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Molecular Mechanisms Driving Prostate Cancer Neuroendocrine Differentiation

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Molecular mechanisms driving prostate cancer neuroendocrine differentiation Submitted by Joseph Edward Sutton Supervisory team: Dr Amy Poole (DoS) Dr Jennifer Fraser Dr Gary Hutchison A thesis submitted in partial fulfilment of the requirements of Edinburgh Napier University, for the award of Doctor of Philosophy. October 2019 School of Applied Sciences Edinburgh Napier University Edinburgh Declaration It is hereby declared that this thesis is the result of the author’s original research. It has been composed by the author and has not been previously submitted for examination which has led to the award of a degree. Signed: II Dedication This thesis is dedicated to my grandfather William ‘Harry’ Russell, who died of stomach cancer in 2014. Thank you for always encouraging me to achieve my ambitions, believing in me and for retaining your incredible positivity and sense of humour, even at the very end of your life. III Acknowledgements First of all, I would like to acknowledge my parents, who dedicated so much effort and energy into helping me to achieve my lifelong ambition of becoming a scientist. From taking me to the Natural History and Science Museums in London as a child, to tolerating my obsession with Jurassic Park and continuing to support me in both of your unique yet equally important ways, thank you. I would also like to thank my PhD supervisors Dr Amy Poole and Dr Jenny Fraser, not only for their excellent scientific guidance but also for their great banter and encouragement along the way. Thank you for seeing some potential in me, taking a chance on me and for helping me to continue my scientific journey. Thank you also to Dr Gary Hutchison and Dr Sharon Vass for their words of wisdom and encouragement. Thank you to my amazing friends at Edinburgh Napier who are too numerous to mention all by name, in particular Lee Curley, Dave Lawson, Liam Ralph and Alva Smith who are the best friends that anyone could ask for. Thank you all (you know who you are) for making these the best years of my life, I will always remember the impromptu barbecues on the meadows, the PGR conferences, the pub quiz victories and of course the times Lewis climbed the Christmas tree and Lee almost lost his hand. Thank you also to the amazing support staff at Napier, especially Tracy for always wishing us a good day and making sure there was always a hot haggis roll waiting on those freezing winter mornings, it makes a difference. I would also like to acknowledge Gareth Southgate for making us believe that one day, football is coming home. Thanks also to Thom, Jonny, Colin, Ed, Phil and Nigel for the company during long writing sessions and weekends in the lab. Finally, and most of all, thank you to Sasha Pereira for all of your love, support, patience and belief during these difficult and often uncertain times. IV Abstract Annually, 11,500 men in the UK die of prostate cancer (PCa). PCa tumours are initially dependent upon androgen receptor (AR) signalling and androgen deprivation therapy (ADT) is highly effective in restricting tumour growth. However, ADT resistance and progression to castrate-resistant prostate cancer (CRPC) is inevitable, usually occurring within three years. CRPC is considerably more aggressive, and metastatic CRPC remains incurable. One mechanism of ADT resistance is neuroendocrine differentiation (NED). NED is common in PCa tumours treated with ADT (30% of cases) and is associated with poorer survival. Prevalence of NED is rising and can be induced by other therapeutics including radiotherapy and chemotherapeutics. However, the precise molecular events underlying NED remain poorly understood. Therefore, an in-depth molecular investigation of NED was conducted using an in vitro system. The first objective was to establish a robust, in vitro model of ADT-induced NED using the PCa cell line, LNCaP. Extensive molecular analysis by qRT-PCR, Western blotting and confocal microscopy revealed the transcription factor, human achaete-scute homolog-1 (hASH1) as a potential key driver of NED. hASH1 localisation shifted from exclusively cytoplasmic to nuclear upon acquisition of NED morphology, concurrent with increased expression of the clinical biomarker neuron specific enolase (NSE) and decreased expression of prostate-specific antigen (PSA). Next, the effects of intermittent (I)ADT on the NED pathway were investigated. AD arrest resulted in reacquisition of epithelial morphology and resurgence of PSA expression. Interestingly, hASH1 was retained in the nucleus alongside NSE upregulation, indicating emergence of a potential ‘hybrid’ phenotype. After a second AD cycle, cells regained NED morphology and maintained hASH1 nuclear localisation. As hASH1 