comparison with normal tissue specimens. Increased AKT activity in FTCs correlated with either activating mutations in Ras or PPARγ/Pax8 gene rearrangements. In addition, Wu et al. (2005) reported a relatively high frequency of PI3KCA gene amplifications in FTCs. PI3K signaling has been shown to play a central role in cancer cell proliferation, antiapoptosis, and motility. Enhanced AKT activity is associated with advanced tumor stage and progression for a number of different malignancies (Kim et al., 2005a). In thyroid cancer, the association between increased AKT activity and tumor size and invasion has been demonstrated for both FTC and PTC, with the exception of tumors with BRAF activating mutations (Shin et al., 2004; Vasko et al., 2004). In addition, a broad role for PI3K signaling has been demonstrated for proliferation, survival, and invasion and motility for PTC, FTC, and anaplastic thyroid cancer cell lines in vitro. In vivo, studies in the TRβ pv/pv mouse that develops metastatic FTC-like thyroid cancer have demonstrated enhanced AKT activation in the primary and metastatic tumor tissue (Kim et al., 2005b). Moreover, primary cultures from TRβ pv/pv primary thyroid cancers display AKT-dependent cell motility in Boyden chambers, consistent with a role for AKT in metastasis in this model.

Therapies to Target PI3K-AKT Pathway Thus, it appears that activation of AKT as well as other PI3K-regulated proteins, including p70S6 kinase and mammalian target of rapamycin (Suh et al., 2003; Miyakawa et al., 2003; Motti et al., 2005; Hay, 2005), play important roles in thyroid cell proliferation and cancer progression. Whereas it is unlikely that any single pathway is alone responsible for tumor progression, PI3K effectors, including AKT, appear to play an important role in this process in thyroid cancer. Pharmacological and molecular inhibition of PI3K and AKT isoforms has been demonstrated to reduce proliferation and motility in a number of human thyroid cancer cell lines in vitro. PI3K and more specific AKT inhibitors reduce thyroid cancer cell cycle progression at G2/M phase transition and are able to induce apoptosis, despite the overall resistance of poorly differentiated thyroid cells to apoptosis in general (Mandal et al., 2005; Braga-Basaria et al., 2004). In addition, Ret/PTC-induced cell motility and expression of osteopontin, a key regulator of epithelial-mesenchymal transition (EMT), has been shown to be mediated by PI3K and AKT (Guarino, 2005). More recently the ability of PPARγ agonists to partially reverse EMT has

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been shown to be in part dependent on up-regulation of PTEN expression with subsequent inhibition of AKT activity (Aiello et al., 2006).

Angiogenesis in Thyroid Cancer High Levels of VEGF in the Serum of Patients with Thyroid Cancer The formation of new blood vessels is a crucial step in determining tumor expansion and is greatly dependent on proangiogenic factors that are produced in a paracrine fashion by tumor cells undergoing hypoxia or mechanical compression. Several growth factors are involved in the process of angiogenesis in malignant tumors; among them, vascular endothelial growth factor (VEGF) appears to be the most prominent. Besides the functional activity of stimulating vascular proliferation and permeability and inducing metastasis, VEGF may function as an apoptotic protector for the newly formed vessels via the PI3K-AKT signaling pathway by activating its receptor, a RTK (Gerber et al., 1998). Significantly increased levels of VEGF have recently been demonstrated in the serum of patients with well differentiated metastatic thyroid tumors compared with lower levels found in patients considered to be in a complete remission (Tuttle et al., 2002). High levels of VEGF have been associated with the occurrence of metastasis (Lennard et al., 2001) and possibly also with a worse prognosis (Klein et al., 2001).

Overexpression of EGFR Family Proteins Four structurally related receptors (Her1, ErbB1, or EGFR; Her2/neu or ErbB2; Her3 or ErbB3; and Her4 or ErbB4) are part of the epidermal growth factor receptor (EGFR) superfamily. When binding to ligands, these receptors either homodimerize or heterodimerize, and trigger pathways that lead to cell cycle progression and anti-apoptosis. EGFR blocking leads to cell cycle arrest in G1, apoptosis, antiangiogenesis, and down-regulation of matrix metalloproteinase, resulting in a decreased incidence of metastases. Her2/neu is the preferred heterodimerization partner for EGFR, and both EGFR and Her2/neu have been implicated in thyroid cancer progression. The EGFR is commonly expressed in differentiated thyroid tumors (Kanamori et al., 1989), and its overexpression has been associated with a worse prognosis (Akslen et al., 1993). Gene amplification of Her2/neu has also been detected in various solid tumors and has been correlated with a poor prognosis. Although several ligands have been identified, the kinase activity of Her2/neu can be activated without any ligand when overexpressed, homodimerized, or heterodimerized (Qian et al., 1994). In differentiated thyroid tumors, Her2/neu has been demonstrated to be up-regulated, particularly in papillary thyroid cancer (Haugen et al., 1992).

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Strategies of Antiangiogenesis Considering the important role of both EGF and VEGF receptors in the development and progression of thyroid cancers, significant attention has been dedicated to new drugs that potentially block pathways related to these receptors. Currently, it is possible to pharmacologically interrupt the activation of these receptors at different levels through neutralization of the ligand with antibodies, blocking the receptor with small molecules, and inhibiting mRNA with antisense compounds or si RNA.

Several Functional Proteins Were Overexpressed in Thyroid Cancer Several proteins have been found to be overexpressed in thyroid neoplasms, but they do not readily fit into currently known signal transduction pathways or cell cycle regulation mechanisms. However, their overexpression or abnormal expression provides some growth or survival advantage. This abnormal expression pattern may also provide useful diagnostic and therapeutic targets.

Thyroid Peroxidase The enzyme thyroid peroxidase (TPO) is present in all thyroid follicular cells and catalyzes iodide oxidation, thyroglobulin iodination, and iodothyronine coupling. Loss of expression is associated with reduction in iodide trapping in thyroid follicles, impairment of thyroid hormone synthesis, and a resultant decrease in autoregulation of most thyroid functions, including growth. TPO expression and mutation seem important in the progression and evaluation of thyroid lesions. Mutations in the TPO gene have been demonstrated in cold thyroid nodules of patients who otherwise lacked germline or somatic mutations of the gene (Krohn and Paschke, 2001). Overexpression of TPO is associated with development of benign thyroid lesions and thyroid malignancy (Christensen et al., 2000; De Micco et al., 1999; Faroux et al., 1997).

Ceruloplasmin and Lactoferrin Ceruloplasmin (CP) and the lactoferrin (LF) are glycoproteins, which share a remarkable amino acid sequence homology and similar biological behavior. CP binds most of the circulating blood copper and LF is an iron-binding glycoprotein. High levels of serum CP and LF have been observed in a number of neoplasms (Song, 1991). Immunohistochemical studies reveals that both CP and LF can be found in all FC but little or no staining in follicular adenoma (FA) (Kondi-Pafiti

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et al., 2000; Tuccari and Barresi, 1987). This data suggest that the expressions of CP and LF may be associated with the progression of thyroid cancer.

DAP4 Dipeptidyl-aminopeptidase 4 (DAP4) is a highly specific membrane-linked serine protease known to be involved in T-cell activation. The activity of this exopeptidase is increased in many cancers (Gonz´alez-C´ampora et al., 1998; Tang et al., 1996; Iwabuchi et al., 1996). DAP4 is absent from normal thyroid tissues but seems to be expressed at high levels in malignant thyroid cells (Zoro et al., 1996). Furthermore, the expression level of DAP4 was also related to the progression of thyroid cancer (Kotani et al., 1992).

HMGI Proteins The high mobility group I (HMGI) family is a class of nuclear proteins which regulate chromatin structure and formation (Ringel, 2000; Chiappetta et al., 1998). Although they have no inherent transcriptional activity, they seem to interact with a number of transcription factors, including the HIPK2 serine threonine kinase, to regulate gene transcription (Chiappetta et al., 2001; Pierantoni et al., 2001). Normal physiologic expression of HMGI is generally limited to embryogenesis, but activation has been reported to occur in experimental cancer models as well as in a number of human malignancies (Bandiera et al., 1998). HMGI protein expression correlates with the appearance of a malignant phenotype in rat thyroid transformed cells (Pierantoni et al., 2001). Transfection with antisense can rescue thyroid cells from exhibiting a malignant phenotype (Berlingieri et al., 1995) and induce apoptosis in ATC cells (Scala et al., 2000). Thus, the expression level of HMGI appears to be correlated with the progression of thyroid cancer.

Molecules Involved in Cellular Differentiation and Adhesion Is Decreased in the Progression of Thyroid Cancer Cellular differentiation, and terminal differentiation in particular, is usually associated with decreasing proliferative potential. Similarly, the establishment of cellular adhesion and interactions requires the exit from active cycling. Epithelial cells show anchorage dependent cell survival, which is mediated by integrin receptor-mediated interactions with the extracellular matrix. Their disruption leads to anoikis, a specialized version of apoptotic cell death (Frisch and Ruoslahti, 1997). It is therefore necessary for neoplastic cells to overcome the growth inhibition of these factors in carcinogenesis.

