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Role of transporters in pancreatic cancer drug resistance Lo, Maisie K.Y. 2007

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ROLE OF TRANSPORTERS IN PANCREATIC CANCER DRUG RESISTANCE by MAISIE K.Y. LO B.Sc., The University of Waterloo, 1999 M.Sc., The University of Waterloo, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA December 2007 © Maisie K.Y. Lo, 2007 ABSTRACT Pancreatic cancer (PC) is known to be highly resistant to chemotherapy. Transporters, which regulate the influx and efflux of substrates across the plasma membrane, may play a role in PC drug resistance. ABC transporters are a large family of transmembrane proteins with diverse physiological functions, several of which play major roles in cancer drug resistance. Given that 90% of PC express a mutant K-ras oncogene and that PC are highly hypoxic, I postulated that constitutive K-ras activation and/or hypoxia may correlate with ABC transporter expression, which in turn may promote drug resistance in PC. Using normal and PC cell lines either overexpressing mutant K-ras or subjected to hypoxic treatment, mRNA expression was profiled for 48 ABC transporters. My findings indicate that expression of mutant K-ras and hypoxic treatment, as well as long-term exposure to chemotherapy, may contribute to the development of drug resistance in PC cells in part by inducing the expression of ABC transporters. Similar to ABC transporters, I investigated whether amino acid transporters would mediate drug resistance in PC. The x: amino acid transporter (x c") mediates cellular uptake of cystine for the biosynthesis of glutathione, a major detoxifying agent. Because the ?c c" has been regulates the growth of various cancer cell types, and x," is expressed in the pancreas, I postulated that the xc- may be involved in growth and drug resistance in PC. The xe" transporter is differentially expressed in normal pancreatic tissues and is overexpressed in PC in vivo. Using PC cell lines, I found that cystine uptake via the N.: was required for growth and survival in response to oxidative stress, and that expression of the x e- correlated with gemcitabine resistance. Accordingly, inhibition of x e" expression via siRNA reduced PC cell proliferation and restored sensitivity to gemcitabine. I also identified the anti-inflammatory drug sulfasalazine as a mixed ii inhibitor of the x, -, which acts to inhibit cell proliferation via reducing x," activity and not by reducing NFKB activity. My findings thus indicate that the ,cc- plays a role in PC growth in part by contributing to glutathione synthesis to promote PC cell proliferation, survival, and drug resistance. iii TABLE OF CONTENTS ABSTRACT ^ ii TABLE OF CONTENTS ^ iv LIST OF TABLES viii LIST OF FIGURES ^ ix LIST OF ABBREVIATIONS ^ xi ACKNOWLEDGEMENTS xiv DEDICATION ^ xvii CHAPTER 1 INTRODUCTION^ 1 1.1 PANCREATIC CANCER 1 1.1.1 Epidemiology ^  1 1.1.2 Classification, location and symptoms ^ 2 1.1.3 Treatment 4 1.1.3.1 Chemotherapy ^ 4 1.1.3.2 Gemcitabine 5 1.1.3.3 Combination chemotherapy ^ 8 1.1.3.4 Combination chemotherapy in pancreatic cancer ^ 9 1.1.4 Characteristics of pancreatic cancer 10 1.1.4.1 K-ras mutation ^  11 1.1.4.1.1 Ras overview ^ 11 1.1.4.1.2 K-ras in pancreatic cancer ^ 12 1.1.4.1.3 Other genetic abnormalities in pancreatic cancer ^ 14 1.1.4.2 Hypoxia ^ 16 1.1.4.2.1 Overview ^ 16 1.1.4.2.2 Hypoxia in pancreatic cancer ^ 18 1.2 DRUG RESISTANCE ^ 19 1.2.1 Overview 19 1.2.2 Gemcitabine resistance mechanisms in pancreatic cancer ^ 20 1.3 ABC TRANSPORTERS ^ 24 1.3.1 Overview 24 1.3.2 Drug resistance transporters in pancreatic cancer^ 26 1.3.3 ABC transporter subfamilies: relationship to drug resistance and their genetic mutations leading to diseases ^ 27 1.3.4 ABC transporters and hypoxia ^ 31 1.4 THE xc- TRANSPORTER^ 32 iv 1.4.1 Overview of the x c- transporter ^ 32 1.4.1.1 Function of the xc- transporter ^ 32 1.4.1.1.1 Importance of GSH in cancer cell growth ^ 35 1.4.1.2 Structure of the xc- transporter ^ 35 1.4.1.3 Regulators of xc- transporter expression ^ 39 1.4.1.4 Transcriptional control of xc" transporter expression ^ 41 1.4.1.5 Expression of the x c" transporter in the pancreas ^ 42 1.4.2 Role of the x c- transporter in cancer ^ 43 1.4.2.1 Requirement for cystine/cysteine in cancer cell growth ^ 43 1.4.2.2 Expression of the x c- transporter in cancer cells ^ 46 1.4.2.3 Role of the x c" transporter in tumor progression and multidrug resistance ^ 47 1.4.2.4 Targeting the x e- transporter 48 1.5 AIMS OF THE STUDY^ 51 CHAPTER 2 MATERIALS AND METHODS^ 54 2.1 TISSUE CULTURE ^ 54 2.1.1 Maintenance of HPDE cell cultures ^ 54 2.1.2 Maintenance of pancreatic cancer cell cultures ^ 54 2.1.3 Growth of cell lines in hypoxia 55 2.1.4 Assessment of growth requirements for exogenous cystine ^ 55 2.2 TRANSIENT TRANSFECTIONS ^ 56 2.2.1 xCT and 4F2hc plasmid constructs ^ 56 2.2.2 xCT and 4F2hc small interfering ribonucleic acid (siRNA) ^ 57 2.2.3 NFKB reporter constructs ^ 57 2.3 DRUG PREPARATION^ 58 2.3.1 Vincristine and Gemcitabine (GEM) ^ 58 2.3.2 Sulfasalazine (SASP) ^ 58 2.4 RIBONUCLEIC ACID (RNA) ANALYSIS 58 2.4.1 RNA isolation 58 2.4.2 Quantitative Reverse Transcriptase Polymerase Chain Reaction (q-RT-PCR) ^ 59 2.5 PROTEIN ANALYSIS ^ 60 2.5.1 Protein isolation and western blotting ^ 60 2.5.2 Immunofluorescence microscopy 61 2.5.3 Immunohistochemistry ^ 62 2.6 CELL SURVIVAL ASSAY 63 2.7 GLUTATHIONE (GSH) ASSAY 63 2.8 RADIOACTIVE UPTAKE ASSAY ^ 64 2.9 NFKB REPORTER ASSAY 65 2.10 TUMORIGENICITY ASSAY 66 2.11 STATISTICAL ANALYSIS^ 66 CHAPTER 3 ABC TRANSPORTER PROFILING IN PANCREATIC CANCER .. 67 3.1 ABSTRACT^ 67 3.2 RESULTS 69 3.2.1 Effect of constitutive K-ras activation on ABC transporter expression in HPDE cells ^ 69 3.2.2 Effect of hypoxia on ABC transporter expression in HPDE cells ^ 72 3.2.3 Effect of combined constitutive K-ras activation and hypoxia on ABC transporter expression in HPDE cells ^ 72 3.2.4 Effect of combined K-ras activation and hypoxia on ABC transporter expression in pancreatic cancer cells ^ 75 3.2.5 MDR1 is not expressed but is inducible in pancreatic cancer cells ^ 77 3.3 DISCUSSION ^ 80 CHAPTER 4 CHARACTERIZING THE xc - TRANSPORTER IN PANCREATIC CANCER ^ 84 4.1 ABSTRACT^ 84 4.2 RESULTS 85 4.2.1 Pancreatic cancer cells depend on extracellular cystine for growth ^ 85 4.2.2 A negative correlation exists between extracellular cystine deprivation and expression of the^transporter ^ 87 4.2.3 Oxidative stress increases x c- transporter expression and GSH levels ^ 89 4.2.4 Expression of x c- transporter in primary human pancreatic cancer specimens 91 4.2.5 A positive correlation exists between the expression level of xCT and resistance towards GEM ^ 91 4.2.6 Overexpression of exogenous xCT increases cystine uptake and increases GEM resistance 93 4.3 DISCUSSION ^ 97 CHAPTER 5 INHIBITING THE x c - TRANSPORTER IN PANCREATIC CANCER^ 102 5.1 ABSTRACT 102 5.2 RESULTS ^ 103 5.2.1 Targeting x c- transporter expression with siRNA ^ 103 5.2.2 Targeting x c" transporter function with SASP 103 5.2.3 Effect of SASP on sensitivity to GEM in vitro and in vivo ^ 107 5.2.4 Kinetic studies of SASP as an inhibitor of the x c- transporter in pancreatic cancer cells ^  113 vi 5.2.5 Investigating the mechanism of SASP action: NFKB activation ^ 115 5.3 DISCUSSION 118 CHAPTER 6 SUMMARY AND FUTURE DIRECTIONS ^ 124 REFERENCES ^ 129 APPENDIX 155 A.1 UBC Animal Care Certificate ^ 156 A.2 UBC BCCA Research Ethics Board Certificate of Approval^ 157 vii LIST OF TABLES CHAPTER 1 Table 1.1^Types of clinical drug resistance behavior classified by tumor type and treatment outcome ^ 21 viii LIST OF FIGURES CHAPTER 1 Figure 1.1^Anatomy of the pancreas ^ 3 Figure 1.2^Chemical structures of deoxycytidine and GEM ^ 6 Figure 1.3^The Ras signaling cascade 13 Figure 1.4^Model for pancreatic cancer development and progression ^ 15 Figure 1.5^Illustration of a radial cross-section view in a tumor^ 17 Figure 1.6^Model of the ABC transporter P-gp (MDR1) ^ 25 Figure 1.7^Chemical structures of cysteine and cystine 33 Figure 1.8 Role of GSH in combating ROS^ 36 Figure 1.9^Illustration of the xc- transporter 38 Figure 1.10 The transsulfuration pathway 44 Figure 1.11 Proposed mechanism for inducing cystine/cysteine starvation in cancer cells ^ 50 Figure 1.12 Chemical structure of SASP 52 CHAPTER 3 CHAPTER 4 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Expression of K-ras protein in HPDE cell lines ^ 70 ABC transporter mRNA expression profile in HPDE cell lines ^ 71 Expression of HIF-1 a protein in HPDE cell lines in response to hypoxia ^ 73 ABC transporter mRNA expression profile in HPDE cell lines cultured in normoxia or hypoxia ^ 74 ABC transporter mRNA expression profile in pancreatic cancer cell lines cultured in normoxia or hypoxia ^ 76 Effect of vincristine on MDR-1/P-gp expression in pancreatic cancer cell lines ^ 78 Pancreatic cancer cells depend on extracellular cystine for growth ^ 86 A negative correlation exists between extracellular cystine deprivation and expression of the x c- transporter ^ 88 Oxidative stress increases x c- transporter expression and GSH levels ^ 90 Expression of the x c- transporter in primary human pancreatic cancer specimens ^ 92 A positive correlation exists between the expression level of xCT and resistance towards GEM ^ 94 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 ix Figure 4.6 Overexpression of exogenous xCT increases cystine uptake and increases GEM resistance ^ 95 Targeting x c- transporter expression with siRNA ^ 104 Targeting xc- transporter function with SASP 106 Effect of SASP on x c- transporter expression and function ^ 108 Effect of SASP on intracellular GSH levels ^ 109 Effect of SASP on sensitivity to GEM in vitro  111 Effect of SASP on sensitivity to GEM in vivo 112 Kinetic studies of SASP as an inhibitor of the xc" transporter in pancreatic cancer cells ^ 114 Investigating the mechanism of SASP action: NFKB activation ^ 117 Model for the role of the x c" transporter in pancreatic cancer cell growth and drug resistance ^ 123 CHAPTER 5 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 5.5 Figure 5.6 Figure 5.7 Figure 5.8 Figure 5.9 x LIST OF ABBREVIATIONS [140]^radiocarbon [3 H] tritium 2-ME^2-mercaptoethanol 4F2hc 4F2 heavy chain 5'FU^5' fluorouracil ABC ATP-binding cassette Akt protein kinase B (Ak-mouse strain, t-transforming) ALD^adrenoleukodystrophy APE apurinic/apyrimidinic endonuclease ARE^antioxidant response element ASC alanine-serine-cysteine ATP^adenosine triphosphate 132m [3-2-microglobulin BCA^bicinchonic acid BCRP breast cancer resistance protein 1 BNIP3^bcl-2/adenovirus E19-interacting protein 3 C carboxy- Ca2+^calcium cDNA complementary deoxyribonucleic acid CEACAM6^carcinoemybronic antigen-related cell adhesion molecule 6 CMV^cytomegalovirus CoCl2 cobalt chloride C t^threshold cycle D aspartic acid DAPI^4',6-diamidino-2-phenylindole dCK deoxycytidine kinase DEM^diethylmaleate dFdC difluorodeoxycytidine dFdCTP^difluorodeoxycytidine-triphosphate DMEM Dulbecco's modified Eagle's medium DNA^deoxyribonucleic acid DNTB 5,5'-dithiobis-2-nitrobenzoic acid E glutamic acid EAATs^excitatory amino acid transporters EGFR epidermal growth factor receptor EpRE^electrophile response element FBS fetal bovine serum G glycine G12V^glycine to valine at position 12 GAPDH glyceraldehyde-3-phosphate dehydrogenase GEM^gemcitabine GSH glutathione xi H Harvey- HEK^human embryonic kidney HIF-la hypoxia inducible factor-1a HPDE^human pancreatic ductal epithelial HSHAT heavy subunit heterodimeric amino acid transporter hxCT^human sodium-independent cystine in exchange for glutamate transporter subunit IC50^half maximal inhibitory concentration K Kirsten- kD kilodalton Keap1^ketch-like ECH-associated protein 1 KSF keratinocyte serum-free LSHAT^light subunit heterodimeric amino acid transporter MDR multidrug resistance MDR1^multidrug resistance 1 MEM minimum essential medium mRNA^messenger ribonucleic acid MRP multidrug resistance protein MSG^monosodium glutamate N amino- N-ras^Neuroblastoma-ras Na +^sodium NBFs nucleotide binding folds NCI-60^national cancer institute for drug screening-60 human cancer cell lines NFKB^nuclear factor KB Nrf2 nuclear factor erythroid 2-related factor 2 PanIN^pancreatic intraepithelial neoplasia pBp plasmid babepuro PBS^phosphate-buffered saline PCR polymerase chain reaction P-gp^permeability glycoprotein P13K phosphatidylinositol 3-kinase PKC^protein kinase C q-RT-PCR^quantitative reverse transcriptase polymerase chain reaction RNA^ribonucleic acid RNAi ribonucleic acid interference RNase^ribonuclease ROS reactive oxygen species RT-PCR^reverse transcriptase polymerase chain reaction siRNA small interfering ribonucleic acid TGFp^transforming growth factor p TM transmembrane domain TNFot,^tumor necrosis factor a V valine VEGF^vascular endothelial growth factor xii sodium-independent transport system for the anionic form of cystine in exchange for glutamate sodium-independent cystine in exchange for glutamate transporter subunit ACKNOWLEDGEMENTS I would like to thank my research supervisor Dr. Victor Ling for giving me the intellectual freedom to learn and discover as a student scientist. The opportunity to learn from you has been priceless, and I will always remember your words of wisdom. I would like to acknowledge my supervisory committee, Drs. Ralph Durand, David Hedley, Sylvia Ng, George Mackie for all their time and insight into my project throughout these years. To Dr. Peter Gout, my collaborator, thank-you for your mentorship in the SASP project and for generously sharing your life experiences with me. I have learned so much and it has definitely been a pleasure working with you. I would like to acknowledge many members of the BCCRC for all their technical support. From the Wang lab: Hui Xue, Jun Guan, Daniel Doxsee for assisting me in animal work; Rebecca Wu for immunohistochemistry; Dr. Y.Z. Wang for his support of the SASP project. From the Vielkind lab: Margaret Sutcliffe and Samina Noorali for various microscopy discussions. From the Karsan lab: Shauna Dauphinee for teaching me NFKB assays; Fred Wong and Linda Chang for lending me solutions. From the Durand lab: Kevin Bennewith and Michelle Wong for assisting me with hypoxia experiments. From the Ng lab: Dr. Sylvia Ng for teaching me how to section OCT blocks. From the Minchinton lab: Alastair Kyle for his generous time in setting up the hypoxic chamber. From the Helgason lab: Amy Tien (a.k.a. FLO princess) for all the FACS experiments. From the Olive Lab: Susan MacPhail for trusting me with radioactive materials. From the Lam Lab: everyone that let me use the nano-drop, everytime! Sincere thanks to all past and present members of the Ling Lab for all the knowledge I have gained from interacting with each and every one of you everyday, as well as all the good xiv times we shared outside the lab. To Barbara Schmidt, thank-you for teaching me tissue culture techniques and how to order reagents. To Christoph BOs and Dennis Leveson-Gower, thank-you for sharing your knowledge of protein assays. To Lin Liu, thank-you for teaching me q-RT-PCR and cloning. To Renxue Wang, thank-you for discussing kinetic assays with me. To Jonathan Sheps, thank-you for all the proof-reading you have done for me throughout these years, from scholarship grants to the final version of this thesis. To Chris Low, thank-you for your skills in animal work. To Tania Kastelic (a.k.a. lab buddy), thank-you for being a great scientific resource whenever I needed advice. To Maria Ho, Scott Zuyderduyn, and Melvin Kwok, my fellow Ling Lab graduate student colleagues, thank-you for being friends. Despite all our unique behaviors and characteristics, I will never forget those mid-week late afternoon hang-outs at City Square discussing just about anything in life. To the BCCRC administrative staff, Rebecca Smith, Marion Kealy, Marlise Reuter and Sue Smith, thank-you for doing all the paper work. To the BC Cancer Agency librarians especially Dianna Hall, for their assistance in various research information issues. To the BCCRC IT staff, for solving all my computer-related problems. From UBC, Mr. Patrick Carew, secretary for the Department of Experimental Medicine, thank-you for always being so professional and for all the little things you have done behind the scenes to make graduate school run smoothly for the students. I am blessed with friends that I have met during graduate school. To Jasmine Kam, Amy Tien, Michelle Wong, Steven Lowe, friends from the Vancouver Cathay Lions Club and friends from GrasPods, thank-you for all the support and fun times. To my friends from afar, Christine Yuen, Linda Nowak, Maria Ibarra, Ildiko Denes, Anita Padham, and Doreen Siu, thank-you for your constant encouragement throughout these years even though we are miles apart. To my new xv friends in San Francisco, Nandhini Ramamoorthi and Suresh Selvaraj, thank-you for being such great role models for me. I am most grateful for my parents, Brian and Katherine Lo, for their unconditional love, financial support and unquestioning faith throughout this whole graduate school process, once and again! To my big brother, Fred Lo, thank-you for persuading me to come back to Vancouver and to pursue my studies here. To my awesome sister-in-law Elaine Lo, thank-you for organizing all the family reunions (especially mom and dad' s 35 th anniversary). To my nephews Atticus and Jonas, thank-you for all your cute smiles and hugs. Last but not least, I would especially like to thank Kevin Leong. Thank-you for your pleasant companionship throughout this whole journey. Your motivation and inspiration as a scientist has been crucial to my success. Thank-you for being the light when I was lost in the tunnel. I owe you the world! Financial support for my Ph.D. studies was provided by a University Graduate Entrance Scholarship from the University of British Columbia, a Postgraduate Scholarship from the National Science and Engineering Research Council, a Doctoral Research Award from the Canadian Institutes of Health Research, a Biomedical Senior Graduate Studentship from the Michael Smith Foundation for Health Research, a B.C. Cancer Foundation Scholarship, and a King Wong Memorial Scholarship. xvi In Memory of Po Po, for her charisma xvii Chapter 1 INTRODUCTION 1.1. PANCREATIC CANCER 1.1.1. Epidemiology Pancreatic cancer is the 13 th most common cancer in the world, and the 2 nd most common gastrointestinal malignancy. It is the 4 th leading cause of cancer-related deaths in North America (Freelove and Walling, 2006), exceeded by lung, colorectal and breast (Jemal et al., 2007) and carries the worst prognosis of any gastrointestinal cancer (Moore et al., 1996). The fatality rate is extremely high, with a 5-year survival rate of less than 5% and a median survival of 3-6 months (Abbruzzese, 2002; Yeo et al., 2002). Hence the number of new deaths from this disease is virtually identical to the number of new cases. In 2007, it is estimated that 37,170 Americans will be diagnosed with pancreatic cancer, and that 33,370 Americans will die of the disease (Jemal et al., 2007). The mortality rate for pancreatic cancer increases rapidly with age (Michaud, 2004). Pancreatic cancer almost never occurs before age 30, after which the rate increases exponentially, reaching a maximum at age 80 (Michaud, 2004). The total number of cases is slightly higher for men than women (Jemal et al., 2007). It affects all ethnic groups, although African-Americans are at a 35% increased risk (Michaud, 2004; Zell et al., 2007). Despite increases in awareness, diagnosis, and access to medical care, the mortality rates for pancreatic cancer are increasing steadily every year (Freelove and Walling, 2006; Michaud, 2004). The exact cause of pancreatic cancer has not yet been identified. Although 5-10% of all cases can be attributed to germline mutations in numerous genes (p16INK4a, BRCA1, BRCA2, 1 LKB1, trypsinogen mutations) (Maitra et al., 2006), etiologic factors such as cigarette smoking, chronic pancreatitis, high-fat diet, alcohol, and diabetes mellitus are likely to contribute to this disease (Michaud, 2004). 1.1.2 Classification, location and symptoms The most common type of pancreatic neoplasm is ductal adenocarcinoma that arises from ductal epithelial cells, which accounts for over 95% of all pancreatic malignancies (Freelove and Walling, 2006; Stelow et al., 2006). The remaining 5% include other subtypes of the exocrine pancreas (e.g. serous cystadenoma, solid pseudopapillary tumor) and tumors of other cell types of the pancreas, such as acinar cell cancers or endocrine gland tumors (Fesinmeyer et al., 2005). These tumors have completely different diagnostic and therapeutic profiles compared to ductal adenocarcinoma, and generally have more favorable prognoses (Fesinmeyer et al., 2005). The term "pancreatic cancer" generally refers to ductal adenocarcinoma (Freelove and Walling, 2006). The pancreas is located within the abdominal cavity, posterior to the stomach and in close association with the duodenum (Varty et al., 2005). The pancreatic duct runs the length of the pancreas and empties into the duodenum in which the common bile duct joins at this point (Varty et al., 2005) (Figure 1.1). The pancreas can be divided into four regions: head, neck, body, and tail (Varty et al., 2005). Over 60% of pancreatic tumors occur in the head of the pancreas (Schima et al., 2007). Importantly, these tumors can metastasize more deeply into the pancreas and surrounding areas such as the lymph nodes, liver and lungs (Sarbia et al., 2007). Pancreatic cancer is considered a silent disease because there are no early detectable symptoms (Freelove and Walling, 2006; Michaud, 2004). As a result, diagnosis of pancreatic cancer is often delayed (Michaud, 2004). In late-stage pancreatic cancer, symptoms can include 2 FIGURE IS REMOVED DUE TO COPYRIGHT ISSUES Figure 1.1: Anatomy of the pancreas. (a) Gross anatomy of the pancreas demonstrating its close relationship with the duodenum and the common bile duct. (b) Histology of the pancreas showing islets of Langherhans (endocrine gland) responsible for glucose homeostasis. The bulk of the pancreas is mainly exocrine gland and consists of acinar cells (*), which are involved in secreting various digestive enzymes into the ducts (arrow). Adapted from: Hezel et al., 2006. 3 pain in the upper abdomen and upper back, jaundice, dark urine, weakness, loss of appetite, nausea, and unexplained weight loss, all of which are common to other digestive illnesses (e.g. gallstones in the common bile duct or peptic ulcers) (Freelove and Walling, 2006). 1.1.3 Treatment Surgery is the only curative treatment currently available for pancreatic cancer patients, but only if the cancer is found at an early, localized, stage (Beger and Rau, 2007; Freelove and Walling, 2006). However, despite potentially curative radical pancreatectomy which involves complete removal of the pancreas, disease recurrence is still common (Akakura et al., 2001; Freelove and Walling, 2006). The Whipple procedure, a major surgical operation which involves the pancreas, duodenum, and other organs, is the most common surgical treatment for cancers involving the head of the pancreas (Beger and Rau, 2007; Freelove and Walling, 2006). Unfortunately, less than 15% of patients are considered operable, with the majority of patients usually entering palliative treatment which may consist of chemotherapy and radiation (Rosenberg, 1997). Even with chemotherapy, radiation therapy, or a combination of both, these treatments have failed to increase the overall survival of patients with late stage pancreatic cancer, as complete remission is extremely rare (Russo et al., 2007). 1.1.3.1 Chemotherapy Although the effectiveness of chemotherapeutics can vary significantly between individual patients, it is evident that the vast majority of pancreatic cancers are resistant to almost all types of chemotherapy drugs. In the past, chemotherapy for pancreatic cancer included use of 5' fluorouracil (5'FU) and cytarabine with little success (Merriman et al., 1996; Rosenberg, 1997; Wolff, 2007). Over the last two decades, many new drugs have been tested for activity against 4 pancreatic cancer and more than 40 phase II clinical trials involving novel agents either in single or combination regimens have been reported (Ahlgren, 1996; Moore et al., 1996; Wolff, 2007). However, none of these trials have shown a response rate greater than 20%, thus failing the standard for further testing (Kokkinakis et al., 1997; Wolff, 2007). It has been a decade since gemcitabine (GEM) has become the standard chemotherapeutic agent for the treatment of locally advanced pancreatic cancer (Wolff, 2007). 1.1.3.2 Gemcitabine GEM [2'2'-difluorodeoxycytidine (dFdC), GemzarTM, Eli-Lilly, Indianapolis, IN], a novel cytidine nucleoside (Figure 1.2), is a cell cycle-dependent (S phase-specific) oncolytic agent of the antimetabolite class (Pearce and Alice Miller, 2005). The general function of antimetabolites is to inhibit macromolecule biosynthesis by competing with endogenous substrates in cellular metabolic processes, leading to competitive starvation and a resultant block in cell replication (Kinsella and Smith, 1998). GEM is first transported via nucleoside transporters into the cell as a prodrug and then phosphorylated to dFdC-monophosphate (dFdCMP) by deoxycytidine kinase (dCK), followed by subsequent phosphorylation to dFdC-diphosphate (dFdCDP) and dFdC-triphosphate (dFdCTP) (Bergman et al., 2003). The dFdCTP is the active form of GEM which competes with deoxycytidine triphosphate (dCTP) for incorporation into deoxyribonucleic acid (DNA) (Gregoire et al., 2002; Plunkett et al., 1995). Following dFdCTP incorporation onto the end of the elongating DNA strand, the incorporation of one additional deoyxnucleotide is required before DNA polymerases are unable to continue DNA synthesis (Plunkett et al., 1995). This action is termed masked-chain termination, which locks the drug in place within the DNA thus 5 a^ b NH2 N \ NCO^0 c...0j OH  NH2 HO deoxycytidine gemcitabine (GEM) Figure 1.2: Chemical structures of deoxycytidine and GEM. GEM is a nucleoside analog in which the hydrogens on the 2' carbon of deoxycytidine are replaced by fluorines. The drug replaces deoxycytidine during synthesis of deoxyribonucleotides required for DNA replication. This incorporation into DNA arrests tumor proliferation and induces apoptosis, as new nucleosides cannot be attached to the nucleoside analog. 6 preventing its removal by proofreading enzymes, resulting in inhibited cell replication (Plunkett et al., 1995). In randomized trials, GEM was the first and only chemotherapeutic agent shown to have significant clinical benefit on survival and disease related symptoms of pancreatic cancer patients. For example, a phase III randomized trial in advanced pancreatic cancer demonstrated that GEM was more effective than 5'FU (Burris et al., 1997). In comparison to cytarabine, GEM exhibits greater intracellular accumulation, higher membrane permeability, higher affinity for dCK, and higher activity against solid tumors (Rosenberg, 1997). In the clinic, the standard systemic dose of GEM is 1000 mg/m 2 administered as a 30 min intravenous infusion once a week. This regimen is currently used as first-line treatment for patients with locally advanced or metastatic diseases such as adenocarcinoma of the pancreas (Pearce and Alice Miller, 2005). However, partial tumor response rates with this regimen are about 15-18%, with a complete response achieved in only 2-4% of all patients (Burris et al., 1997; Kindler, 2005). Indeed, it has been reported that no more than 25% of patients with pancreatic cancer will benefit from GEM (Burris et al., 1997). GEM in combination with other chemotherapy drugs such as paclitaxel or carboplatin have been implicated for treatments of metastatic breast cancer and ovarian cancer, respectively (Pearce and Alice Miller, 2005). Despite an improved outcome in objective response rates, these combination treatments have had little impact on the prolongation of life (El-Rayes and Philip, 2003). Therefore, considerable efforts are underway to further our understanding of the mechanism of action of GEM and to discover ways to improve its efficacy, including its use in combination chemotherapy. 