Mono-ubiquitination mediated regulation of KMT5A and its role in prostate cancer

Abstract

Abstract: Prostate cancer (PC) is the most common cancer and the second cause of cancer related death in men. Central to this, is the role of the androgen receptor (AR) which acts as a transcription factor, regulating the expression of genes required for normal prostate growth and cancer development. Consequently, the AR remains the primary target for therapeutic intervention. However, these treatments become ineffective, resulting in castrate resistant prostate cancer (CRPC) which generally retains AR expression. The AR interacts with several co-regulatory proteins which can perturb AR-targeted therapies in CRPC. Targeting these co-regulatory proteins to indirectly target the AR signalling cascade may prove beneficial. Recently, our group identified KMT5A as a potential regulator of AR through selective siRNA library screening. KMT5A is a lysine methyltransferase that mono-methylates histone 4 lysine 20 and non-histone proteins, including p53. Using a relevant in vitro CRPC model it was shown that KMT5A acquires AR co-activator activity which is in contrast to androgen sensitive models where KMT5A co-represses AR activity. This highlights the importance of studying KMT5A regulation. KMT5A protein levels are tightly regulated by multiple E3 ligases for cell cycle-dependent poly-ubiquitination-mediated degradation. KMT5A poly-ubiquitination by E3 ligases SCFβ-TRCF, CRL4Cdt2 and APCCdh1 promotes degradation in G1, S and late mitosis cell cycle phases, respectively. Moreover, the Skp2 E3 ligase has been suggested to play a role in KMT5A ubiquitination and degradation but direct supporting evidence is currently absent. Additionally, Skp2 is suggested to directly regulate the AR signaling pathway. It is also unknown whether KMT5A could be modified directly by ubiquitination without promoting its degradation. As such, we aimed to investigate KMT5A mono-ubiquitination and the role of Skp2 in regulating KMT5A as well as independently regulating the AR signaling cascade. Mono-ubiquitinated KMT5A was demonstrated in a panel of PC cell lines. Its existence was further confirmed by performing ubiquitination assays in COS7 cells. Furthermore, the KMT5A C-terminal SET domain was identified as the target for mono-ubiquitination. Moreover, mono-ubiquitinated KMT5A was highly enriched in S phase cells, coincident with extremely low levels of unmodified KMT5A. Mono-ubiquitinated KMT5A was exclusively cytoplasmic and its abundance was greatly enhanced by Skp2, but not associated with protein turnover. Together, this data suggests that cell cycle-dependent KMT5A mono-ubiquitination is an important mechanism to diminish nuclear, unmodified KMT5A levels to facilitate cell cycle progression. Thus, insight for the physiological significance of mono-ubiquitinated KMT5A ii may provide a novel therapeutic target to indirectly target the AR. Finally, Skp2 was not found to have a direct effect on AR signaling.