The Role of Plasma Membrane Calcium Pump during Pancreatic Cancer A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2019 PISHYAPORN SRITANGOS School of Medical Sciences/ Division of Cancer Sciences Table of Contents List of Figures ........................................................................................................................... 6 List of Tables ............................................................................................................................. 8 List of Abbreviations ................................................................................................................ 9 Abstract ................................................................................................................................... 13 Declaration .............................................................................................................................. 14 Copyright Statement ............................................................................................................... 14 Acknowledgements ................................................................................................................ 15 Chapter 1 - Introductions ....................................................................................................... 16 1.1 Pancreatic ductal adenocarcinoma ......................................................................................... 16 1.2 Cancer metabolism in PDAC .................................................................................................... 19 1.2.1 Cell metabolism – Glycolysis and Oxidative Phosphorylation ............................................... 19 1.2.2 The Warburg effect – a shift towards aerobic glycolysis ........................................................ 20 1.2.3 Metabolic reprogramming in PDAC ........................................................................................ 23 1.2.3.1 Oncogenes activation in PDAC....................................................................................... 23 1.2.3.2 Inactivation of tumour suppressor genes in PDAC ......................................................... 24 1.3 Functional coupling between glycolysis and Ca2+ signalling ............................................... 26 1.4 Ca2+ signalling and its remodelling in cancer ........................................................................ 28 1.4.1 Ca2+ – a versatile signalling molecule..................................................................................... 28 2+ 1.4.1.1 Elevation of [Ca ]i .......................................................................................................... 30 2+ 1.4.1.2 Reduction of [Ca ]i ......................................................................................................... 30 1.4.2 Remodelling of Ca2+ signalling modulates cancer hallmarks ................................................. 31 1.5 Plasma membrane calcium ATPases ...................................................................................... 34 1.5.1 Plasma membrane calcium ATPase isoform 4 (PMCA4) ...................................................... 36 1.6 Caveolae – a potential intersection between PMCA and glycolytic enzymes? .................. 37 1.6.1 Caveolae ................................................................................................................................ 37 1.6.1.1 Caveolae structural composition ..................................................................................... 38 1.6.1.2 Functional role of the caveolae ....................................................................................... 38 1.6.2 Caveolin .................................................................................................................................. 39 1.6.3 PMCA localisation in the caveolae and potential coupling to glycolytic enzyme ................... 40 1.7 PKM2 – an oncogenic glycolytic enzyme ............................................................................... 41 1.7.1 Regulation of PKM2 oligomeric states ................................................................................... 42 1.7.2 Non-canonical role of PKM2 ................................................................................................... 44 2 1.8 Summary .................................................................................................................................... 45 1.8.1 Overarching Hypothesis ......................................................................................................... 46 1.8.2 Experimental objectives and specific aims ............................................................................. 47 1.9 Journal/Alternative format thesis ............................................................................................ 49 Chapter 2 – General Methods and Optimizations ................................................................. 51 2.1 Materials and methods .................................................................................................................. 51 2.1.1 Cell culture.............................................................................................................................. 51 2.1.2 Chemicals and reagents ......................................................................................................... 51 2.1.3 Cell surface biotinylation ........................................................................................................ 51 2.1.4 Sucrose gradient ultracentrifugation ...................................................................................... 51 2.1.5 Western immunoblot .............................................................................................................. 51 2.1.6 Sulforhodamine B (SRB) protein quantification assay ........................................................... 53 2.1.7 Cell count kit-8 (WST-8) viability assay .................................................................................. 53 2.1.8 ATP-luciferase ATP quantification and cell viability assay ..................................................... 53 2.1.9 Live-cell fluorescence microscopy ......................................................................................... 54 2.1.11 Fura-2 calcium concentration calibration ............................................................................... 55 2.1.12 Calcium overload assay ......................................................................................................... 56 2.1.13 Calcium clearance assay ....................................................................................................... 57 2.1.14 GO-ATeam ATP-FRET reporter assay .................................................................................. 58 2.1.15 Immunofluorescence imaging ................................................................................................ 59 2.1.16 RT-qPCR ................................................................................................................................ 59 2.1.17 Gap closure cell migration assay ........................................................................................... 60 2.1.18 siRNA expression knockdown ................................................................................................ 60 2.1.19 Caspase 3/7 cleavage apoptosis assay ................................................................................. 61 2.1.20 BirA* proximity Bio-Identification ............................................................................................ 61 2.1.20.1 Generating stably transfected BirA*-fusion cell lines ...................................................... 61 2.1.20.2 Sample processing and enrichment ............................................................................... 62 2.1.20.3 Data processing and analysis ......................................................................................... 62 2.1.21 Datamining ............................................................................................................................. 63 2.1.22 Agilent Seahorse cell metabolism assays .............................................................................. 63 2.1.23 Statistical Analysis .................................................................................................................. 64 2.2 Methods Optimization ............................................................................................................... 66 2.2.1 Comparing sulforhodamine B (SRB) and cell count kit-8 (WST-8) cell viability assay .......... 66 2.2.2 Optimization of gap closure cell migration assay ................................................................... 69 2.2.3 siRNA expression knockdown, a necessary tool to study the role of PMCA4 and Cav-1 ..... 71 3 2.2.3.1 Selective PMCA4 inhibitors failed to inhibit PMCA activity in MIAPaCa-2 cells predominantly expressing PMCA4 ............................................................................ 71 2.2.3.2 siRNA expression knockdown optimization ............................................................. 73 2.2.4 Analysis of ATP production rate from existing Mito Stress Test data .................................... 76 Chapter 3 – PMCA4 is important for cell migration and apoptotic resistance of MIAPaCa-2 pancreatic