7 1.1.3.3 Combination chemotherapy The goal of combination chemotherapy is to improve therapeutic effects compared to using a single agent treatment (Chou and Rideout, 1991). The overall combined effects can increase therapeutic index, increase drug efficacy, and decrease drug toxicity (Chou and Rideout, 1991). Furthermore, combination chemotherapy may reduce or delay the development of drug resistance since cancers are less likely to be resistant to multiple agents with different mechanisms of action (Chou and Rideout, 1991). Because drugs may have different toxicity profiles, this may allow the use of each agent at its maximum tolerated dosage, hence maximizing the effectiveness of each drug in combination (Chou and Rideout, 1991). However, to minimize side-effects, cytotoxic drugs used in combination regimens should have non- overlapping toxicities. Drug combination outcomes can be classified into four types: synergistic, additive, antagonistic, and potentiative, with the latter demonstrating that one drug exhibits no effect on its own yet enhances the effect of another drug (Chou and Rideout, 1991). There are very complex mathematical models that exist to quantify drug combination effects (Chou and Rideout, 1991); however, these are beyond the scope of this thesis. It is important to evaluate drug interactions as each drug in combination has its own effect and concentration-response curve, and can be influenced by pharmacokinetic (process by which the drug is absorbed, distributed, metabolized and eliminated by the body) and pharmacodynamic (study of the effects of drugs on living organisms) factors. In order to study the effect of drug combinations, factors such as drug concentration or schedule of drug administration to kill cells at different cell cycle phases must be varied for each drug one at a time (Chou and Rideout, 1991). Afterwards, in-depth biochemical and molecular biological 8 analyses can be performed to ascertain the mechanism behind the interaction observed (Chou and Rideout, 1991). 1.1.3.4 Combination chemotherapy in pancreatic cancer According to the Goldie-Coldman hypothesis, the use of combination drug therapy increases tumor kill (Chou and Rideout, 1991). Over the last decade, combination chemotherapy has been a major research theme in the treatment of pancreatic cancer, with GEM as the core combination drug (O'Reilly and Abou-Alfa, 2007). Although no major breakthrough has been made beyond the use of GEM as a single agent, considerable efforts and potentially promising approaches are underway (O'Reilly and Abou-Alfa, 2007). For pancreatic cancer patients deemed healthy enough to withstand a rigorous chemotherapeutic regimen, a GEM-based fluoropyrimidine or platinum combination is considered a standard treatment (O'Reilly and Abou-Alfa, 2007). Prior to the discovery of GEM in 1997, 5'FU was considered the most active single agent in the treatment of advanced pancreatic cancer (Cascinu et al., 1993; Raderer et al., 1997). However, the logical initial combination chemotherapy using 5'FU plus GEM was disappointing, with most phase III trials showing no significant difference in overall survival (Berlin et al., 2002). Many combination chemotherapy studies using GEM have also been disappointing showing no significant increase in survival rates. For example, the topoisomerase-1 inhibitors irinotecan and exatecan mesylate, although found to have single-agent activity in pancreatic cancer (Wagener et al., 1995), failed to increase overall survival when used in combination with GEM (Abou-Alfa et al., 2006; Rocha Lima et al., 2004). The multi-targeted anti-folate drug premetrexed, when used in combination with GEM, demonstrated in vitro synergy, but no survival benefit when compared to GEM single-agent in phase III trials (Kindler, 2002). In 9 addition, phase III trials using the platinum-based drug oxaliplatin plus GEM (Louvet et al., 2005) and using the oral fluoropyrimidine capecitabine (xeloda) plus GEM (Herrmann et al., 2007) both failed to improve overall survival. Multiple drug cytotoxic combinations continue to be studied extensively in the treatment of pancreatic cancer. A regimen involving GEM in combination with docetaxel and capecitabine has gained significant popularity based on high response and survival rates (Fogelman et al., 2004). Another multiple drug combination involving cisplatin, epirubicin, 5'FU, and GEM, however, yielded no clear results with varying toxic side effects (Reni et al., 2005). Combination chemotherapy with GEM and targeted therapeutics have also been put to the test. Bevacizumab, a humanized monoclonal antibody that inhibits vascular endothelial growth factor (VEGF), did not exhibit improved efficacy in combination with GEM (Ducreux et al., 2007). In addition, cetuximab, a monoclonal antibody that targets epidermal growth factor receptor (EGFR), yielded no clear benefit when used in combination with GEM in different clinical trials (Hochster et al., 2006; O'Reilly and Abou-Alfa, 2007; Xiong et al., 2004). Thus far, GEM in combination with erlotinib, an EGFR tyrosine kinase inhibitor, has provided a promising alternate option to single-agent GEM (Moore et al., 2007). Interestingly, manifestation of a drug-induced rash was found to correlate with better patient survival outcomes from this combination (Ducreux et al., 2007). The development of novel targeted therapeutics will undoubtedly result in additional therapeutic regimens with which to treat pancreatic cancer. 1.1.4 Characteristics of pancreatic cancer Several notable characteristics are more prevalent in cancers of the pancreas than in those of other organs. In the following sections, mutation in the Kirsten (K)-ras oncogene, a genetic abnormality observed in over 90% of all pancreatic cancer cases, will be discussed. In 10 addition, the role of hypoxia in pancreatic cancer will be examined, as this microenvironmental factor has been shown to contribute to the pathogenesis of this disease. 1.1.4.1 K-ras mutation Like many cancers, pancreatic cancer is a genetic disease, characterized by a distinct pattern of prevalent genetic mutations and chromosomal abnormalities that disrupt specific cellular controls. Many of these abnormalities have been identified to account for the invasive and metastatic nature of pancreatic cancer. 1 .1.4.1 .1 Ras overview The Ras proteins are ubiquitous in all eukaryotes and are a highly homologous group of monomeric, membrane-localized guanosine triphosphatases of approximately 21 kD in size (Ellis and Clark, 2000). Ras proteins function as molecular switches linking receptor and non- receptor tyrosine kinase activities to downstream cytoplasmic or nuclear events (Friday and Adjei, 2005). The Ras family of proteins consists of Harvey (H)-ras, Neuroblastoma (N)-ras, and K-ras members, of which two alternatively spliced forms of K-ras have been identified, 4A and 4B (Friday and Adjei, 2005). The Ras genes possess the ability to transform cells (Chang et al., 1982). Although the Ras proteins exhibit a high degree of homology, they are not functionally equivalent as deletion of the K-ras gene, but not H- or N-ras genes, results in embryonic lethality (Reuther and Der, 2000). The Ras proteins exist in two distinct functional states, active or inactive, that are regulated by accessory proteins in a well-defined cycle (Lowy and Willumsen, 1993). The active Ras proteins are key players in numerous signaling pathways such as the extracellular signal-regulated kinase pathway, which induces cell proliferation, and the 11 phosphoinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which mediates cell survival and protection from apoptosis (Friday and Adjei, 2005) (Figure 1.3). 1.1.4.1.2 K-ras in pancreatic cancer Mutation in the K-ras gene, but not the H-ras or N-ras genes, is observed in over 90% of pancreatic cancers (Dergham et al., 1997). Although mutation in the K-ras gene exhibits the highest prevalence in pancreatic cancers compared to all other human cancers (Hruban et al., 1993; Rozenblum et al., 1997), the frequency of K-ras mutation is not high in all types of pancreatic cancer, but only in ductal adenocarcinoma (Goggins et al., 1998). The K-ras mutation is a single point mutation which can occur in codons 12, 13, and less frequently in codons 59, 61, and 63, all of which lie in close proximity to each other in the folded protein (Friday and Adjei, 2005; Pellegata et al., 1994). Common point mutations in codon 12 include the conversion of glycine (G) to aspartic acid (D), glutamic acid (E), or valine (V) (Friday and Adjei, 2005). As a result of the point mutation, the normal K-ras proto-oncogene is converted to an oncogene, and the protein becomes constitutively active in its ability to transmit growth factor-initiated signaling cascades, resulting in the loss of a normal regulatory mechanism for growth, differentiation and apoptosis (Friday and Adjei, 2005). To date, many transgenic mouse models of pancreatic cancer have been generated by exploiting the acinar-specific rat elastase promoter (Ornitz et al., 1987a; Ornitz et al., 1987b; Quaife et al., 1987) or the mature ductal epithelium cytokeratin 19 promoter (Brembeck et al., 2001; Deramaudt and Rustgi, 2005). The most outstanding mouse model to date was developed by crossing mice expressing a Cre-activated K-ras G12D allele knocked into the endogenous wild- type K-ras locus (Deramaudt and Rustgi, 2005; Jackson et al., 2001) with mice expressing pancreatic-specific Cre recombinase (Deramaudt and Rustgi, 2005; Hingorani et al., 2003). The 12 Upstream signal (Growth factor ligand) Plasma membrane Downstream signaling cascade )3°Q5°4 Expression of downstream genes Figure 1.3: The Ras signaling cascade. Ras proteins operate as binary switches, binding GDP in their inactive state and GTP in their active state. Inactive GDP-bound Ras is converted to active GTP-bound Ras by a guanine nucleotide exchange factor (GEE). Active GTP-bound Ras is converted back to an inactive state by a GTPase activating protein (GAP), which promotes hydrolysis of GTP back to GDP by Ras via release of a phosphate (P). Amino acid substitutions caused by oncogenic point mutations in Ras block this cycle by inactivating the intrinsic GTPase activity of Ras (broken arrow), thus trapping Ras in its constitutively active signaling state. 13 Cre recombinase was expressed through an islet-specific homeobox-1 promoter or an acinar- specific locus knock-in. Both lines resulted in the development of pancreatic tumors. Importantly, the developed lesions were able to recapitulate the histological characteristics of human Pancreatic Intraepithelial Neoplasia (PanIN) lesions (Hruban et al., 2001) and pancreatic ductal adenocarcinomas (Deramaudt and Rustgi, 2005; Hingorani et al., 2003). Other cell line models aimed at increasing K-ras expression have been made. In particular, a normal human pancreatic ductal epithelial (HPDE) cell line was transduced with the K-ras GI2v mutation, resulting in increased proliferation, survival, and tumorigenesis in mice (Qian et al., 2005). 1.1.4.1.3 Other genetic abnormalities in pancreatic cancer Additional genes that are mutated at high frequency in pancreatic cancers include the p16, p53, and mothers against decapentaplegic-4 (DPC4/SMAD4) tumor suppressor genes, with an increasing number of genes being discovered that exhibit lower mutation frequencies including breast cancer 2 (BRCA2), retinoblastoma 1 (RB1), transforming growth factor-13 (TGF-(3) receptors-1 and -2 (TGFf3R1 and TGF(3R2), human epidermal growth factor receptor/neuroglioblastoma (Her-2/neu), (3-catenin, mitogen-activated protein kinase-4 (MKK4), serine/threonine protein kinase-1 1 (LKB1/STK11), p300, and DNA mismatch repair genes (Kern and Hruban, 2001). In addition, numerous receptor tyrosine kinases are amplified in pancreatic cancers (Hezel et al., 2006; Ko, 2007). It is believed that the sum total of genetic changes contributes to pancreatic cancer development and progression (Figure 1.4). Hence pancreatic cancers arise as a result of both high and low frequency genetic mutations, which manifest in the deregulation of signal pathways that control cell proliferation, death and motility, thus contributing to the aggressive phenotype seen in the clinic (Kern and Hruban, 2001). 14 I— Normal^PanIN-IA^Paoli\ -113 —I I----- Panli\-2   Paull\ 3 ^ ^K ras /6^ DP( '4 Figure 1.4: Model for pancreatic cancer development and progression. The model illustrates changes that occur during the conversion of normal pancreatic ductal cells to noninvasive pancreatic precursor lesions. These changes include conversion from cuboidal to columnar epithelium, loss of cell polarity, nuclear hyperchromasia and atypia, papillary folding, and cell shedding into the lumen. The precursor lesions are referred to as Pancreatic Intraepithelial Neoplasias (PanlNs). Genetic alterations in K-ras and HER-2/neu occur during the early stage (PanIN-1A and PanIN-1B), with mutations in p16 occurring mid-stage (PanIN-2) and inactivation of DPC4 and p53 occurring during the end stage (PanIN-3). Adapted from: Hruban et al., 2001. 15 Microenvironmental factors, however, also play an important role in the progression of pancreatic cancer. Indeed, pancreatic tumors have been reported to be one of the most hypoxic solid tumors observed in the clinic (Cui et al., 2007; Ide et al., 2007). 1.1.4.2 Hypoxia 1.1.4.2.1 Overview Hypoxia arises when tumor cells proliferating around the vascular core outgrow their initial blood supply resulting in inadequate perfusion (Liao and Johnson, 2007) (Figure 1.5). Cells in these areas are more acidic and nutrient-starved than those in well-vascularized areas (Swietach et al., 2007). Tumors react to hypoxia by stimulating the growth of new blood vessels from the existing vasculature via a process known as angiogenesis (Liao and Johnson, 2007). However, tumor blood vessels are different from those in normal tissues because they can be tortuous with sluggish and irregular blood flow which leads to less efficient oxygen delivery, therefore perpetuating the hypoxic tendency of tumors (Durand, 2001). Hypoxia contributes to tumor drug resistance in several ways. Hypoxic cells of solid tumors are located further away from blood vessels, in regions of poor drug delivery (Durand, 2001). In addition, hypoxic cells are non-proliferating or slowly proliferating, which renders them resistant to most chemotherapeutic drugs that target rapidly dividing tumor cells (Airley and Mobasheri, 2007; Brown and Giaccia, 1998). Radiation treatment also fails to kill hypoxic cells since oxygen is required to generate reactive oxygen species (ROS) that cause DNA damage-induced cell death (Airley and Mobasheri, 2007; Brown and Giaccia, 1998). Hence hypoxic solid tumors are differentially resistant to cancer treatment. When cancer cells are exposed to hypoxia, the hypoxia inducible factor-la (HIF-la) transcription factor becomes stabilized and is known to upregulate several genes that promote 16 02 Aerobic^Hypoxic --> Necrotic Figure 1.5: Illustration of a radial cross-section view in a tumor. Theoretically, oxygen levels decrease from an aerobic to a hypoxic state as a function of distance from the blood vessel, with necrosis occuring approximately 150 p.m from the blood vessel. 17 survival in low oxygen conditions (Airley and Mobasheri, 2007). These include glycolysis enzymes, which can allow adenosine triphosphate (ATP) synthesis in an anaerobic environment, and VEGF, which promotes angiogenesis (Airley and Mobasheri, 2007; Calzada and del Peso, 2007). Hence HIF-1 a plays an important role in solid tumor formation and survival by promoting anaerobic metabolism and angiogenesis. 1.1.4.2.2 Hypoxia in pancreatic cancer Intratumoral oxygen measurements using polarographic needle electrodes have conclusively identified areas of hypoxia in a wide variety of solid tumors including the pancreas (Evans and Koch, 2003). Although angiogenesis is required for the growth of solid tumors, measurement of blood flow via angiography demonstrates that most pancreatic cancers are either highly avascular or have aberrant microcirculation due to structural abnormalities of tumor vessels (Yassa et al., 1997). Hence blood supply is poor in pancreatic tumors, and pancreatic cancer cells are continuously exposed to severe hypoxia and nutrient deprivation compared with other well-vascularized tumor cells (Akakura et al., 2001). Although tumor hypoxia acts as a physiological barrier to cell survival, it paradoxically drives malignant progression by selecting for cells that can adapt and proliferate in a hypoxic environment (Feldman et al., 2005). Studies aimed at addressing how pancreatic cancer cells retain the potential to proliferate and survive in severely hypoxic and nutrient-deprived conditions are being performed (Xie et al., 2006). Nevertheless, several studies have also reported a positive correlation between blood vessel density and pancreatic cancer progression (Ikeda et al., 1999; Karademir et al., 2000; Shibaji et al., 2003), thus demonstrating an important role for angiogenesis in the pathogenesis of pancreatic cancer. 18 Interestingly, it has been reported that 15 out of 20 human pancreatic cancer cell lines examined were found to constitutively express HIF- 1 a (Akakura et al., 2001). The pancreatic cell lines with constitutive expression of HIF- 1 a were more resistant to apoptosis induced by hypoxia and glucose deprivation than those without constitutive expression of HIF-la (Akakura et al., 2001). Importantly, transfection of HIF-la into cells that did not exhibit constitutive HIF- la expression rendered them resistant to apoptosis and increased their tumorigenicity (Akakura et al., 2001). Moreover, the pancreatic cell lines with constitutive expression of HIF-1 a expressed higher levels of the anaerobic metabolism-associated genes such as glucose transporter 1 (Glutl) and aldolase (Akakura et al., 2001). These findings suggest that constitutive expression of HIF-la may contribute to the survival and proliferation of pancreatic cancer cells in hypoxic and nutrient-deprived conditions by the activation of anaerobic metabolism (Akakura et al., 2001). In addition to exhibiting activated K-ras gene mutations and elevated hypoxia levels, pancreatic cancers are distinct from other solid tumors in their intrinsic drug resistance. These characteristics all play important roles in the disease pathogenesis of pancreatic cancer. 1.2 DRUG RESISTANCE 1.2.1 Overview Drug resistance is the most important reason for cancer treatment failure (Huang, 2007). Numerous factors can contribute to the development of drug resistance, such as increased drug transporter expression, decreased drug transport inward through the plasma membrane, decreased drug activation, increased drug metabolism and detoxification, altered expression of target proteins, increased DNA repair mechanisms, and dysregulation of the apoptotic pathway 19 (O'Connor, 2007; Pinedo and Giaccone, 1997). Because genetic instability can render sensitive tumor cells drug-resistant during continual drug treatment (Duesberg et al., 2007; Zalatnai and Molnar, 2007), it is important to develop novel therapeutics to restore drug sensitivity. Drug resistance is a major problem in the treatment of pancreatic cancer (Borst, 1999; Clary and Lyerly, 1998; Zalatnai and Molnar, 2007). Over the past two decades, numerous studies have attempted to determine why pancreatic cancers are so difficult to treat compared to other solid tumors (Bhattacharyya and Lemoine, 2006; Borst, 1999). Table 1.1 illustrates the different types of organ-specific cancers and their clinical drug resistance classification. It has been found that a broad spectrum of drug resistance mechanisms are inherent to pancreatic cancer rather than acquired during the course of initially successfully chemotherapy (Bhattacharyya and Lemoine, 2006). However, the mechanisms involved are often complex and poorly understood (Bhattacharyya and Lemoine, 2006; Zalatnai and Molnar, 2007). This thesis focuses on resistance to GEM, the chemotherapeutic drug used for first-line therapy in the treatment of pancreatic cancer (O'Reilly and Abou-Alfa, 2007). 1.2.2 Gemcitabine resistance mechanisms in pancreatic cancer Numerous genes can affect GEM resistance, including those related to nucleoside transport and metabolism, as well as those involved in the intracellular manipulation of GEM in cancer cells. Among these genes are human equilibrative nucleoside transporters 1 and 2 (hENT I , hENT2) and concentrative nucleoside transporters 1 and 3 (hCNT1, hCNT3) that mediate GEM uptake into cells (Podgorska et al., 2005; Rauchwerger et al., 2000), M1 or M2 subunits of ribonucleoside reductase (Duxbury et al., 2004d), and dCK that phosphorylates and activates GEM from a prodrug form (Galmarini et al., 2004). 20 Clinical behavior^Tumor Type^Type of drug^Treatment resistance outcome Responds completely Hodgkin's lymphoma acute leukemia^None^Curable with teratoma (usually) chemotherapy ovarian Initial response, but followed by relapse breast 01^Induced Non-Hodgkin's^resistance lymphoma Chemotherapy gives symptomatic improvement or short increase in survival, not curable Resistant melanoma kidney pancreas Intrinsic resistance Chemotherapy usually ineffective Table 1.1: Types of clinical drug resistance behavior classified by tumor type and treatment outcome. Adapted from: (Abeloff et al., 2004; Bernal, 1997) 21 Other mechanisms of GEM chemoresistance involve the modulation of apoptosis-related genes. GEM-resistant pancreatic cancer cell lines have been shown to overexpress the anti- apoptotic protein B-cell lymphoma-xL (Bcl-xL) (Schniewind et al., 2004; Sharma et al., 2005; Shi et al., 2002), and pancreatic cancers exhibit upregulated expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) (Dong et al., 2005; Sun et al., 2002). Transfection of the pro- apoptotic gene bax into human pancreatic cancer cells increased GEM sensitivity (Xu et al., 2002). Furthermore, the anti-apoptotic protein p8 is specifically overexpressed in GEM-resistant pancreatic cell lines, and forced expression of p8 in GEM-sensitive pancreatic cells increased their resistance to apoptosis (Giroux et al., 2006). Interleukin-113 and nitric oxide have also been reported to contribute to GEM chemoresistance via inactivation of the caspase machinery (Muerkoster et al., 2006), and X-linked inhibitor of apoptosis (XIAP), when inhibited, increased GEM-related apoptosis (Li et al., 2006). The caspase inhibitory factor survivin was found to be overexpressed in malignant pancreatic ductal tumors with an associated increase in resistance to chemotherapy-induced apoptosis (Satoh et al., 2001). In a microarray study, the hypoxia-induced pro-apoptotic gene Bcl-2/adenovirus E1B-interacting protein 3 (BNIP3) was found to be downregulated in pancreatic cancer samples and GEM-resistant pancreatic cancer cell lines, and knockdown of BNIP3 resulted in an increase in GEM resistance (Akada et al., 2005; Erkan et al., 2005). Other players that regulate GEM resistance in pancreatic cancer involve proliferation pathways that include cellular-sarcoma (c-Src) (Duxbury et al., 2004c; Duxbury et al., 2004e) and focal adhesion kinase (FAK) (Duxbury et al., 2004b), both of which play a role in activating the PI3K/Akt pathway. Hypoxia within pancreatic tumors was reported to induce resistance to GEM mainly by activating the PI3K/Akt and NFKB pathways, as well as partially through 22 activating the mitogen-activated protein kinase (MAPK) pathway (Yokoi and Fidler, 2004). Furthermore, inhibition of the PI3K/Akt pathway with PI3K inhibitors (such as LY292002 and wortmannin) significantly increased GEM-induced apoptosis in vitro (Ng et al., 2000) and in vivo (Ng et al., 2001). Modulation of DNA repair pathways has also been reported to play a role in GEM resistance in pancreatic cancers. Apurinic/apyrimidinic endonuclease (APE), an enzyme involved in DNA base excision repair, was induced by treatment with GEM, while treatment with antisense oligonucleotides targeting APE was able to sensitize pancreatic cancer cells to GEM (Lau et al., 2004). The transcription factor nuclear factor-K-B (NFKB), involved in stress adaptation, cell cycle control and inflammation, was also reported to play a role in GEM resistance. Pancreatic cancer cell lines expressing constitutive NFKB were resistant to GEM (Arlt et al., 2003). Furthermore, 70% of pancreatic cancers examined expressed constitutively active NFKB (Arlt et al., 2003). The carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM 6) plays a role in metastasis and is a mediator of apoptosis (Duxbury et al., 2004a). In drug resistance studies, CEACAM6 overexpression in a pancreatic cancer cell line increased GEM resistance, whereas CEACAM6 silencing with siRNA increased GEM sensitivity (Duxbury et al., 2004a). Despite advancements in our understanding of the mechanisms that contribute to drug resistance in pancreatic cancer, drug resistance remains a major obstacle in pancreatic cancer therapy. Hence the elucidation of therapeutically-targetable mediators of drug resistance in pancreatic cancer is necessary. In this thesis, the roles of two types of transporters in pancreatic cancer drug resistance are examined: the energy-dependent ATP-binding cassette (ABC) 23 transporters and the non-energy-dependent x e- (transport system for the anionic form of cystine in exchange for glutamate) amino acid transporter. 1.3 ABC TRANSPORTERS 1.3.1 Overview The ABC transporter genes represent one of the largest family of transmembrane (TM) membrane proteins (Dean and Allikmets, 2001). These proteins utilize ATP to drive the transport of a wide variety of substrates (such as metabolic products, lipids, sterols, and drugs) across extracellular and intracellular membranes (Dean and Allikmets, 2001). Family members are classified based on sequence similarity and organization of their ATP-binding domains, also known as nucleotide-binding folds (NBFs) (Dean and Allikmets, 2001). The NBFs contain characteristic motifs (denoted Walker A and B motifs), separated by approximately 90-120 amino acids, are found in most ATP-binding proteins (Dean and Allikmets, 2001). ABC genes also contain an additional conserved signature (C) motif element, located upstream of the Walker B site. Unlike the Walker A and B motifs, which are found in other proteins which hydrolyze ATP, the C motif is unique to ABC transporters (Dean and Allikmets, 2001; Lepper et al., 2005). ABC transporters may be classified as half transporters or full transporters (Higgins, 2007; Lepper et al., 2005). Full transporters are functional proteins that contain typically two NBFs located in the cytoplasm and two TM domains which function to localize the transporter protein, bind substrates and transfer energy to transport substrates across membranes (Higgins, 2007; Lepper et al., 2005) (Figure 1.6). Half transporters consists of only one TM and one NBF and must combine with another half transporter, in a homodimeric or heterodimeric fashion, to gain function (Higgins, 2007; Lepper et al., 2005). ABCs are all unidirectional, with the majority 24 L sugar chain• extracellular space plasma membrane \ NBF ^(3) \s '77\ I^/./ ♦ cytoplasm ♦ • substrate • Figure 1.6: Model of the ABC transporter P-gp (MDR1). P-gp is a full transporter containing 12 transmembrane domains that form a pore in the plasma membrane. Two nearly identical domains protrude into the cell, with each domain containing nucleotide binding factors (NBFs) that bind ATP, resulting in the efflux of substrate from the cytoplasm to the extracellular space. Sugar chains are attached to the extracellular portions of the transporter. 