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Modulators of Differentiation Some studies have identified two important transcription factors, Pax-8 and thyroid transcription factor 1 (TTF-1) and both participate in the regulation of thyroglobulin (TG), TPO, sodium iodide symporter (NIS), all of which are central to thyroid development and function (Chun et al., 1998; Komatsu et al., 2001; Schmitt et al., 2001; Esposito et al., 1998; Kogai et al., 2001; Pasca et al., 2000). Pax-8 is a paired-domain transcription factor expressed in the developing and adult thyroid and is essential for thyroid follicular cell development and regulation thyroid gene expression (Moretti et al., 2000; Puglisi et al., 2000). Mutations of Pax-8 have been described in thyroid dysgenesis and congenital hypothyroidism (Macchia et al., 1998). Analysis of homozygous Pax-8 knockout mice demonstrates that this transcription factor is required for the formation of follicular cells in the thyroid, providing cues for the differentiation of endoderm primordia (Mansouri et al., 1998). The expression of Pax-8 is statistically different between benign and malignant thyroid diseases, and noticeable decrease in Pax-8 expression correlates with increasing aggressiveness of tumors (Fabbro et al., 1994; Ros et al., 1999). Pax-8 may also follow a paradigm in follicular thyroid neoplasia similar to RET/PTC and TRK-T1 rearrangements in PTC. A chromosomal translocation forming a fusion protein between Pax-8 and PPARγ1 has been described in FC but not in benign lesions, FA, or PTC (Kroll et al., 2000). TTF-1 is a homeodomain-containing protein expressed in embryonic diencephalon, thyroid, and lung (Pasca et al., 2000). Mice lacking TTF-1 lack thyroid follicular cells and parafollicular C-cells and fail to develop a thyroid gland (Mansouri et al., 1998). Like Pax-8 expression, TTF-1 is lower in thyroid carcinomas than in adenomas (Ros et al., 1999).

Adhesion Molecules The initial step in aggressive tumor behavior is the dissociation of neoplastic cells from the primary tumor as a result of broken cell–cell adhesion. The cell adhesion system includes the cadherin family, a group of functionally related glycoproteins which are frequently absent or decreased in various epithelial tumor cells (Baloch et al., 2001). E-cadherin (uvomorulin) is the dominant member of this family, expressed by epithelial cells and essential in maintaining cell polarity and epithelial integrity. There is evidence to suggest that decrease in E-cadherin is important specifically in epithelial thyroid tumors. Early, less aggressive FC shows greater E-cadherin expression compared to widely invasive FC (Kato et al., 2002). The reduction of E-cadherin levels is found in 100% of ATC and 50% of follicular lesions (Naito et al., 2001). Several studies have shown that decreased E-cadherin expression was associated with advanced tumors, higher rates of synchronous lymph node involvement, and distant metastases in thyroid cancer (Walgenbach et al., 1998; von Wasielewski et al., 1997; Scheumman et al., 1995). Aberrant methylation of the E-cadherin 5’CpG island may be associated with the reduced

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expression of E-cadherin, develpoment and pregression of thyroid cancer (Graff et al., 1998). The galectins are a growing family of proteins, which have been implicated in regulation of cellular growth, differentiation, and malignant transformation in a number of tissues (Gasbarri et al., 1999; Fern´andez et al., 1997; Bartolazzi et al., 2001). Increased expression of galectins has been shown in transformed thyroid cells and thyroid carcinomas, and distribution of these proteins in cancer samples has been shown to localize to tumors, but not adjacent regions of normal thyroid (Gasbarri et al., 1999; Fern´andez et al., 1997; Xu et al., 1995). Most studies of thyroid tumors have investigated expression of galectin-3 that acts as a cell death suppressor interfering with a Bcl-2-related apoptotic pathway (Akahani et al., 1997).

Estrogen Is a Proliferative Factor for Thyroid Cancer The incidence of thyroid carcinoma is three times higher in women than in men during reproductive years. The therapeutic use of estrogens has increased risk for thyroid cancer. The human thyroid gland appears to have the potential for both estrogen synthesis and intracrine or paracrine estrogen responsiveness, which seem to be greater in women than men and may become enhanced with the process of tumorigenesis (Dalla et al., 1998). These data suggest that female sex hormone is an etiological factor for the development and progression of thyroid carcinoma. There are two types of estrogen receptor (ER), ERα and ERβ. They may locate in nucleus, cell membrane, or other sites of cells (Levin, 2005). ERα is proliferative, whereas ERβ is proapoptotic (Cho et al., 2007). Data about ERs expression in thyroid cancer are controversial. Some studies showed increased ERs expression in thyroid cancer (Kawabata et al., 2003; Bl´echet et al., 2007). Others demonstrated no difference of expression between cancer and normal tissues, and even loss with the development of thyroid cancer (Egawa et al., 2001; Tavangar et al., 2007). However, there is evidence show that estrogen can stimulate proliferation of thyroid cancer cells through ERα-ERK pathway (Zeng et al., 2007). Otherwise, estrogen can also induce proliferation of some thyroid carcinoma cells which have not functional estrogen receptors, through activation of ERK and PI3K pathways (Vivacqua et al., 2006). These results suggest that there may be other molecules which have ability to bind estrogen and transduce proliferation signal of estrogen. Currently, G protein-coupled receptor 30 (GPR30), a seven transmembrane protein, has been demonstrated to mediate proliferative role of estrogen (as shown in Fig 15.1). SiRNA for GPR30 can significantly attenuate pERK1/2 expression and proliferation of thyroid cancer cells (Vivacqua et al., 2006). Therefore, study on estrogen binding proteins, with exception of ER, is an emerging research area for development and progression of thyroid cancer, and will provide effective therapeutic target for thyroid carcinoma.

360 Fig. 15.1 Proliferative role of estrogen. Estrogen stimulates growth of thyroid cancer cells through activation of ERK and PI3K pathways by binding ERα and GPR30

Z.-M. Liu and G.G. Chen Estrogen Extracellular GPR30

ER˞ Intracellular

ERK, PI3K/AKT ER˞

Expression of Genes associated growth Nucleus

Mutation of p53 is Common and Is a Target of Gene Therapy in Thyroid Cancer The p53 protein is an important nuclear transcription factor that can upregulate the expression of p21(Zedenius et al., 1996), and controls G1 to S phase progression. Its activation allows for repair of DNA mismatches that can occur normally over time or in response to some external events, such as exposure to radiation. When the damage occurs to a certain degree, p53 can activate a cascade of events that results in apoptosis. Mutant p53 proteins can be caused by missense mutations in the coding regions of the p53 gene in either one (heterozygous mutation) or both (homozygous mutation) alleles. Heterozygous mutations can result in reduced function of the normal p53 by binding to the normal protein or by inhibiting activity directly (dominant negative effect). Homozygous mutations result in the production of p53 proteins with reduced or absent activity. In both cases, inhibition of normal p53 activity results in more rapid cell cycle progression, thus not allowing for appropriate DNA repair or apoptosis in response to cellular damages. Inactivating mutations of p53 are among the most common gene mutations found in human malignancies. Malignant cells bearing wild-type p53 are typically more susceptible to a broad range of chemotherapeutic agents compared with cells expressing mutant forms of p53. In addition, p53 mutations are generally more common in poorly differentiated cancers in most forms of solid tumors. Similar to other cancers, p53 mutations are more frequently found in poorly differentiated form of thyroid cancer, ATC, compared with well differentiated cancers. A direct role for p53 mutation in thyroid cancer is supported by data demonstrating that reexpression of wild-type (wt) p53 in a thyroid cancer cell line harboring a p53 mutation leads to growth arrest (Kim et al., 2001). Moreover, reexpression of wt p53 can render thyroid cancer cells more sensitive to chemotherapy (Fagin et al., 1996). Therefore, restoration of wt p53 expression has been used in a variety of thyroid cancer models as well as in human clinical trials (Spitzweg and Morris, 2004).

