THE ROLE OF RNF6 IN REGULATION OF CELL CYCLE PROGRESSION IN PROSTATE CANCER

Abstract

Prostate cancer is one of the most commonly diagnosed cancers in men worldwide. Ubiquitin E3 ligases play an important role in carcinogenesis by modulating the expression levels of important cell cycle checkpoint proteins. The CDK2 inhibitor p27 (Kip1) is an important regulator of the G1/S checkpoint. Reduced nuclear p27 levels have previously been shown to be an independent predictor of decreased time between prostatectomy and biochemical recurrence. Here we investigated the role of the E3 ubiquitin ligase RNF6 in cell cycle progression in prostate cancer. Our data demonstrate that RNF6 can promote cell cycle progression by modulating the levels of p27. Knockdown of RNF6 led to an increase in the stability of p27 and to the arrest of cells in the G1 phase. RNF6 interacted with p27 via its KIL domain and this interaction was found to be phosphorylation independent. RNF6 enhanced the ubiquitination and subsequent degradation of p27 in the early G0/G1 phase of the cell cycle. Knockdown of RNF6 by short hairpin RNA led to the inhibition of the CDK2/Cyclin E complex thereby reducing the phosphorylation of the retinoblastoma protein (pRb) and to subsequent decrease in cell cycle progression and proliferation. In addition to its role in cell cycle regulation, RNF6 is also involved in the modulation of PARP-1 activity. Both PARP-1 and CDK2 play an important role in DNA repair. Since RNF6 regulates the activities of both, CDK2 and PARP-1, we hypothesized that combining a PARP-1/2 inhibitor Veliparib to a CDK2 inhibitor Dinaciclib might sensitize prostate cancer cells to the CDK2 inhibitor and enhance cytotoxicity. Treatment of LNCaP and CWR-R1 cells with Dinaciclib at pharmacologically relevant dose levels, led to a significant induction of apoptosis. Simultaneously treating DU145 cells with a PARP-1 inhibitor, Veliparib, led to enhanced PARP-1 cleavage which is consistent with apoptosis. In summary, we have provided evidence that p27 is a substrate for RNF6. Dinaciclib alone or in combination with Veliparib could be used as a therapeutic intervention in prostate cancer.