25 moving compounds from the cytoplasm to the extracellular space or into an intracellular organellar compartment for transport to other organs or excretion. The ABC transporter family in humans is divided into seven subfamilies (ABCA, ABCB, ABCC, ABCD, ABCE, ABCF and ABCG) (Dean and Allikmets, 2001; Zhang, 2007). To date, 49 members have been identified (Zhang, 2007). 1.3.2 Drug resistance transporters in pancreatic cancer Extensive studies have been conducted on individual ABC transporter members regarding their role in chemoresistance (Gottesman et al., 2002). The major transporters involved in drug resistance are the multidrug resistance 1 (MDR1, also known as ABCB1) protein, the multidrug resistance proteins 1 (MRP1, also known as ABCC1), and the breast cancer resistance protein 1 (BCRP1, also known as the mitoxantrone-resistance 1 (MXR1) protein or ABCG2) (Gottesman et al., 2002). The MDR1 gene encodes the plasma membrane protein permeability-glycoprotein (P-gp), which functions in cancer cells as a drug efflux pump leading to decreased intracellular drug accumulation, thus conferring multidrug resistance (Gottesman et al., 2002). Its role in pancreatic cancer, however, remains debatable. Various studies have reported either the presence or absence of MDR1/P-gp in pancreatic cancer cell lines (Miller et al., 1996; Zhao et al., 2004a). One study examining numerous cancer types reported elevated MDR1 expression in untreated, intrinsically drug-resistant pancreatic tumors (Goldstein et al., 1989). Despite additional reports of MDR-1/P- gp expression in pancreatic cancers, expression failed to predict biological activity in vivo or in the clinic (Lu et al., 2000; Sagol et al., 2005; Suwa et al., 1996). Nonetheless, P-gp-expressing pancreatic cancer cells were found to be resistant to taxotere, a P-gp drug substrate, while 26 addition of the P-gp inhibitor verapamil was able to re-sensitize the cells to drug treatment (Liu et al., 2001). In addition to the MDRI gene, the MRP genes also confer drug resistance in pancreatic cancers (Miller et al., 1996). MRP3 and MRP5 were found to be overexpressed in pancreatic cancer samples, with a positive correlation detected between expression and tumor grade (Konig et al., 2005). An association between BCRP1-mediated drug resistance and pancreatic cancer, however, has not been reported. 1.3.3 ABC transporter subfamilies: relationship to drug resistance and their genetic mutations leading to diseases There are 12 members in the ABCA subfamily (Dean et al., 2001) of which seven (ABCA1, ABCA2, ABCA3, ABCA4, ABCA5, ABCA7, and ABCAl2) have been identified by microarray technology to play a potential role in drug resistance using drug resistant cell lines (Zhang, 2007). Expression of ABCA1 and ABCA4 are associated with inherited human diseases (Dean et al., 2001). Specifically, the development of Tangier disease has been linked to mutations in ABCA1, which prevent the export of intracellular cholesterol and phospholipids out of the cell, leading to cholesterol accumulation in body tissues, reduced biosynthesis of high density lipoprotein, and ultimately coronary artery disease (Attie et al., 2001; Oram and Lawn, 2001). ABCA4 (also known as ABCR) is expressed exclusively in retina photoreceptor cells and transports essential retina-specific substrates across the photoreceptor cell membrane (Allikmets et al., 1997). Mutations in ABCA4 can be found in patients with Stargardt disease (juvenile macular degeneration), age-related macular degeneration, and retinosis pigmentosa; diseases that ultimately lead to blindness (Allikmets et al., 1997). 27 The ABCB subfamily consists of 11 members (Dean et al., 2001), of which all members with the exception of ABCB5 and ABCB7 have been vaguely associated with drug resistance using microarray technology and drug resistant cell lines (Zhang, 2007). ABCB1 (also known as MDR I) is the first identified and most commonly known multidrug resistance transporter of this subfamily (Dean et al., 2001). ABCB2 (also known as transporter associated with antigen processing 1 (TAP1)) and ABCB3 (also known as transporter associated with antigen processing 2 (TAP2)), expressed mainly in the endoplasmic reticulum, are responsible for transporting peptides for antigen presentation from the major histocompatibility complex (Momburg et al., 1994). Mutations in these genes may result in celiac disease, an autoimmune disorder of the small intestines (Momburg et al., 1994). Four members of this subfamily are expressed exclusively in the mitochondria (ABCB6, ABCB7, ABCB8 and ABCB10) and function to transports phospholipids, metal ions, and peptides into mitochondrial membranes (Allikmets et al., 1999; Lill and Kispal, 2001). Mutations in ABCB6 can result in lethal neonatal metabolic syndrome due to a lack of mitochondrial function (Krishnamurthy et al., 2006), and mutations in ABCB7 are associated with sideroblastic anemia, an abnormal production of red blood cells due to dysfunctional heme produced in the mitochondria (Allikmets et al., 1999). Mutations in ABCBI 1 (also known as bile salt export pump (BSEP) or sister of P-gp), a transporter expressed exclusively in liver hepatocyte membranes, are associated with inherited cholestasis liver disease which manifests at early infancy as a result of dysfunctional bile flow from the liver to the duodenum (Alonso et al., 1994; Lam et al., 2005; Strautnieks et al., 1998). The ABCC subfamily is the largest subfamily (Dean et al., 2001) and consists of 13 members. Some of the more commonly known members include ABCC1-6 (also known as MRP 1-6), ABCC 10-11 (also known as MRP7-8), ABCC7 (also known as cystic fibrosis 28 transmembrane conductance regulator (CFTR)), ABCC8 (also known as sulfonylurea receptor 1 (SUR1)), and ABCC9 (also known as sulfonylurea receptor 2 (SUR2)). Ten out of the 13 members, with the exception of ABCC7, ABCC10, and ABCC12, have been reported to be associated with drug resistance (Zhang, 2007). ABCC1 is the most studied multidrug resistant protein of the group and is rated next to MDR1 for its ability to efflux different classes of chemotherapeutics contributing to multidrug resistance in cancer patients (Dean et al., 2001). Both ABCC1 and ABCC2 transport anionic conjugates with glutathione (GSH), glucuronoside, and sulfate, as well as other hydrophobic compounds such as anticancer drugs (Dean et al., 2001). ABCC4 and ABCC5 both transport cyclic nucleotides and some nucleoside monophophate analogues (Dean et al., 2001). Mutations in ABCC6 are associated with Pseudoxanthoma elasticum, an inherited disorder characterized by the mineralization of elastic fibers that affects the skin, eyes and blood vessels (Le Saux et al., 2000). ABCC7 is a chloride channel transporter that regulates other transport pathways (Quinton, 1999). Mutations in this gene are associated with cystic fibrosis, a common inherited condition affecting the lungs and digestive function, as well as congenital bilateral aplasia of the vas deferens, a condition causing infertility (Dean et al., 1990; Pignatti et al., 1995). ABCC8 is found exclusively in the islet cells of the endocrine pancreas. Mutations of this protein may cause inherited hyperinsulinemic hypoglycemia infancy, a condition characterized by unregulated and high insulin secretion, as well as non-insulin- dependent diabetes mellitus, characterized by defective insulin secretion (Pignatti et al., 1995). Mutations in ABCC12 have been associated with choreoathetosis, a disorder characterized by the occurrence of involuntary movements (Tammur et al., 2001). Four members comprise the ABCD subfamily: ABCD1 (also known as adrenoleukodystrophy protein (ALDP)), ABCD2 (also known as adrenoleukodystrophy-related 29 (ALDR), ABCD3 (also known as proxisomal membrane protein, 70 kDa (PMP70)), and ABCD4 (also known as P7OR) (Dean et al., 2001; Kemp and Wanders, 2007; Wanders et al., 2007). Two of the four members (ABCD3 and ABCD4) have been identified to potentially contribute to drug resistance by the use of microarray technology and drug resistant cell lines, however, without functional validation (Yasui et al., 2004; Zhang, 2007). All members of the ABCD subfamily are expressed intracellularly in peroxisomal membranes and their functions range from the transport and catabolism of very long chain fatty acids to peroxisome biogenesis (Albet et al., 1997). Mutations in ABCD1 and ABCD2 are associated with adrenoleukodystrophy, an inherited demyelinating nervous system disorder (Mosser et al., 1993; Pujol et al., 2000). As well, mutations in ABCD2 and ABCD3 are associated with Zellweger syndrome, a genetic congenital lethal disorder characterized by a reduction or absence of peroxisomes, resulting in inhibited (3- oxidization of long fatty acids and decreased body toxin removal (Kemp and Wanders, 2007; Wanders et al., 2007). The ABCE subfamily consists of only one member, ABCE1 (also known as the ribonuclease L (RNase L) inhibitor) which lacks TM domains and hence is not or part of a transporter (Dean et al., 2001). ABCE1 functions to block the activity of RNase L, an enzyme involved in viral interferon signaling (Dean et al., 2001; Zhao et al., 2004b). The ABCF subfamily consists of three members (Dean et al., 2001). ABCF1, which lacks TM domains and not considered a transporter, has been implicated in the regulation of tumor necrosis factor-a (TNF-a) signaling (Richard et al., 1998). ABCF2 and ABCF3 have no known function, but have both been identified in microarray studies to play a potential role in drug resistance using drug resistant cell lines. However their functions are yet to be validated (Gillet et al., 2004; Yasui et al., 2004). 30 The ABCG subfamily, commonly referred to as the white subfamily, consists of six members (Dean et al., 2001). ABCG1 is involved in macrophage cholesterol efflux. As well, it has been postulated to be involved in drug resistance in microarray studies that have yet to be functionally validated (Gillet et al., 2004). ABCG2 (also known as BCRP), the most-studied member of this subfamily because of its drug resistance phenotype, is responsible for the efflux of mitoxantrone and anthracyclines, among other substrates (Allikmets et al., 1998; Miyake et al., 1999). ABCG5 (also known as Sterolin-1) and ABCG8 (also known as Sterolin-2) both function to limit intestinal absorption and promote biliary excretion of sterols (Berge et al., 2000; Lee et al., 2001). Mutations in either of these genes contribute to sitosterolemia, an extremely rare inherited lipid metabolic disorder, characterized by sterol accumulation and atherosclerosis (Salen et al., 1997). 1.3.4 ABC transporters and hypoxia A number of hypoxia-responsive genes are associated with tumor growth. Recently, it was demonstrated that the MDR1 gene product P-gp is induced by hypoxia (Comerford et al., 2002). Epithelial cells exposed to hypoxia exhibited a 7-fold increase in MDR1 gene expression (Comerford et al., 2002). Moreover, enodthelial and epithelial cultured cell monolayers as well as multicellular spheroids (developed from KB cells) subjected to hypoxia exhibited increased resistance to doxorubicin when compared to normoxic conditions (Comerford et al., 2002). The MDR1 gene promoter contains a HIF- 1 a binding site, and inhibiting HIF-la expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-induced MDR1 expression (Comerford et al., 2002). These data collectively indicate that hypoxia-induced P-gp expression may represent a mechanism by which tumor cells develop resistance towards chemotherapeutics. 31 1.4 THE xc" TRANSPORTER 1.4.1 Overview of the xc - transporter Plasma membrane amino acid transporters allow regulated bidirectional transfer of specific amino acids across the plasma membrane. As such they have essential roles in the maintenance and proper functioning of numerous amino acid-dependent cellular processes, including protein synthesis, energy metabolism, and cell preservation (Chillaron et al., 2001; Christensen, 1990). These transporters are essential for cells that cannot sufficiently synthesize certain amino acids and hence require their uptake from the extracellular space for growth and viability. This thesis focuses on the xc cystine/glutamate antiporter, a major plasma membrane transporter for cystine and glutamate. 1.4.1.1 Function of the x e' transporter The^transporter was first described in 1980 by Bannai and Kitamura as a sodium (Na+)-independent transport system for L-cystine and L-glutamate in human fibroblasts (Bannai and Kitamura, 1980). The x c- designation was subsequently assigned by Makowske and Christensen (Makowske and Christensen, 1982). The ?c c- transporter is an obligate, electroneutral anionic antiporter for cystine and glutamate. Cell growth is dependent on extracellular cystine or cysteine, the reduced form of the amino acid (Figure 1.7) (Eagle et al., 1966; Gmunder et al., 1990; Iglehart et al., 1977; Ishii et al., 1981b; Uren and Lazarus, 1979). In the extracellular milieu, cysteine is readily oxidized to cystine which consequently is the predominant form of the amino acid in the circulation and particularly in culture media (Toohey, 1975). Cystine is transported into cells in exchange for intracellular glutamate with a stoichiometry of 1:1 (Bannai, 1986; Chillaron et al., 2001; Christensen, 1990). Once inside a cell, cystine is rapidly reduced to 32 ^COOH^COO.H H 2 N—C—H^H2N—C—H `^ `` —~^^ HS —CH 2 Cysteine Cysteine 21-1 J 21-1 COOH^COOH H 2 N—C—H H 2 N—O—H Cystine Figure t7: Chemical structures of cysteine and cystine. Extracellularly, two cysteine molecules oxidize to form one cystine molecule. Intracellularly, cystine is readily reduced to two cysteine molecules. 33 cysteine (Bannai and Ishii, 1988; Christensen, 1990). As a result, the intracellular levels of cystine are much lower than its extracellular levels. In contrast, the intracellular levels of glutamate are in general much higher than its extracellular levels, a result of glutamine uptake via the alanine-serine-cysteine (ASC) transporter system. This glutamate concentration gradient is thought to provide, at least in part, the driving force of the cystine/glutamate exchange by x c - (Bannai and Ishii, 1988; Christensen, 1990). The x c- transporter can also mediate cellular uptake of levorotary (L)-glutamate, and xc - activity can be quantified using either L-cystine or L- glutamate as a substrate; in both instances, the uptake is chloride (C1 -)-dependent and Nat- independent (Patel et al., 2004). In view of similar affinities of cystine and glutamate for x c- , L- cystine uptake by this exchange system is potently inhibited by L-glutamate (e.g., monosodium glutamate (MSG)) and vice versa (Bannai, 1986). The xc" antiporter has two major functions. One function of the x c - transporter is to mediate the cellular uptake of cystine (with subsequent conversion to cysteine) for general protein biosynthesis. In particular, cysteine is a rate-limiting precursor in the biosynthesis of GSH, a tripeptide thiol consisting of glutamate, cysteine, and glycine. GSH plays a critical role in cellular defenses against oxidative stress as a free radical scavenger and detoxifying agent (Griffith, 1999). A second function of the x c- transporter is to maintain the redox balance between extracellular cystine and cysteine. In the extracellular milieu, cysteine is rapidly oxidized to cystine. Somatic cells such as fibroblasts, activated macrophages, and dendritic cells, can take up cystine via the x e - transporter, reduce it internally to cysteine and secrete cysteine into the extracellular compartment for uptake by other cells not expressing the xc - transporter, thus closing the loop in the redox cycling of the amino acid (Angelini et al., 2002; Bannai and 34 Ishii, 1988; Christensen, 1990; Eck and Droge, 1989; Edinger and Thompson, 2002; Gmunder et al., 1990; Sido et al., 2000). 1.4.1.1.1 Importance of GSH in cancer cell growth GSH, a major intracellular antioxidant, is a tripetide thiol synthesized from cysteine, glutamate, and glycine by the sequential actions of the enzymes gamma-glutamylcysteine synthetase and GSH synthetase (Estrela et al., 2006). GSH acts by specifically protecting the thiol groups of proteins to minimize oxidative stress-induced disulfide bond cross-linkages with DNA, RNA and lipid membrane moieties (Estrela et al., 2006). GSH also plays a major role in cell proliferation, by removing ROS that are constantly produced in the mitochondria as by- products of cellular respiration (Griffith, 1999). Moreover, GSH participates in the removal of toxic substances (e.g. drugs) that enter the cell by forming GSH-drug conjugates, resulting in the shuttling of these conjugates out of the cell via multi-drug resistance proteins (Asakura et al., 1997; Leitner et al., 2007; Morrow et al., 2006). Hence GSH is vital for tumor growth, survival, and drug-related resistance. Indeed, drug resistance in cancer cells has been shown to correlate with higher GSH levels (Townsend and Tew, 2003). Figure 1.8 illustrates the role of GSH in counteracting the major damaging effects of ROS. Of importance to this thesis is the fact that GSH biosynthesis is dependent on the limiting precursor amino acid cysteine, which is transported into the cell as cystine via the xc- transporter. 1.4.1.2 Structure of the x c" transporter The^transporter is a member of a family of heteromeric amino acid transporters (HATs). These transporters are composed of a heavy subunit (HSHAT) and a light subunit (LSHAT) coupled via a disulfide bridge. The HSHAT is involved in trafficking of the heterodimer to the plasma membrane, whereas the LSHAT confers transport and substrate 35 Cu./Fe2 ' 2 Catalase Thioredoxin peroxiredoxin GS-X GSH-S-transferases GSH Generation of ROS Superoxide dismutases ^► H202^H20+ 0 Exogenous sources: Chemotherapy drugs Radiation Inflammation GSH reductase' GS-SG peroxidaseEndogenous sources: Mitochontria electron transport chain NADPH oxidase Xanthine oxidase L-arginine Nitric oxide synthases ^► NO'^ON00 'OH + OH -  Activate proto-oncogenes Inactivate tumor suppressor genes Incomplete DNA repair Angiogenesis stimulation Lipid peroxidation Nitrotyrosine formation Figure 1.8: Role of GSH in combating ROS. GSH can detoxify toxic chemicals (X = molecules that bind GSH forming drug conjugates) as well as react with hydrogen peroxide (H 202) to prevent the formation of the hydroxyl radical (•OH), the major damaging ROS. Another major damaging ROS, peroxynitrite (ON00 -) is formed from L-arginine. Adapted from: (Karihtala and Soini, 2007). 36 specificity. In the case of the human x c - transporter, a HSHAT, designated 4F2 heavy chain (4F2hc) (also known as cluster of differentiation 98 (CD98)), is coupled to xCT, a member of the human LSHAT family conferring specificity for cystine. The 4F2hc subunit is a type II membrane glycoprotein commonly expressed in cells since it acts as a subunit for various amino acid transporters (Chillaron et al., 2001; Verrey et al., 2004). It may be noted that the 4F2hc subunit of x: can be replaced by the basic (0,+) amino acid transporter (rBAT), another HSHAT, with retention of activity (Fernandez et al., 2006; Wang et al., 2003) (Figure 1.9). The molecular nature of the transporter was elucidated in 1999 by Sato and co- workers (Sato et al., 1999). They isolated cDNA encoding the mouse x e" transporter from mouse peritoneal macrophages treated with diethylmaleate (DEM) and lipopolysaccharide, two potent inducers of x c - activity. Expression of mouse xe - activity in Xenopus oocytes was found to require two cDNA transcripts, one coding for 4F2hc and the other for xCT. Subsequently, the cDNA for human xCT (hxCT) was identified (Bassi et al., 2001; Kim et al., 2001; Sato et al., 2000). The full-length hxCT gene was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from an undifferentiated human teratocarcinoma cell line; the nucleotide sequence translated into a 501-amino acid predicted protein product with 89% identity and 93% similarity to mouse xCT (Bassi et al., 2001). To achieve expression of x c - activity in Xenopus oocytes, expression of both hxCT and 4F2hc subunits was required (Bassi et al., 2001; Kim et al., 2001; Sato et al., 1999). Similarly, cystine/glutamate transport function of cloned hxCT in human retinal cells was shown to be dependent on co-expression of 4F2hc (Bridges et al., 2001). Variants of hxCT cDNA have been reported (Kim et al., 2001; Sato et al., 2000), including a functional splice variant, hxCTb, showing differences in the carboxyl (C)-terminal region and degree of expression in a variety of tissues (Kim et al., 2001). The Human Genome Mapping Workshop approved name for hxCT is 37 Figure 1.9: Illustration of the xc - transporter. The transporter is composed of two subunits joined by a disulfide bond. The 4F2hc subunit is a common subunit for various amino acid transporters, while the xCT subunit confers specificity for cystine uptake. Under physiological conditions, one intracellular molecule of glutamate is exchanged for one extracellular molecule of cystine. 38 Solute Carrier Family 7, member 11 (SLC7A11). Thus far, ten members of the LSHAT family have been identified (SLC7A5 -11, Asc-2, AGT-1 and arpAT) (Fernandez et al., 2005; Verrey et al., 2004). Extensive research has been conducted on x c- membrane topology and substrate binding sites (Gasol et al., 2004; Jimenez-Vidal et al., 2004). The studies focused on the xCT subunit, which is considered responsible for the transport of cystine and glutamate. The 4F2hc subunit, which is predicted to have only a single TM domain, is presumably incapable of transport activity by itself (Jimenez-Vidal et al., 2004; Palacin et al., 2000). Glutamate transport by 4F2hc- hxCT heterodimers in Xenopus oocytes was found to be markedly inhibited by thiol-modifying mercurial reagents suggesting a role for cysteine residues in xCT function. Using heterodimers formed by 4F2hc with hxCT cysteine mutants generated via site-directed mutagenesis, it was found that Cys 327 in the 8 th putative TM domain of xCT is a functionally important residue, accessible from the aqueous extracellular compartment and structurally linked to the permeation pathway and/or substrate binding site (Jimenez-Vidal et al., 2004). Based on accessibility of single xCT cysteines to 3-(N-maleimidyl-propionyl)biocytin, a topological model was proposed for xCT consisting of 12 TM domains with both the amino (N)- and C-termini located inside the cell; the intracellular locations of the termini were subsequently confirmed by immunofluorescence. Furthermore, a re-entrant loop with substrate-restricted accessibility was revealed within intracellular loops 2 and 3 (Gasol et al., 2004). 1.4.1.3 Regulators of x c" transporter expression Glutamate is the main exchange substrate in the x, --mediated uptake of cystine, with an affinity for^similar to that of cystine; as such it is a potent, highly specific inhibitor of cystine uptake. Likewise, cystine competitively inhibits glutamate uptake via the^transporter (Bannai 39 and Kitamura, 1980). Other potent inhibitors of the x c- transporter include a-aminoadipate, a- aminopimelate, homocysteate, (S)-4-carboxyphenylglycine, L-serine-O-sulphate, ibotenate, (R,S)-4-bromohomoibotenate and quisqualate (Bannai and Kitamura, 1981; Patel et al., 2004). In addition, x,"-mediated cystine uptake can be inhibited by ultraviolet-B irradiation (Zhu and Bowden, 2004) and by L-lactate (Koyama et al., 2000). Certain anti-inflammatory drugs have also been reported to have xe"-inhibitory activity (Bannai and Kasuga, 1985). In a search for x c - inhibitors potentially useful as novel anticancer agents, it was observed that sulfasalazine, a disease-modifying anti-rheumatic drug, is a potent and quite specific inhibitor of the transporter (Gout et al., 2001). The pharmacology and kinetic properties of the x c" transporter have been extensively studied using L-tritiated ([ 3 H])-glutamate to determine cellular uptake of this amino acid and glutamate efflux as a measure of substrate activity (Patel et al., 2004). A wide variety of cystine, aspartate, and glutamate analogues were examined for glutamate uptake-inhibitory activity. In addition to identifying a number of competitive inhibitors, these uptake blockers could be further classified as either alternative substrate inhibitors (e.g., ibotenate) or non-substrate inhibitors [e.g., (L)-4-carboxyphenylglycine]. Interestingly, the latter compound, a cyclic glutamate analogue with very little substrate activity, was one of the most potent competitive inhibitors, suggesting that distinct structural features of the transporter may control the actual binding and transport of a substrate into the cell. It is noteworthy that substrate inhibitors of the x e- transporter, while inhibiting cystine/glutamate uptake, could potentially increase the likelihood of excitotoxic injury by way of an xc - exchange-mediated glutamate efflux leading to increased extracellular levels of glutamate (Patel et al., 2004). In view of this, non-substrate x e - inhibitors that induce intracellular GSH depletion without glutamate efflux, could be more useful for therapeutic 40 applications aimed at reducing growth and/or drug resistance of malignant cells dependent on function. Upregulation of xCT-mRNA expression leading to increases in xc transporter expression and GSH levels can be obtained in a variety of cell systems by numerous stimuli, including electrophilic agents such as DEM (Bannai, 1984a; Kim et al., 2001; Tomi et al., 2003; Tomi et al., 2002), oxygen (Bannai et al., 1989), bacterial lipopolysaccharide (Sato et al., 1995), nitric oxide (Bridges et al., 2001; Dun et al., 2006; Li et al., 1999; Sato et al., 1999; Watanabe and Bannai, 1987), nuclear factor erythroid 2-related factor 2 (Nrf2) overexpression (Shih et al., 2003) and by the transactivator (Tat) protein (Bridges et al., 2004). It should be noted that the induction of GSH synthesis can depend on the concentration of the stimulating agent. Specifically, DEM at low concentrations (0.1 mM) can increase transporter expression and GSH levels, but at higher concentrations (1 mM) can act as an oxidative stressor and thus deplete GSH levels, as seen in human fibroblast cultures (Bannai, 1984a). Induction of xCT expression has been reported to occur in macrophages and dendritic cells during the immune response, resulting from antigen presentation to lymphocytes (Angelini et al., 2002; Gmunder et al., 1990; Sido et al., 2000). Increased expression of both xc - subunits has been reported for astrocytes following incubation with dibutyryl-cyclic adenosine monophosphate (Gochenauer and Robinson, 2001). Increased xCT expression can also result from deprivation of cystine or other amino acids and involves transcriptional control mediated by amino acid response elements (Sato et al., 2004). 