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Trpml1 Regulates Prostate Cancer Cell Proliferation Through the Modulation of Oxidative Stress and Cell Cycle

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Trpml1 Regulates Prostate Cancer Cell Proliferation Through the Modulation of Oxidative Stress and Cell Cycle TRPML1 REGULATES PROSTATE CANCER CELL PROLIFERATION THROUGH THE MODULATION OF OXIDATIVE STRESS AND CELL CYCLE by Andra Mihaela Sterea Submitted in partial fulfilment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia August 2018 © Copyright by Andra Mihaela Sterea, 2018 Dedicated to Mușca și Stuf ii Table of Contents List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................. viii Abstract .......................................................................................................................................... x List of Abbreviations and Symbols Used ................................................................................... xi Acknowledgements .................................................................................................................... xiv CHAPTER I: INTRODUCTION ................................................................................................ 1 1.1 Subchapter I: Cancer, A Historical Account and Current Knowledge ............................ 1 1.1.1 History of Cancer ........................................................................................................... 1 1.1.2 Current Knowledge and Challenges .............................................................................. 2 1.1.3 The Molecular Mechanisms of Cancer and the Basis for the Famous “Hallmarks of Cancer” ................................................................................................................................... 5 1.1.3.1 PI3K/AKT/mTOR Pathway .................................................................................... 6 1.1.3.2 JNK Signaling Pathway .......................................................................................... 7 1.1.3.3 ERK Pathway .......................................................................................................... 8 1.1.3.4 Autophagy ............................................................................................................... 8 1.1.3.5 Generation of Reactive Oxygen and Nitrogen Species........................................... 9 1.1.3.6 DNA Damage and Repair ..................................................................................... 11 1.1.3.6.1 Homologous Recombination ......................................................................... 12 1.1.3.6.2 Mismatch Repair ............................................................................................ 12 1.1.3.6.3 Nucleotide Excision Repair ........................................................................... 13 1.1.3.6.4 DNA Strand Crosslink Repair ....................................................................... 13 1.1.3.6.5 Nonhomologous End-Joining ........................................................................ 14 1.1.3.7 Apoptosis .............................................................................................................. 14 1.2 Subchapter II: A Comprehensive Description of Prostate Cancer ................................. 19 1.2.1 Physiology of the Prostate Gland ................................................................................. 19 1.2.2 Prostate Cancer Epidemiology ..................................................................................... 20 1.2.3 Diagnosis...................................................................................................................... 21 1.2.4 Biomarkers ................................................................................................................... 22 iii 1.2.4.1 PSA Testing .......................................................................................................... 22 1.2.4.2 Emerging “omics”-Related Biomarkers ............................................................... 27 1.2.4.3 Immune System-Based Biomarkers ...................................................................... 28 1.2.5 Tumor Biopsy Analysis ............................................................................................... 29 1.2.5.1 Grading ................................................................................................................. 29 1.2.5.2 Staging .................................................................................................................. 29 1.2.6 Driver Mutations .......................................................................................................... 30 1.2.6.1 Genes Alterations in PCa and Other Cancers ....................................................... 30 1.2.6.3 Mutations Specific to PCa .................................................................................... 32 1.3 Subchapter III: Prostate Cancer Therapies and Patient Care ......................................... 33 1.3.1 Current Treatments ...................................................................................................... 34 1.3.2 Emerging Therapies ..................................................................................................... 34 1.3.2.1 TRP Ion Channel-Based Therapies for Cancer ..................................................... 35 1.3.2.2 TRP Ion Channel-Based Therapies for Prostate Cancer ....................................... 36 1.4 Subchapter IV: Lysosomal TRP Channels and Their Role in Carcinogenesis ............. 36 1.4.1 Lysosome Physiology .................................................................................................. 36 1.4.2 TRP Channels .............................................................................................................. 42 1.4.2.1 TRPML1 ............................................................................................................... 42 1.5 Subchapter V: Project Rationale and Objectives ................................................................ 48 1.5.1 Rationale and Hypothesis ............................................................................................ 48 1.5.2 Objectives .................................................................................................................... 49 CHAPTER II: MATERIALS AND METHODS ..................................................................... 50 2.1 Cell Lines ....................................................................................................................................... 50 2.2 Reagents ......................................................................................................................................... 50 2.3 Mucolipin-Specific Inhibitor (ML-SI) Treatment ............................................................... 51 2.4 TRPML1 Knockdown ................................................................................................................ 52 2.5 RT-qPCR ....................................................................................................................................... 52 2.5.1 Primer Sequence Table ................................................................................................ 53 iv 2.6 Western Blot ................................................................................................................................. 53 2.7 Flow Cytometry ........................................................................................................................... 55 2.7.1 CFSE (5(6)-Carboxyfluorescein N-Hydroxysuccinimidyl Ester) Staining ................. 55 2.7.2 Propidium Iodide (PI) Staining .................................................................................... 56 2.7.3 DAF-FM and H2DCFDA Staining............................................................................... 57 2.7.4 Annexin V/7AAD Staining .......................................................................................... 57 2.8 MTS Assay .................................................................................................................................... 58 2.9 Confocal Microscopy .................................................................................................................. 59 2.10 Calcium Imaging ....................................................................................................................... 59 2.11 Bioinformatics Analysis ........................................................................................................... 60 2.11.1 Kaplan Meier Survival Analysis ................................................................................ 61 2.11.2 CCLE Gene Expression ............................................................................................. 61 2.11.3 Co-Expression Analysis ............................................................................................. 61 2.12 Statistics ....................................................................................................................................... 61 CHAPTER III: RESULTS ......................................................................................................... 63 3.1 The Loss of TRPML1 Decreases Cell Proliferation in PCa Cell Lines in a Dose- Dependent Manner ............................................................................................................................ 63 3.2 Knockdown of TRPML1 in PCa Leads
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