drives the development of GABAergic neurons and GABA has previously been implicated in PCa growth and invasion, a comprehensive characterisation of GABA receptor subunit expression in PCa cells was undertaken. Differential expression between androgen-sensitive and androgen resistant cells was discovered and indicated PCa GABAergic signalling may be modulated by androgen availability. V Abbreviations °C - Celsius AD - Androgen deprived AD1 - 1st AD cycle AD2 - 2nd AD cycle ADT - Androgen deprivation therapy ANOVA - Analysis of variance AR - Androgen Receptor ARE - Androgen response element ATCC - American type culture collection BLAST - Basic local alignment search tool BPH - Benign prostate hyperplasia BSA - Bovine serum albumin BZ - Benzodiazepine C - Control cAD - Constant AD CCC - Cation-chloride co-transporters cDNA - Complimentary DNA CgA - Chromogranin-A CREB - cAMP response element-binding protein CNS – Central nervous system CRPC - castrate-resistant prostate cancer CS-FBS - Charcoal stripped fetal bovine serum CSC - Cancer stem cell Ctrl - Control DEPC - diethylpyrocarbonate DHT - Dihydrotestosterone DMSO - dimethyl sulphide DNA - Deoxyribonucleic acid DRE - Digital rectal examination ECM - Extracellular matrix EDTA - Ethylenediaminetetraacetic acid EGF - Epidermal growth factor EGFR - Epidermal growth factor receptor VI ELISA - Enzyme-linked immunosorbent assay EMT - Epithelial to mesenchymal transition ER - Estrogen receptor FBS - Foetal bovine serum GABA - -aminobutyric acid GABA-T - GABA transaminase GABARs - GABA receptors GAD1 - Glutamate decarboxylase 1 GAD2 - Glutamate decarboxylase 2 GBP – Gabapentin GRP – Gastrin releasing peptide H&E - Haematoxylin and eosin H2O - Water hASH1 - Human achaete-scute homolog-1 IADT - Intermittent androgen deprivation IGF-1 - Insulin-like growth factor 1 IHC - Immunohistochemistry KCC - K+-Cl- co- transporters KDa - Kilodalton LSB - loading sample buffer mAb - monoclonal antibody MMP - Matrix metalloproteinase mRNA - messenger RNA MTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MW - molecular weight NB - Neuroblastoma NE - Neuroendocrine NED - Neuroendocrine differentiation NEPC - Neuroendocrine Prostate Cancer NICE - National Institute for Health and Care Excellence NKCC - Na+-K+-2Cl- co-transporters NSE - Neuron specific enolase PBS - Phosphate buffered saline VII PBS-T - Phosphate buffered saline-Tween PCa - Prostate Cancer PCPT – Prostate Cancer Prevention Trial PDX - Patient Derived Xenograft PNS – Peripheral nervous system PSA - Prostate specific antigen PTEN - Phosphatase and tensin homolog PTOV1 - Prostate Tumour Overexpressed 1 qRT-PCR - Quantitative reverse transcription-polymerase chain reaction REST - RE1-silencing transcription factor RIN - RNA integrity number RNA - Ribonucleic Acid RNAi - Ribonucleic acid interference RPMI - Roswell park memorial institute SCLC - Small cell lung cancer SDS - Sodium Dodecyl Sulphate SDS-PAGE - Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis SRD5A2 - steroid-5α-reductase, α-polypeptide 2 TAPS - Total androgen program suppression TEMED - N,N,N′,N′-Tetramethyl-ethylenediamine TIMP – Tissue inhibitor of metalloproteinase UK – United Kingdom VFTD - Venus fly trap domain VGCCs - Voltage-gated calcium channels VIII CONTENTS Page No. 1. Introduction ................................................................................................... 1 1.1 Prostate cancer epidemiology ............................................................ 1 1.1.1 Endogenous and exogenous risk factors .............................. 2 1.1.2 Androgen steroidogenesis .................................................... 3 1.1.3 Anti-androgen therapeutic targets and total androgen blockade ................................................................................................... 3 1.1.4 Prostate anatomy and physiology ......................................... 4 1.1.5 Androgen dependent growth of healthy prostate .................. 6 1.1.6 Neuroendocrine cells in benign and malignant prostate ....... 7 1.1.7 Prognostic factors and diagnostic tools ................................ 8 1.1.8 Current treatment strategies ............................................... 10 1.2 Progression to castrate-resistant prostate cancer ........................... 15 1.2.1 Molecular mechanisms of castrate-resistant prostate cancer ............................................................................................................... 15 1.2.1.1 Androgen receptor ............................................... 15 1.2.1.2 Hypersensitive pathway ........................................ 16 1.2.1.3 Promiscuous pathway .......................................... 16 1.2.1.4 Outlaw pathway ................................................... 17 1.2.1.5 Endogenous androgen synthesis .........................
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