1.4.1.4 Transcriptional control of x c - transporter expression The response of the xCT gene to oxidative stress or electrophilic agents is mediated by a cis-acting transcriptional regulatory element in its promoter region designated the "Antioxidant Response Element" (ARE) or "Electrophile Response Element" (EpRE). Mutational analysis of 41 the ARE/EpRE has shown that it is critically involved in the response to agents such as DEM (Sasaki et al., 2002). Inducible expression of the xCT gene is primarily regulated by the binding of the Nrf-2 transcription factor to the ARE (Lee and Johnson, 2004). Under basal conditions, Nrf2 protein is sequestered in the cytoplasm by Kelch-like ECH-associated protein 1 (Keapl), a negative regulator protein associated with the actin cytoskeleton (Mann et al., 2007). Nrf2 induction results in its dissociation from Keapl, followed by Nrf2 translocation to the nucleus where it binds to ARE sequences (Mann et al., 2007). In astrocytes, overexpression of Nrf2 in vitro resulted in marked increases in xCT messenger ribonucleic acid (mRNA) levels and expression of the xc transporter, as well as coordinated upregulation of proteins involved in GSH biosynthesis (e.g. y-glutamyl cysteine synthetase, GSH synthase), function (e.g. GSH-S- transferase, GSH reductase), and export (e.g. MRPI) (Shih et al., 2003). In another study, astrocytes infected with tsl (a mutant of the Moloney murine leukemia virus) were found to mobilize their thiol redox defenses by upregulating levels of Nrf2 protein as well as its targets, including the xe- transporter (Qiang et al., 2004). ARE-mediated gene expression can be negatively regulated by transcription factors such as c-Maf (Dhakshinamoorthy and Jaiswal, 2002) and Bachl (Dhakshinamoorthy et al., 2005). Activation of Nrf2 can confer protection against many human diseases, and thus a proper understanding of Nrf2 regulation appears crucial in the development of drugs for therapeutic intervention (Zhang, 2006). On the other hand, targeting Nrf2 may assist in decreasing the expression of the x e- transporter and GSH biosynthetic genes in malignant target cells and hence lower their drug resistance. 1.4.1.5 Expression of the x," transporter in the pancreas Several studies have examined expression of the x c - transporter in the normal pancreas. In the normal human pancreatic ductal cell line PaTu 8902, two distinct mechanisms for L-cystine 42 uptake have been reported. Specifically, up to 60% of cystine uptake by this cell line was found to be mediated by the x c - transporter, with the remainder being mediated by y-glutamyl transpeptidase, an enzyme also located on the outer surface of the plasma membrane (Sweiry et al., 1995). In a separate study, xCT mRNA was found to be predominantly expressed in the normal pancreas, specifically in the islet cell population (Bassi et al., 2001). Importantly, treatment of cultured pancreatic AR42J acinar and pTC3 islet cells with the antioxidant response-activating agent DEM led to cystine uptake predominantly mediated by the xc - transporter (Sato et al., 1998). These findings suggest that upregulation of x e - transporter activity may contribute to cellular antioxidant defense mechanisms during the pathogenesis of pancreatic disease, including pancreatic cancer. 1.4.2 Role of the x e" transporter in cancer 1.4.2.1 Requirement for cystine/cysteine in cancer cell growth Cysteine is traditionally viewed as a nutritionally non-essential amino acid since it is synthesized in the body, primarily by the liver, from L-methionine via the transsulfuration pathway (Ishii et al., 2004) (Figure 1.10). Certain cancers, including leukemias and lymphomas, are incapable of synthesizing cysteine (Eagle et al., 1966; Gout et al., 1997; Iglehart et al., 1977). This may be due to a deficiency in y-cystathionase, the last enzyme in the transsulfuration pathway (Rosado et al., 2007; Uren and Lazarus, 1979). To maintain growth and viability, such cells and tissues depend on uptake of cysteine from their microenvironment. A growth requirement for extracellular cystine can be readily demonstrated for cultured cells by transferring them to culture medium specifically deficient in the amino acid and monitoring their growth (Lo et al., 2006) or by omitting cystine uptake enhancers (e.g., 2-mercaptoethanol (2- 43 L-methionine S-Adenosyl-L-methionine 1 S-Adenosyl-L-homocysteine adenosyl-homocysteinase L-homocysteine i cystathlonme-p-synthase cystathionine y-cystathonase L-cysteine glutathione a-ketobutyrate propionyl-CoA Figure 1.10: The transsulfuration pathway. This pathway illustrates the biosynthesis of L-cysteine which is found mainly in the liver. L-methionine is a nutritionally essential amino acid in the diet that can be converted to the nutritionally non-essential amino acid L-cysteine through this pathway. Cystathionine is a pre-cursor of L-cysteine which is cleaved by y-cystathionase to form L-cysteine. Cells that do not express enzymes in the transsulfuration pathway may be sensitive to cystine/cysteine starvation therapy. 44 ME)) from the medium (Gout et al., 1997). Blood plasma contains relatively high concentrations of cystine (100-200 JIM half-cystine), but only 10-20 JIM cysteine; the cysteine concentrations are extremely low in comparison with the plasma concentrations of other protein-forming amino acids (Chawla et al., 1984; Gmunder et al., 1991; Saetre and Rabenstein, 1978). While lymphoid cells can readily take up cysteine via other amino acid transporters, such as the ASC transport system (Christensen, 1990), the in vivo levels of circulating cysteine are apparently too low to sustain their proliferation (Gout et al., 2003). Moreover, lymphoid cells do not generally express a plasma membrane cystine transporter and hence cannot take up cystine. Rather, these cells acquire the necessary amounts of the amino acid from transient increases in the levels of cysteine in their microenvironment as it is secreted by neighboring somatic cells (fibroblasts, activated macrophages or dendritic cells) - a process based on xe- transporter-mediated uptake of cystine from the circulation (Angelini et al., 2002; Edinger and Thompson, 2002; Gmunder et al., 1990; Ishii et al., 1981b; Sido et al., 2000). Tumor-associated macrophages (TAMs) have been reported to promote cancer growth via secretion of a variety of factors, including cytokines and angiogenic factors (Bingle et al., 2002). Similarly, such stromal cells could promote the growth and drug resistance of certain cancers via secretion of cysteine essential for GSH/protein biosynthesis - a process based on x c" transporter function. Alternatively, cancer cells expressing the transporter may take up the amino acid directly. Given that the concentration of intracellular cysteine is only 1-10% that of GSH in most normal tissues and cultured cells, intracellular cysteine levels are inherently more difficult to quantify than intracellular GSH levels (Koch and Evans, 1996). Of interest, cysteine levels in several cancers, including human esophageal (Evans et al., 2002) and cervical (Vukovic et al., 2000) cancers, as well as a variety of rodent tumors grown in vivo, have been reported to be 45 much higher compared to their adjacent non-malignant tissues. Furthermore, recent studies have suggested that cysteine, in addition to GSH, may play a role in the pathogenesis and treatment response of cancers. Specifically, cysteine is considered a low molecular weight thiol-containing compound known to protect cells from the toxic effects of ionizing radiation and ROS (Evans et al., 2002; Koch and Evans, 1996). Thus, it is possible that high x e" transporter expression and activity may be responsible for the high levels of cysteine reported in these studies. 1.4.2.2 Expression of the x c" transporter in cancer cells Expression of the x e- transporter has been demonstrated in numerous cultured cancer cell lines, including rat and human hepatoma cells (Bannai, 1984b; Maechler and Wollheim, 1999; Makowske and Christensen, 1982), rat and human lymphoma cells (Gout et al., 2001), human glioma cells (Chung et al., 2005) and human colon (Bassi et al., 2001), breast (Narang et al., 2003) and prostate (Doxsee et al., 2007) cancer cells. In some cases, the presence of the ?c c- transporter in cultured cell lines reflects its regular expression in their tissues of origin, such as normal pancreatic tissue (Bassi et al., 2001). However, the presence of the transporter in cultured cells, when the tissues of origin do not normally express it, may be a manifestation of cells circumventing cystine starvation. Whereas cells in vivo have access not only to cystine but also to cysteine, culture media such as Minimum Essential Medium (MEM) and Fischer's medium only provide cystine [cysteine would be rapidly converted to cystine via autoxidation (Toohey, 1975)]. Establishment in such media of cell lines from cancer tissues requiring extracellular cystine/cysteine but lacking cystine transporters may therefore reflect cellular adaptation, leading to xc- transporter expression or to outgrowth of an existing subpopulation of ?c c- transporter- expressing cells. Indeed, this has been reported for lymphoma cells (Gout, 1987; Hishinuma et al., 1986) and a variety of normal mammalian cells (Ishii et al., 1992). Consistent with this 46 notion is the recent observation that human peripheral neutrophils did not express the x e- transporter until the cells were cultured in vitro (Sakakura et al., 2007). It may be noted that certain culture media (e.g. RPMI-1640) are supplemented with cystine at levels markedly exceeding those in MEM or human plasma, in an effort to enhance cystine uptake and culture growth. Cellular uptake of cystine can also be enhanced by additives such as 2-ME (Ishii et al., 1981a). Hence the xc - transporter is essential for the maintenance of a variety of experimental cancers that require extracellular cystine/cysteine for growth. 1.4.2.3 Role of the xc - transporter in tumor progression and multidrug resistance Early evidence of a role for the x c - transporter in tumor progression came from a rat prolactin-dependent Nb2 lymphoma cell line and its sublines, used as an in vitro and in vivo model for the malignant progression of lymphomas (Gout et al., 1994). These sublines had clonally developed from the parent line with an increasing number of chromosomal alterations at each progression step (Horsman et al., 1991). The emergence of x c - transporter expression in these sublines augmented their cystine uptake and GSH-generating capabilities. Importantly, xe - transporter expression resulted in enhanced growth autonomy (Gout et al., 1997) and increased resistance against oxidative stress (Meyer et al., 1998), two important features of malignant progression. Another important feature of cancer progression is the development of multidrug resistance. The x e - transporter can contribute to the development of drug resistance by mediating cellular uptake of cystine to enhance GSH biosynthesis, as found for ovarian (Okuno et al., 2003) and lung (Huang et al., 2005) cancer cells. GSH plays a major role in the protection of cells from drug-induced oxidative stress by mediating cellular detoxification of drugs and their extrusion 47 via multidrug resistance proteins (Filipits et al., 2005; Haimeur et al., 2002; Yadav et al., 2007; Yang et al., 2006). GSH has been shown to induce a conformational change within MRP I which is essential for drug interaction and extrusion (Uchino et al., 2002). Conversely, depletion of intracellular GSH levels can cause marked inhibition of cell growth and survival, and reversal of drug resistance (Schnelldorfer et al., 2000; Vanhoefer et al., 1996). In view of this, inhibition of the xe. transporter leading to GSH depletion would provide an alternative avenue for overcoming drug resistance, as previously suggested (Gout et al., 2003). In a pharmacogenomics study, microarrays were used to analyze the expression of membrane transporters in 60 human cancer cell lines, used by the National Cancer Institute for drug screening (NCI-60), and expression of the transporter was linked with the potencies of 1,400 candidate anticancer drugs (Huang and Sadee, 2006). Importantly, 39 drugs exhibited positive expression correlations with the x: transporter, whereas 296 drugs exhibited negative correlations. Such pharmacogenomic studies may be used to predict mechanisms of drug sensitivity and resistance and provide insights for selecting optimal drug regimens for combination chemotherapy (Dai et al., 2007). 1.4.2.4 Targeting the 'cc' transporter Development of more effective strategies for treating drug-resistant cancers is of major importance in cancer management. The finding that lymphoma and leukemia cells critically depend on extracellular cystine/cysteine for growth (Iglehart et al., 1977) suggested that selective depletion of this amino acid may provide a useful therapeutic approach for such malignancies. The xc - transporter appears to be critically involved in GSH-based drug resistance (Huang et al., 2005; Kagami et al., 2007; Narang et al., 2007; Okuno et al., 2003). GSH has an important role in cancer cell proliferation and multidrug resistance. Given that cysteine is a rate-limiting 48 precursor for the synthesis of GSH (Griffith, 1999) and that intracellular GSH has a short half- life (Griffith, 1999), cysteine deficiency may lead to GSH depletion followed by growth arrest and reduced drug resistance. Indeed, cystine/cysteine starvation of cancer cells, by specifically blocking the function of the x c - transporter, has been suggested as a potential therapy for cancers that are critically dependent on uptake of the amino acid for growth and viability (Gout, 1997; Gout et al., 1997) (Figure 1.11). Support for this approach has been demonstrated in vitro. MSG, a highly specific inhibitor of the ?c c - transporter (Bannai and Kitamura, 1980), markedly reduced cystine uptake by lymphoma cells and almost completely arrested their proliferation (Gout et al., 1997). Unfortunately, the neurotoxic nature of MSG precluded its use as a therapeutic agent (Choi, 1988). In search of a therapeutic compound for the inhibition of x c- transporter activity, it was discovered that the Food and Drug Administration (FDA)-approved anti-inflammatory drug sulfasalazine (SASP) potently inhibited transporter-mediated uptake of cystine by cancer cells. Using rat and human lymphoma cells that are dependent on proper x e- transporter function for growth, SASP treatment, at a concentration found in patients' sera, induced cystine starvation with subsequent proliferation arrest and cell lysis (Gout et al., 2001; Gout et al., 2003). Importantly, intraperitoneal administration of SASP resulted in substantial growth arrest of established lymphoma allografts without major toxicity to the rat hosts (Gout et al., 2001; Gout et al., 2003). Using human DU-145 and PC-3 prostate cancer cells, SASP treatment depleted GSH levels in vitro, and intraperitoneal administration of SASP substantially reduced xenograft growth in severe combined immunodeficiency (SCID) mice (Doxsee et al., 2007). SASP has also been shown to inhibit the growth of glioma (Robe et al., 2004) and lung adenocarcinoma (Lay et 49 Cancer cell Proliferation Cystine .♦ Oxidation Cyst° i ne  Activated macrophage/dendritic cell Figure 1.11: Proposed mechanism for inducing cystine/cysteine starvation in cancer cells. Cystine/cysteine starvation therapy may be useful for targeting cancer cells that cannot adequately synthesize the amino acid and whose glutathione levels and growth critically depend on uptake of the amino acid from their micro-environment. By using a specific inhibitor of the x c- transporter (INHIB), the uptake of cystine by activated tumor-associated macrophages/dendritic cells can be inhibited, leading to reduced secretion of cysteine into the microenvironment of the cancer cells and hence lowered cysteine uptake by these cells. The inhibitor will also impair cystine uptake by cancer cells that express the >cc ' transporter. INHIB-induced cysteine deficiency of the cancer cells would readily lead to their intracellular glutathione depletion and subsequent growth arrest and/or reduced drug resistance. 50 al., 2007) xenografts in vivo. Of importance to this thesis, a tumor-sensitizing effect of SASP has been reported in human pancreatic cancer xenografts (Muerkoster et al., 2003). Taken together, the above observations suggest that inhibition of the x e- transporter by SASP could form a viable chemotherapeutic strategy for the treatment of a variety of cancers, especially in combination with conventional drugs (Figure 1.12). Indeed, SASP significantly enhanced the growth-inhibitory activity of doxorubicin against human MDA-MB-231 mammary cancer cells in vitro (Narang et al., 2007) and PC-3 prostate cancer xenografts in vivo (Kagami et al., 2007), and clinical trials using SASP in combination with melphalan showed some success for the treatment of ovarian cancer (Gupta et al., 1995). 1.5 AIMS OF THE STUDY It is well known that some members of the ABC transporter family of membrane proteins play an important role in the regulation of cancer cell survival and drug resistance (Dean et al., 2001). ABC transporter profiling studies aimed at identifying drug resistant phenotypes in pancreatic cancer, however, are lacking. Given that the majority of pancreatic adenocarcinoma are characterized by the presence of activating K-ras mutations (Dergham et al., 1997) and a very hypoxic microenvironment (Akakura et al., 2001), and that pancreatic cancer is one of the most drug resistant cancers (Zalatnai and Molnar, 2007), I sought to determine whether K-ras mutations and/or hypoxia would influence ABC transporter expression and hence drug resistance in pancreatic cancers. Chapter 3 examines the effects of constitutive K-ras activation and hypoxia on ABC transporter expression in HPDE cell lines and human pancreatic cancer cell lines, in an effort to determine whether ABC transporters are responsible for the intrinsic drug resistant phenotype of pancreatic cancers commonly observed in the clinic. 51 NHS02 N=N sulfasalazine (SASP) Figure 1.12: Chemical structure of SASP. SASP is classified as a sulfonamide (RSO 2 NH 2) drug. It is composed of two metabolites: the antibiotic 5-aminosalicylic acid (5'ASA) and the antibacterial sulfapyridine. These metabolites are joined together by an azo bridge (R-N=N-R). SASP and its metabolite 5'ASA are poorly absorbed into the bloodstream. The sulfapyridine metabolite, however, is easily absorbed into the bloodstream. The mechanism of action for treatment in ulcerative colitis and rheumatoid arthritis is not clearly understood. If taken orally, SASP will be reduced in the gut by bacterial cleavage of the azo bridge, resulting in reduced anti-cancer action. 52 In addition to ABC transporters, amino acid transporters may mediate drug resistance in pancreatic cancer. It has been well documented that cystine is the limiting substrate for the production of the major antioxidant GSH, which plays a role in cancer cell proliferation and drug resistance. The x c - transporter, a major plasma membrane transporter for cystine and glutamate, is expressed in normal pancreatic cells (Bassi et al., 2001; Sato et al., 1998). A functional role for the xc" transporter in pancreatic cancer cell proliferation and drug resistance, however, has not been well-studied. In Chapter 4, I characterize the role of the x c- transporter in pancreatic cancer cell growth and drug resistance. In Chapter 5, I investigate the x e- transporter as a potential therapeutic target in the treatment of pancreatic cancer. Overall, three major hypotheses were tested in this thesis: (1) Constitutive K-ras activation and hypoxia influence ABC transporter expression in pancreatic cancer; (2) The xc - transporter promotes growth, survival, and GEM-resistance in pancreatic cancer cells; (3) Inhibition of the xc" transporter attenuates pancreatic cancer cell growth in vitro and in vivo. 53 Chapter 2 MATERIALS AND METHODS 2.1 TISSUE CULTURE All cells were maintained in a humidified chamber at 37 °C in an atmosphere of 95% air and 5% CO2. For passaging cells, 0.25% trypsin solution (Gibco, Gaithersburg, MD) was used. For harvesting cells, a scraper was used to physically detach cells. 2.1.1 Maintenance of HPDE cell cultures The normal human pancreatic ductal epithelial cell line HPDE6-E6E76c7 (HPDE) and its derivatives HPDE-pBp, HPDE-pBp-Kras G12V, and HPDE-pBp-KrasG12vT (gift from Dr. Ming- Sound Tsao, University of Toronto, Princess Margaret Hospital, Toronto, ON, Canada) were routinely maintained in monolayer cultures in keratinocyte serum-free (KSF) medium supplemented by epidermal growth factor and bovine pituitary extract (Life Technologies, Grand Island, NY), as previously reported by the supplier (Ouyang et al., 2000). Briefly, these cell lines were established by retroviral transduction of pBabepuro-K-ras4B G12v and the corresponding empty vector pBabepuro into primary cultures of normal pancreatic ductal epithelial cells, followed by puromycin selection (Qian et al., 2005). 2.1.2 Maintenance of pancreatic cancer cell cultures The human pancreatic cancer cell lines (MIA PaCa-2, PANC-1, and BxPC-3) were a gift from Dr. David Hedley (University of Toronto, Princess Margaret Hospital, Toronto, ON, Canada). For the experiments in Chapter 3, these cell lines were routinely maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 15% fetal bovine 54 serum (FBS). For the experiments in Chapters 4 and 5, these cell lines were routinely maintained in MEM specifically containing 0.1 mM cystine (StemCell Technologies, Vancouver, BC, Canada) and supplemented with 10% FBS (Invitrogen, Carlsbad, CA) and 3.6 g/L glucose (Sigma, Oakville, ON, Canada). 2.1.3 Growth of cell lines in hypoxia Cells were cultured in T75 flasks in either KSF medium supplemented with growth factors (HPDE cell lines) or in DMEM supplemented with 10% FBS (pancreatic cancer cell lines). The T75 flasks were placed in a 37 °C water bath and sealed with a rubber stopper connected to a gas regulator apparatus (courtesy of Ralph E. Durand, British Columbia Cancer Research Centre, Vancouver, BC, Canada). The gas regulator apparatus was connected to tanks filled with pre-mixed gases (normoxic: 20% 02/5%CO2/75%N 2 or hypoxic: 1%02/5% CO2/94% N2; Praxair, Danbury, CT), and gas release was adjusted to a flow rate of 60 cc/min. Culture flasks were gently agitated at regular intervals to facilitate the dissolving of gas into the culture media. Cells were cultured in normoxic or hypoxic conditions for 24 h. 2.1.4 Assessment of growth requirements for exogenous cystine Cells were plated in 96-well plates at 1,000 cells/well (in triplicate) in the appropriate maintenance media. Following incubation overnight to allow cell attachment, media were removed quickly with an aspirator and the cells were washed gently three times with phosphate buffered saline (PBS). Cells were then overlaid with cystine- and methionine-deficient RPMI- 1640 medium (Sigma) supplemented with 10% FBS and 2 mM glutamine, in the presence or absence of cystine (0.1 mM), methionine (0.1 mM), and/or cystathionine (0.15 mM) in various combinations. Following 72 h of incubation, a neutral red uptake assay was performed to 55 determine cell survival. Note: Because normal cystine concentrations in human blood plasma range between 0.1-0.2 mM (Chawla et al., 1984; Gmunder et al., 1991), therefore we defined the normal cystine concentration to be 0.1 mM cystine. Accordingly, cystine concentrations lower than 0.1 mM or higher than 0.2 mM were considered as low or high cystine concentrations, respectively. 2.2 TRANSIENT TRANSFECTIONS 2.2.1 xCT and 4F2hc plasmid constructs Plasmid constructs of mouse xCT and 4F2hc cDNAs cloned into the pcDNA3.1 ± vector (gift of Andy Shih, University of British Columbia, Vancouver, BC, Canada) were transformed into the E.coli strain DH5a, and plasmid DNA harvested using a Qiagen Maxiprep kit according to manufacturer's recommendations. For xc - transporter overexpression studies, pancreatic cancer cells were plated at a density of 0.5x10 3 cells/well in 96-well plates. After 24 h, wells were aspirated and 125 of fresh media were added. Cells were transiently transfected with 0.1 [tg DNA (empty vector pcDNA3.l control, pcDNA3.1-xCT, or pcDNA3.1-4F2hc) diluted in 12.5 pt 150 mM NaC1 and 0.75 111_, ExGen 500 in vitro transfection reagent (Fermentas, Burlington, ON, Canada) for 24 h. Wells were then aspirated and GEM diluted in medium was added to each well for 72 h, followed by a neutral red uptake assay to determine cell survival/proliferation. Each sample was done in triplicate. For transient transfection of cells used for radioactive uptake assays, cells were plated at a density of 2x10 3 cells/well in 24-well plates, and transfections/treatments were performed as for 96-well plates but with all volume measurements increased four times. Each treatment sample was done either in triplicate or quadruplicate. 56 2.2.2 xCT and 4F2hc small interfering ribonucleic acid (siRNA) Pre-designed ON-TARGET plusTM SMART pool siRNAs against human xCT (accession number NM 014331) and human 4F2hc (accession number NM 001012661) were purchased from Dharmacon (Lafayette, CO). For xe- transporter knock-down expression studies, MIA PaCa-2 and PANC-1 cells were plated at a density of 0.5x10 3 cells/well in 96-well plates overnight. For each well, two solutions were prepared: (1) 20 nM siRNA diluted in 25 pit of serum free media, and (2) 0.3 !IL of Lipofectamine 2000TM Reagent (Invitrogen) diluted in 25 uL of serum free media. Following incubation of each solution for 5 min at room temperature, the solutions were mixed together and incubated for an additional 20 min at room temperature, after which the combined solution was added to each well. After 24 h incubation, wells were aspirated and GEM diluted in media was added to the wells for 72 h, followed by a neutral red uptake assay to determine cell survival/proliferation. Each sample was done in triplicate. For transient transfection of cells used for radioactive uptake assays, cells were plated at a density of 2,000 cells/well in 24-well plates, and transfections/treatments were performed as for 96-well plates but with all volume measurements increased four times. Each treatment sample was done either in triplicate or quadruplicate. 2.2.3 NFKB reporter constructs In 24-well plates, MIA PaCa-2 or PANC-1 cells were plated at 4,000 cells/well overnight. Cells were then co-transfected overnight with 300 ng/well pGL3-empty luciferase reporter vector or pGL3-NFIc13 promoter luciferase reporter (Promega, Madison, WI, USA) and 7.5 ng/well pRL-cytomegalovirus (CMV) (internal control vector) (Promega) using 3µL/well of ExGen 500 in vitro transfection reagent (Fermentas) according to manufacturer's recommendations. NFic13 luciferase activity was assessed 24 h post transfection. 57 2.3 DRUG PREPARATION 2.3.1 Vincristine and Gemcitabine (GEM) Vincristine (Sigma) was dissolved in PBS at a stock concentration of 10 mM. GEM (BC Cancer Agency Pharmacy, Vancouver, BC, Canada) was dissolved in 0.9% NaCl at a stock concentration of 33 mM. Drug solutions were stored at -20 °C. For in vivo studies, GEM was prepared in PBS at a stock solution of 12 mg/mL. 2.3.2 Sulfasalazine (SASP) For in vitro studies, a 10 mM SASP solution was made by dissolving 40.1 mg SASP (Sigma) in 4 mL of 0.1 N NaOH and 5.77 mL of PBS. The solution was adjusted to pH 7.5 using 1 N HC1, filter-sterilized, kept away from light and used within 24 h. For in vivo studies, SASP solutions were prepared fresh daily at 20 mg/mL using 400 mg SASP in 15 mL of 0.1 N NaOH and 4.8 mL of PBS (pH 7.2). The solution was adjusted to pH 8.0 using 1 N HC1 and filter- sterilized. 2.4 RIBONUCLEIC ACID (RNA) ANALYSIS 2.4.1 RNA isolation In Chapter 3, isolation of total RNA from cultured cells in vitro or tumor cells in vivo was performed using an RNA isolation kit (Stratagene, La Jolla, CA) according to manufacturer's recommendations. RNA integrity was verified by capillary electrophoresis using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). In Chapters 4 and 5, isolation of total RNA from cultured cells in vitro or tumor cells in vivo was performed using an RNeasy Micro kit (Qiagen) according to manufacturer's recommendations. Quantification of RNA 58 concentration was determined using an ND-1000 spectrophotometer (Nano-Drop, Wilmington, DE). 