Characterisation of deubiquitinase enzymes involved in androgen receptor regulation in prostate cancer

Abstract

The androgen receptor (AR) is a key transcription factor in prostate cancer (CaP) growth and metastases, and is the main target for CaP treatment via the use of anti-hormonal therapies. Unfortunately, this treatment is only effective in the short-term and re-growth of the tumour results in most cases, termed castrate-resistant CaP (CRCaP), this is refractory to additional chemotherapies and hence fatal. Expression and reactivation of the AR is commonly seen in CRCaP and acts as a driver of advanced CaP growth suggesting the receptor remains a suitable target for next generation CaP therapies. Ubiquitination represents one of the numerous post-translational modifications that are vital for many cellular processes, including transcription and regulation of protein stability. The AR is a target for ubiquitination by several E3 ubiquitin ligase enzymes that can lead to either increased activity or degradation depending on the E3 ligase involved. Importantly, the persistence of the AR in CRCaP suggests that deciphering the mechanisms that regulate AR stability in this disease state may provide extremely useful and novel therapies. A major knowledge gap exists however, in our understanding of the reversal of ubiquitination by deubiquitinase enzymes (DUBs) within the AR signalling cascade that could be extremely important for the evidenced aberrant AR function in CRCaP. Therefore, the aim of this study was to characterise deubiquitinase enzymes (DUBs) involved in controlling AR activity and establishing potential roles of the enzymes in CaP development. A comprehensive siRNA library screen investigating the role of each DUB enzyme in AR signalling using the androgen-dependent LNCaP CaP cell line was undertaken and ubiquitin specific protease (USP) 12 (USP12) and USP10 were identified as regulators of AR activity. USP12 depletion resulted in reduced expression of androgen-responsive genes, suggesting USP12 is an activator of AR-mediated transcription, a notion confirmed in luciferase reporter assays. USP12 depletion failed to affect AR protein levels, but attenuated receptor recruitment to target gene promoters suggesting it is required to facilitate activated AR promoter binding. Studies of the phenotypic effects of USP12 knockdown revealed reduced LNCaP proliferation, increased cell cycle arrest and apoptosis, suggesting that USP12 inhibition could hold therapeutic potential in prostate cancer. In contrast, USP10 depletion resulted in increased expression of androgen-response genes, and repressed AR transcriptional activity in luciferase reporter assays. Unlike USP12, which interacted with AR when over-expressed in COS-7 cells, USP10 did not interact with the receptor, leading to the hypothesis that it may exert its effects through deubiquitination and stabilisation of the AR co-repressor, and E3 ubiquitin ligase, MDM2. A preliminary study to investigate the effects of MDM2 and USP10 co-transfection was unfortunately unsuccessful but further optimisation would assist in elucidating the role of the DUB in the AR signalling cascade. In conclusion, two DUB enzymes, USP12 and USP10, were identified to be important in modulation of AR activity. USP12 acts as co-activator of the AR and may be a potential therapeutic target in CaP. USP10, on the other hand, is a co-repressor of the AR and may act through MDM2.