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Alteration of Apoptotic Pathways in Thyroid Cancer Telomerase Activity Is Raised in Thyroid Cancer Telomerase is responsible for maintaining the length of chromosomal telomeres by adding back (TTAGGG)n hexamer repeats in a dynamic equilibrium with the sequences lost during DNA replication. It is a specialized reverse transcriptase with an integral RNA moiety that serves as its own template, and is detectable in germ cells, stem cell containing tissues with rapid cellular turnover, immortal cell lines, and cancer cells, but is undetectable in most normal differentiated somatic tissues (Kim et al., 1994; Shay and Bacchetti, 1997). Telomerase activity is essential to cell survival during neoplastic progression, possibly by delaying cellular senescence long enough to allow the accumulation of the multiple genetic alterations required for the malignant phenotype (Oshimura and Barrett, 1997; Kiyono et al., 1998). While there are alternative, telomerase-independent mechanisms that allow the maintenance of viable telomeres and cellular immortality (Tsutsui et al., 2002; Stavropoulos et al., 2002), they appear to be used only occasionally in human cancer. RT-PCR of human telomerase reverse transcriptase (hTERT) revealed its expression in 89% of papillary, 79% of follicular, 80% of anaplastic and 100% of medullary carcinoma cases (Asaad et al., 2006). Moreover, immunohistochemical staining of hTERT showed that expression rate of follicular carcinoma was much higher than follicular adenoma (Wang et al., 2005), suggesting that hTERT expression was associated with the progression of thyroid cancer. Since telomerase is known to be activated in thyroid cancer cells compared with normal cells, the therapeutic strategies directed against thyroid cancer would include the targeting of telomerase. Several approaches including antisense and immunotherapy directed against hTERT have been used both in vitro and in vivo, and the telomerase promoter has also been tested to direct cytotoxic therapy toward the cancer cells (Zeiger and Meeker, 2004; Takeda et al., 2003).

Upregulation of Antiapoptotic Molecules The Bcl family has been associated with thyroid lesions, but the relationship of Bcl family expression and malignant behavior remains controversial. Both the antiapoptotic Bcl-x and the pro-apoptotic Bax have been associated with FC (Branet et al., 1996). Some studies have claimed a direct correlation between Bcl-2 expression and differentiation (Branet et al., 1996) , supporting a promoting role in the growth of thyroid tumors, while others have found an increased level of Bcl2 expression in undifferentiated thyroid tumors (Pollina et al., 1996). Treatment with a Bcl-2 antisense compound can block Bcl-2 expression and activity, resulting in cell death. Therefore, Bcl-2 may be a promising therapeutic target for thyroid cancer.

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In addition, Bcl-2-associated athanogene (BAG) family proteins represent an evolutionarily conserved group of heat shock protein 70 (HSP70)-binding cochaperones. BAG proteins are characterized by their interaction with a variety of intracellular proteins, such as HSPs, steroid hormone receptors, Bcl-2, Raf-1, and other molecules involved in regulating protein folding and a number of cellular processes including proliferation and apoptosis (Takayama et al., 1997; Takayama et al., 1999; Takayama and Reed, 2001). Six BAG members have been identified in the human and mouse genomes (Takayama et al., 1997; Takayama et al., 1999; Takayama and Reed, 2001) . BAG3 forms a complex with HSP70, a protein able to modulate apoptosis by interfering with cytochrome c release, apoptosome assembly and other events in the death process (Li et al., 2000; Ravagnan et al., 2001; Saleh et al., 2000). BAG3 has also been reported to bind the antiapoptotic protein Bcl2, demonstrating synergism with Bcl-2 in preventing cell death (Liao et al., 2001; Romano et al., 2003). In support of its antiapoptotic function, the expression of BAG3 is elevated in some leukemia and solid tumors including thyroid tumors (Takayama et al., 1997; Takayama et al., 1999; Takayama and Reed, 2001). BAG3 is expressed in papillary, follicular, and anaplastic thyroid carcinoma tissues, but not detectable in specimens of normal thyroid or goiters (Chiappetta et al., 2007). These results suggest that the expression of this protein is associated with the development and progression of thyroid cancer and that BAG3 may also be an emerging therapeutic target for thyroid cancer.

Fas Signaling Is Diverted from Apoptosis to Proliferation in Thyroid Cancer Death receptor-mediated apoptosis can be induced through the activation of any of the family of death receptors, such as Fas, tumor necrosis factor (TNF) receptor, TNF-related apoptosis inducing ligand (TRAIL) receptor by their respective ligands (Locksley et al., 2001). This pathway plays a prominent role in immunosurveillance and cell-mediated cytotoxicity. Binding of the Fas ligand (FasL)to Fas induces the formation of the death-inducing signaling complex (DISC), a complex formed by the cross-linked Fas, Fas-associated death domain (FADD), and the zymogen form of caspase-8. At the DISC, pro-caspase-8 oligomerizes and is activated by autocatalysis (Muzio et al., 1998) and functions to activate caspases. Thyroid cancer is a human malignancy with a strong immunoregulatory component. Overexpression of Fas and the presence of lymphocytic infiltrates are common and intriguing observations in these cancers (Mitsiades et al., 1999). Despite Fas expression, thyroid cancers are not only resistant to Fas-mediated apoptosis but are also hyperproliferative (Arscott et al., 1999; Arscott et al., 1997; Irmler et al., 1997; Poulaki et al., 2002). Exactly how this pathway is altered in thyroid cancers is currently not known. Nevertheless, several genetic aberrations can be postulated to explain this abnormality, including the inhibition of caspase-8

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caspase-8

FLIP FLIP

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Fig. 15.2 Regulation of death receptor-mediated apoptosis by caspase-8 and FLIP. Caspase-8 is recruited to DISC upon the activation of death receptors by ligand binding. This recruitment results in the activation of caspase-8 and eventually apoptosis. FLIP is an enzymatically dead structural analogue of caspase-8 and it competes for the binding of caspase-8 on the DISC. This dominant-negative inhibition limits caspase-8 activation, resulting in the inhibition of death receptor-induced apoptosis

by Fas-associated death domain-like interleukin-1β-converting enzyme inhibitory protein (FLIP) (as shown in Fig. 15.2). FLIP is an enzymatically inactive caspase8 homologue (Goltsev et al., 1997). As a result of this homology, FLIP exerts dominant-negative effects to inhibit the activation of caspase-8. The increased level of FLIP can result in the activation of extracellular signal-regulated kinase (ERK) and NF-κB signaling pathways after T-cell receptor ligation (Kataoka et al., 2000). Overexpression of FLIP has been reported in a variety of cancers including thyroid cancer (Dutton et al., 2006; Mitsiades et al., 2006). Inhibition of FLIP expression with a FLIP antisense oligonucleotide has shown to result in significant sensitization to Fas-mediated apoptosis (Varfolomeev et al., 1998). As the majority of human cancers express death receptors, targeting these receptors by agonist monoclonal antibodies may be an effective strategy in many tumor types. However, death receptor signaling in some cancers including thyroid cancer may be altered by the components of the pathway, for example FLIP, to impart survival impetus to neoplastic cells, which may have a dangerous consequence. Therapeutic strategies relying on the activation of the death receptor pathway rather than the correction of altered components are likely to be counterproductive. Therefore, it may be prudent in these cases to use small-molecule inhibitors of targets such as FLIP as an adjuvant to TRAIL and other death receptor–targeting therapeutic agents to enhance their versatility to embrace tumors in question. These findings also underscore the necessity to understand specific mutations in tumors so as to tailor a rational tumor therapy for individual tumor genotype. That is where the future of cancer chemotherapy belongs.

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Selenium Deficiency Is a Risk Factor for Thyroid Cancer Development The essential micronutrient selenium (Se) occurs in the form of the amino acid selenocysteine in selenoproteins which exert various effects, while maintaining the cell reduction-oxidation balance. All three deiodinases that convert thyroxine (T4) into triiodothyronine (T3) contain selenocysteine, illustrating how the production of the active thyroid hormone is dependent on Se status (K¨ohrle et al., 2000). The selenoenzyme families of glutathione peroxidases (GPx) and thioredoxin reductases (TRx) possess powerful antioxidant properties and form a complex defense system that protects thyrocytes from oxidative damage (Zagrodzki et al., 2001; Brown and Arthur, 2001). The Food and Drug Administration (FDA) has determined that there is sufficient evidence to warrant a qualified health claim for Se and cancer and new evidence suggests that Se is implicated in thyroid tumor genesis (Trumbo, 2005). The Janus Serum Bank, a long-term project that searches for parameters in the sera that might be associated with cancer development, has found that low levels of Se in serum constitutes a risk factor for thyroid cancer development (Jellum et al., 1993). Compared with the concentrations of Se in thyroid tissues of patients with various thyroid diseases, the lowest mean Se level is observed in those with thyroid cancer (Kucharzewski et al., 2002). This may indicate that thyroid cancer is with low defense mechanisms and/or impaired detoxifying capacity, which may be of importance in cases with mutations of the ras oncogene (Sugawara et al., 2002), which can be the initial step in tumor genesis. Moreover, the activation of ras oncogene forms superoxide radical (Namba et al., 1990b). Thus, thyroid tumors containing mutated ras oncogene may generate large amounts of reactive oxygen species (ROS). Furthermore, the concentration of serum Se has been found significantly lower in cases developing thyroid cancer as compared to controls (Glattre et al., 1989), indicating the increased risk of thyroid cancer in persons with low serum Se levels. Therefore, any increase in Se intake can potentially influence the incidence of thyroid cancer. In many parts of the world, the main source of Se is diet. Nutritional Se repletion results in rapid accumulation of Se in endocrine tissues, reproductive organs and the brain (K¨ohrle et al., 2005; K¨ohrle et al., 2005). Therefore, food systems-based approaches should be seriously considered as a means of increasing Se in food products to improve Se status in populations whose intake is low and thus to prevent the occurrence of thyroid cancer.