2.4.2 Quantitative Reverse Transcriptase-Polymerase Chain Reaction (q-RT-PCR) 5 lig of RNA was used to synthesize first strand cDNA using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA), according to manufacturer's recommendations. Q-RT-PCR reactions were performed in 384-well plates using SYBR® Green PCR Master Mix (Applied Biosystems) in a volume of 15 1AL (containing 50 ng of cDNA). Fluorescence emission was detected for each PCR cycle on a ABI Prism 7900 Sequence Detection System (Applied Biosystems) and threshold cycle (C t) values were determined based on a 40 cycle reaction. Thermal cycling conditions were 50 °C for 2 min and 95 °C for 5 min, followed by 40 cycles of 15 s at 95 °C, 30s at 58°C, and 30s at 72°C. C t values were defined as the PCR cycle number at which the fluorescence signal increased above a preset background threshold value. Average C t values from duplicate PCR reactions were normalized to average C t values for the housekeeping gene P-2-microglobulin ((32M) from the same cDNA preparations. The relative mRNA expression of a gene was calculated as follows: Average C t ((32M) - Average C t (gene) = d; mRNA expression relative to p2M = 2^(d). Each q-RT-PCR reaction was done in duplicate. In Chapter 3, primers were designed with the Primer Express Software (Applied Biosystems, Foster City, CA). In Chapters 4 and 5, primer sequences are as follows: mouse 4F2hc: forward 5' -GAGGACAGGCTTTTGATTGCAG-3' , reverse 5'- AGGTAGGAGCTGGTCAACAGCA-3'; mouse xCT: forward 5'- GAGGACAGGCTTTTGATTGCAG-3', reverse 5'-AGGTAGGAGCTGGTCAACAGCA-3'; mouse B2M: forward 5'-CACCCCCACTGAGAGACTGATACA-3', reverse 5'- TGATGCTTGATCACATGTCTCG-3'; human 4F2hc: forward 5'- 59 AGTGCCAACATGACTGTGAAG-3', reverse 5'-CCTTACTCCGCTGGTCACTCAG-3'; human^xCT:^forward^5'-TGCTGGCTGGTTTTACCTCAAC-3',^reverse^5'- CCAATGGTGACAATGGCCAT-3';^human^B2M:^forward^5'- ACCATGTGACTTTGTCACAGCC-3', reverse 5'-AATCCAAATGCGGCATCTTC-3'. 2.5 PROTEIN ANALYSIS 2.5.1 Protein isolation and western blotting Cell cultures were harvested and washed twice in PBS and resuspended in 500 [it protease inhibitor solution (1 tablet of Complete Protease Inhibitor Cocktail dissolved in 25 mL of PBS) (Roche, Basel, Switzerland). Cells were lysed by sonication and centrifuged at 500xg for 5 min. The pellet was recovered as the nuclei fraction, while the cloudy supernatant was centrifuged at 48,000xg for 3 h at 4 °C. The resultant supernatant was recovered as the cytosolic fraction and the pellet, resuspended in 1X PBS, was recovered as the crude membrane fraction. To determine HIF- 1 a protein expression in hypoxia assays, 100 11,M cobalt chloride (CoC12; Sigma) prepared in IX tris-buffered saline was added to the cultured cells before harvest, followed by two washes in 100 uM CoCl2 before resuspending in protease inhibitor solution. Protein concentration was determined using the Bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL), with bovine serum albumin as a standard. Protein samples (10 ug) were electrophoresed under reducing conditions on NuPAGE 4-12% Bis-Tris gels with 3-(N- morpholino)propanesulfonic acid (MOPS) running buffer (Invitrogen) and transferred to 0.45- µm nitrocellulose membranes (Invitrogen). Membranes were blocked overnight at 4 °C with 10 % skim milk in 1X PBS, followed by incubation with the following primary antibodies: mouse anti- human c-K-ras (Oncogene, San Diego, CA; 1:1000 dilution), mouse anti-human HIF-1 a (BD 60 Transduction Laboratories, Mississauga, Ontario, Canada; 1:2000 dilution), rabbit anti-mouse xCT (Trans Genic, Kumamoto, Japan; 1:100 dilution), mouse anti-human 4F2hc (Chemicon, Temecula, CA; 1:250 dilution), mouse anti-human a-tubulin (Sigma, 1:10,000 dilution). After incubation for 1 h at room temperature, membranes were washed with PBS and incubated with the following secondary antibodies: horse radish peroxidase-linked anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:10,000 dilution) or horse radish peroxidase- linked anti-rabbit IgG (Jackson ImmunoResearch Laboratories; 1:2,000 dilution). After incubation for 1 h, Amersham ECL Plus TM Western blotting detection reagents (GE Healthcare, Piscataway, NJ) were used for visualization. 2.5.2 Immunofluorescence microscopy Pancreatic cancer cells used for immunocytochemistry were cultured on glass chamber slides. Cells were washed twice in PBS and fixed in 4% paraformaldehyde (Sigma) for 10 min. Following three washes in PBS and 30 min incubation in blocking solution (PBS plus 5% goat serum plus 0.1% Triton X-100), cells were incubated with rabbit anti-mouse xCT antibody (Trans Genic Inc.; 1:50 dilution) or mouse anti-human 4F2hc antibody (Chemicon; 1:200 dilution), diluted in blocking solution for 45 min at 4 °C. Cells were then rinsed three times with blocking solution and incubated with either goat anti-mouse or goat anti-rabbit AlexaFluor 594 (Inv itrogen; 1:100 dilution) for 30 min at room temperature in the dark. Finally, cells were washed three times with PBS, stained with 4'6-diamidino-2-phenylindole (DAPI; Sigma), mounted with Vectashield® (Vector Laboratories, Burlingame, CA), and sealed with nail polish. Immunofluorescence was detected with an Axiovert 40 imaging microscope (Carl Zeiss Canada, Toronto, ON, Canada), and images were captured with a AxioCam HRC digital camera (Carl 61 Zeiss). Merged images were generated using Adobe PhotoShop (Adobe Systems, Mountain View, CA). Primary human pancreatic tumors and corresponding normal human pancreatic tissues (gift of Dr. Sylvia Ng, BC Cancer Agency, Vancouver, BC, Canada) were embedded and frozen in TissueTek® optimal cutting temperature compound (Somagen Diagnostics, Edmonton, Alberta, Canada), sectioned at 5 thickness using a CM1850 UV cryostat (Leica, Wetzlar, Germany), and mounted on Fisherbrand Plus microscope slides. Subsequently, these samples were subjected to the same immunohistochemical staining procedures as described for culture cells. 2.5.3 Immunohistochemistry Human pancreatic cancer xenografts were harvested from Rag-2M mice, fixed in 10% neutral buffered formalin overnight at room temperature, dehydrated, and embedded in paraffin. 7 pun sections were prepared using a microtome and mounted on glass slides. Sections were dewaxed in Histoclear (National Diagnostic, Atlanta, GA) and hydrated in graded alcohol solutions and distilled water. Endogenous peroxidase activity was quenched with 0.5% hydrogen peroxide in methanol for 30 min followed by washing in PBS (pH 7.4), and non-specific binding was blocked by incubating in 5% normal goat serum in PBS for 30 min. Sections were then incubated with the following primary antibodies: mouse anti-human Ki67 (Immunotech, Westbrook, ME, USA), mouse anti-human activated caspase-3 (Cell Signaling Technology, Danvers, MA). Nonimmune mouse immunoglobulin G (IgG; Zymed Corp., South San Francisco, CA) was used as a negative control. After incubation overnight at 4 °C, sections were washed with PBS and incubated with the appropriate biotinylated secondary anti-mouse IgG (Amersham International, Arlington Heights, IL; 1:300 dilution) for 30 min at room temperature. Sections 62 were washed three times in PBS, 10 min per wash, and incubated with avidin-biotin complex (Vector Laboratories) for 30 min at room temperature. Following an additional 30 min of washing in PBS, immunreactivitity was visualized using 3'3'-diaminobenzidine in PBS and 0.03% hydrogen peroxide. Sections were counterstained with hematoxylin, and slides were mounted with Permount® (Fisher Scientific). Immunhistochemistry was examined using an Axiovert 40 (Carl Zeiss) imaging microscope and images captured using an AxioCam HRC (Carl Zeiss) digital camera. 2.6 CELL SURVIVAL ASSAY Pancreatic cancer cell proliferation and viability were determined by neutral red uptake. Cells were plated in 96-well plates at 1x10 4 cells/well. Following incubation overnight to allow for cell attachment, treatments were administered as dictated by the specific experiment. Wells were aspirated and incubated with 100 pt 0.0025% neutral red dye (Sigma) in cell culture medium. Empty wells were also incubated with neutral red dye to allow for background absorbance correction. After 4 h of incubation, wells were aspirated and 100 !AL 1% acetic acid in 50% ethanol were added to solubilize the intracellular neutral red dye. Absorbance was determined at 550 nm. Each sample was done in triplicate. 2.7 GLUTATHIONE (GSH) ASSAY Total GSH levels were measured using the ApoGSH TM GSH Colorimetric Detection Kit (BioVision, Mountain View, CA) according to manufacturer's recommendations. This assay is based on the GSH recycling system by 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and GSH reductase. Briefly, DTNB and GSH react to generate 2-nitro-5-thiobenzoic acid and oxidized 63 GSH (GSSG). Because 2-nitro-5-thiobenzoic acid is a yellow colored product, GSH concentration can be determined by measuring absorbance at 412 nm. 2.8 RADIOACTIVE UPTAKE ASSAY All the uptake assays in this study were performed in an Na t-free environment, so that uptake activity specific to the x, - transporter could be detected (given that the x e - transporter is the only known Nat-independent transporter with a high substrate affinity for cystine and glutamate). Xe- transporter uptake activity was measured in cultured cells as described previously (Shih and Murphy 2001). Briefly, cells were plated in 24-well plates at 5x10 5 cells/well and incubated overnight. Following cell attachment, medium was removed quickly by aspiration and adherent cells were washed with and pre-incubated in 1 mL/well Nat-free Buffer A consisting of 140 mM N-methyl-D-glucamine, 5.4 mM KCI, 0.4 mM KH2PO4, 10 mM HEPES, 5 mM D- glucose, 1.8 mM CaC12, 0.8 mM MgSO4 (pH 7.4) for 20 min at 37 °C. The medium was then replaced with 300 tit Natfree Buffer A containing 33 nM L-[ 3H]-glutamate (49 Ci/mmol) (Amersham Pharmacia/GE Healthcare, Pittsburg, PA) in the presence or absence of 1 1.IM unlabeled amino acid competitors (L-glutamate, L-cystine) or non-competitors (L-leucine) for 20 min at 37°C. Uptake was terminated by washing three times with ice-cold Na t-free Buffer A, after which cells were solublized with 200 ttt 0.5% Triton X-100 in 0.1 M potassium phosphate buffer (pH 7.0). To determine intracellular L-[ 3H]-glutamate uptake, 100 [It of cell lysate was mixed with 5 mL of scintillation cocktail (Fisher, Pittsburg, PA), and radioactivity was measured using a LKB Wallac 1214 Rackbeta (American Instrument Exchange, Inc., Haverhill, MA) liquid scintillation counter. A 10 1.11, aliquot of cell lysate was used in a BCA protein assay kit (Pierce) to determine protein concentration, with bovine serum albumin as a standard. In experiments involving 24 h pre-incubation with SASP and 2-ME, 112 nM L-[ 14C]-cystine (300 64 mCi/mmol) (Perkin Elmer and Analytical Sciences, Waltham, MA) was used instead of L-[ 3 1-1]- glutamate because 2-ME reacts with cystine as it enters the cells via the leucine transporter (Ishii et al., 1981a). To determine kinetic measurements, both L4 3 1-1]-glutamate and unlabelled L-glutamate were added to the medium to give a final L-glutamate concentration range from 10-700 p,M. Apparent Vmax and Km were determined from the double reciprocal plot of uptake rate versus substrate concentration (Lineweaver-Burk plot). 2.9 NFKB REPORTER ASSAY To determine NFKB activity, pancreatic cancer cells were transfected with NFKB luciferase reporter constructs. One day post transfection, the medium was removed and cells were treated with 10 ng/mL TNF-a to induce NFKB activity. Following 3 h incubation, the medium was removed and the cells were treated with various concentrations of SASP or MSG with or without 2-ME. Following 24 h incubation, cells were washed and NFKB luciferase activity was determined using the Dual-Luciferase ® Reporter Assay System (Promega). 40 1AL of passive lysis buffter was added to each well, and the plate was placed at -80 °C for 1 h to ensure complete cell lysis. Following thawing of the plate at room temperature, cells were mechanically detached from the well with a cell scraper. 10 1AL of lysate was added to a 5 mL glass tube containing 50 1AL Luciferase Assay Reagent II and mixed by hand vigorously. The tube was then inserted into a luminometer to obtain a firefly luciferase reading. Afterwards, 50 JAL of Stop and Glo® Reagent was added to the tube, mixed, and inserted into the luminometer to obtain a renilla luciferase internal control reading. All samples were performed in triplicate, and final luciferase 65 activity was determined by taking the average of all the ratios of firefly luciferase to renilla luciferase readings. 2.10 TUMORIGENICITY ASSAY PANC-1 or MIA PaCa-2 cells (3x10 6 cells/100 [tiL) were subcutaneously injected into both dorsal flanks of each Rag-2M mouse. Tumor xenografts were allowed to grow for about three weeks until tumors reached an average volume of approximately 50 mm 3 , and mice were randomized into control and treatment groups (six mice/group). PBS control, SASP (250 mg/kg/twice daily; i.p., prepared fresh every day), GEM (120 mg/kg/once per week; i.p.), or a combination of SASP and GEM were administered for 14 days. Tumor volume was assessed pre- and post-treatment via caliper measurements, and determined using the formula: volume (mm3) = 0.52 x length x width x height (in mm). At the end of treatment, animals were sacrificed and tumors were harvested for histological analyses. Mice were provided with food and water ad libitum, and animal health was monitored daily for signs of stress, including weight loss or abnormal behavior. Animal experiments were carried out in accordance with the guidelines of the Canadian Council on Animal care. 2.11 STATISTICAL ANALYSIS Student's t-test was used to determine statistical significance unless otherwise stated, with P < 0.05 considered significant. 66 Chapter 3 ABC TRANSPORTER PROFILING IN PANCREATIC CANCER 3.1 ABSTRACT The ABC transporters are a large family of transmembrane proteins with diverse physiological functions, several of which have been found to play major roles in cancer drug resistance. Pancreatic cancer is highly resistant to chemotherapeutic drugs (Zalatnai and Molnar, 2007). Given that 90% of pancreatic tumors express a mutant K-ras oncogene (Hruban et al., 1999) and that pancreatic tumors are highly hypoxic (Garcea et al., 2006; Sun et al., 2007), I postulated that constitutive K-ras activation and/or hypoxia may correlate with ABC transporter expression, which in turn may promote intrinsic drug resistance in pancreatic cancers. To answer this question, I utilized HPDE cells overexpressing mutant K-ras, as well as HPDE cells subjected to hypoxic treatment, and performed mRNA expression profiling for 48 ABC transporters. The current studies identified specific ABC transporters that were induced in response to constitutive K-ras activation and/or hypoxic treatment. I also performed mRNA expression profiling for the 48 ABC transporters in three commonly used pancreatic cancer cell lines, MIA PaCa-2, PANC-1, and BxPC-3, which express either wildtype or mutant K-ras. Although PANC-1 cells, which express mutant K-ras, exhibited the highest upregulation of global ABC transporter expression, no clear correlation could be detected between global ABC transporter expression and expression of constitutively active K-ras. Moreover, hypoxic treatment induced differential effects on ABC transporter expression in all three pancreatic cancer cell lines. Interestingly, all three cell lines did not express the common MDR1 ABC transporter, a major player in drug resistance, and hence lacked intrinsic resistance to vincristine. 67 However, in response to long term treatment with low dose vincristine, MDR1 expression was upregulated, thus demonstrating that drug resistance can be induced in pancreatic cancer cells. Taken together, my results suggest that expression of mutant K-ras and hypoxic treatment, as well as long-term exposure to chemotherapy, may contribute to drug resistance in pancreatic cancer cells in part by inducing the expression of ABC transporters. 68 3.2 RESULTS 3.2.1 Effect of constitutive K-ras activation on ABC transporter expression in HPDE cells To determine whether expression of a constitutively active K-ras protein would affect ABC transporter expression in pancreatic cells, we used the following HPDE cell lines: HPDE parental cells (mock), HPDE cells stably transfected with the empty vector pBabepuro (pBp), HPDE cells stably transfected with pBp encoding a constitutively active K-ras protein with the mutation G12V(pBp-K-ras G12V), and the latter cell line derived from a tumor in vivo (pBp-K- rasGl2VT) (Qian et al., 2005). I confirmed K-ras protein overexpression in K-ras-transfected cell lines (Figure 3.1). I next performed mRNA expression profiling for 48 ABC transporters in these four HPDE cell lines using q-RT-PCR, and determined gene expression relative to the housekeeping gene p2m. Mutated K-ras-expressing cell lines did not exhibit an overall global increase in ABC transporter expression (Figure 3.2). However, several ABC transporters were overexpressed in both pBp-K-ras GI2v and pBp-K-rasGI2vT cell lines, including ABC50, ABCA2, ABCA7, ABCB6, ABCF3, AIdP, MRP1, MRP7, PMP70, TAP1, and whitel (Figure 3.2). Importantly, although numerous ABC transporters were not expressed in any of the four HPDE cell lines, I did not detect any ABC transporters that exhibited reduced expression in the K-ras GI2v cell lines compared to control cell lines (Figure 3.2). These findings suggest that constitutively active K- ras expression may correlate with induced expression of specific ABC transporters in pancreatic epithelial cells. 69 O 2 K-ras tubulin 23 kD 52 kD Figure 3.1: Expression of K-ras protein in HPDE cell lines. Western blots for K-ras and tubulin protein expression. Mock: parental HPDE cell line; pBp: HPDE cell line transduced with the empty vector pBabepuro; pBp-K-ras' v: HPDE cell line transduced with pBabepuro _ K_rasGi2v; K_ rasG121-: op_Krasc121 cell line derived from a tumor. 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' c X  §  E  x a a>  a a )m ^ x z -a <  a ) oz  ( -T 3 ' u=  -t--... 70  ( si-, ce -0  E 5 E  2 c') 4_ • -1:3 7:2 ,..o^ .c  a) _E2 0 C  (1) a -10> 0  a >  . 0  —  -c 0 ..c  a ) (0 c 2 -_:-. . Do  - um mw vs a) ,c ... I- 0 ^ ..c a) C (..) a I t ) ^p - ' C O  a c_) 0 ct) <  >2 N  c  > N  „ 6 o 7 = ,5 7 .) ca a  (12 . -  a A., cz c L . a Y  m  0 m  . - ■  >  a.)L- a ) a>  a- m  a ) a it 'C  c , _c 2 71 3.2.2 Effect of hypoxia on ABC transporter expression in HPDE cells To determine whether hypoxic treatment would affect ABC transporter expression in pancreatic cells, HPDE-pBp control cells were incubated in normoxic or hypoxic conditions. Cells cultured in an hypoxic environment exhibited upregulated protein expression of the hypoxia-inducible transcription factor HIF- 1 a (Liao and Johnson, 2007), thus confirming the induction of a hypoxic response (Figure 3.3). No observable difference in cell proliferation was observed after 24 h hypoxic treatment when compared with normoxic control (data not shown), indicating that hypoxic treatment did not induce a cytostatic or cytotoxic response in cells. Upon comparing HPDE-pBp cells cultured in normoxic or hypoxic conditions, several ABC transporter genes were found to be upregulated in response to hypoxia, including ABC50, ACBA2, ABCA7, ABCB6, ABCB8, AldP, tapl, and whitel (Figure 3.4). Interestingly, hypoxic treatment downregulated the expression of ABCA5, ABCB10, ABCF2, ABCF3, bcrpl, MRP2, MRP4, and RNase L (Figure 3.4). Taken together, our data thus far demonstrate that either constitutive K-ras activation alone (Figure 3.2) or hypoxic treatment alone (Figure 3.4) is sufficient to induce expression of six ABC transporters: ABC50, ABCA2, ABCB6, AldP, tapl, and whitel. 3.2.3 Effect of combined constitutive K-ras activation and hypoxia on ABC transporter expression in HPDE cells Having demonstrated that six ABC transporters exhibit upregulated expression in response to either constitutive K-ras activation or hypoxic treatment alone, we sought to determine whether constitutive K-ras activation, in combination with hypoxic treatment, would upregulate the same six ABC transporters to a greater extent than constitutive K-ras activation alone. To this end, we incubated HPDE-pBp-Kras G12V cells (which express constitutively active 72 HPDE pBp^pBp-K-rasG12v co^co X co^x ?^ >- Q ^? C5 o.. >.^t) Z I Z 0 0. 98 kD 52 kD HIF-1a tubulin Figure 3.3: Expression of HIF-1u, protein in HPDE cell lines in response to hypoxia. Western blots for HIF-1 a and tubulin protein expression. pBp: HPDE cell line transduced with the empty vector pBabepuro; pBp-K-ras c' 12v: HPDE cell line transduced with pBabepuro-K-ras'. Results are representative of two independent experiments. 73 x oa < -, ,-, ^ C a .  n a  Q . ta ^ Calit-Im C a d ZaPqm y e ?.ins g e e s I, 0 1 -IM LI ns del^ o , • ca. u" a ) ,o u,,, _ c a ) .c a ) so . co  o ll a. , Ce0 .c  co a.) c -..^ a) . 7. , (I)^ s.-- 7  a) a ) D  C CO CO a  A  5  - , a , x  T ! ti « = a • —  c c0 .0ce. -0  c ca a x E =  x co -0 -00_ ^ c-70-,...,^ G @ ^ () 22 7 5"<- ca^ C C ) to  c ^ 0  5 ' < - ^ t e l i q M ^ <  c o  0 E  0 O ^ c o  (t3 E  c6 .1- 0  •-,..7... c z 1 g2 ---- > > _ C  2 6gdw d ^ .. >. 1-.3 O  -Z 6 diw ^ C C  0 c  o 9 ` ° 6  .. ,̀4 ., s2 0 Ld iw ^ .th 2 ,-0- ' > x  c o O ,_  >  -'' 9d . ^ 0. —  ci, 0.) ,0 >, 0 ...c, 2 « © . 0  . c... .... ^ a ... o  f. qd.iw ^ 0  00 a) ca^ = -.E' cd.lw ^ 9  u i -0  a) o i  -0  E zdiw ^ E  fl. a ) - c. -_ L. I ^ a.) 1.d.ku^ 2  •  E - / 0  . _ipw^ .c 6,_ , 0 zs c2, a ) c 1.80IAJ^ a)" 3  ° Juo^ c  ■  v) E 0_ ,-- o dasq^ 5  C O  e, 0  c i. -- 4--a) 1,d.ioq ^ fi) L i : -2 -  C O a) z ,_  , c  a  uj eiPIV == 4.04)^ ..c ° clPIV ^ a) al c.) u ) t)  CO  co_ d0f3V^ L a  c `  4 0  — C d 0 8 V ^ c O  c P o C L  t g ))) ^ _ = ^ I  a ) Z d 0 9 V  • >  o ) a ) -^ 1  0 0 O V 6 8 0 9 V 9809 V L8 0 9 V ^ a ) cl- ^ / .z.: E^ D . 0  -C  _c 2  ' t - -,' C L (I)  C O  73 C7 7 ) § ' a ) )) -C 0 =  (3 ) > , C  -C 9 9 3 ^ co^ ..c _8 a.) -r) 9 9 0 8 V ^ 1 .... (1 ) ,-, a  0  s''' 22 x ^ a) a) 01.809V ^ 0  10cn  > 6VOSEIV^ <  c  .--a -;' 2 Z  e 2 C 4 -'-' c 'cy.)- 9 V 0 8 V ^ E  c u  — .S o ro zvoav ^ tf> —  (7 ) C:1 A c v t  -ci) - ( i ) c O 0  2  .° C I. u j 0 . ,,, 7) gV O S V ^ 0C  '-'' c l) 2 ^ 1  ZV O E IV ^ C L < 0:3 -,^ Ct. l^ 1- -L. ^ x 4...^ Z  a) - _— ^ 0  6 - eL ,,,, 1..vosv o3 o 00 E  -, C II.V39V ^ < ^ . co ,.., I.VD 9V ^ e t .g(15 :  cf!- ^  OSOEN ^ o  —  CD 0 .3  ,-, C  > 6. ,..... e  0..) o ^ o ^ 0 ^ o ^ 0 ^ o ^ 0 ^ =  (/ ) c o  2 co^ it) ^ -1. ^ cr,^ C•4^ ..--^ 0 5  C  a )  - ir. .....2  `E).. Z  I c D  al 00 G O • 0  •  0 E  0 v) co ,....- C L  M  al 0  > , L .. L .. 74 \ 3 r * D w 9 m  a w u q s s d a  e u a o K-ras) in normoxic or hypoxic conditions and profiled ABC transporter expression via q-RT- PCR. We detected an induction of HIF- 1 a protein expression in hypoxia-treated cells (Figure 3.3), thus confirming the induction of a hypoxic response. Remarkably, constitutive K-ras activation in combination with hypoxic treatment resulted in a marked downregulation of five of the six ABC transporters that exhibited upregulation in response to K-ras activation alone: ABC50, ABCA2, ABCB6, tap 1 , and white 1 (Figure 3.4). Of interest, numerous other ABC transporters also exhibited a similar downregulation in expression in response to combined K-ras activation and hypoxic treatment, including ABCF2, ABCF3, MRP4, MRP5, PMP69, and RNase L (Figure 3.4). The only ABC transporter that exhibited upregulated expression in response to combined K-ras activation and hypoxic treatment to a greater extent than K-ras activation alone was AIdP (Figure 3.4). These data suggest that hypoxia may function to negatively regulate the induction of ABC transporter expression by constitutive K-ras activation. 3.2.4 Effect of combined K-ras activation and hypoxia on ABC transporter expression in pancreatic cancer cells Two commonly used human pancreatic cancer cell lines, MIA PaCa-2 and PANC-1, have previously been shown to contain a mutated hyperactive K-ras gene, with a third cell line BxPC- 3 possessing a wild-type K-ras gene (Nakada et al., 2001). Given the differential status of K-ras activation among these three cell lines, we cultured cells under normoxic or hypoxic conditions to determine if any correlations in ABC transporter expression with respect to K-ras status could be detected. The MIA PaCa-2 cell line, which expresses constitutively active K-ras (Nakada et al., 2001), did not exhibit a clear trend towards upregulation or downregulation of ABC transporters in response to hypoxic treatment (Figure 3.5a). The PANC-1 cell line, which also expresses 75 76 ABC transporters ^ BxPC-3 (Normoxia) ■ BxPC-3 (Hypoxia) 0 7 CD N < <C.)^< <CO CO U 0 co co< c0 CO < << < CD I,- CO CT) 0 0 CV (,) 0_ CO CO CO m m ^LL^26 O 00 0030 CO CO CO CO^< 03 CO^CO< < < < < < ABC transporters ^ MIA PaCa-2 (Normoxia) ■ MIA PaCa-2 (Hypoxia) -0(2 1; c4 1? '?? 1? 1? NE? c.E. 2 2 2 2 2 2 2 E E y a a ccc 0^ 0 10 0 7 0 CV N LO CO h.- CO CO I"- co a) o 0 N co a_ O (-) Q< 0 0 0 C.)^C..) C.)^ (cij u_ L.L. v e- C.) 0 ;".7, o Loa.^-- < < < < < ca ca ca X) M 00 0 0 caa0c a caa3cacac 0300 <c0a3 -- ao < <a0M««««<c0 « <<^ < (,)^C,")^r-- co o o^cv •-• 0,1 -ts EL 2- e-^p.- e- e- e-^18_ rs-^2. 2 2 2 2 2 2 2 2 2^E E (i) a_^cce 500 - 400 - 300 - 200 100 0 EL 6 -0 co 2 2 ABC transporters Figure 3.5: ABC transporter mRNA expression profile in pancreatic cancer cell lines cultured in normoxia or hypoxia. Q-RT-PCR data showing mRNA expression relative to pan in (a) MIA PaCa-2, (b) PANC-1, and (c) BxPC-3. 37 of the 48 ABC transporters profiled are shown. The omitted 11 ABC transporters exhibited no detectable expression. Error bars were omitted due to the variation of the data in individual experiments. However, similar expression patterns were observed thus, the results shown are from one experiment representative of three independent experiments. O °• • "- N N^N- CO CO h- CO a) 0 0 N < < < < < CO CO al CO^0 U_ <<U00C..)00(..)0 0 00 0000 021,20 0000 ca m a0C0 ca a0 ca a00<00 < < oo « « « « < a:3 << < < Le. 2 2 2 2 2 2 2 §_ ..7-1 7_ '-c,_ c•la. ''- a.) 0 m co a) 0 CO I-- H.. —co c Cr it 600 500 - 400 - 300 - 200 100 - 0 600 - C CO I--,^I_ L b 1200 1000 800 - 600 - 400 - 200 - 0 0 PANC-1 (Normoxia) ■ PANC-1 (Hypoxia) Ili 1 11 111 constitutively active K-ras (Nakada et al., 2001), exhibited upregulation of a majority of ABC transporters in response to hypoxic treatment, with the exception of ABCB 10 and MRP3 (Figure 3.5b). In contrast, the BxPC-3 cell line, which expresses wildtype K-ras, exhibited downregulation of a majority of ABC transporters in response to hypoxia, with the exception of ABCA7, MRP5, and MRP7 (Figure 3.5c). Hence hypoxia induced differential effects on ABC transporter expression in all three pancreatic cancer cell lines, with no clear correlation detected between global ABC transporter expression and expression of constitutively active K-ras. Interestingly, although the drug resistance-related ABC transporters BCRP and MRP1 exhibited expression in all three cell lines (Figure 3.5a-c), the drug resistance-related ABC transporter MDR1 was not detected in any of the pancreatic cancer cell lines tested (Figure 3.5a-c). Hence neither hypoxic stress nor K-ras activation was sufficient to induce MDRI transporter expression. 