Functional analysis of the histone methyltransferase SET9 in androgen receptor regulation and prostate cancer

Abstract

Prostate cancer is the leading cause of cancer-related death in western men and results in approximately 10,000 deaths in the UK per year (Cancer Research UK). The androgen receptor (AR) plays a prominent role in both androgen-dependent (AD) and androgen-independent (AI) disease, but treatments that attempt to inactivate the receptor are in-effective. There is a requirement therefore to develop new therapies that permanently disrupt AR function and attenuate disease progression. Hence, identification of new targets within the AR signalling cascade is vital. Numerous AR co-regulators have been identified that regulate AR activity and several of these proteins have been suggested to play a role in the progression of AD and AI disease. In this project, using CaP cell lines, different aspects of the histone methyltransferase enzyme SET9 were studied including its phenotypic influence, expression dynamics and also the molecular mechanisms it mediates in CaP cells. Our previous data demonstrated that SET9 enhances AR activity by directly methylating the receptor at lysine (K) residue 632 in the KLKK motif within the hinge domain of the receptor, and this affected co-activator-AR interaction in LNCaP prostate cancer cells. To assess the physiological role of SET9 in CaP cells, SET9 expression in LNCaP cells was reduced by siRNA interference and the effects on proliferation and apoptosis were investigated. Interestingly, SET9 knockdown reduced LNCaP cell proliferation and up-regulated apoptosis, implicating a role for SET9 in driving CaP progression. Moreover, a combination of SET9 knockdown and treatment with the DNA-damaging agent Doxorubicin in LNCaP cells synergised to increase apoptosis suggesting SET9 may be a potential therapeutic target for advanced CaP. Using a GFP-SET9 fusion protein and immunofluorescence, incorporating an anti-SET9 antibody, SET9 was demonstrated to be predominantly cytoplasmic in LNCaP and U2OS cells, suggesting additional, non-nuclear roles for SET9. To address this issue, and also to explore novel mechanisms of SET9 regulation, the enzyme was immunoprecipitated from LNCaP cells and the immunoprecipitated material was subjected to in solution based protein separation (OFF-GEL fractionation) followed by LC-MS/MS analysis to identify novel SET9 interacting proteins. Amongst several SET9 interacting partners, FXR1 was identified as a pro-apoptotic protein that facilitates SET9 knockdown mediated apoptosis in response to Doxorubicin. A predominantly cytoplasmic co-localisation pattern was confirmed for FXR1 using confocal microscopy, which was consistent with the data obtained from prostate clinical specimen using immunohistochemistry. Moreover, FXR1 was shown to function through AR to repress SET9 mediated co-activation of AR in reporter assays. More surprisingly, FXR1 displayed potent repressive effects on AR without the induction of SET9. In summary, this data highlights SET9 as a novel AR co-regulator that is important for prostate cancer cell growth. Further characterisation of SET9- interacting proteins including FXR1 may also provide novel protein targets for CaP therapy.