NIS Expression and Therapeutic Potential in Thyroid Cancer Sodium iodide symporter (NIS), an intrinsic plasma membrane glycoprotein, mediates the active transport of iodide at the basolateral membrane of thyroid follicular cells. Functional NIS expression in the thyroid gland is responsible for thyroidal accumulation of iodide, an essential constituent of the thyroid hormones T3 and T4. Due to the expression of NIS, thyroid follicular cells have unique ability to trap and

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concentrate iodide and this feature allows imaging as well as highly effective therapy of differentiated thyroid carcinomas and their metastases by administration of radioiodine after total or near-total thyroidectomy, thereby improving the prognosis of thyroid cancer patients significantly (Mazzaferri, 1996). However, defects in the genetic basis for NIS, including hypermethylation of the NIS gene promoter (Venkataraman et al., 1999) and mutations in NIS gene that result in functional abnormalities, have been demonstrated in tall cell and columnar variants of papillary cancer, and Hurthle cell cancer, ATC and MTC (Spitzweg and Morris, 2002). Since these poorly differentiated and undifferentiated thyroid cancers can not concentrate iodine due to defects in NIS, treatment with radioiodine is ineffective and have poor prognosis. Fortunately, cloning of the NIS gene (Smanik et al., 1996, Dai et al., 1996) has paved the way for the development of a novel cytoreductive gene therapy strategy for the treatment of these cancers based on NIS gene transfer followed by radioiodine therapy. Targeted expression of functional NIS in cancer cells would enable these cells to regain their ability to concentrate iodide from plasma, and would, therefore, offer the possibility of radioiodine therapy. Application of the NIS gene as a gene therapeutic tool therefore extends the diagnostic and therapeutic use of 131 I and the extensive experience with radioiodine in the management of differentiated thyroid cancer to the treatment of poorly differentiated, anaplastic, and medullary thyroid cancer. Lee et al. (Lee et al., 2003) have used a recombinant adenovirus to transfect a panel of undifferentiated thyroid carcinoma cell lines (ARO, FRO, NPA) with the NIS gene and demonstrated significant iodide accumulation in these cells. Finally, in medullary thyroid cancer, a therapeutic effect of radioiodine has been demonstrated following induction of tissue-specific iodide uptake activity by calcitonin promoter-directed NIS gene transfer in vitro (Cengic et al., 2005). Taken together, these studies demonstrate the potential of NIS as a novel therapeutic gene allowing 131 I therapy of dedifferentiated follicular cell-derived thyroid carcinomas and medullary thyroid cancer after NIS gene transfer.

Conclusions It is generally accepted that the carcinogenic process proceeds through multiple discrete steps, since experimental data suggest that a single oncogenic mutation is not sufficient to induce malignant transformation. Disruptions including combinations of hyperfunctional proliferative factors with downregulated growth suppressive factors lead to a proliferative advantage and clonal expansion in one or multiple foci. As the proliferating cells approach their replicative limits and accumulate genetic damage, the neoplastic clones must achieve immortalization and overcome apoptotic signals to continue their growth. Like other cancers, thyroid carcinogenesis is a complex process. A large number of genetic events, molecular abnormalities, alterations of signaling pathway have been identified in the development and progression of various thyroid neoplasms, Furthermore, it is highly likely that the advent of

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large-scale genomic screening will bring forth an abundance of additional data that will likely to result in advances in the management of thyroid cancer in the future.

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Index

A ABT-737, 209, 228 Ac-DEVD-CHO, 262 Acute lymphoblastic leukemia, ALL, 196–198, 203–205 Acute myeloid leukemia, AML, 77, 96, 180, 204, 208, 209, 275, 280 Ac-YVAD-CHO, 262 Adenoma, 78–81, 83, 84, 86–89, 92, 349, 356, 361 Adenomatous polyposis coli, AdPC, 78, 82, 83, 88, 89, 163, 164, 166, 167, 285 Adenosine tri-phosphate, ATP, 5, 12, 30, 38, 39, 94, 113, 115, 174, 254, 299, 328, 329, 335–337, 340–345 Adenovirus E1A, 277 Adenyl cyclase, AC, 350, 351 Aldehyde dehydrogenase, 11 alfa-interferon, 112 Alkylating agents, 112 Allelic imbalance, AI, 116–118 Alpha-fetoprotein, AFP, 228 Alpha-Tocopherol, 309, 313 Alternative-reading frame protein, ARF, 274, 275 3-aminobenzamide, 3-AB, 336, 341 AMP-activated protein kinase, AMPK 30, 38 Anaphase-promoting complex, APC, 163, 276 Anoikis, 228, 357 Antagomir, 66, 67 Antennapedia, 329, 341 Anti-apoptotic protein, 4, 9, 28, 31, 40, 85, 92, 96, 127, 129, 136, 140, 174, 177, 200, 300 Antigen presentation, 36 Antimycin, 228 Antisense oligonucleotides, ASO, 15, 40, 96, 114, 209, 223, 226–228, 240, 301, 363 Apicidin, 178 Apoptosis inducing factor, 4

Apoptosome, 5, 115, 116, 118, 255, 299, 362 Apoptotic inducing factors, AIF, 298 Apoptotic protease-activating factor 1, APAF-1, 2, 5, 111, 113, 115–119, 130, 174, 222, 228, 254, 298, 299, 301 A proliferation-inducing ligand, APRIL, 199 Arachidonic acid, AA, 92, 252, 256, 261, 265, 308, 314 Arsenic compound, 35 ATP-binding cassette, ABC, 12 Autophagy, 3, 25, 27–30, 34, 36–38, 41, 60 Autopsy, 80 Azacytidine, 34, 226 5-aza-deoxycytidine, 179, 279 B Bacillus calmette-guerin–BCG vaccine, BCG, 36, 40 Bad, 9, 10, 32, 129, 134, 141, 146, 170, 219–222, 225, 259, 300, 311 Bak, 9, 10, 30, 39, 55, 56, 129, 134, 163, 174, 175, 219–222, 224, 225, 227, 228, 254, 255, 284, 286, 300, 333, 335 Barrett’s Esophagus, BE, 128, 141–145, 147 Base excision repair, BER, 165 Basic fibroblast growth factor, bFGF, 193, 195–197 Basic helix-loop-helix transcription factor, 276, 284, 286 Bax, 5, 9, 10, 30, 32, 39, 55, 56, 59, 61, 86, 87, 92, 97, 98, 129, 130, 134, 135, 139, 140, 144, 146, 163, 164, 167, 170, 174, 175, 178, 197, 208, 209, 219–222, 224, 225, 227, 228, 239, 254, 255, 261, 262, 264, 284, 286, 300, 301, 309, 333, 335, 361 B cell activating-factor of the tumor necrosis factor family, BAFF, 193, 195, 199, 200, 209, 210

375

376 Bcl-2, 4, 5, 7, 9–11, 31, 87, 97, 98, 114, 134, 140, 143–144, 146, 170, 174, 177, 197, 203, 208–209, 219–229, 239, 240–241, 244, 255, 284, 362 Bcl-2/adenovirus E1B 19 kDa interacting prote´ın, Bnip, 178, 219, 220, 222 Bcl-2-associated athanogene, BAG, 362 Bcl-2 interacting killer, Bik, 219–221, 226, 228, 255 Bcl-2 interacting mediator of cell death, Bim, 3, 114, 115, 164, 170, 179 Bcl-2-modifying factor, Bmf, 219, 220, 222, 228, 229 Bcl-2-related ovarian killer, Bok/Mtd, 219, 220 Bcl-w, 219–224, 228 Bcl-x, 134, 146, 223, 301, 312, 361 Bcl-XL , 9, 10, 32, 92, 96, 98, 129, 134, 138–140, 144, 146, 198, 200, 219–224, 226, 228, 240, 255, 256, 262, 264, 286, 300, 301 Bcl-XS, 9, 134, 219, 220, 223, 224, 226, 227, 301 Beclin-1/ATG6, 36 Bee venom, BV, 262 Benzamide, 34 1-Benzylimidazole, 1-BI, 262 Beta-Carotene, 309, 313 Beta-Catenin, 14, 164, 228, 285 Bfl-1/A1, 200, 219–221, 223, 224, 228, 229 Bid, 10, 129, 141, 219–222, 225, 254, 255, 299 Biomarkers, 11, 12, 14, 26, 111, 117, 145, 147, 164, 175, 301, 310, 311 Bladder cancer, 25–28, 30–36, 38–40 BOC-Asp-FMK, 333, 334, 342 Bok, 221, 224, 229 Bombesin, BN, 257 Bone marrow stromal cell, BMSC, 193–205, 210 Bortezomib, 210, 255 BRD7, 275, 278, 279 Breast cancer–Neoplasms, 79 C C225, 307, 311 CA 125, 310 Calmodulin (CaM)-regulated Ser/Thr kinase, DAPK, 35 Calpain, 27, 38, 39 Calpain 3, p94, 38 Calpain 9, 38 Calpain 10, 38 Camptothecin, 303, 339 Cancer stem cell, 1, 3, 11–15, 157, 180–182

