Characterisation of the co-chaperone small glutamine-rich tetratricopeptide repeat containing protein alpha as a regulator of androgen receptor activity in prostate cancer cells.

Abstract

Prostate cancer remains one of the leading causes of cancer related morbidity and mortality in Australian men. The androgen receptor (AR) is an intracellular transcription factor that mediates the biological actions of circulating androgens to drive the growth and survival of prostate cancer cells. However, current treatment options for non-localised, advanced stage prostate cancer invariably fail, which is a consequence of continued AR signalling during all stages of disease progression. Therefore, understanding the regulatory mechanisms of AR action is essential for the development of more effective therapies. Molecular regulation of the AR can occur during the process of protein maturation. This includes the incorporation of tetratricopeptide repeat (TPR) containing co-chaperones into the heat shock protein 90 (Hsp90) molecular chaperone complex, which collectively acts to generate AR proteins capable of high affinity ligand binding, nuclear translocation and gene regulation. The co-chaperone small glutamine-rich TPR containing protein alpha (SGTA) acts to restrict AR nuclear translocation and thereby regulate AR transcriptional activity. The clinical implications of SGTA are evident by a decline in protein levels with prostate cancer progression. The loss of SGTA may therefore disrupt the regulatory process of AR cytoplasmic retention, and thus contribute to continued AR activity. However the precise regions of SGTA that mediate these effects or how SGTA may contribute to prostate cancer proliferation have yet to be elucidated. The aims of this thesis were to further characterise the mechanisms of SGTA action on AR signalling, to further define the biological actions of SGTA in prostate cancer cells and to determine the functional consequences these actions have on prostate cancer growth. SGTA is unique amongst the steroid receptor associated TPR co-chaperones through its ability to form homodimers. Therefore, the requirement of the SGTA homodimerisation domain may be essential in deciphering how SGTA affects AR activity. Deletion mapping combined with structural prediction analysis revealed that the first 80 amino acids of the amino terminus are required to form SGTA homodimers, as well as maintain SGTA protein steady state levels. Further studies also confirmed that SGTA homodimerisation occurs independently of the Hsp90 interacting TPR domain or glutamine-rich carboxyl terminus, implying that interactions between SGTA and the Hsp90 molecular chaperone complex do not contribute to the formation of SGTA homodimers. Moreover, inhibition of SGTA homodimerisation did not alter the ability of SGTA to inhibit AR activity. These studies demonstrate that while the SGTA homodimerisation is characterised by conserved structural elements within the first 80 amino acids, SGTA can sufficiently act as a monomer to inhibit AR activity. Further investigations into the mechanisms of how SGTA affects AR transactivation activity demonstrated that SGTA restricts the ligand sensitivity of AR activity across multiple androgen responsive luciferase reporters in a low hormone environment. These studies also revealed that the strongest inhibitory effect by SGTA was observed on those loci that exhibited the greatest fold change to androgen treatment. Additionally, the effects of SGTA on AR are not mediated through the homodimerisation, TPR or glutamine-rich domains. These results suggest that SGTA has the ability to constrain the sensitivity of AR activation to ligand in a castrate environment and therefore SGTA may act as a buffer against aberrant AR responses. To determine the functional consequences of modulating SGTA levels in prostate cancer cells, both SGTA over expression and knockdown models were developed. Modulation of SGTA levels in prostate cancer cell lines decreased proliferation. This effect was not a result of altered AR activity, as determined by expression of the androgen regulated gene prostate specific antigen (PSA). Collectively, these results suggest that the impact of SGTA on prostate cancer cell proliferation is independent of AR signalling. Microarray analysis was used to determine the effects of SGTA knockdown on the prostate genome, which demonstrated that cells depleted of SGTA differentially regulated the expression of genes involved in cell cycle regulation. Specifically, genes with decreased expression included several within the Class I phophoinositide-3 kinase (PI3 kinase) pathway, a critical pro-proliferation signalling pathway in prostate cancer. Consequently, knockdown of SGTA resulted in decreased Akt phosphorylation at the threonine 308 (T308) and serine 473 (S473) residues, which are key indicators of active PI3 kinase signalling. Collectively, these studies demonstrated that SGTA is able to modulate the expression and/or activity of multiple signalling pathways within prostate cancer cells to influence cell proliferation. The findings from this thesis provide novel insights into the structural and functional complexity of SGTA action on AR activity in prostate cancer cells. Importantly, this highlights the potential mechanistic consequences that SGTA may have for AR and other signalling pathways that occur during prostate cancer pathogenesis and provides the basis for further investigations evaluating SGTA as a novel therapeutic target in prostate cancer.