3.2.5 MDR1 is not expressed but is inducible in pancreatic cancer cells The MDR1 gene encodes for the drug resistance-related ABC transporter protein P- glycoprotein (P-gp) (Endicott et al., 1991). Given that primary pancreatic tumors have been reported to express P-gp without previous drug treatment, we were surprised to find that the untreated pancreatic cancer cell lines used in our study did not express MDRI. To obtain functional verification that P-gp protein was not expressed in the pancreatic cancer cell lines used in our study, we treated MIA PaCa-2, PANC-1, and BxPC-3 cells with vincristine and measured cell survival. MIA PaCa-2 and BxPC-3, in addition to the vincristine-sensitive control cell line SKOV3, underwent cell death in response to vincristine treatment, while PANC-1 showed moderate resistance to vincristine up to 1 mM (Figure 3.6a). In contrast, MDR1/P-gp- overexpressing SKVCR2.0 ovarian carcinoma cells were resistant to vincristine treatment 77 MIA PaCa-2 PANC-1 BxPC-3 SKVCR0.1 SKVCR0.2 SKVCR2.0 10 9 8 7 6 5 4 3 2 1 0 •Vincristme20^El Control 0^18^• Vincristine 16 0 a MIA PaCa-2 PANC-1 OxPC-3 SKOV3 -4*--SKVCR2 0 0.0 -5^-4^-3^-2^-1^0 log [Vincristinel (pM) 1 4-) 0 8 Lc) in 0.6 0.4 Tt 0.2 e ,0 e^<b* -<•c' .ef^\. 1, (1,- ...j,:"^; ,c• -̀'-c.@ I; a'^■._^.--..---\\C.) C1 N N 6, rb 'q '4' e4j4'ClCi <'`‘' Ce'e:OVV' 'R)*) '_(Ci:' (2'; •<‘^e 6^6\,\<,, ON ON „§) 0^c,\+- c,ssss^■L` d 60 120 100 80 60 50 40 30 20 Figure 3.6: Effect of vincristine on MDR-1/P-gp expression in pancreatic cancer cell lines. (a) Neutral red uptake assay for survival of pancreatic (MIA PaCa-2, PANC-1 and BxPC-3) and ovarian (SKOV3 and SKVCR2.0) cancer cell lines treated with increasing concentrations of vincristine for 72 h. (b) Q-RT-PCR for MDR1 expression in pancreatic cancer cell lines treated with vincristine. Results shown are representative of three independent experiments. (c) Q-RT-PCR for MDR1 expression relative to control SKOV3 ovarian cancer cell lines treated with vincristine. Results shown are representative of three independent experiments. (d) Western blot for P-gp protein expression in pancreatic cancer cell lines treated with vincristine for 2 months. Total membrane protein stained with Coomassie Blue is shown below. (e) Western blot for P-gp protein expression in ovarian cancer cell lines treated with vincristine for 2 months. Total membrane protein stained with Coomassie Blue is shown below. kD 170 kD 120 ^ -4-170 kD 100 80 60 50 40 30 20 78 (Bradley et al., 1989) (Figure 3.6a). Hence pancreatic cancer cells that lack MDR1 expression are sensitive to vincristine treatment. I next determined whether MDR1 expression could be induced in the three pancreatic cancer cell lines used in our study, by treating the cells with low-dose vincristine (10 nM) over a 2 month time period (fresh medium and drug was administered twice a week) to promote the development of drug resistance. All three pancreatic cancer cell lines upregulated MDR1 mRNA expression in response to vincristine treatment, with PANC-1 cells exhibiting the greatest induction (Figure 3.6b). In contrast, vincristine-sensitive SKOV3 cells expressed minimal MDRI levels, with the vincristine-resistant cell lines SKVCR0.1, SKVCR0.2, and SKVCR2.0 expressing increasing levels of MDR1 in accordance with increasing levels of vincristine resistance (SKVCR2.0>SKVCR0.2>SKVCR0.1) (Figure 3.6c). Induction of P-gp protein in the vincristine-treated cell lines (Figure 3.6d,e) was confirmed, and a correlation between P-gp protein expression and MDRI mRNA expression was observed (Figure 3.6b-e). Hence MDR1/P- gp expression with subsequent vincristine resistance can be induced in pancreatic cancer cell l ines. 79 3.3 DISCUSSION To date, many studies have been performed using a variety of methods to profile the expression of ABC transporter family members in numerous drug resistant cell lines and clinical samples (Zhang, 2007). Despite these studies, the functions of many members of the ABC transporter family still remain unknown or not fully understood. In pancreatic cancer, one of the most intrinsically drug resistant cancers, knowledge of the role of ABC transporter expression in disease pathology is severely limited. Indeed, none of the NCI-60 cell lines used in predicting drug sensitivity and resistance in microarray experiments are pancreatic in origin (Ross and Doyle, 2004; Szakacs et al., 2004). Hence, in the present study I chose to use a variety of pancreatic cell lines in our q-RT-PCR ABC profiling studies. The K-ras point mutation G12V, which results in a constitutively active K-ras protein, is present in over 90% of pancreatic cancer and occurs at a very early stage of cancer development (Almoguera et al., 1988). Immortalized HPDE cells stably transfected with the K-ras G12v oncogene have been reported to be tumorigenic (Qian et al., 2005). Moreover, HPDE-K-ras G12v cells exhibit gene expression changes that closely resemble those observed in pancreatic ductal adenocarcinomas (Qian et al., 2005). Specifically, microarray expression profiling studies identified 584 genes whose expression levels were specifically upregulated by the K-ras oncogene, of which 42 have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors (Qian et al., 2005). However, none of the 42 genes mentioned in this report were from the ABC transporter family (Qian et al., 2005). It is also unknown whether any of the remaining 542 genes might be from the ABC transporter family. Furthermore, microarray technology is less sensitive and less quantitative than the q-RT-PCR method used in my experiments. In my study, expression of constitutively active K-ras in HPDE 80 cells induced the expression of 11 out of 48 ABC transporters examined. These findings suggest that expression of constitutively active K-ras may correlate with a specific ABC transporter expression profile in transformed pancreatic epithelial cells. Pancreatic cancer is very hypoxic in nature (Garcea et al., 2006; Sun et al., 2007). By incubating HPDE-pBp control cells under hypoxic conditions, I found that eight ABC transporters were overexpressed. Hence hypoxia, independent of K-ras activation, is sufficient to induce the expression of specific ABC transporters. However, by incubating HPDE-pBp-K- raS GI2V cells under hypoxic conditions (i.e. combined K-ras activation and hypoxic treatment), an overall downregulation of ABC transporters was observed, suggesting that hypoxia may function to negatively regulate the induction of ABC transporter expression by constitutive K-ras activation. The only exception was the ABC transporter AIdP, which was upregulated in the presence of both K-rasG12v and hypoxia-induced HIF-1 a overexpression. AIdP (also known as ABCD1 or ALD) is one of four ABC transporters that functions to transport fatty acids into peroxisomes for (3-oxidation (Gartner et al., 2002; Roerig et al., 2001). AIdP is mutated in adrenoleukodystrophy (ALD), a condition characterized by the inability to process long-chain fatty acids in the brain, thus adversely affecting the growth and development of myelin (Gartner et al., 2002; Roerig et al., 2001). Although I observed AIdP induction in response to K-ras activation and hypoxic treatment in transformed pancreatic epithelial cells, its potential function in pancreatic tumorigenesis remains to be explored. I next sought to determine whether the ABC transporter expression profiles we observed in HPDE cell lines would also be found in pancreatic cancer cell lines. Using three pancreatic cancer cell lines (MIA PaCa-2, PANC-1, and BxPC-3), no definite pattern of ABC transporter expression could be attributed to the presence or absence of a K-ras mutation. ABC transporter 81 expression profiles were not shared between the HPDE-pBp-K-ras G12V cell line and the mutated K-ras-expressing MIA PaCa-2 and PANC-1 cell lines. Several reasons could account for these observations. Firstly, the media used to culture the HPDE cell lines and pancreatic cancer cell lines were different. The HPDE cells were cultured in KSF media with supplements, while the pancreatic cancer cell lines were cultured in DMEM containing serum. Indeed, incubating HPDE cell lines in DMEM containing serum resulted in a change in morphology to a more differentiated phenotype (data not shown). Given that growth factors in the culture medium can influence morphological characteristics of a specific cell line, it is likely that such growth factors also affect gene expression in response to stressful environments such as hypoxia. Secondly, it has been reported previously that the pancreatic cancer cell lines PANC-1 and BxPC-3 constitutively express HIF-la, the transcription factor normally induced in response to hypoxic treatment (Akakura et al., 2001). Given that HIF-la is already expressed in normoxic PANC-1 and BxPC-3 cells, it is difficult to determine the effect hypoxic treatment would have on gene expression in these cell lines. It is possible that hypoxia would induce a greater increase in HIF-1 a expression levels, thus further upregulating downstream target genes. On the other hand, HIF-la expression levels may already be beyond threshold levels in constitutive HIFI a- expressing cells, thus minimizing the effects of hypoxic treatment on gene expression. Moreover, constitutive and inducible HIF-la activation may play different roles at different stages of pancreatic cancer development and progression (Xie et al., 2006). Thirdly, it is possible that other signaling pathways, in addition to the K-ras and HIF-la signaling pathways, may regulate ABC transporter expression in pancreatic cancers. Other 82 genetic mutations may function to promote or antagonize K-ras- and/or HIF-1 a-induced ABC transporter expression. In an endothelial cell line, treatment with hypoxia induced HIF-1 a expression with a subsequent increase in MDRI (ABCB1) expression and P-gp (protein of MDR1) function, resulting in increased drug resistance (Comerford et al., 2002). In my studies, however, treatment with hypoxia did not influence the expression of MDRI in pancreatic cancer cell lines. Furthermore, it was observed that the pancreatic cancer cell lines were not resistant to vincristine treatment, thus confirming the absence of MDRI expression. These results were surprising since clinical pancreatic cancer tissues are often reported to express detectable levels P-gp (O'Driscoll et al., 2007), which is believed to mediate their drug resistant phenotype as seen in the clinic. The fact that MDRI expression was not detected in any of the pancreatic cancer cell lines tested under normoxic or hypoxic treatment suggests that these pancreatic cancer cell lines may not accurately represent primary pancreatic cancer cells, particularly with respect to intrinsic drug resistance. Nonetheless, we demonstrate that long-term treatment with vincristine is sufficient to induce MDR1 expression. The aim of the present study was to investigate two characteristic traits of pancreatic cancer, K-ras mutation and hypoxia-induced HIF- 1 a expression, to determine whether they contribute to drug resistance via the induction of ABC transporter expression. The findings suggest that expression of mutant K-ras and hypoxic treatment, as well as long-term exposure to chemotherapy, may contribute to the development of drug resistance in pancreatic cancer cells in part by inducing the expression of ABC transporters. The relative importance of the induced ABC transporters, however, remains to be determined. Clearly additional studies examining the role of ABC transporters in pancreatic cancer progression and drug resistance are warranted. 83 Chapter 4 CHARACTERIZING THE xc" TRANSPORTER IN PANCREATIC CANCER 4.1 ABSTRACT The xc - transporter plays a role in the biosynthesis of GSH by enabling the uptake of extracellular cystine into the cell (Verrey et al., 2004). Because the x c" transporter has been shown to regulate the growth of various cancer cell types (Doxsee et al., 2007; Narang et al., 2003), and xc - is expressed in the pancreas (Bassi et al., 2001; Kim et al., 2001), I postulated that the x: transporter may be involved in growth and drug resistance in pancreatic cancer. To answer this question, I first characterized the expression of the x c - transporter in pancreatic cancer cell lines subjected to cystine-depletion and oxidative stress factors. My studies indicate that cultured pancreatic cancer cells depend on the x c - transporter for the uptake of cystine to survive and can modulate its expression to accommodate growth needs. Pancreatic cancer cells also depend on the xe . transporter for the biosynthesis of GSH to survive in oxidative stress conditions. I show that the x c - transporter protein is differentially expressed in normal pancreatic tissues and is overexpressed in pancreatic cancers in vivo. It was also demonstrated that mRNA expression of the x, - transporter correlates with gemcitabine resistance in pancreatic cancer cell lines. Thus, my results suggest that the x, - transporter may play a role in pancreatic tumor growth in part by contributing to the synthesis of GSH to promote cancer cell proliferation, survival, and drug resistance. 84 4.2 RESULTS 4.2.1 Pancreatic cancer cells depend on extracellular cystine for growth It is known that cysteine, a reduced alternative of cystine, is a non-essential amino acid in the human diet since tissues in the body such as the liver can generate cysteine from the transsulfuration pathway (Brosnan and Brosnan, 2006; Stabler et al., 1993). This pathway involves the metabolism of methionine, a nutritionally essential amino acid, to a cystathionine intermediate and its subsequent cleavage by y-cystathionase to a-ketobutyrate, resulting in the production of intracellular cysteine (Brosnan and Brosnan, 2006; Stabler et al., 1993). In contrast to normal cells, certain cancer cells cannot generate their own cysteine/cystine and hence depend on extracellular sources to sustain growth and survival (Gout et al., 2001; Iglehart et al., 1977). To determine if pancreatic cancer cells require exogenous cystine for growth and survival, the pancreatic cancer cell lines MIA PaCa-2, PANC-1, and BxPC-3 were cultured in medium supplemented with all possible combinations of cystine, methionine, and/or cystathionine. Survival and robust growth of all three cancer cell lines was observed only in cultures containing both methionine and cystine (Figure 4.1), demonstrating that the absence of either amino acid inhibited survival and proliferation in vitro. Cystathionine, which can substitute for cystine in some cell systems (Uren and Lazarus, 1979), failed to promote cell survival/growth when added to cystine-deficient and methionine-containing cultures (Figure 4.1). These results indicate that pancreatic cancer cell lines are dependent on the uptake of cystine from their microenvironment for growth and survival, suggesting that the enzymes involved in the transsulfuration pathway may not be present or activated in these cells. 85 ■ MIA PaCa-2 • PANC-1 ^ BxPC-3 Methionine Cystine Cystathionine 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Figure 4.1: Pancreatic cancer cells depend on extracellular cystine for growth. Neutral red uptake assay for cell proliferation in MIA PaCa-2, PANC-1, and BxPC-3 cells incubated in medium containing methionine (0.1 mM), cystine (0.1 mM), and/or cystathionine (0.15 mM), in all possible combinations for 72 h. Data represent the mean ± SEM from three independent experiments. *P < 0.01. 86 4.2.2 A negative correlation exists between extracellular cystine deprivation and expression of the x c" transporter Extracellular cystine is transported into the cell via the x e- transporter (Bannai, 1984b). In order to determine whether extracellular cystine concentrations affect the expression of the x, - transporter, pancreatic cancer cells were incubated in medium containing low (0.01 mM), normal (0.1 mM) or high (1.0 mM) levels of cystine for up to 72 h. Cells in media containing low cystine exhibited signs of death after 72 h (data not shown). The x, - transporter is structurally composed of a xCT light subunit which confers substrate specificity and a 4F2hc heavy subunit which is a common subunit of many amino acid transporters (Bassi et al., 2001; Sato et al., 1999). The mRNA expression levels of the xCT and 4F2hc subunits at varying cystine concentrations were determined by q-RT-PCR. In response to low cystine concentrations, xCT mRNA was elevated in two of the three cell lines (MIA PaCa-2 and PANC-1), while 4F2hc mRNA was elevated in all three cell lines (MIA PaCa-2, PANC-1 and BxPC-3) (Figure 4.2a). Expression of 4F2hc protein in all three cell lines was determined by western blot analysis and yielded a similar increased expression level in response to low cystine concentration (Figure 4.2b). Unfortunately, no satisfactory anti-human xCT antibody for western blotting was available at the time these experiments were conducted, thus precluding the examination of xCT protein expression in our studies. These findings demonstrate that inverse correlations exist between extracellular cystine concentration and both xCT and 4F2hc expression in some pancreatic cancer cell lines, suggesting that these cells can modulate the expression of x c - transporter to accommodate their growth needs. 87 BxPC-3 PANC-1 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 xCT 4F2hc 0.01^0.1^1 [cystine] (mM) 0.01^0.1^1 [cystine] (mM) 0.01^0.1^1 [cystine] (mM) b BxPC-3^PANC-1^MIA PaCa-2 2-2 4.0 3.5 cl.)x^cr) 3.0 Cll 2.5 a) M 2.0 1.5 >'2 1.0 0.5 71-3 0.0 MIA PaCa-2 ■ xCT 4F2hc 2^22^22 E^E 0 0 0 0 2 E 2 2 E^E --o o 2 E a 47 kD 52 kD 4F2hc tubulin Figure 4.2: A negative correlation exists between extracellular cystine deprivation and expression of the x c - transporter. (a) Q-RT-PCR for expression of xCT or 4F2hc mRNA in MIA PaCa-2, PANC-1, and BxPC-3 cell lines incubated in varying cystine concentrations (0.01, 0.1 or 1 mM) for 72 h. Data represent the mean ± SEM from three independent experiments. *Ip 5 0.05. (b) Western blot for expression of 4F2hc or tubulin in MIA PaCa-2, PANC-1, and BxPC-3 cell lines incubated in varying cystine concentrations (0.01, 0.1 or 1 mM) for 72 h. Results are representative of two independent experiments. 88 4.2.3 Oxidative stress increases x c" transporter expression and GSH levels The oxidative stressor, DEM, is commonly used to regulate intracellular GSH levels (Bannai, 1984a; Hosoya et al., 2002; Kim et al., 2001) and is often used to induce the expression of stress response-related genes. All three pancreatic cancer cell lines were treated with 1 mM DEM for 24 h, and an increase in total intracellular GSH levels in response to DEM treatment was confirmed (Figure 4.3a). In a blood brain barrier cell line, treatment with DEM increased GSH levels with a corresponding increase in xCT mRNA expression (Hosoya et al., 2002). To determine whether the DEM-induced increase in GSH levels in pancreatic cancer cells corresponded with an increase in x e. transporter expression, mRNA expression levels of xCT and 4F2hc were determined by q-RT-PCR. xCT mRNA expression was significantly upregulated in all three cell lines in response to DEM (Figure 4.3b). In contrast, 4F2hc mRNA remained at control levels (Figure 4.3b), consistent with previous studies that reported no effect of DEM on 4F2hc mRNA expression in a rat retinal capillary endothelial cell line (Tomi et al., 2002) and a human retinal pigment epithelial cell line (Bridges et al., 2001). Corresponding 4F2hc protein levels also remained unchanged in response to DEM treatment (Figure 4.3c). To assess xCT protein expression, immunofluorescent staining with an anti-human xCT antibody (not ideal for use in western blotting) was performed on pancreatic cancer cell lines. Increased xCT protein levels were observed in all three pancreatic cancer cells in response to DEM treatment, with the BxPC-3 cell line exhibiting a clear localization of the xCT protein to the plasma membrane upon treatment with DEM (Figure 4.3d). These findings suggest that pancreatic cancer cells, in response to oxidative stress, upregulate expression of the x e - transporter by inducing xCT (but not 4F2hc) subunit expression, resulting in a corresponding increase in GSH synthesis. This 89 a 6.0 5.0 4.0 3.0 2.0 1.0 0.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 6 • ra) 5 > c a) co 4— • 0fn^3 • -3 2 1 0 BxPC-3 • xCT^* 4F2hc MIA PaCa-2 ■ xCT 0 4F2hc Control DEMti q <Z;,# * ■ Control 0 DEM PANC-1 ■ xCT 4F2hc 15_ s'E' ^E^"E' Wo WoW • 0 0 0 0 0 52 kD Control DEMControl DEM Figure 4.3: Oxidative stress increases x c - transporter expression and GSH levels. (a) Intracellular GSH levels in MIA PaCa-2, PANC-1, and BxPC-3 cell lines either untreated or treated with 1 mM DEM for 24 h. Data represent the mean ± SEM from at least two independent experiments. *P < 0.05 using the Wilcoxon rank-sum test. (b) Q-RT-PCR for xCT and 4F2hc mRNA expression in MIA PaCa-2, PANC-1, and BxPC-3 cell lines either untreated or treated with 1 mM DEM for 24 h. Data represent the mean ± SEM from at leas+t two independent experiments. *P < 0.05 (c) Western blot for expression of 4F2hc and tubulin in MIA PaCa-2, PANC-1, and BxPC-3 cell lines either untreated or treated with 1 mM DEM for 24 h. Results are representative of two independent experiments. (d) Immunofluorescence for xCT (red) and DAPI (blue) in MIA PaCa-2, PANC-1, and BxPC-3 cell lines either untreated or treated with 1 mM DEM for 24 h. Results are representative of two independent experiments. 90 increase in GSH synthesis may in turn enable pancreatic cancer cells to survive in the presence of elevated levels of ROS. 4.2.4 Expression of the xc" transporter in primary human pancreatic cancer specimens Having demonstrated that pancreatic cancer cell lines express the x: transporter, the expression of the x c - transporter in primary human pancreatic cancer tissues was assessed. Due to difficulties in obtaining fresh human pancreatic cancer tissues, only two patient samples were examined in this study. Both pancreatic cancer patient specimens with corresponding normal pancreatic tissue from the same patient were stained with anti-xCT antibody by immunofluorescence. In normal pancreatic tissue, xCT protein expression is primarily localized to ductal cells, not acinar cells (Figure 4.4). Importantly, pancreatic ductal adenocarcinomas exhibit overexpression of xCT protein. The distinct histological features of the two pancreatic cancer specimens, including atypical epithelial architecture, papillary folding, and intraluminal cell shedding, indicate that these specimens are likely invasive pancreatic ductal adenocarcinomas. Hence pancreatic cancer exhibits upregulated expression of xCT protein, suggesting that elevated xc - transporter expression may play a role in the pathogenesis of pancreatic cancer. 4.2.5 A positive correlation exists between the expression level of xCT and resistance towards GEM Thus far we have demonstrated that elevated x c - transporter expression promotes pancreatic cancer cell growth by promoting the uptake of extracellular cystine, which in turn functions to maintain high levels of intracellular GSH to promote cancer cell survival in the presence of oxidative stress. Among the three pancreatic cancer cell lines used in this study, PANC-1 cells expressed the highest relative level of xCT mRNA compared to that of BxPC-3 91 0Normal acinar cells A ,TLY, Pancreatic ductal adenocarcinoma Figure 4.4: Expression of the x c - transporter in primary human pancreatic cancer specimens. Immunofluorescence for xCT (red) and DAPI (blue) in normal ductal cells, normal acinar cells, and pancreatic ductal adenocarcinoma cells from two primary human pancreatic cancer patient specimens. 92 and MIA PaCa-2 (Figure 4.5a), suggesting that PANC-1 cells may exhibit the greatest resistance towards oxidative stress-induced cell death. GEM, the most common chemotherapeutic agent for the treatment of pancreatic cancer (Maehara et al., 2004), induces cell death via a mechanism that can involve oxidative stress (Donadelli et al., 2007; Maehara et al., 2004). To determine whether a relationship exists between the expression level of the x c" transporter and resistance towards GEM, we treated pancreatic cancer cells with GEM and determined the half maximal inhibitory concentration (IC50) of GEM using neutral red assays. The IC50 of GEM was highest in PANC-1 cells (>50 1.IM) compared to MIA PaCa-2 (-0.03 11M) and BxPC-3 (0.01 1.1M) cells (Figure 4.5b). The higher resistance of the PANC-1 cells to GEM may correspond with the higher number of cystine transporters per cell as suggested by relative xCT expression levels. Hence a negative correlation exists between the expression level of xCT and sensitivity to GEM; PANC-1 cells, which have highest relative expression of xCT, are the least sensitive to GEM, while MIA PaCa-2 cells, which have the lowest relative expression of xCT, are the most sensitive to GEM. 4.2.6 Overexpression of exogenous xCT increases cystine uptake and increases GEM resistance To determine if exogenous expression of the ?c c- transporter can increase GEM drug resistance, xCT and 4F2hc cDNAs were transfected into the pancreatic cell lines MIA PaCa-2 and PANC-1. Increased xCT and 4F2hc mRNA expression was confirmed by semi-quantitative RT-PCR and q-RT-PCR in transfected MIA PaCa-2 (Figure 4.6a-c) and PANC-1 (data not shown) cell lines. Using a radioactive glutamate uptake assay, MIA PaCa-2 cells transfected with either xCT cDNA alone or both xCT and 4F2hc cDNA exhibited increased x e - transporter 93 bN c; c.‘"-^c5'• 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -4^-3^-2^-1^0 log [GEM] (NM) PaCa-2 PA NC- 1 -A— BxPC-3 * * ** * a^5.0 4.5 '06  a_f 4.0 c.) 3.5-a • cl.) 3.0 C CU co" 2.5- >( "c' 2.0 > o 1.5 • 1.0 CY^0.5 - ^ 0.0 ^ Figure 4.5: A positive correlation exists between the expression level of xCT and resistance towards GEM. (a) Q-RT-PCR for xCT expression in MIA PaCa-2, PANC-1, and BxPC-3 cell lines. Data represent the mean ± SEM from three independent experiments. *P < 0.05. (b) Neutral red uptake assay for cell proliferation in Mia PaCa-2, PANC-1, and BxPC-3 cells treated with increasing concentrations of GEM for 72 h. Data represent the mean ± SEM from three independent experiments. *P < 0.01. 94 5 10 - 9v), El2 0 8 7 6 • c0.1^5 -c 4 a) la 3> 21.--O• 1 0^ •^ 0 As ‘^N;^ 4. 0 'V 0 3 N' 0^ 0N- \-\- Q(' 9 (9 9° c2 b • 16 0^14 f12 •^ 12 x a)^10a) • c 8cts X L 6O 0 4$2 • - 2ct NControl DGEM d a 100 bp -+ xCT ^ 4F2hc c,").^'15‘^'15`#^p-̀ 13' 4 41\'^I)* "D' N' OAP GOB^ 2\''\ 0QGQGO^QGQGO 1.8 1.6 - 1.4 - 1.2 1.0 - 0.8 - 0.6 - 0.4 - 0.2 - 0.0  ■ MIA PaCa-2 PANC-1 e 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 T "a^,1,\'` ^0 1\ c, \-.'-- 1\'^t)^-4-Cj (1,N‘ 0^ 'y N N/ ,t)‘‹ '`i.'^cl-^p[-b:^i'r'' N0^\-' `b. QUO ^BOO9 9`'. ,,;>^C)^ri,,C‘('^' b,<< 0 '5. N'g^\->'''^,i,•'5 .^ \ - 0^\.^'' '5.Q 0 (9 \.\S?-.Q^g 0 Q Figure 4.6: Overexpression of exogenous xCT increases glutamate uptake and increases GEM resistance. (a) RT-PCR for xCT and 4F2hc expression in MIA PaCa-2 cells transfected with pcDNA3.1 empty vector, pcDNA3.1-xCT plasmid, or pcDNA3.1-4F2hc plasmid. (b,c) Q-RT-PCR for xCT (b) or 4F2hc (c) expression in MIA PaCa-2 cells transfected with pcDNA3.1 empty vector, pcDNA3.1-xCT plasmid, or pcDNA3.1-4F2hc plasmid. *P < 0.001. (d) [3 1-1]-glutamate uptake assay in MIA PaCa-2 and PANC-1 cells transfected with pcDNA3.1 empty vector, pcDNA3.1-xCT plasmid, and/or pcDNA3.1-4F2hc plasmid. *fp < 0.01 compared to respective cell line pcDNA3.1 control. (e) Neutral red uptake assay for survival of untreated or GEM-treated (0.01 pM) MIA PaCa-2 cells transfected with pcDNA3.1 empty vector, pcDNA3.1-xCT plasmid, and/or pcDNA3.1-4F2hc plasmid. *fp .̂ 0.05 compared to pcDNA3.1 control; #P 0.05 compared to pcDNA3.1 GEM treatment. All data represent the mean ± SEM from three independent experiments. 