Index Carboplatin, 63, 66 Carcinoembryonic antigen, CEA, 310 Carcinogen, 26, 33, 76, 159, 252, 254, 263, 273, 349, 350, 353 Carcinogenic agents, 35, 347, 349 Caspase, 301–304, 309, 310, 328–335, 342, 344, 345, 362 Caspase 2, 5, 33, 130, 301 Caspase 3, 4, 5, 8, 9, 31–33, 61, 91, 94, 97, 129, 130, 136, 138, 164, 204, 221, 225, 255, 260, 261, 262, 264, 298, 299, 301–303, 308, 328, 329, 332, 333, 336, 343, 344 Caspase 6, 7, 8, 61, 129, 130, 298, 299, 301 Caspase 7, 4, 5, 7–9, 91, 97, 129, 130, 255, 298, 299, 301, 303, 328, 343 Caspase 8, 4–8, 85, 88, 91, 94, 97, 129, 130, 164, 254, 260, 298, 299, 301, 303, 333, 335, 342, 362, 363 Caspase 9, 4, 5, 7–9, 33, 91, 97, 113, 115, 116, 118, 119, 130, 138, 145, 164, 222, 226, 254, 255, 262, 298, 301, 302, 311 Caspase 10, 4, 5, 130, 164, 254, 301 Caspase 12, 226 Caspase recruitment domain, CARD, 115 Cathepsin, 39 CD4, 36 CD24, 11, 12 CD34, 12, 329 CD38, 12 CD44, 11–15, 276, 283 CD45, 12 CD55, 12 CD95, FAS-R, Fas receptor, 4, 30, 94, 131, 174, 302 CD133, 12, 13 CDC20, 282 CDH1, 159, 163, 164, 166, 167, 170, 173, 282 cDNA microarray, 157, 158 Celecoxib, 159–261, 256, 264, 265, 303 Cell adhesion molecules, 78, 83, 160, 164, 202, 210, 279, 280 Cell cycle, 1, 5, 6, 25, 34, 35, 51–56, 58, 62, 66, 67, 79, 83, 87, 91, 118, 128, 131, 135, 139, 141, 147, 163, 164, 166, 167, 176, 178, 179, 199, 202, 210, 220, 227, 238–240, 242, 253, 254, 259, 262, 273–279, 281, 283, 285, 286, 303, 304, 308–310, 313, 324, 353–356, 360 Cell cycle proteins, 194, 205 Cell-cycle regulators, 52, 54, 128, 163, 164, 361

Index Cell death, 1–3, 5, 6, 9, 13, 15, 25–30, 29, 34, 35, 39, 41, 58, 60–62, 83, 84, 86–90, 98, 113, 120, 127, 129–131, 134, 137, 138, 140, 148, 157, 164, 173–176, 178, 194, 208, 219, 222, 225–227, 237, 238, 240, 251, 253–255, 260, 262, 264, 265, 276, 282, 285, 296, 299, 301, 305–307, 309, 327–331, 333–337, 341–345, 357, 359, 361, 362 Cell division cycle, CDC, 222, 282 Cell proliferation, 2, 12, 13, 37, 53, 78, 84, 86–88, 90, 93, 97, 129, 131, 166, 197–200, 232, 238, 243, 252, 253, 256, 264, 265, 275, 278, 280, 282, 283, 285, 286, 305, 308, 309, 313, 314, 354 Cell survival, 4, 13, 14, 29, 30, 37, 63, 86, 89, 116, 143, 177, 181, 194–205, 207–210, 221, 224, 227, 276, 278, 284, 302, 305, 307–309, 311, 314, 357, 361 Cellular FLICE-like inhibitory protein, c-FLIP, 6, 94, 203, 300, 303, 342 Cellular senescence, 36, 58, 238, 239 Ceruloplasmin, CP, 349, 356, 357 Cervical cancer, 51–54, 57–67 Cervical intraepithelial neoplasia, CIN, 54, 57, 61 Chemoprevention, 32, 127, 257, 263 Chemotherapy, 2, 3, 6, 9, 12, 14, 26, 31, 39, 41, 42, 51, 52, 57–63, 65, 75–98, 111–118, 120, 127, 136–141, 145–148, 161–163, 175, 179, 181, 194, 200, 203, 204, 207–209, 220, 225, 227, 237, 239, 240, 253, 263, 273, 277, 286, 296, 298, 301, 303, 304, 315, 327, 339, 360, 363 Chenodeoxycholic acid, 76 Chromosomal instability, 56, 166 Chromosome 9, 38 Chromosome segregation, 82, 166, 282 Chronic lymphocytic leukemia, CLL, 170, 177, 196, 197, 199, 200, 203, 208, 209, 239 Chronic myelocytic leukemia, CML, 203 c-IAP1, 4, 8, 15, 140, 200 c-IAP2, 8, 15, 140, 200 cis-diamminedichloroplatinum, Cisplatin, 51, 57, 61, 62, 63, 66, 79, 114, 115, 137, 162, 163, 177, 226, 244, 255, 261, 277, 283, 303 c-jun kinase, 33, 62, 65, 82, 227 c-kit; v-kit Hardy-Zuckerman, 4 feline sarcoma viral oncogene homolog, KIT, 164 Clusterin, CLU, 237, 240, 242, 244, 245, 311 c-met; hepatocyte growth factor receptor, MET, 163, 164, 349, 351, 352

377 c-myc; v-myc avian myelocytomatosis viral oncogene homolog, MYC, 41, 55, 56, 164, 170, 223, 252, 253, 276, 277, 281 Colorectal cancer, CRC, 75–84, 89–95, 97, 98, 159, 164 Complementary DNA, CDNA, 163, 172 COX-2 inhibitors, 251, 257–261, 263–265, 314 Curcumin (diferuloylmethane), 32, 33 Cyclic adenosine monophosphate, cAMP, 305, 350, 351 Cyclin A, 55 Cyclin D1, 33, 82, 276, 278, 281, 284, 285 Cyclin-dependent kinase, CDK, 61, 97, 135, 164, 167, 169, 176, 238, 239, 274–278, 281, 284–286, 304 Cyclin E, CCNE1, 55, 164 Cycloheximide, 303 Cyclooxygenase, COX, 92, 228, 251–253, 256–265, 313, 314 Cyclophilin D, 30 Cyclophosphamide, 177 Cysteine protease, 28, 38, 254 Cytarabine, 204, 208 Cytochrome c, 2–5, 7, 9, 27, 30, 31, 33, 85, 92, 94, 113, 115, 116, 119, 129, 130, 134, 140, 141, 164, 174–176, 178, 221, 222, 225, 228, 254, 255, 259, 260, 298, 299, 301, 314, 339, 362 D Dacarbazine, DTIC, 112, 114 Death effector domains, DED, 4, 56, 130, 298 Death-inducing signaling complex, DISC, 4, 6, 55, 85, 94, 129, 298, 299, 303, 362, 363 Death receptor 4/Death receptor 5, DR4/DR5, DR5, 4, 40, 131, 132, 137, 142, 164, 174, 178, 254, 260, 298 Decitabine, 34, 179 Decoy receptors, 4, 40, 132, 137, 300 Decoy receptors 1 (DcR1 or TRAIL-R3), TRID, 4, 132, 137 Decoy receptors 2 (DcR2 or TRAIL-R4), TRUNDD, 4, 132 Deguelin, 260 Deleted in colorectal cancer, DCC, 83, 163, 164 5-deoxyazacytidine, 274 Deoxyribonucleic acid, DNA, 2, 4–7, 12, 25–27, 32, 34, 38, 39, 41, 51–54, 56–60, 62–67, 78, 79, 82–85, 93, 96, 113–115, 117, 129, 131, 135, 139, 144, 157, 159, 163, 165–179, 171–179, 200, 206, 207, 238–240, 242, 252–254, 259, 261,

