Molecular mechanisms driving prostate cancer neuroendocrine differentiation

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