title: The investigation of androgen receptor phosphorylation and inflammation in prostate cancer
Creators: name: McAllister, Milly Joy
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: prostate cancer, phosphorylation, inflammation.

description: Prostate cancer 10-year survival rates have tripled over the last 40 years within the UK. Unfortunately, despite increases in diagnostics and effective treatment strategies, over 300,000 men die from prostate cancer per year worldwide. Additionally, these treatments associate with side effects from the un-personalised treatment approaches used. Therefore, it is evident that specific and differentiating biomarkers are required to provide accurate treatment approaches for prostate cancer patients. The androgen receptor is a main focus in prostate cancer as 80-90% of diagnoses are androgen dependent. Once activated, AR activity promotes pro-tumourigenic activity and reduces survival. However, whilst androgen-deprivation therapy is widely used, response rates remain poor due to the development of castrate resistant disease. AR phosphorylation has been associated with androgen-independent activation and castrate resistance. AR serine 81 phosphorylation is activated by DHT resulting in increased AR transcriptional ability and elevated cell growth. However, various serine residues become activated androgen-independently and are proposed as potential mechanisms for castrate resistance. One such phosphorylation site is serine 213, a putative substrate for Akt activated via PI3K, which promotes transcriptional activity and receptor stability in prostate cancer. Furthermore, the idea of combined AR phosphorylation at multiple sites providing a poorer prognosis has since been proposed, therefore leading to our hypothesis that patients expressing pARser81 and pARser213 have a reduced overall survival but would benefit from dual targeting androgen-dependent and Akt-driven AR activation. Immunohistochemistry and/ or immunofluorescence on a cohort of matched hormone naïve and castrate resistant tissue identified reduced pARser81 expression and increased pARser213 expression following the development of castrate resistance suggesting an androgen-independent pathway reactivates AR signalling. At castrate resistance, high pARser81 expression showed a trend towards reduced time to death from biochemical relapse (p=0.068) suggesting patients who still have this site phosphorylated have a more aggressive disease. However, since androgens are depleted from these patients it is hypothesised that serine 81 may also be phosphorylated via non-canonical signalling. Additionally, high pARser213 expression reduced time to death from biochemical relapse. We observed combined expression of pARser81 and pARser213 reduced time to death from biochemical relapse suggesting a cumulative effect of AR phosphorylation and multiple pathways acting upon AR following the development of castrate resistance alters survival rates. Upstream of PI3K/AKT signalling is epidermal growth factor receptor activated by epidermal growth factor that stimulates intracellular pro-tumourigenic signalling. We observed an increase in pARser81 and pARser213 following EGF stimulation in androgen-independent prostate cancer cells. We observed a correlation between AR phosphorylation sites and EGFRvIII, a constitutively active EGFR. Interestingly, EGFRvIII expression reduces cyclin dependent kinase inhibitor levels, an inhibitor of cell cycle progression. As CDKs activate pARser81 expression and EGFRvIII expression reduces CDK inhibition, this could reactivate AR at castrate resistance. We observed reduced DHT induced pARser81 expression following treatment with Enzalutamide or BKM120 (PI3K inhibitor). Furthermore, following EGF stimulation, BKM120 treatment reduced pARser81 expression, confirming pARser81 can occur androgen-dependently and androgen-independently. pARser213 expression reduced following EGF stimulation and BKM120 treatment. However, neither Enzalutamide nor BKM120 reduced cell viability as mono-treatments but when combined cell viability reduced by 10 days. Therefore, we hypothesis that for this combined treatment approach to be successful, participants must be preselected based on their combined pARser81 and pARser213 expression. Genes associated with inflammation were differentially expressed between tumours with high and low AR expression/ AR amplification including those involved in interleukin signalling and chemokine receptors suggesting inflammation plays a pro-tumourigenic effect in prostate cancer and promotes AR activity. Inflammation within the tumour microenvironment now classifies as a hallmark of cancer. However, its effects in prostate cancer remain controversial. Therefore, an aim of this thesis was to determine if inflammation associates with AR phosphorylation and aggressive disease. We observed increased expression of inflammatory genes in prostate cancer. Hormone naïve CD3, CD8, and CD68 infiltration reduced cancer specific survival and associated with tumour cytoplasmic AR expression. Additionally, elevated Ki67+ proliferation associated with elevated CD3, CD8, and CD68 infiltration suggesting highly proliferative tissues experience elevated inflammatory infiltrate. This data suggests inflammation in prostate cancer associates with a worse prognosis contradictory to the protective roles observed in other cancers. To conclude, the data presented within this thesis suggest androgen receptor serine 81 and serine 213 phosphorylation play important roles in the development of castrate resistant prostate cancer. Additionally, further in vitro and in vivo studies will be needed to fully investigate the combined effects of targeting AR and PI3K/AKT signaling at castrate resistance. Finally, of great interest to prostate cancer patients will be the in-depth investigation into the roles of inflammation and the introduction on immunotherapies into treatment regimens to reduce the inflammatory burden we have described within this thesis.