378 274–278, 280–283, 286, 296, 298, 299, 304, 307, 308, 310, 332–334, 344, 352, 360, 361 Depsipeptide, 34, 178 Dexamethasone, 198, 199 Diabetes mellitus, 38 2, 3-dimethoxycinnamoyl azide, DMCA, 35 Dipeptidyl-aminopeptidase, DAP4, 349, 357 Direct IAP binding protein with low pI, DIABLO, 304 Disease progression, 112, 224, 352 DNA damage, 4–6, 25, 38, 39, 41, 51, 57–60, 62, 64, 65–67, 83, 85, 115, 129, 135, 139, 166, 171–173, 175, 176, 178, 207, 228, 238, 239, 275–278, 280–283, 298 DNA distortion, 62 DNA methyl trasferase, DNMT1, 173, 179 DNA repair, 12, 51, 57, 62, 78, 83, 93, 131, 157, 165, 166, 206, 238–240, 242, 254, 281, 360, 361 Docetaxel, 162, 163, 264 Double-stranded DNA breaks, 352 Doxorubicin, DOX, 12, 15, 40, 59, 66, 145, 202, 226, 227, 244, 263, 264 Drug resistance, 12, 13, 25, 89, 94, 139, 193, 194, 199–203, 205–208, 211, 239, 259, 296, 327, 328 Ductal carcinoma in situ, DCIS, 3, 8, 12, 13, 15 E E2F transcription factor 1; retinoblastomaassociated protein, E2F1, 55, 164, 170, 276, 282–284, 349, 359 E-cadherin, CDH1, 159, 163, 164, 166, 167, 170, 172, 173 Ectodysplasin-A receptor, EDAR, 131 Effector caspases, 5, 129, 130, 140, 222, 299 Eicosanoid, 256 Embryogenesis, 221, 357 Embryonic stem cells, 13 Epidemiology, 76, 128, 158, 160 Epidermal growth factor, EGF, 163, 165, 257, 279, 285, 311 Epidermal growth factor receptor, EGFR, 13, 54, 55, 96, 257, 265, 276, 286, 307–309, 311, 355, 356 Epigallocatechin-3-gallate, EGCG, 32, 308 Epigenetic marker, 157 Epirubicin, 162, 163 Epstein-Barr virus, EBV, 157, 159, 168, 173, 209, 273, 276, 282, 283 ErbB1, 355 ErbB2, 163, 164, 173, 355

Index ErbB3, 355 ErbB4, 355 Esophageal carcinoma, 127, 128 Estrogen, 12, 359 Estrogen receptor, ER, 10, 240, 359, 360 Etoposide, VP-16, 66, 181, 204, 262 Extracellular matrix, ECM, 11, 14, 90, 128, 193–195, 201, 202, 207, 283, 357 Extracellular signal-regulated kinase, ERK, 146, 265, 279, 309, 312, 359, 360, 363 F Familial adenomatous polyposis, FAP, 77, 81 Farnesylation, 146, 353 Farnesyl transferase, 353 Fas, 3, 4, 6, 7, 9, 30, 31, 39, 40, 54, 55, 85, 94, 129, 131–133, 138, 142, 143, 145, 146, 174, 198, 201, 202, 221, 222, 225, 226, 253, 254, 264, 298–301, 303, 306, 329, 333–335, 342, 344, 362, 363 Fas-associated death domain, FADD, 4, 6, 55, 56, 129, 174, 254, 298–300, 302, 303, 344, 362 Fas-associated death domain-like interleukin1β-converting enzyme inhibitory protein, FLIP, 201, 210, 302, 303, 349, 363 Fas-associated phosphatase-1, FAP-1, 300 Fas decoy receptors, DcR3, 300 Fas L, Fas ligand, 4, 30, 39, 40, 129, 132, 143, 299, 362 Fibroblast growth factor, FGF, 165, 196, 197 Fibronectin, FN, 194, 195, 202–204, 206, 207, 283 FK228, 34, 35 Flavonoid, 309, 314 Flavopiridol, 147 Fludarabine, 197 Fluoride, 305, 306 5-Fluorouracil, 5-FU, 79, 92, 93, 95, 147, 161–163, 225, 301 Folinic acid, 79 Follicular adenoma, FA, 349, 351, 353, 356, 358, 361 Follicular carcinoma, FC, 347–350, 352, 353, 356, 358, 361 Forskolin, 257 Fragile histidine triad, FHIT, 164 Frame shift mutation, 135, 167 Free-radical scavengers, 342, 343 G G1/S –specific cyclin D2, CCND2, 164 G3139, 177, 209, 227 Galectins, 359

Index Gastric cancer, 38, 157–182, 278 Gastrin, GAST, 168, 175 Gastrin-releasing peptide, GRP, 257 Gastroesophageal reflux disease, GERD, 128, 141, 144 Geldanamycin, 197 Gemcitabine, 32, 33, 66, 264, 301, 304, 307, 308, 311, 312, 314 Gene amplification, 165, 354, 355 Gene silencing, 64, 167, 168 Gene therapy, 40, 152, 228, 274, 277, 279–281, 287, 353, 360, 365 Genetic marker, 157 Genetic predisposition, 160, 347, 348 Genetics, 130 Genistein, 309, 314 Genomic instability, 52, 53, 55, 56, 90, 166, 281 Gingerol, 209, 314 Glioblastoma, 12, 181, 304 Glutathione peroxidase, GPx, 314, 364 Glutathione S-transferase π, GSTP1, 173 Glycogen synthase kinase 3, GSK3, 311 GN963, 312 Goitrogens, 350 G protein-coupled receptor 30, GPR30, 349, 359, 360 G-protein-coupled receptors, 261, 263, 295, 305, 359 G protein subunit alpha, Gsp, 349–351 Growth factor signaling pathways, 2, 28 GTPase-activating protein, GAP, 38 H H2AX, 283 Half life, 5, 79, 238 Hamartin, 38 Hamartoma syndrome, 353 Harakiri, Hrk/DP5, 219, 220, 226 Heat shock protein, HSP, 115, 116, 197, 311, 362 Helicobacter pylori, 157, 159, 160, 166, 168, 171, 172, 175, 180 Hematopoietic stem cell, HSC, 193 Hepatitis B virus X protein, 225 Hepatocyte growth factor/scatter factor, HGF/SF, 164, 221, 352 Her1, 309, 355 Her2, 15, 164, 277, 355 Her3, 355 Her4, 355 Hereditary non-polyposis colonic cancer, HNPCC, 77

379 Heterogeneity, 26 High-mobility group B1, HMGB1, 41, 42 High mobility group I, HMGI, 349, 357 HIPK2, 357 Histone acetyltransferase, HAT, 34, 168 Histone deacetylase, HDAC, 34, 55, 168, 169, 178, 209, 210 Histone deacetylase inhibitors, HDACIs, 34, 178 Hrk, 219, 220, 222, 226, 229 Human papillomavirus, HPV, 51–61, 63–65, 67 Human telomerase reverse transcriptase, hTERT, 55, 349, 361 Hyaluronan, 14 Hydroxamic acid, 34, 178 Hyperglycemia, 313 Hypoxia, 4, 29, 115, 355 Hypoxia-inducible factor 1 alpha Subunit, HIF-1α, 163 I IC50 , 15, 206, 264 Iloprost, 263 ILP-2, 140 Immunoblotting, 329 Immunohistochemistry, IHC, 91, 116, 135, 144, 244, 277 Immunosuppression, 31 Immunotherapy, 26, 36, 112, 113, 118, 119, 296, 304, 361 Indomethacin, 264 Inflammatory bowel disease, 76 Inhibitor of apoptosis proteins, IAP, 2, 8, 9, 15, 27, 94, 111, 114, 115, 118, 119, 140, 141, 147, 164, 175–177, 200, 256, 282, 302, 303 Inhibitor of differentiation, Id1, 276, 283, 286 Initiator caspases, 4, 5, 7, 130, 298, 299 Inositol hexakisphosphate kinase-2, 306 Inositol phosphates, 295 InsP5 , 306, 307 InsP6 , 302, 305–307 InsP7 , 306, 307 Insulin-like growth factor binding protein, IGFBP, 92, 97 Insulin-like growth factor, IGF-I, 91, 96–98 Insulin-like growth factor receptor, IGF-I R, 91, 96 Insulin resistance, 309, 313 Interferon regulatory factor-3, IRF-3, 54–56 Interleukin 1 family member 6, ε, IL1F6, 165 Interleukin 1 family member 8, ξ , IL1F8, 165 Interleukin-1, IL-1, 257 Interleukin 1, α, IL1A, 165

