TRPM2 ION CHANNEL REGULATES CANCER CELL SURVIVAL IN A CALCIUM-DEPENDENT MANNER by Shekoufeh Almasi Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Dalhousie University Halifax, Nova Scotia September 2018 © Copyright by Shekoufeh Almasi, 2018 TABLE OF CONTENTS TABLE OF CONTENTS .................................................................................................. ii LIST OF TABLES ............................................................................................................. x LIST OF FIGURES .......................................................................................................... xi ABSTRACT ..................................................................................................................... x i v LIST OF ABBREVIATIONS AND SYMBOLS USED ............................................... xv ACKNOWLEDGMENTS .............................................................................................. xxi CHAPTER 1: INTRODUCTION ..................................................................................... 1 1.1 CANCER AND RISK FACTORS ............................................................................ 1 1.2 THE BURDEN OF CANCER .................................................................................. 2 1.3 CANCER TREATMENT OPTIONS ....................................................................... 4 1.4 MEMBRANE TRANSPORT PROTEINS IN CANCER ......................................... 8 1.5 TRANSIENT RECEPTOR POTENTIAL (TRP) FAMILY OF ION CHANNELS AND CANCER THERAPY ............................................................. 11 1.6 TRANSIENT RECEPTOR POTENTIAL MELASTATIN (TRPM) SUBFAMILY OF ION CHANNELS IN CANCER ............................................... 14 1.7 TRANSIENT RECEPTOR POTENTIAL MELASTATIN-2 (TRPM2) STRUCTURE AND FUNCTION .......................................................................... 16 1.7.1 TRPM2 and insulin secretion ............................................................................ 20 1.7.2 TRPM2 and immune response ........................................................................... 21 ii 1.7.3 TRPM2 and cancer ............................................................................................ 22 1.8 HYPOTHESIS ........................................................................................................ 24 CHAPTER 2: MATERIALS AND METHODS ........................................................... 25 2.1 CELL CULTURE ................................................................................................... 25 2.2 GENERATION OF STABLE GENE-KNOCKDOWN CELL LINES .................. 27 2.3 RT-QPCR ................................................................................................................ 28 2.4 PATCH CLAMP ..................................................................................................... 30 2.5 CALCIUM IMAGING ........................................................................................... 30 2.6 MITOCHONDRIAL CALCIUM LEVEL .............................................................. 30 2.7 MITOCHONDRIAL MEMBRANE POTENTIAL ................................................ 31 2.8 WESTERN BLOT ................................................................................................... 31 2.9 TRYPAN BLUE CELL COUNTING ..................................................................... 34 2.10 MTT VIABILITY ASSAY .................................................................................... 34 2.11 CELL PROLIFERATION ASSAY ....................................................................... 35 2.12 ANNEXIN V/7-AAD BINDING ASSAY............................................................. 35 2.13 CELL CYCLE ANALYSIS ................................................................................... 36 2.14 AUTOPHAGY DETECTION ASSAY ................................................................. 36 iii 2.15 INTRACELLULAR REACTIVE OXYGEN SPECIES DETECTION ASSAY .. 36 2.16 MITOCHONDRIAL SUPEROXIDE DETECTION ASSAY............................... 37 2.17 INTRACELLULAR NITRIC OXIDE DETECTION ASSAY ............................. 37 2.18 EXTRACELLULAR FLUX ANALYSIS ............................................................. 38 2.19 GAP CLOSURE OR WOUND HEALING ASSAY ............................................. 38 2.20 CELL MIGRATION AND INVASION ASSAY .................................................. 39 2.21 MORPHOLOGICAL ANALYSIS OF MITOCHONDRIA .................................. 39 2.22 ANIMAL STUDY ................................................................................................. 40 2.23 SURVIVAL CURVES AND GENE EXPRESSION DATA ................................ 40 2.24 CALCULATION OF IC50 .................................................................................... 41 2.25 REAGENTS ........................................................................................................... 41 2.26 STATISTICAL ANALYSIS .................................................................................. 41 CHAPTER 3: TRPM2 CHANNEL-MEDIATED REGULATION OF AUTOPHAGY MAINTAINS MITOCHONDRIAL FUNCTION AND PROMOTES GASTRIC CANCER CELL SURVIVAL VIA THE JNK SIGNALING PATHWAY ................................................................................... 42 3.1 ABSTRACT ............................................................................................................ 43 3.2 INTRODUCTION .................................................................................................. 44 3.3 RESULTS ............................................................................................................... 46 iv 3.3.1 TRPM2 expression is negatively correlated with the overall survival of gastric cancer patients ............................................................................................... 46 3.3.2 TRPM2 is functionally expressed in gastric cancer cells .................................. 47 3.3.3 TRPM2 downregulation decreases cancer cell survival and enhances apoptosis in gastric cancer cells ................................................................................ 48 3.3.4 TRPM2 downregulation hampers mitochondrial function ................................ 52 3.3.5 TRPM2 downregulation alters the autophagy process in gastric cancer cells . 5 4 3.3.6 TRPM2 regulates autophagy in a JNK-dependent and mTOR-independent manner……………………………………………………………………………………………………………………... 55 3.3.7 TRPM2 downregulation sensitizes gastric cancer cells to paclitaxel and doxorubicin ................................................................................................................. 58 3.4 DISCUSSION ......................................................................................................... 59 3.5 CONFLICT OF INTEREST ................................................................................... 64 3.6 SUMMARY OF CHAPTER 3 AND TRANSITION TO CHAPTER 4 ................ 64 CHAPTER 4: TRPM2 ION CHANNEL CONTROLS MIGRATION/INVASION ABILITY OF GASTRIC CANCER CELLS THROUGH REGULATING AKT SIGNALING PATHWAY ......................................................................... 65 4.1 ABSTRACT ............................................................................................................ 66 4.2 INTRODUCTION .................................................................................................. 66 4.3 RESULTS ............................................................................................................... 68 4.3.1 TRPM2 activation elicits cytosolic calcium elevation in AGS cells .................. 68 v 4.3.2 Genetic silencing of TRPM2 inhibits migration and invasion abilities of AGS cells .................................................................................................................... 71 4.3.3 PTEN/Akt signaling pathway is required for TRPM2-mediated migration and invasion ability of AGS cells....................................................................................... 72 4.3.4 TRPM2 silencing inhibits tumor formation ability of AGS cells in NOD/SCID mice ....................................................................................................... 74 4.4 DISCUSSION ......................................................................................................... 76 4.5 CONFLICT OF INTEREST ................................................................................... 78 4.6 SUMMARY OF CHAPTER 4 AND TRANSITION TO CHAPTER 5 ................ 78 CHAPTER 5: SILENCING OF TRPM2 ION CHANNEL PROMOTES MITOCHONDRIAL-DEPENDENT APOPTOSIS IN TRIPLE NEGATIVE BREAST CANCER CELLS ......................................... 80 5.1 ABSTRACT ............................................................................................................ 81 5.2 INTRODUCTION .................................................................................................. 82 5.3 RESULTS ............................................................................................................... 84 5.3.1 TRPM2 expression is correlated with the poor survival of breast cancer patients............................................................................................................ 84 5.3.2 TRPM2 is overexpressed in triple negative breast cancer cell lines and it could be successfully downregulated using TRC lentiviral system ...........................