95 activity (Figure 4.6d). Transfection of 4F2hc cDNA alone, however, was not sufficient to upregulate x, - transporter activity (Figure 4.6d). Furthermore, transfection of both xCT and 4F2hc cDNA was required to increase GEM resistance in MIA PaCa-2 cells (Figure 4.6e). Hence upregulation of xCT subunit expression is necessary to upregulate x: transporter activity, with a corresponding increase in GEM resistance. These results suggest that overexpression of the x, - transporter, as seen in primary pancreatic ductal adenocarcinoma specimens, may increase pancreatic cancer cell survival in response to GEM treatment. 96 4.3 DISCUSSION The x," transporter has been shown in various cancers to supply cells with cystine for GSH synthesis resulting in increased proliferation and GSH-related drug resistance (Doxsee et al., 2007; Narang et al., 2003). The goal of this project was to investigate whether a similar system was also used by pancreatic cancer cells for growth, survival and drug-related resistance. In the present study, the human pancreatic cancer cell lines MIA PaCa-2, PANC-1, and BxPC-3 were determined to be critically dependent on extracellular cystine for growth (Figure 4.1). When cultured in media in the absence of either methionine, a nutritionally essential amino acid, or cystine, a non-nutritionally essential amino acid, pancreatic cancer cells were unable to survive and proliferate, indicating that these cells were unable to make their own intracellular cysteine from methionine. This finding demonstrates that the biochemical cysteine synthesis pathway known as the transsulfuration pathway does not participate in alleviating cystine depletion. Some cell types, such as neurons and astrocytes, do not rely on the ?c c" transporter for growth (Chung et al., 2005). As such, these cells (i) may possess a functional transsulfuration pathway, (ii) may express other transporters that mediate the transport of cystine or cysteine into the cell (e.g. excitatory amino acid transporters (EAATs)) (McBean and Flynn, 2001), or (iii) may not express the x e" transporter but instead may rely solely on the uptake of extracellular cysteine secreted from other cell types (Chung et al., 2005). The potential utility of screening tumor biopsies for the presence of enzymes in the transsulfuration pathway (i.e. for 1 ,- cystathionase) to determine whether a patient may benefit from cystine starvation therapy remains a possibility. Because our results indicate that pancreatic cancer cell lines are dependent on extracellular cystine/cysteine for growth, these cell lines may exhibit sensitivity towards depleted 97 cysteine/cystine levels in the media. Indeed, at least two of the three cell lines tested showed an inverse correlation between a low extracellular cystine environment (0.01 mM) and expression of the transporter, suggesting that these cells can modulate their expression of the x c - transporter to accommodate their growth needs. Consistent with published reports, cell proliferation is strongly associated with cystine/cysteine availability and intracellular GSH levels (Godwin et al., 1992; Noda et al., 2002). Besides manipulating cysteine/cystine levels in the extracellular microenvironment to effect a change in x e - transporter expression, the addition of the oxidative stressor DEM was also used. In accordance with other studies (Bannai, 1984a; Hosoya et al., 2002; Kim et al., 2001), DEM treatment induced xCT mRNA expression with a corresponding increase in total GSH, indicating that increased intracellular GSH synthesis enables pancreatic cancer cells to survive in the presence of oxidative stress. In human embryonic kidney (HEK) cells, xCT expression has been reported at the plasma membrane (Shih and Murphy, 2001). Of interest is the BxPC-3 cell line which, upon treatment with DEM, clearly exhibited localization of the xCT subunit to the plasma membrane (Figure 4.3d). The 4F2hc subunit is known to translocate other light subunits to the plasma membrane for functional activity to occur (Chillaron et al., 2001; Verrey et al., 2004), suggesting that xCT subunits located in the plasma membrane of BxPC-3 cells are likely functional. The fact that prominent xCT protein localization to the plasma membrane was not seen in the other cell lines tested may be explained by the observation that BxPC-3 cells morphologically grow in tighter groups of cells, facilitating the visualization of xCT protein at cell-cell (intercellular) plasma membrane junctions. In contrast to BxPC-3 cells, DEM induced upregulation of xCT protein in MIA PaCa-2 and PANC-1 cells within the intracellular compartment (Figure 4.3d). Indeed, subcellular localization studies of xCT in HEK cells have 98 reported xCT expression not only at the plasma membrane, but also at intracellular membranes such as the lysomsomal or endosomal membranes (Shih and Murphy, 2001). The functional significance of intracellular xCT expression, however, remains to be determined. Although the use of the oxidative stressor DEM was able to increase xCT expression, it did not have an effect on 4F2hc mRNA or protein expression (Figure 4.3b,c). The xCT promoter region contains an ARE that regulates transcription of the gene (Ishii et al., 2000; Wasserman and Fahl, 1997). In contrast, the presence of an ARE has not been reported in the 4F2hc promoter region, suggesting a possible explanation for the observed difference in response to oxidative stress for the two genes. Alternatively, basal levels of 4F2hc may be much higher than that of xCT, potentially rendering changes in 4F2hc expression undetectable. Indeed, in a blood- retinal barrier cell line, mRNA levels of 4F2hc were reported to be 56-fold higher than that of xCT (Tomi et al., 2002). This suggests that 4F2hc mRNA in some cells is in excess, most likely due to the fact that 4F2hc is a common component of many other amino acid transport systems. Among normal tissues, xCT is expressed predominantly in normal pancreas (Bassi et al., 2001; Kim et al., 2001), specifically in the islet cell population (Bassi et al., 2001). Furthermore, xCT has also been reported to exhibit higher expression in an acinar cell line compared to a pancreatic islet cell line (Sato et al., 1998). The present study shows that xCT is expressed in normal pancreatic tissues preferentially in the ductal cells compared to the acinar cells. Importantly, in primary human pancreatic ductal adenocarcinoma specimens, xCT protein is overexpressed throughout cancerous ductal structures (Figure 4.4). Given that the x e - transporter is overexpressed in pancreatic cancers, and that expression of the x e - transporter can be induced in pancreatic cancer cell lines in response to oxidative stress, our findings implicate the x, - transporter as an important mediator of pancreatic cancer cell proliferation and survival. 99 The higher xCT expression by the PANC-1 cells suggest that they have more x e- cystine transporters per cell than the other cells and hence a greater ability to take up cystine from the microenvironment, likely synthesizing more GSH when needed. It has been reported that NIH3T3 cells treated with sublethal levels of DEM to induce ROS defense genes such as xCT and glutathione-S-transferase al (GSTa 1) are associated with higher resistance to various apoptotic stimuli such as buthionine sulfoximide, ultraviolet, and etoposide (Faraonio et al., 2006). Moreover, HT22 hippocampal cells resistant to oxidative stress exhibited a 7-fold overexpression of xCT mRNA, with a concomitant increase in resistance to glutamate-induced oxidative stress and hydrogen peroxide-induced oxidative stress (Lewerenz et al., 2006). In the present study, although PANC-1 cells express a 3.5-fold higher level of xCT mRNA compared to the MIA PaCa-2 and BxPC-3 cell lines, PANC-1 cells are 1000X more resistant to GEM. This suggests that GEM resistance cannot be solely explained by higher xCT mRNA expression, but rather that additional GEM resistance mechanisms are at play. Nonetheless, overexpressing exogenous xCT and 4F2hc in pancreatic cancer cells resulted in an overall increase in the GEM resistant phenotype (Figure 4.6). The fact that overexpressing the x e- transporter was able to increase cell survival even in the absence of GEM treatment highlights the importance of the x, - transporter as a regulator of pancreatic cancer cell proliferation and survival. My results demonstrate that pancreatic cancer cells exhibit upregulated expression of the xe - transporter, which promotes the uptake of extracellular cystine for the synthesis of GSH. In turn, elevated intracellular GSH levels promote pancreatic cancer cell growth and survival in response to the production of ROS by oxidative stressors, including the chemotherapeutic agent GEM. Hence drug resistance in pancreatic cancer may be explained in part by the overexpression 100 of the x, - transporter, raising the possibility that selectively targeting the x e - transporter may be of therapeutic potential. 101 Chapter 5 INHIBITING THE xc - TRANSPORTER IN PANCREATIC CANCER 5.1 ABSTRACT Pancreatic cancer is notorious for its drug resistant phenotype (Hezel et al., 2006; Zalatnai and Molnar, 2007). In the previous chapter, it was demonstrated that the 'cc - transporter mediates uptake of extracellular cystine for the synthesis of GSH in pancreatic cancer cells, and that expression of the x e" transporter is elevated in primary human pancreatic cancer. In response to elevated levels of intracellular GSH, pancreatic cancer cells exhibit enhanced cell growth and protection from oxidative stress when exposed to DEM or the chemotherapeutic drug GEM. I next investigated the effect of inhibiting the xc - transporter on pancreatic cancer cell growth and drug resistance using two approaches: (i) siRNA against the xe" transporter, and (ii) treatment with SASP, an anti-inflammatory drug that specifically inhibits the ?c c - transporter. I also determined the effect of SASP in combination with GEM on pancreatic cancer cell growth in vitro and in vivo. Furthermore, the kinetic mechanism of SASP as a x e - transporter inhibitor was assessed, as well as its cell growth inhibitory action in relation to the NFKB pathway. In terms of kinetic mechanism, I identify SASP as a mixed inhibitor of the x e- transporter, which acts to inhibit pancreatic cancer cell proliferation via a mechanism that involves reduced xc - transporter activity and not reduced NFKB activity. Importantly, my studies demonstrate that inhibiting x c " transporter expression or function reduces pancreatic cancer cell proliferation concomitant with a restoration of sensitivity to GEM. Hence inhibition of the x, - transporter may be a useful strategy to sensitize drug-resistant pancreatic cancer cells to chemotherapy. 102 5.2 RESULTS 5.2.1 Targeting x c" transporter expression with siRNA Using pooled siRNAs targeted against xCT or 4F2hc, expression of endogenous xCT or 4F2hc was downregulated in MIA PaCa-2 and PANC-1 cell lines (Figure 5.la,b), and inhibition of xe - transporter function was confirmed by a reduction in L-[ 3H]-glutamate uptake (Figure 5.1c). To determine whether reduced xc transporter function correlated with increased sensitivity to GEM, PANC-1 cells transfected with either xCT siRNA or 4F2hc siRNA were treated with GEM at 100 ?AM for 72 h and cell survival was determined by neutral red assay. Whereas 4F2hc- downregulation did not decrease cell survival in response to GEM treatment, xCT siRNA elicited a modest decrease in cell survival, compared to control cells transfected with the mock treatment (Figure 5.1d). Of note, downregulated expression of either xCT or 4F2hc alone, independent of GEM treatment, resulted in decreased PANC-1 cell survival, indicating a requirement for the x e- transporter in pancreatic cancer cell survival (Figure 5.1d). Hence downregulating expression of the transporter, in particular the xCT subunit of the transporter, decreases pancreatic cancer cell survival by inhibiting cystine uptake with a concomitant increase in GEM sensitivity. 5.2.2 Targeting x c" transporter function with SASP It has previously been shown that SASP, an non-steroidal anti-inflammatory drug used for the treatment of rheumatoid arthritis and inflammatory bowel disease, is an effective inhibitor of the transporter (Gout et al., 2001). When used in vitro at patient-tolerable levels (0.1-0.3 mM) (G101-14), SASP inhibited the growth of various cancer cell types by specific inhibition of cystine uptake (Gout et al., 2001; Narang et al., 2003). Importantly, SASP treatment via 103 00. 0) X C a) a) C 0 0 X 1:3 > 0 a 1.6 - 1.4 - 1.2 - 1.0 - 0.8 - 0.6 - 0.4 0.2 0.0 Ili MIA PaCa-2 l PANC-1 PANC-1 II Control 0 GEM ^■ MIA PaCa-2c^1.2 o PANC-1.....o I^ o^1.0Q, ' .._-,-,..,x c7) 0.8 ^ *^ C.) 0 0.6 r ^.0 . r-- (NI 0LI- -cry v 0 0.4 - 0 ......, --1-111-- ,^ tc;cu^ 0.2 ^CC ^- 0.0 ^ ■ MIA P aCa-2 0 PANC-1 Control^xCT^4F2hc ^ Control ^ xCT ^ 4F2hc siRNA siRNA siRNA siRNA siRNA siRNA c^ d 1.2 1.0ro a -8, 0.8 -5 0.6 c 0 0.4 0.2 0.0 Control^xCT^4F2hc siRNA siRNA siRNA 1.5 _ '701, 1.0 C> -5 n o 0.5 0.0 `caper ti  o , s1/40 ry Figure 5.1: Targeting xc- transporter expression with siRNA. (a,b) Q-RT-PCR for xCT (a) or 4F2hc (b) expression in MIA PaCa-2 and PANC-1 cells transfected with control siRNA or siRNAs targeting xCT or 4F2hc. *P < 0.05. (c) ['I-IF-glutamate uptake in MIA PaCa-2 and PANC-1 cells transfected with control siRNA or siRNAs targeting xCT or 4F2hc. *P < 0.01. (d) Neutral red uptake assay for cell survival in Panc-1 cells either untreated or treated with 100 pM GEM for 72 h. Cells were subjected to mock transfection, transfection reagent alone, or transfection with control siRNA or siRNAs targeting xCT or 4F2hc. *P 0.05 compared to control mock; #P s 0.05 compared to GEM mock. All data represent the mean ± SEM from three independent experiments. 104 intraperitoneal administration in vivo can inhibit the growth of certain tumor xenografts, such as prostate cancer (Doxsee et al., 2007). To determine whether SASP would inhibit the growth of pancreatic cancer cells, neutral red uptake assays were performed. SASP treatment of MIA PaCa-2, PANC-1, and BxPC-3 cell lines yielded IC 50s of 0.01, 0.05 and 0.35 mM, respectively (Figure 5.2a). 2-ME allows for cellular uptake of cystine (as a mixed disulfide of 2-ME and cysteine) via the leucine transporter (Ishii et al., 1981a), thus acting via an alternate route for cystine transport that is not inhibited by glutamate (Ishii et al., 1981a). Addition of 2-ME was able to negate the growth inhibitory effects of SASP at concentrations of 0.2 and 0.5 mM (Figure 5.2b). Hence growth inhibition by SASP was due to decreased xj transporter-mediated cystine uptake, as previously observed with lymphoma (Gout et al., 2001; Gout et al., 1997), breast (Narang et al., 2003), and prostate cancer cell lines (Doxsee et al., 2007; Narang et al., 2003). Of note, the BxPC-3 cell line exhibited the highest 10 50 with SASP, suggesting that this cell line may contain additional mechanisms that promote cell proliferation in cystine-depleted conditions. MSG is a specific inhibitor of the xc transporter since the uptake of cystine can be competitively inhibited by glutamate (Bannai, 1986). Consistent with this, pancreatic cancer cells treated with MSG exhibited inhibited growth, with the addition of 2-ME to MSG-treated samples resulting in a complete restoration of growth (Figure 5.2c). Moreover, the relative sensitivities of the three pancreatic cancer cell lines towards SASP and MSG were similar, with MIA PaCa-2 and PANC-1 cell lines exhibiting greater sensitivity to both MSG and SASP compared to BxPC-3 cells (Figure 5.2b,c). Taken together, our findings demonstrate that SASP inhibits the growth of pancreatic cancer cells by targeting the x, - transporter. 105 ■ MIA PaCa-2 El PANC-1 08xPC-3 * b^2.0 - 1.8 - 1.6 - co 0,1.4 - 1.2 1 .0 - 12' 3 0.8 - - 0.6 0.4 0.2 0.0 2-ME SASP (0.2 mM) SASP (0.5 mM) * * * C^2.0 1.8 1.6 c0 0, 1.4 - c. ;12 cts^1.2 <13 -5 1 . 0 - a.113. 2o 0.8 - 0.6 0.4 - 0.2 - 0.0 2-ME MSG (0.8 mM) MSG (4.0 mM) ■ MIA PaCa-2 O PANC-1 ^ BxPC-3 * * -2 -1 . 5 -1 -0 . 5 0 0.5^1.5 tog [SASP] (mM) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -25 Figure 5.2: Targeting x c . transporter function with SASP. (a) Neutral red uptake assay for cell proliferation in MIA PaCa-2, PANC-1, and BxPC-3 cells treated with increasing concentrations of SASP for 72 h. (b) Neutral red uptake assay for cell proliferation in MIA PaCa-2, PANC-1, and BxPC-3 cells treated with SASP (0.2 or 0.5 mM) or 2-ME (66 vt.M) in various combinations for 72 h. *P 0.05. (c) Neutral red uptake assay for cell proliferation in MIA PaCa-2, PANC-1, and BxPC-3 cells treated with MSG (0.8 or 4.0 mM) or 2-ME (66 ktM) in various combinations for 72 h. *P s 0.05. All data represent the meant SEM from three independent experiments. 106 I next determined whether the inhibitory effect of SASP on pancreatic cancer cell growth was due to inhibited ?cc - transporter expression and/or function. Although SASP did not affect xCT or 4F2hc mRNA expression in the three pancreatic cancer cell lines (Figure 5.3a,b), SASP used at either 0.2 mM or 0.5 mM decreased the uptake of L-radioactive carbon 14 ([ 14C])-cystine in MIA PaCa-2 and PANC-1 cells (Figure 5.3c). This inhibition of L-[ 14C]-cystine uptake was completely prevented and in fact was increased by the addition of 2-ME (66 1AM) (Figure 5.3c). Furthermore, competitive substrates of L-[ 14C]-cystine (such as unradiolabelled L-glutamate and L-cystine) decreased the uptake of L-[ 14C]-cystine, whereas non-substrates (such as L-leucine) had no effect on L-[' 4C]-cystine uptake (Figure 5.3c). Hence the mechanism of action of SASP in pancreatic cancer cells involves the inhibition of ?c c" transporter function, but not expression. To determine whether SASP-inhibited transporter function resulted in reduced intracellular GSH levels, pancreatic cancer cells were incubated with SASP in the absence and presence of 2-ME for 24 h. MIA PaCa-2 and PANC-1 cells treated with SASP at 0.15 mM and 0.5 mM exhibited reduced GSH synthesis, which could be completely prevented by the addition of 2-ME (66 viM) (Figure 5.4a). Given that cells treated with SASP remained viable for up to 48 h (Figure 5.4b), these data suggest that inhibiting the function of the 'c c" transporter with SASP is sufficient to inhibit GSH synthesis within 24 h, and that the inhibition of GSH synthesis precedes a reduction in cell survival. 5.2.3 Effect of SASP on sensitivity to GEM in vitro and in vivo Thus far we have demonstrated that SASP is able to deplete GSH levels in pancreatic cancer cells. Given that depleted GSH levels can increase drug sensitivity in various cell types (Doxsee et al., 2007; Narang et al., 2003), we determined whether SASP-mediated GSH depletion would result in enhanced sensitivity to GEM. The pancreatic cancer cell lines MIA 107 QP b ■ Control^a^ ■ Control 0 SASP .v)_ c) 2.0^0 SASP 2 Q. Iii 1.5 x C)a) c ^ co.c _c 1.0 -CA t., L41)^0.5 -72.-'>74al 715 •^ 0 . 0 N Ci-6';1"^\-C1 '''^<i?"^e:.-#.ee a • 2.0 2 -a—) 1.5 ■ MIA PaCa-2 0 PANC-1^*C^4.5 Y^4.0 0_ 0 3.5• c5) 3.0 a) a,c^2.5 -0u 2.0 (-? o^1.5 - to 0.5 - 0.0 2-ME L-Glutamate L-Cystine L-Leucine SASP (0,2 mM) SASP (0.5 mM) Figure 5.3: Effect of SASP on xc - transporter expression and function. (a,b) Q-RT-PCR for xCT (a) or 4F2hc (b) expression in MIA PaCa-2, PANC-1, and BxPC-3 cells either untreated or treated with 0.2 mM SASP for 48 h. (c) [' 4C)-cystine uptake in MIA PaCa-2 and PANC-1 cells treated with SASP (0.2 or 0.5 mM) in the absence or presence of 2-ME (66 4M), competitive substrate (1 mM glutamate or cystine) or non-substrate (1 mM leucine) for 24 h. *P 5_ 0.05. All data represent the mean ± SEM from three independent experiments. 108 a 1.6 - 1.4 • 1.2 -(T.1 cn> ca) co^1.0 -.c• (..)^0.8 - o • 0.6 0.4 0.2 0.0 2-ME SASP (0.15 mM) SASP (0.5 mM) ■ MIA PaCa-2 PANC-1 ■ MIA PaCa-2 PANC-1 ^ BxPC-3 b 1.6 1.4 • 1.2 a)• 1.0 f, 0.8 M 0.6 0.4 0.2 0.0 0^24^48^72 Time treated with 0.2 mM SASP (h) Figure 5.4: Effect of SASP on intracellular GSH levels. (a) Intracellular GSH levels in MIA PaCa-2 and PANC-1 cells treated with SASP (0.15 mM or 0.5 mM) and 2-ME (66 It.M) for 24 h. (b) Neutral red uptake assay for cell survival in MIA PaCa-2, PANC-1, and BxPC-3 cells treated with 0.2 mM SASP for 0, 24, 48, and 72 h. All data represent the mean ± SEM from three independent experiments. *P 0.05. 109 PaCa-2, PANC-1, and BxPC-3 were subjected to pre-treatment with SASP for 24 h (resulting in depleted GSH levels without a reduction in cell survival (Figure 5.4a,b)) followed by incubation with GEM for an additional 72 h. Pre-treatment with 0.1 mM SASP alone for 24 h depleted GSH levels (Figure 5.4a) without affecting the survival of all three cell types (Figure 5.5). Treatment with 10 nM GEM alone for 72 h caused significant cell death in MIA PaCa-2 and BxPC-3 cell lines (Figure 5.5). In contrast, GEM treatment exerted a reduced effect on PANC-1 cell survival (Figure 5.5), thus indicating that PANC-1 cells are relatively resistant to GEM. Importantly, 0.1 mM SASP pre-treatment followed by 10 nM GEM treatment increased cell death in all three cell lines, including the GEM-resistant PANC-1 cell line, to a greater extent than GEM alone (Figure 5.5). Hence SASP treatment, via depletion of GSH levels, can increase pancreatic cancer cell sensitivity to GEM in vitro. I next determined whether SASP would enhance pancreatic cancer cell sensitivity to GEM in vivo. To this end, mice bearing actively growing MIA PaCa-2 and PANC-1 subcutaneous xenografts were treated with PBS control, SASP alone, GEM alone, or SASP and GEM in combination. To avoid intra-intestinal cleavage of SASP to sulfapyridine and 5-amino- salicylic acid (Klotz, 1985), which would decrease the inhibitory activity of SASP (Gout et al., 2001), all treatments were administered by intraperitoneal injection. MIA PaCa-2 xenografts treated with either SASP or GEM alone exhibited inhibited growth, with combination treatment inhibiting tumor growth to the greatest extent (Figure 5.6a). In contrast, PANC-1 xenografts were relatively resistant to treatment with SASP or GEM alone (Figure 5.6a). Importantly, combination treatment with SASP and GEM resulted in marked inhibition of PANC-1 xenograft growth (Figure 5.6a). Hence SASP treatment increased the in vivo sensitivity of pancreatic cancer cells to GEM. MIA PaCa-2 and PANC-1 xenografts treated with SASP and GEM alone 110 1.2 1.0 7-0 En 0.8 .> -5 0.6 (1) 0 0.4 - 0.2 - 0.0 ^ SASP GEM  ■ MIA PaCa-2 PANC-1 ^ BxPC-3 ** * Figure 5.5: Effect of SASP on sensitivity to GEM in vitro. Neutral red uptake assay for cell survival in MIA PaCa-2, PANC-1, and BxPC-3 cells incubated in MEM (control), 0.1 mM SASP alone at time 0-24 h, 10 nM GEM alone at time 24-96 h, or 0.1 mM SASP at time 0-24h followed by 10 nM GEM at time 24-96 h. Data represent the mean ± SEM from three independent experiments. #P 0.05, 0.01 and "P < 0.001. 111 a^MIA PaCa-2 xenografts^PANG-1 xenografts 2.5 2.0 1.5 1.0 0.5 0.0 c,y \I\ C'< Control GEM • * GEM+SASPC 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 co c° SASP b 70 60 12 50 40 co 30 %E. 20 u) o 10 0 80 70 60 50 40 30 20 10 0 c;q^4^4.0\ c)(^c,4 CP^cY. \-1\ MIA PaCa-2 xenografts ■ KI67o Caspase-3 PANG-1 xenografts • Kr67 caCaspase-3 • • • a Figure 5.6: Effect of SASP on sensitivity to GEM in vivo. Rag-2M mice harboring established subcutaneous MIA PaCa-2 or PANC-1 xenografts (grown for five weeks after tumor implantations; average tumor volume of 50 mm 3) were given intraperitoneal injections of PBS control (twice daily for two weeks; n=6), SASP (250 mg/kg; twice daily for two weeks), GEM (120 mg/kg; once weekly for two weeks; n=6), or GEM+SASP (n=6). (a) Tumor volumes of MIA PaCa-2 and PANC-1 xenografts. < 0.05. (b) Quantification of Ki67 and active caspase-3 staining in MIA PaCa-2 and PANC-1 xenografts.*P < 0.01. (c) Immunohistochemistry for active caspase-3 in MIA PaCa-2 and PANC-1 xenografts. 112 or in combination did not exhibit reduced staining for the proliferation marker Ki67 compared to treatment controls (Figure 5.6b). Rather, GEM treatment alone or in combination with SASP increased the percentage of activated caspase3-positive cells in both xenograft types (Figure 5.6b,c). Taken together, our findings demonstrate that SASP treatment, via inhibiting xj transporter function to effect GSH depletion, may be a useful mechanism to restore GEM sensitivity in GEM-resistant pancreatic cancer cells. 5.2.4 Kinetic studies of SASP as an inhibitor of the x c - transporter in pancreatic cancer cells Enzyme inhibitors can be classified into four major types: competitive, non-competitive, uncompetitive, and mixed (Whiteley, 2000; Zhang and Wong, 2005). I sought to determine if the inhibitory activity of SASP on x c . transporter function could be classified into one of these types. To accomplish this, I first performed time course experiments to establish experimental conditions for the steady-state (i.e. linear) transport of L-[ 3H]-glutamate into MIA PaCa-2 and PANC-1 cell lines. In both cell lines, uptake of L-[ 3H]-glutamate increased linearly for the first 30 min of incubation at 37 °C (Figure 5.7a,b). As expected, SASP pre-treatment for 1 h inhibited L.-CM-glutamate uptake by both MIA PaCa-2 and PANC-1 cells (Figure 5.7a,b). Of note, PANC-1 cells reached a higher L-[ 3 H]-glutamate uptake rate after 30 min incubation compared to MIA PaCa-2 cells (Figure 5.7a,b), consistent with our earlier findings demonstrating greater xCT mRNA expression in PANC-1 cells relative to MIA PaCa-2 cells (Figure 4.5a). I therefore used PANC-1 cells for subsequent kinetic studies of SASP inhibition. PANC-1 cells, either untreated or pre-treated with 0.5 mM SASP for 1 h, were incubated with L-[ 3 H]-glutamate at concentrations ranging from 10-700 jAM for 30 min, and L-[ 3H]- glutamate uptake was measured. In the absence of SASP pre-treatment, accumulation of L-[ 3M- 113 Mia PaCa-2 350 ♦Control 300 • SASP 250 200 150 100 50 0 0 5 10 15 20 25 30 35 Time (min) a Panc-1 600 *Control 500 • SASP 400 300 200 100 0 0 5 10 15 20 25 30 35 Time (min) Panc-1 N a> — .1c E won E rn 10 9 8 7 6 5 4 3 2 1 0 0 —4—Control —s—SASP 200 400 600 800 [glutamate]pM ♦ Control 782^■ SASP a) _Ne C_ S2 cy) E E  3as 7 25. •E' 9' ca- 2 d -0.04 -0^0 0 0.02 0.04 0.06 0.08 0.10 0.12 -1^1 / [glutamate] (1.1M) Figure 5.7: Kinetic studies of SASP as an inhibitor of the x c - transporter in pancreatic cancer cells. (a,b) [3H]-glutamate (33 nM) uptake in Mia PaCa-2 (a) or Panc-1 (b) cells either untreated or treated with 0.5 mM SASP over time. Each data point represents the mean ± SEM (n=3). (c) [3H]-glutamate uptake in Panc-1 cells either untreated or treated with 0.5 mM SASP. Varying concentrations of [3H]-glutamate and cold glutamate were added to cells to give a total glutamate concentration of 700 1AM. Each data point represents the mean ± SEM (n=3). (d) Lineweaver-Burk plot of initial velocity as a function of substrate concentration for the transport of [ 3H]-glutamate by the xc- transporter in Panc-1 cells. 