380 Interleukin-4, IL-4, 13 Interleukin-6, IL-6, 193, 195–199, 201, 209, 210, 314 Interleukin-8, IL-8, 256, 314 Intestinal trefoil factor, ITF, 92, 93, 164 Invasive ductal carcinoma, 12 Ion channels, 31 Isorhamnetin, 138 IκB kinase, IKK, 200, 252, 256 J JTE-522, 264 K Keratinocyte, 13, 53, 55, 56, 61, 278 Ketoconazole, 262 Kirsten rat sarcoma viral oncogene homolog, KRAS, 82, 83, 89, 163, 164 L L-165041, 264 Lactoferrin, LF, 349, 356, 357 Laminin, 283 Laryngeal cancer, 227–245, 306 Latent membrane protein 1, LMP1, 276, 282–284, 286 Lenalidomide, 210 Leucovorin, LV, 161–163 Leukemia, 12, 34, 35, 136, 164, 170, 177, 179, 180, 193, 194, 196, 197, 203–205, 208, 209, 275, 280, 362 Limb-girdle muscular dystrophy, 38 Lipopolysaccharide, 257 5-lipoxygenase, 5-LOX, 265 Lithocholic acid, 76 Locked nucleic acid, LNA, 179 Loss of heterozygosity, LOH, 116, 117, 141, 165–167, 174, 275, 279, 280 Lung cancer, 7, 12, 239, 251–265, 275, 277, 279, 280, 340 Lymphoma, 34, 35, 42, 164, 174, 193, 194, 196, 197, 199–202, 205, 206, 208, 210, 227, 239, 282, 329, 347, 348 M Mammalian target of rapamycin, mTOR, 30, 37, 38, 354 Matrix metalloproteinase, 355 Mcl-1, 219–222, 224, 226–228, 300 Melanoma, 34, 97, 111–121, 177, 198, 239, 262, 302, 348 Melphalan, 202, 203, 205, 206, 210 Menstrual cycle, 2 Mesothelin, 311

Index messenger RNA, mRNA, 31, 32, 35, 54, 59, 60, 63–66, 97, 113, 116, 143, 146, 169–172, 197, 209, 222, 225, 226, 240, 242, 244, 263, 304, 314, 350, 356 Methylation, 7, 34, 114, 141, 165, 167–169, 173, 179, 226, 274, 275, 310, 358 4-(methylnitrosoamino)-1-(3-pyridyl)1-butanone, NNK, 252, 253, 259 MHC class I, 54 Microphthalmia-associated transcription factor, MITF, 114 microRNA, miRNA, 66, 113, 157, 165, 166, 169, 170, 179–182 Microsatellite instability, 157, 165–167, 175 Microsomal prostaglandin E synthase, mPGES, 262 Minimal residual disease, MRD, 193, 194, 201, 208, 211 miR-21, 66, 165, 179, 181 miR-34, 66, 165, 170 Mismatch repair ATPase MutS family, MSH, 63, 78, 163, 165, 167 Mitochondria, 2–4, 9, 10, 27, 85, 89, 94, 114, 118, 129, 130, 134, 136, 142, 164, 174, 176, 221, 222, 224, 228, 238, 254, 259, 263, 298, 299, 301, 304, 339 Mitochondrial membrane, 10 Mitogen-activated protein kinase, MAPK, 28, 197, 198, 252, 253, 279, 283, 312 Mitomycin, 66, 147 Mitotic arrest deficient 2, MAD2, 276, 282, 283 Mitoxantrone, 66, 202, 205–207 MnTMPyP, 337, 338, 341, 342 Mothers against decapentaplegic (MAD) Drosophila homolog, MADDH, 164, 173, 174 Mouse double minute 2 homolog, MDM2, 58, 59, 65, 164, 176, 177, 238, 275, 277, 280, 311 Msx1, 285 Mucin, (oligometric mucus/gel-forming), MUC, 164, 310, 311 Mucosa-associated lymphoid tissue, MALT, 348 Multiple endocrine neoplasia, MEN, 348 Multiple inositol polyphosphate phosphatases, MINPP, 307 Multiple Myeloma, MM, 34, 177, 193, 194, 196–200, 202, 205, 206, 208–210 Munc18-1-interacting protein 2, MINT2, 173 Mutation, 3, 6, 11, 12, 38, 56, 60, 62, 75, 77, 78, 82, 83, 88–90, 92, 94, 95, 98,

Index 114, 115, 135, 136, 138, 141, 144, 145, 147, 159, 163, 165–167, 171, 173–175, 180, 196, 201, 205, 225, 238, 241, 242, 252, 274, 275, 280, 283, 285, 307, 311, 349–354, 356, 358, 360, 363–365 MutL human homolog DNA mismatch repair, MLH1, 163, 165, 167, 173 MutY human homolog base excision repair, MYH, 77, 165, 171 Myelodysplastic syndrome, 197 Myeloid cell leukemia-1, Mcl-1, 24, 219–222, 226–228, 300 N NADPH oxidase activator, Noxa, 10, 59, 115, 141, 219, 220, 226 Nanoparticles, 179 Nasopharyngeal Carcinoma, NPC, 273–277 Necrosis, 3, 4, 25, 27, 29, 30, 38, 39, 41, 42, 56, 60, 84, 85, 117, 129, 131, 165, 174, 199, 222, 225, 254, 257, 297, 298, 305, 327–329, 331, 332, 336–345, 362, 911 Neo-adjuvant treatment, 78 Nerve growth factor receptor, NGFR, 131 Neural cell adhesion molecule, N-CAM, 83 Neuroblastoma, 7, 34, 304 Neurogenic locus notch, NOTCH, 181, 204, 205 Neuronal apoptosis-inhibitory protein, NAIP, 8, 140 NF-κB; Nuclear factor kappa light polypeptide gene enhancer in B-cells, NFKB1, 174, 175, 177, 205, 252, 313 NGX6, 275, 279 Nimesulide, 71, 259, 260, 264 Nip3-like protein X, Nix, 10, 222, 226 Nitric oxide, NO, 222, 305, 314 Nitrosamines, 33, 76 N-nitroso-bis(2-oxopropyl)amine, 260 N-nitrosomorpholine, 227 Non-Hodgkin’s lymphoma, NHL, 196, 197, 202, 209, 227, 239 Non-small cell lung cancer, NSCLC, 7, 251–253, 255–257, 261–264, 280 Non-steriodal anti-inflammatory drugs, NSAID, 92, 257, 259 N-(phosphonacetyl)-L-aspartic acid, PALA, 79 NS-398, 252, 259–261, 264 Nuclear hormone receptor, 263 Nutrition, 194, 295

381 O O-6-methylguanine-DNA methyltransferase, MGMT, 173 Oblimersen sodium, 114, 177, 209, 240 Oil A, 309, 310 Oligonucleotide microarray, 206 Oncogene, 9, 51, 60, 62, 82, 89, 131, 157, 164, 169, 170, 174, 181, 254, 276, 283–285, 313, 351, 352, 364 Oncogenic transformation, 2 Oncoproteins, 51, 54, 55, 63, 78, 352 ONO-NT-126, 261 Onyx-015, 95 Open reading frame, ORF, 54, 135 OSI-774, 307 Osteopontin, 283, 354 Oxaliplatin, 63, 66, 89, 93, 95, 141, 147, 148 Oxidative stress, 4, 129, 173, 313, 314 8-oxo-7, 8-dihydro 2 deoxyguanosine, 8-oxoG, 171 P p14ARF , 173, 274, 275, 277 p15; INK4B; cyclin-dependent kinase inhibitor 2b, CDKN2B, 164, 167, 176, 281 p16; INK4A; cyclin-dependent kinase inhibitor 2A, CDKN2A, 34, 163, 164, 167, 173, 274, 275, 277, 286, 310 p21, 13, 33, 35, 55, 59, 60, 135, 138, 202, 227, 239, 259, 261, 276, 286, 309, 310, 328, 360 p21 WAF1/Cip1-dependent pathway -CDKN1A protein, human, 13, 163, 164, 169, 176, 205 p27; kip1; cyclin-dependent kinase inhibitor 1B, CDKN1B, 55, 164, 176, 202, 275–277, 284, 286, 309, 310 p50, 32, 200, 252, 255, 256 p52, 200, 205, 255 p53, 5, 6, 12, 30, 35, 41, 51–67, 78, 81, 83, 87, 89, 90, 94–96, 111, 113–116, 135, 136, 138, 139, 144–147, 163, 164, 166, 169, 170, 173, 176, 177, 221, 223, 224, 226–228, 237–243, 245, 252, 253, 259, 262, 264, 275, 277, 278, 280, 281, 308–311, 314, 327–344, 349, 360 p53 upregulated modulator of apoptosis, PUMA, 10, 29, 59, 141, 164, 220, 226, 228, 333, 335 p65, 32, 33, 252, 256 p70S6 kinase, 354 p73, 60, 164, 173 Paclitaxel, PAC, 32, 66, 140, 226, 244, 284–286, 299, 327, 333, 334, 339, 340

