Targeting PCNA/AR interaction inhibits AR-mediated signaling in castration resistant prostate cancer cells

Abstract

We previously showed that proliferating cell nuclear antigen (PCNA) interacts with androgen receptor (AR) through a PIP-box (PIP-box4) at the N-terminus of AR and regulates AR activity. In this study, we further investigated PCNA/AR interaction. We identified a second PIP-box (PIP-box592) in the DNA binding domain of AR and found that dihydrotestosterone enhances the binding of full-length AR (AR-FL) but not a constitutively active variant (AR-V7) to PCNA. Treatment with R9-AR-PIP, a PIP-box4-mimicking small peptide, inhibits the PCNA/AR interaction, AR occupancy at the androgen response element (ARE) in PSA and p21 genes, and expression of AR target genes, and induces cytotoxicity in AR-positive castration-resistant prostate cancer (CRPC) cells. R9-AR-PIP also significantly inhibits transcriptional activity of AR-FL upon dihydrotestosterone stimulation and the constitutive activity of AR-V7. Moreover, R9-AR-PIP and PCNA-I1S, a small molecule PCNA inhibitor, inhibit the ARE occupancy by AR-FL and AR-Vs in CCNA2 gene that encodes cyclin A2 and cyclin A2 expression. Finally, we found that cyclin A2 is overexpressed in all CRPC cells examined, suggesting that it may contribute to the development of CRPC. These data indicate that targeting PCNA/AR interaction inhibits both AR-FL- and AR-Vs-mediated signaling and implicates it could be a novel therapeutic strategy against CRPC.

Keywords: AR splicing variants; CRPC; PCNA; PCNA inhibitors; androgen receptor.

Figure 1. The functional domains of AR-FL and AR-Vs.

PIP box4 and PIP box592 are indicated. Abbreviations: NLS: nuclear localization signal; NTD: N-terminal domain; DBD: DNA binding domain; LBD: ligand binding domain (LBD).

Figure 2. Identification of PIP-box592 at the DNA binding domain of AR.

(A) The cell extracts from PC-3 transfected with HA-AR-FL, HA-AR-NTD, or HA-AR-V7 expression vectors and (B) the cell extracts from PC-3 transfected with HA-AR-V7, HA-AR-V7-PIP4m, or HA-AR-V7-PIP592m expression vectors were subjected to GST-PCNA pull-down assay (Upper panel). The transfected PC-3 cell extracts (25 μg) were served as loading control (Lower panel). HA-tag and PCNA antibodies were used for immunoblot analysis.

Figure 3. Dihydrotestosterone enhances the binding of AR-FL to PCNA.

(A) PC-3 cells transfected with AR-FL or AR-V7 expression vector and (B) LNCaP and 22Rv1 cells, were treated without or with dihydrotestosterone (DHT) (10⁻⁸M) in stripped medium overnight. The cell extracts (1 mg) from these cells were subjected to GST-PCNA pull-down assay. AR(441) antibody was used for immunoblot analysis. Input of GST-PCNA for the pull-down assay was indicated. PC-3 cell extracts (25 μg) were served as loading control for GST pull-down (A, lower panel). (C, D) The cell extracts (1 mg) from LNCaP cells (C) without or with dihydrotestosterone and/or R9-AR-PIP (20 μM) treatment overnight were subjected to Co-Immunoprecipitation (Co-IP) using AR antibody. The cell extracts (1 mg) from 22Rv1 cells (D) were subjected to GST-PCNA pull-down assay in the absence and presence of 30 μM T2AA. PCNA and AR antibodies were used for immunoblot analysis.

Figure 4. R9-AR-PIP inhibits transcriptional activity of AR-FL and AR-Vs.

PC-3 cells transfected with PSA(4.3)-Luc reporter as well as expression vector HA-AR-FL, HA-AR-V7, or HA-AR-V7-PIP4+592m for 4 hours respectively, and then incubated without (control) or with R9-AR-PIP (20 μM) and/or dihydrotestosterone (DHT) (10⁻⁸M) for 24 hours, followed by luciferase assay.

Figure 5. R9-AR-PIP and PCNA-I1S inhibit the chromatin association and the ARE occupancy by AR-FL and AR-Vs.

(A) 22Rv1 cells in the stripped medium treated without or with dihydrotestosterone (DHT, 10⁻⁸M), R9-AR-PIP (R9-PIP, 20 μM), T2AA (5 μM), and/or PCNA-I1S (I1S, 1 μM) for 24 hours. Fifty mg chromatin-bound nuclear proteins were subjected to immunoblot analysis using AR(441), PCNA, and Histone H1 antibodies. LNCaP (B) or R1-D567 (C) cells in the stripped medium were treated without or with dihydrotestosterone (10⁻⁸M), R9-AR-PIP (20 μM), PCNA-I1S (1 μM), or Enzalutamide (Enz, 20 μM) overnight. These cells were subjected chromatin immunoprecipitation (ChIP) assay.

Figure 6. Colocalization of AR-FL and AR-V7 with PCNA.

PC-3 cells in stripped medium were transfected with expression vector AR-FL (A) or AR-V7 (B) for 18 hours, incubated either in medium (CTR) or treated with PCNA-I1S (1 μM), T2AA (20 μM), or R9-AR-PIP (20 μM) for 1 hour, then followed by incubation in medium (Med) or stimulation with dihydrotestosterone (DHT) (10 nM) for 4 hours. Subsequently, the cells were stained with fluorophore-labeled anti-AR reacting with both AR-FL and AR-V7 (Alexa Fluor594, red color) and ani-PCNA (Alexa Fluor488, green color) antibodies.

Figure 7. The differential cytotoxic effects of PCNA inhibitors in prostate cancer cells varying with AR expression status.

(A) Cells were plated into 12-well plates at 300–500 cells/well. After an overnight incubation, the cells were treated with enzalutamide (Enz, 20 μM), R9-AR-PIP (R9-PIP, 20 μM), or Enz plus R9-AR-PIP. Twenty-four hours later, the cells were cultured in fresh medium for up to 2 weeks. The colonies formed by these cells were stained with crystal violet and counted using ImageJ with Colony Counter plug-in. (B) Representative images of the assay. ns, not significant; ^* p < 0.05; ^*** p < 0.001.

Figure 8. Dihydrotestosterone regulates cyclin A2 expression in AR-FL expressing cells.

(A) Cell extracts from androgen dependent LNCaP (AR-FL⁺) cells and 4 lines of CRPC cells LNCaP-AI (AR-FL⁺), 22Rv1(AR-FL⁺/AR-V7⁺), R1-D567 (ARv567es), and PC-3(AR⁻) were prepared. LNCaP or R1-D567 cells were cultured in stripped medium for 1 day and then treated without or with dihydrotestosterone (DHT) (B), as well as R9-AR-PIP (20 μM) or PCNA-I1S (1 μM) (C) for 24 hours. The cell extracts from these cells were subjected to immunoblot analysis using AR, cyclin A, and GAPDH antibodies.