114 glutamate occurs in a concentration-dependent manner and the curve approaches an asymptote (Figure 5.7c), thus indicating saturation of the binding site on the x e - transporter. Pre-treatment with SASP, however, causes a marked reduction in L-[ 3H]-glutamate uptake, resulting in an uptake curve that does not approach saturation (Figure 5.7c). By plotting the double reciprocal (Lineweaver-Burk) of the uptake data in Figure 5.7c, the kinetic parameters K m (substrate concentration at which the uptake rate is half-maximal) and Vmax (maximum uptake rate) can be determined. K m was determined to be —112 [1.1\4 and —154 tiM in the absence or presence of SASP, respectively (Figure 5.7d), thus confirming that SASP inhibited x e - transporter function. Consistent with this finding, Vmax was reduced by SASP treatment (-4.7 pmols/mg protein/min and --3.1 pmols/mg protein/min in the absence or presence of SASP, respectively) (Figure 5.7d). Importantly, the two trend lines of L-[ 31-11-glutamate uptake in the absence or presence of SASP did not intersect the ordinate (a characteristic of competitive inhibitors) (Whiteley, 2000) or the abscissas (a characteristic of non-competitive inhibitors) (Whiteley, 2000) (Figure 5.7d), thus demonstrating that SASP functioned as a mixed inhibitor (Figure 5.7c). These data indicate that SASP inhibits xc - transporter function by binding to both the transporter itself as well as the transporter-substrate complex, without directly interacting with the substrate binding site. 5.2.5 Investigating the mechanism of SASP action: NFKB activation Our results demonstrate that SASP exhibits anti-neoplastic activity in pancreatic cancer cells by inhibiting the function of the 'c c - transporter, resulting in reduced cystine uptake with a corresponding decrease in intracellular GSH levels. Previous studies have suggested that the anti-neoplastic action of SASP in various cancer cell types involves the inhibition of NFKB activation (Lay et al., 2007; Muerkoster et al., 2003; Robe et al., 2004). I therefore sought to determine whether the inhibition of pancreatic cancer cell proliferation by SASP was due to 115 inhibited NFKB activation in addition to inhibited x c - transporter function. Incubation of MIA PaCa-2 cells with SASP for 24 h reduced both total GSH levels (Figure 5.4a) and TNFa-induced NFKB activity (Figure 5.8a), with no observable loss of cell viability (Figure 5.4b). Inclusion of 2-ME in SASP-containing cultures was sufficient to maintain GSH at control levels (Figure 5.4a) and prevent growth arrest (Figure 5.2b); however, NFKB activity remained attenuated (Figure 5.8a). Similar results were obtained with PANC-1 cells (Figures 5.2b, 5.4a,b, and 5.8b). Thus cultures containing both SASP and 2-ME exhibited normal growth despite significantly reduced NFKB activity. Growth of MIA PaCa-2 and PANC-1 cells was also arrested by MSG, a potent inhibitor of the xe- transporter (Figure 5.2c), without affecting NFKB activation (Figure 5.8a,b). Taken together, these results demonstrate that growth arrest of pancreatic cancer cells by SASP, at relatively low concentrations (0.2 mM), is primarily due to inhibition of xc - transporter function but not inhibition of NFKB activation. 116 •(-)TNF-a (+) TNF-cx Panc-1 ■ (-) TNF-a o (+) TNF-a * Mia PaCa-2 0.10 5- 0.09 2 0.08 0.07 0.06 Cts^0.05 z 0.04 z 03 0.03 0.02 0.01 0.00 2-ME MSG (10 mM) SASP (0.2 mM) SASP (0.5 mM) b 0.7 - '5- 0.6 2 0.5 5 cL r) a 0.4 -m S efl z 0.1 - 0.0 ^ 2-ME MSG (10 mM) SASP (0.2 mM) SASP (0.5 mM) Figure 5.8: Investigating the mechanism of SASP action: NFKB activation. (a,b) NFKB-promoter luciferase assay in Mia PaCa-2 (a) or Panc-1 (b) cells either untreated or treated with TNF-a for 3 h to induce NFKB activation, then treated with SASP (0.2 or 0.5 mM) or MSG (10 mM) in the absence or presence of 2-ME for 24 h. Data represent the mean ± SEM from three independent experiments. N.D.: not determined. *P < 0.05 with respect to no treatment controls. 117 5.3 Discussion Pancreatic cancer is highly drug resistant and thus the development of novel therapies to restore sensitivity to chemotherapeutic drugs is mandated. I initially used siRNA targeted to xCT or 4F2hc to assess if pancreatic cancer cells could be sensitized to GEM, the chemotherapeutic drug currently used in first-line pancreatic cancer treatment. Using the pancreatic cancer cell line PANC-1, which is highly resistant to GEM treatment, I demonstrated that downregulating transporter expression (via siRNA targeted to the xCT subunit of the transporter) was sufficient to effect a decrease in GEM resistance, although the degree of drug sensitization was minimal (Figure 5.1d). Hence inhibiting expression of the^transporter results in a partial restoration of pancreatic cancer cell sensitivity to GEM. It has been reported that certain anti-inflammatory drugs exert an inhibitory action on x e - transporter function by inhibiting the uptake of cystine (Bannai and Kasuga, 1985). SASP, an anti-inflammatory drug previously shown to specifically target x," transporter function in various cancer cell types (Doxsee et al., 2007; Gout et al., 2001; Narang et al., 2003), was used to determine whether inhibition of x e- transporter function, rather than expression, would be sufficient to restore pancreatic cancer cell sensitivity to GEM. Without affecting x e - transporter expression, SASP treatment alone markedly inhibited xj transporter function, resulting in decreased cystine uptake, decreased intracellular GSH levels, and ultimately decreased pancreatic cancer cell proliferation and survival (Figures 5.2-5.4). Importantly, combination SASP and GEM treatment resulted in a greater inhibition of pancreatic cancer cell survival in vitro and tumor growth in vivo when compared to SASP or GEM alone (Figures 5.5,5.6). However, combination SASP and GEM treatment induced apoptosis to the same degree as GEM treatment alone (Figure 5.6c). This discrepancy between different tumor burdens and similar 118 apoptotic levels may suggest that combination SASP and GEM treatment may be targeting a minor cell population for apoptosis that is not affected by GEM treatment alone, and that the apoptosis of this minor population may not be reflected in the overall apoptotic levels for the tumor as a whole. One possible cell population includes endothelial cells, which are essential for angiogenesis and hence tumor growth (Hicklin and Ellis, 2005). A second cell population may be tumor-associated macrophages, which have been reported to promote tumor growth in various cancer types (Loberg et al., 2007; Porta et al., 2007). By inducing the apoptosis of these minor cell populations, combination SASP and GEM treatment, in contrast to SASP treatment alone, may effect an overall decrease in tumor burden without a detectable increase in apoptosis. An alternative explanation for the observed discrepancy between tumor burdens and apoptotic levels may be the elimination of pancreatic cancer stem cells by combination SASP and GEM treatment. Cancer stem cells have recently been identified in pancreatic tumors, and are believed to give rise to more differentiated progeny that comprise the bulk of the tumor (Li et al., 2007). The possibility that pancreatic cancer stem cells may be rendered sensitive to apoptosis induced by combination SASP and GEM treatment, but not by GEM treatment alone, may explain the observed reduction in tumor burden without a detectable increase in tumor apoptosis. Nonetheless, our data demonstrate that SASP treatment, by inhibiting the function of the x c - transporter, is sufficient to enhance GEM sensitivity in pancreatic cancer cells. The underlying mechanism of action of SASP as an anticancer agent has not been precisely defined. We have demonstrated that SASP can inhibit the function of the transporter in pancreatic cancer cells. To determine if this inhibition occurs via a competitive, non- competitive, un-competitive, or mixed mechanism, enzyme kinetic assays were performed. My data demonstrate that SASP functions as a mixed inhibitor to block x e - transporter function, with 119 0.5 mM SASP decreasing the Vmax and increasing the Km in a glutamate uptake assay. Of interest, the non-steroid anti-inflammatory drug indomethacin was found to be a non-competitive inhibitor of the x: transporter, with 0.5 mM indomethacin decreasing the Vmax while having little effect on the K m in a cystine uptake assay (Bannai and Kasuga, 1985). Taken together, these findings demonstrate that small molecule inhibitors of the x: transporter can act via different mechanisms to yield a similar block in transporter function. Glutamate and cystine are interchangeable substrates for the x: transporter (Bannai, 1986; Murphy et al., 1989; Sato et al., 1999). The physiological concentration gradient of glutamate, which is much higher intracellularly than extracellularly, provides the driving force for cystine uptake in exchange for glutamate in a normal in vivo situation (Bannai and Ishii, 1988; Christensen, 1990). When added to culture media in vitro, this glutamate gradient is reversed and hence the x: transporter functions to promote glutamate uptake. I used L-0-11-glutamate instead of L-[ 14 C]-cystine whenever possible in our uptake assays for several reasons. One reason is that radioactive cystine has been reported to quickly convert to L-cysteine within the cell and to be released into the culture medium (Bannai and Ishii, 1982; Ye et al., 1999). Subsequently, the L- cysteine in the media can be oxidized to cystine that can again become a substrate for the x: transporter (Bannai and Ishii, 1982; Bannai and Ishii, 1988; Ye et al., 1999). This constant cycling of cystine and cysteine would preclude an accurate study of x: transporter activity. A second reason is that glutamate used in a Natindependent experimental system would favor the uptake of glutamate by the x: transporter since other glutamate influx transporters are largely Nat-dependent (such as ASC and EAATs) (Verrey et al., 2004). The use of L-[ 3 1-1]-glutamate would eliminate these variables. 120 Using PANC-1 cells, the Km for glutamate uptake via the transporter was determined to be 112 ktM. This value is smaller than that obtained for glutamate uptake via the x: transporter in a mouse fibroblast cell line (K m = 200 [tM) (Bannai and Kitamura, 1980). This difference in Km constants suggests that glutamate exhibits a higher affinity for the xe" transporter in pancreatic cancer cells compared to fibroblasts. However, additional factors that may influence Km , such as species type, cell type, incubation time, and experimental technique cannot be ruled out. The current study and others have demonstrated that SASP inhibits the function of the x e" transporter, resulting in enhanced cancer cell apoptosis (Chung et al., 2005; Doxsee et al., 2007; Gout et al., 2001; Narang et al., 2007). Recent studies, however, have identified SASP as an NFKB inhibitor that can restore drug sensitivity in a variety of drug-resistant cancers (Lay et al., 2007; Muerkoster et al., 2003; Robe et al., 2004) by inhibiting the NFKB signaling pathway at multiple steps (Yamamoto and Gaynor, 2001). I therefore determined whether suppression of pancreatic cancer cell proliferation in response to SASP-mediated inhibition of x e- transporter function also involved the inhibition of NFKB activation. My results indicated that although SASP inhibited both x c- transporter function and NFKB activation, the effect of SASP on pancreatic cancer cell proliferation was due to inhibited x e - transporter function (resulting in reduced cystine import with subsequent GSH depletion), and not due to inhibited NFKB activation. In accordance with my findings, SASP has been reported to augment cancer therapy in a NFKB-independent manner (Hermisson and Weller, 2003; Olivier et al., 2006). Of interest, inhibition of NFKB with SASP has previously been shown to sensitize pancreatic cancer cells to apoptosis induced by etoposide and doxorubicin in vitro (Arlt et al., 2003) and etoposide and GEM in vivo (Muerkoster et al., 2003). Although my NFKB studies would seem to contradict 121 these earlier findings, it is important to note that these earlier studies did not demonstrate a causal role for SASP-mediated inhibition of NFKB activation in the sensitization to drug. Indeed, SASP is not a specific inhibitor of NFKB, but rather can also inhibit activation of, the EGFR and ERBB2/neu signaling pathways (Lay et al., 2007). In this study, I demonstrated that inhibiting x e - transporter expression with siRNA was sufficient to sensitize pancreatic cancer cells to GEM in vitro. Moreover, using the anti- inflammatory drug SASP, I showed that inhibiting x: transporter function restored pancreatic cancer cell sensitivity to GEM in vitro and in vivo. SASP was determined as a mixed inhibitor of the xe - transporter, which acts to inhibit pancreatic cancer cell proliferation via a mechanism that involves reduced x e- transporter activity and not reduced NFKB activity. Taken together, my results demonstrate a key role for the x e- transporter in pancreatic cancer cell growth and drug resistance (Figure 5.9), and provide a rationale for the use of combination chemotherapy with SASP and gemcitabine in the treatment of pancreatic cancer. 122 Cysteine ♦ ♦ oxidation Cystine SASP Cystine Pancreatic cancer cell^GSH biosynthesis I proliferation I^GEM resistance I Figure 5.9: Model for the role of the x c - transporter in pancreatic cancer cell growth and drug resistance. In the extracellular space, cysteine is oxidized to cystine, which in turn is transported into the cell via the xc transporter to promote GSH biosynthesis and hence proliferation and GEM resistance. Treatment with SASP, an inhibitor of xc - transporter function, attenuates cystine uptake. In turn, GSH biosynthesis is reduced, leading to decreased pancreatic cancer cell proliferation and decreased GEM resistance. 123 Chapter 6 SUMMARY AND FUTURE DIRECTIONS The overall goal of this thesis was to investigate the role of transporters in pancreatic cancer drug resistance. In Chapter 3, I investigated a potential role for ABC transporters in the development of intrinsic and induced drug resistance. Pancreatic tumors are characterized by constitutive K-ras activation (Hruban et al., 1999) and a very hypoxic microenvironment (Garcea et al., 2006; Sun et al., 2007). Given that both of these characteristics are reported to increase pancreatic cancer cell drug resistance (Giovannetti et al., 2006; Yokoi and Fidler, 2004), I investigated whether these characteristics, either alone or in combination, would be associated with the expression of specific ABC transporters. Using HPDE cells, I identified specific ABC transporters that were induced in response to either constitutive K-ras activation or hypoxic treatment alone. Interestingly, the majority of these transporters exhibited downregulated expression in response to combined K-ras activation and hypoxic treatment, when compared to K-ras activation alone. A similar ABC transporter expression profile, however, was not evident in pancreatic cancer cell lines. Moreover, hypoxia induced differential effects on ABC transporter expression in the different pancreatic cancer cell lines examined, with no clear correlation detected between global ABC transporter expression and expression of constitutively active K-ras. Interestingly, all the pancreatic cancer cell lines examined did not express the common MDR1 ABC transporter, a major player in drug resistance (Ling et al., 1997). Consequently, these cell lines are sensitive to vincristine. Long term treatment with low dose vincristine, however, upregulated MDR1 expression, thus demonstrating that drug resistance can be induced in pancreatic cancer cells. My results suggest that intrinsic drug resistance may be 124 associated with constitutive K-ras activation and/or hypoxia, and that induced drug resistance may be associated with long-term exposure to chemotherapy. Importantly, I identified ABC transporters as potential mediators of intrinsic and induced drug resistance in pancreatic cancer cells. Although many ABC transporter profiling studies have been performed to date, studies that specifically investigate ABC transporter expression in pancreatic cancer are lacking. I have used a q-RT-PCR-based approach to profile 48 ABC transporters in numerous pancreatic cell lines. With the development of microarray-based assays, the expression of large numbers of genes in numerous cell lines can be profiled with relative ease. Additionally, such assays have proven invaluable in the screening of drugs for potential treatment efficacy (Szakacs et al., 2004). Employing a microarray-based approach to investige ABC transporter expression in pancreatic cancer would undoubtedly lead to new insights into pancreatic cancer drug resistance. Moreover, additional studies may enable us to determine the relative importance of individual ABC transporters to the drug resistant phenotype. In addition to the ABC transporter family, amino acid transporters may play a role in drug resistance. In Chapter 4, I investigated the amino acid transporter )(,-, which mediates cellular uptake of cystine, as a potential mediator of pancreatic cancer drug resistance. I demonstrated that pancreatic cancer cell lines and primary tumors exhibited upregulated expression of the x c- transporter, which promoted the uptake of extracellular cystine for the synthesis of GSH. In turn, elevated intracellular GSH levels promoted pancreatic cancer cell growth and survival in response to oxidative stressors. Importantly, I showed that expression of the x c - transporter conferred resistance to GEM, a chemotherapeutic drug that has been shown to 125 induce oxidative stress (Donadelli et al., 2007; Maehara et al., 2004). Hence drug resistance in pancreatic cancer may be explained in part by the overexpression of the x e - transporter. Having demonstrated a role for the x e- transporter in pancreatic cancer drug resistance, I next determined whether targeting the xe- transporter would be of therapeutic potential. In Chapter 5, I demonstrated that inhibiting x e - transporter expression with siRNA was sufficient to sensitize pancreatic cancer cells to GEM in vitro. Moreover, using the anti-inflammatory drug SASP, it was demonstrated that inhibiting x e- transporter function was able to re-sensitize pancreatic cancer cells to GEM in vitro and in vivo. Investigation into the kinetic mechanism of SASP action revealed SASP to be a mixed inhibitor of the transporter, which functioned to inhibit pancreatic cancer cell growth via a mechanism that involved reduced xe - transporter activity but not reduced NFKB activity. Taken together, the results from Chapters 4 and 5 demonstrated a key role for the transporter in pancreatic cancer cell growth and drug resistance, and provided a rationale for the use of combination chemotherapy with SASP in the treatment of pancreatic cancer. Although I have identified an important role for the ?c c- transporter in pancreatic cancer drug resistance, the exact location of the x e" transporter within cells remains undetermined. By immunofluorescence microscopy, I have detected xe - transporter expression both at the plasma membrane as well as in intracellular compartments. More detailed analyses with confocal microscopy will be required to obtain a more precise localization of the transporter within the cell, and reveal important insights into the mechanism of action of SASP. My studies have demonstrated that combination therapy with SASP and GEM, compared to either treatment alone, induced a greater inhibition of pancreatic tumor growth in vivo. It would be important to determine whether SASP can enhanced the antitumor activity of other 126 chemotherapeutic drugs. We chose to use GEM in our studies because it is the standard chemotherapeutic drug used for first-line pancreatic cancer treatment (Wolff, 2007). Although GEM is a potent inhibitor of pancreatic cancer cell survival, induction of oxidative stress is but one of its many mechanisms of action (Maehara et al., 2004; Russo et al., 2007). Given that SASP functions to sensitize cells to oxidative stress, it is anticipated that SASP, when used in combination with a drug that specifically induces oxidative stress, would result in a greater anti- tumor response. Another factor that may influence the anti-tumor activity of SASP is the specific in vivo tumor model used to assess efficacy. I used pancreatic cancer cell xenografts grown subcutaneously on the backs of mice to assess efficacy, because this model allowed monitoring tumor growth over time. Subcutaneous tumor models, however, may yield different results compared to more clinically-relevant orthotopic tumor models. Whether SASP would exhibit altered anti-tumor activity in pancreatic tumors grown in the microenvironment of the pancreas remains to be determined. Results presented in this thesis examine the expression of transporters in pancreatic cancer cells, and the relationship between transporter expression and drug resistance. Studies have demonstrated that within a given tumor, a rare population of cells, termed cancer stem/initiating cells, can give rise to all the cell types found within a tumor (Li et al., 2007). Importantly, these rare cancer stem/initiating cells are highly tumorigenic, whereas cells that comprise the bulk of the tumor are not. One feature of cancer stem/initiating cells is their intrinsic ability to efflux drugs (Lou and Dean, 2007). This ability is mediated by a member of the ABC transporter family, ABCG2 (bcrp 1), which exhibits preferential expression in cancer stem/initiating cell populations (Lou and Dean, 2007). Of importance to this thesis, a recent 127 study has identified pancreatic cancer stem cells within primary human pancreatic adenocarcinomas grown as xenografts in mice (Li et al., 2007). Although this study did not report ABCG2 expression within pancreatic cancer stem/initiating cells, it is tempting to speculate that ABCG2, as well as other ABC transporters, may confer drug resistance to this highly tumorigenic population. Moreover, it is possible that the x e- transporter may also promote drug resistance in these cells. Determining whether pancreatic cancer stem/initiating cells exhibit preferential expression of the transporter would reveal important insights into the anti-tumor effects of SASP. In addition to investigating methods to enhance SASP efficacy, it is equally important to investigate potential adverse effects associated with SASP treatment. Given that the x e - transporter is expressed in several normal tissues including the brain (Bassi et al., 2001), the use of SASP at anti-tumor concentrations may deplete GSH levels in normal cells and render them susceptible to oxidative stress. Toxicity may therefore limit the use of SASP as an anti-cancer drug. However, given that SASP is FDA-approved for use as an anti-inflammatory drug, and that I did not observe toxicity (based on changes in weight, behavior, visual signs of stress, and spleen and liver histopathology) in the animal hosts used in my in vivo studies, toxicity may not be an issue. Detailed histopathological analyses of animal hosts would be necessary to validate SASP as a viable anti-cancer drug. In conclusion, the studies presented in this thesis identify a role for ABC transporters and the transporter in promoting pancreatic cancer cell growth and drug resistance. Importantly, my findings suggest that targeting these transporters may effect a re-sensitization of pancreatic tumor cells to chemotherapy, and provide proof-of-principle for SASP and in combination with GEM as a novel therapeutic in the treatment of pancreatic cancer. 128 REFERENCES Abbruzzese, J. L. (2002). New applications of gemcitabine and future directions in the management of pancreatic cancer. 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Biochim Biophys Acta 1773, 1095-1106 Epub 2007 May 1022. 133 or in combination did not exhibit reduced staining for the proliferation marker Ki67 compared to treatment controls (Figure 5.6b). Rather, GEM treatment alone or in combination with SASP increased the percentage of activated caspase3-positive cells in both xenograft types (Figure 5.6b,c). Taken together, our findings demonstrate that SASP treatment, via inhibiting x e- transporter function to effect GSH depletion, may be a useful mechanism to restore GEM sensitivity in GEM-resistant pancreatic cancer cells. 5.2.4 Kinetic studies of SASP as an inhibitor of the x c" transporter in pancreatic cancer cells Enzyme inhibitors can be classified into four major types: competitive, non-competitive, uncompetitive, and mixed (Whiteley, 2000; Zhang and Wong, 2005). I sought to determine if the inhibitory activity of SASP on x c - transporter function could be classified into one of these types. To accomplish this, I first performed time course experiments to establish experimental conditions for the steady-state (i.e. linear) transport of L43 1-11-glutamate into MIA PaCa-2 and PANC-1 cell lines. In both cell lines, uptake of L-[ 3H]-glutamate increased linearly for the first 30 min of incubation at 37 °C (Figure 5.7a,b). As expected, SASP pre-treatment for 1 h inhibited L-[3 H]-glutamate uptake by both MIA PaCa-2 and PANC-1 cells (Figure 5.7a,b). Of note, PANC-1 cells reached a higher L-N-glutamate uptake rate after 30 min incubation compared to MIA PaCa-2 cells (Figure 5.7a,b), consistent with our earlier findings demonstrating greater xCT mRNA expression in PANC-1 cells relative to MIA PaCa-2 cells (Figure 4.5a). I therefore used PANC-1 cells for subsequent kinetic studies of SASP inhibition. 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Gout Department: Surgery Animals Approved:^Mice SCID and CD-1 733 Start Date:^March 1, 2005^ Approval Date: October 3, 2005 Funding Sources: Funding Agency: Funding Title: Unfunded title: Canadian Institutes of Health Research Development of a function-blocking peptide for treatment of cancer N/A The University of British Columbia Animal Care Certificate The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 156 Page 1 of 2  UBC BCCA Research Ethics Board Fairmont Medical Building (6th Floor) 614 - 750 West Broadway Vancouver, BC V5Z 1H5 Tel: (604) 877-6284 Fax: (604) 708-2132 E-mail: reb@bccancerbc.ca Website: http://www.bccancerbc.ca > Research Ethics RISe: http://rise.ubc.ca UBC BC Cancer Agency(v)^r_Altt tt'SCARLii University of British Columbia - British Columbia Cancer Agency Research Ethics Board (UBC BCCA REB) Certificate of Expedited Approval: Annual Renewal PRINCIPAL INVESTIGATOR: Sylvia Ng INSTITUTION / DEPARTMENT: BCCA/Systemic Therapy - VA (BCCA) REB NUMBER: H05 -60127 INSTITUTION(S) WHERE RESEARCH WILL BE CARRIED OUT: Institution^ I^ Site^ ] Vancouver Coastal Health (VCHRINCHA) Vancouver General Hospital BC Cancer Agency^ Vancouver BCCA Other locations where the research will be conducted: N/A PRINCIPAL INVESTIGATOR FOR EACH ADDITIONAL PARTICIPATING BCCA CENTRE: Vancouver:^Sylvia Ng^ Vancouver Island:^N/A Fraser Valley: N/A Southern Interior: N/A SPONSORING AGENCIES AND COORDINATING GROUPS: 1)British Columbia Cancer Agency - Identification Of Biomarkers To Predict Treatment Response And Preclinical Evaluation Of New Treatments For Pancreatic Cancer Using Primary Pancreatic Cancer Xenografts 2) National Cancer Institute of Canada PROJECT TITLE: Identification Of Biomarkers To Predict Treatment Response And Preclinical Evaluation Of New Treatments For Pancreatic Cancer Using Prima^Pancreatic Cancer Xeno • rafts APPROVAL DATE: ^ EXPIRY DATE OF THIS APPROVAL: July 30, 2007 July 30, 2008 CERTIFICATION: 1. The membership of the UBC BCCA REB complies with the membership requirements for research ethics boards defined in Division 5 of the Food and Drug Regulations of Canada. 2. The UBC BCCA REB carries out its functions in a manner fully consistent with Good Clinical Practices. 3. The UBC BCCA REB has reviewed and approved the research project named on this Certificate of Approval including any associated consent form and taken the action noted above. This research project is to be conducted by the provincial investigator named above. This review and the associated minutes of the UBC BCCA REB have been documented electronically and in writing. The UBC BCCA Research Ethics Board has reviewed the documentation for the above named project. The research study as presented in documentation, was found to be acceptable on ethical grounds for research involving human subjects and was approved for renewal by the UBC BCCA REB. 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