382 Parathyroid hormone related peptide, PTHrP, 305 Pathology, 77, 157, 160, 242 Pax-8, 349, 358 Peroxisome proliferator-activated receptor delta, PPARdelta, 263, 264 Peroxisome proliferator-activated receptorgamma, PPARγ, 257, 259, 260, 349, 354, 358 Phagocytosis, 84, 95 Phorbol esters, 257, 312 Phosphatase and tensin homolog, PTEN, 163, 164, 170, 173, 181, 284, 307, 311, 312, 349, 353–355 Phosphatidylinositol 3-kinase, PI3K, 32, 36, 227 Phosphoethanolamine, 28 Phospholipase A2, 308 Phospholipase C, PLC, 302, 305, 306, 350 Phytic acid, 308, 309 Pirh2, 65 Platelet-derived growth factor, PDGF, 165, 257, 312 Platinum, 57, 63 Platinum compounds, 60, 61, 112, 255 Poly-ADP ribose polymerase, PARP, 27, 32, 33, 35, 39, 140, 260, 262, 265, 284, 308, 328, 329, 333, 336, 341, 343 Polychemotherapy, 163 Polycomb group ring finger, BMI1, 181 Polyphenols, 309 Post-translational modification, 168, 280 Pro-apoptotic proteins, 4, 8–10, 15, 53, 59, 88, 92, 129, 222, 224, 301, 311 Procaspase-activating compound-1, PAC-1, 299 Progesterone, PR, 10, 12 Proliferation cell nuclear antigen, PCNA, 223 Promyelocytic leukemia, 275, 280 Prostacyclin I2 , PGI2 , 262, 263 Prostacyclin synthase, PGIS, 262, 263 Prostaglandin E2 , PGE2 , 252, 253, 256, 257, 259, 260, 262, 263–265 Prostaglandin H2 , PGH2 , 92, 253, 256, 261, 262 Prostaglandins, 92, 251–253, 256, 257, 259, 261–263, 265 Prostaglandin synthase, PGES, 256 Protein kinase c, 205, 312 Protein kinase, PKB, 35, 37, 58, 296, 302, 306, 308, 309, 353 Proto-oncogenes, 78, 82, 351, 352

Index pS2; gastrointestinal trefoil protein 1, TFF1, 163, 164, 167, 168, 171–173, 175 Pyroxamide, 178 R Radiotherapy, 2, 39, 51, 57, 58, 60, 62, 75, 78, 79, 86, 93, 111, 113, 138, 139, 237, 241–243, 245, 273, 286, 296, 301, 315 Raf kinase, 352 RAGE receptor, 41 Rapamycin, 30, 38, 354 Ras association RalGDS/AF-6 domain family 1, RASSF1A, 173, 275, 280, 283 Reactive oxygen species, ROS, 28, 30, 39, 41, 85, 171, 172, 174, 178, 327, 337, 341–345, 364 Receptor death domains, 3 Receptor-interacting protein kinase 1, RIPK1, 27, 30, 39 Receptor tyrosine kinase, RTK, 284, 349, 351–353, 355 Resveratrol, 309, 314 Retinoblastoma, Rb, 54, 55, 238, 239, 253, 274, 278, 279, 285, 286 Retinoic acid, 308 Ret/PTC, 349, 351, 352, 354, 358 Ribonucleic acid, RNA, 34, 41, 63, 64, 146, 169, 172, 207, 223, 244, 303, 304, 335, 356, 361 Ribonucleotide reductase, 62, 139 Risk factor, 26, 54, 80, 142, 157, 159, 160, 295, 312, 364 RNA interference, RNAi, 15, 51, 59, 63–67, 276, 283, 285, 304 RNases, 64 Runt-related transcription factor 3, RUNX3, 163, 164, 166–169, 176, 178, 179 S S-1452, 261 Second mitochondria-derived activator of caspase, SMAC, 118, 129, 164, 176, 177, 304 Selenium, Se, 347–349, 364 Senescence, 58, 60, 64, 66, 238, 239, 361 Serglycin, 283 Serine/threonine protein kinase/Pritein kinase B, Akt/PKB, 37, 296, 302, 306, 308, 309, 353 Shikonin, 262 Short-chain fatty acids, 34 Side effects, 40, 63, 79, 113, 161, 264, 265 14-3-3 sigma, 275, 278, 284

Index Signaling Transduction- Signal Transduction, 90, 142, 145, 166, 226, 265, 273, 304, 305, 350, 353, 356 Signal transducers and activators of transcription, STAT, 198, 283 Silibinin, 33 Small interfering RNA, siRNA, 15, 40, 63–66, 139 Small mothers against decapentaplegic, SMAD, 83, 90 SN-38, 264 Sodium arsenite, 35 Sodium butyrate, 34, 178 Sodium fluoride, 305, 306 Sodium iodide symporter, NIS, 349, 358, 364, 365 Sonic hedgehog, SHH, 181 Spermidine/ spermine N1-acetyltransferase, SSAT, 264 Spike, 219, 220, 222 Src kinases, 312 Statins, 146 Stem cells, 1, 3, 11–15, 82, 85–87, 157, 180–182, 193, 329, 361 Stratifin, 275, 278 Suberoylanilide hydroxamic acid, SAHA, 34, 178 Superoxide radical, 364 Survival rate, 57, 63, 78, 131, 135, 162, 224, 253, 255, 265, 282, 295, 296, 310, 347, 348 Survivin; baculoviral IAP repeat-containing 5, BIRC5, 8, 9, 13–15, 31–33, 40, 56, 91, 96–98, 111, 114, 118, 119, 140, 141, 147, 148, 163, 164, 175, 176, 181, 256, 276, 282, 303, 304, 311 T Tarceva, 307, 309 Taxanes, 112 Taxotere, 264 T-cell factor, TCF, 285 Telomerase reverse transcriptase, 165 Terminal deoxynucleotidyl transferase-dUTP nick end labeling, TUNEL, 338–340 Thalidomide, 210 Thioredoxin reductases, TRx, 59, 364 Threonine kinases, 35, 296, 357 Thromboxane, 261, 262 Thromboxane A2 , THXA2 , 256, 261 Thromboxane B2 , THXB2 , 261 Thromboxane receptor, TXR, 256, 261, 262 Thromboxane synthase, THXS, 256, 257, 261–263

383 Thymidine, 79 Thyroglobulin, TG, 356, 358 Thyroid, 307, 347–366 Thyroid cancer, TC, 284, 347–366 Thyroid peroxidase, TPO, 349, 356, 358 Thyroid stimulating hormone, TSH, 349, 350 Thyroid transcription factor 1, TTF-1, 358 Thyroxine, T4, 350, 364 Tiron, 337, 338, 341, 342 TNF receptor associated factors 1 and 2, TRAF1/2, TRAF2, 4, 27, 30, 36, 39 TNF-related apoptosis-inducing ligand, TRAIL, 3, 4, 6, 7, 15, 32, 39, 40, 54, 55, 85, 90, 94, 129, 131, 132, 137, 138, 142, 145, 164, 174, 176–178, 201, 225–227, 253, 254, 256, 265, 298–303, 362, 363 TNF α receptors, 13 Toll-like receptors, 41, 54 Topoisomerase I and II, 60, 202, 206, 207 Topotecan, 57, 66, 327, 339, 340 TRAIL associated death domain, TRADD, 4, 129, 254, 298 TRAIL receptor, 3, 4, 7, 39, 40, 94, 129, 131, 132, 300, 362 Transcriptional coactivator CBP/p300, 55, 56 Transforming growth factor α, TGF- α, 223, 310, 311 Transforming growth factor-beta, TGF-β, 90, 163, 165, 193, 195, 196, 199, 209, 223, 225, 226, 308–310 Transforming growth factor-β receptors, TGFBR, 90, 196 Transitional cell carcinoma, TCC, 25–42 Trapoxin, 178 Trefoil factor, 163 Trichostatin A, 34, 178 Triiodothyronine, T3, 34, 178 Truncated Bid, tBid, 129, 130, 221, 222, 224, 228, 299 Tuberin, 38 Tumor burden, 3, 133 Tumor necrosis factor receptor, TNFR, 3, 4, 6, 30, 39, 56, 129, 131, 254, 298, 362 Tumor necrosis factor, TNF, 30, 32, 33, 40, 174, 177, 178, 199, 225, 253, 254, 298 Tumor necrosis factor α, TNF α, 4, 13, 15, 85, 94, 165, 168, 176, 222, 226, 257, 298, 299, 305, 306 Tumor promoters, 257 Tumor protein p73, P73, 60, 164, 173

384 Tumor suppressor gene, 5, 116, 131, 135, 157, 163, 164, 166, 167, 169, 241, 254, 275, 279–281, 310, 311, 313 Tumor suppressor in lung cancer, TSLC1, 275, 279, 280 Twist, 176, 284, 285 U Ubiquitin, 9, 38, 54, 55, 65, 276, 282 Ulcerative colitis, 76 V v-Akt, 284 Vascular endothelial growth factor, VEGF, 82, 163, 165, 193, 195–297, 199, 201, 209, 210, 256, 276, 285, 286, 349, 355 VEGF receptor, 197, 209, 276, 285, 286, 356 Vinca alkaloids, 112 Vitamin C, 33 Vitamin K3, 33

Index Vitamins, 33 v-raf murine sarcoma viral oncogene homolog B1, B-RAF, 114 W Wikyungtang, 262 X X-linked inhibitor of apoptosis protein, XIAP, 8, 9, 15, 40, 114, 115, 118, 140, 175, 176, 200, 205, 300, 303 Y Yeast two-hybrid, 221, 222 Z ZD1839, 137, 138, 265 ZD6474, 276, 286 Zebularine, 34