Figure 6.

A feed-forward AR-V-PARP regulatory loop facilitates AR-V activity in CRPC. (A) PARPBP and PARP2 mRNA levels were analysed by quantitative RT-PCR in CWR22Rv1-AR-EK and CWR22Rv1 cells transfected with control (siScr) or AR (siARex1) siRNAs for 48 h; CWR22Rv1 cells were also grown in the presence and absence of 10 μM enzalutamide. Data represents the average of three independent experiments ± SD (*P< 0.05 as determined using a two-tailed Student's t-test). (B) Cellular PARP activity was assessed by immunoblotting in CWR22Rv1-AR-EK and CWR22Rv1 cells depleted of AR (siARexon1) using an anti-PAR antibody. Lysates were also probed for PARP1, AR and α-tubulin antibodies. (C) CWR22Rv1-AR-EK cells were treated with and without either 1 and 10 μM talazoparib (Talaz) or olaparib (Olap) for 96 hours before cell count analysis. Data represents the average of three independent experiments ± SD (**P< 0.01, as determined using a two-tailed Student's t-test). (D) LNCaP cells transduced with control or AR-V7-expressing lentivirus for 24 h and then treated with 1 μM talazoparib (Talaz) for an additional 24 h were subject to quantitative RT-PCR to assess expression of AR-target (upper panel) and DDR-associated (lower panel) genes. Data represents the average of three independent experiments ± SD (**P< 0.01 as determined using a two-tailed Student t-test). Lower immunoblot image demonstrates ectopic expression of AR-V7 in LNCaP cells transduced with AR-V7-expressing lentivirus. (E) Diagrammatic representation of interplay between AR-Vs, the DDR pathway and PARP activity in cells. Expression of AR-V-regulated genes, including those involved in the DDR, is enhanced by PARP1/2. The ability of AR-Vs to up-regulate PARPBP and PARP2 expression, which enhance cellular PARP activity, potentiates the existence of a feed-forward regulatory loop in CRPC.