Stilbene induced inhibition of androgen receptor dimerization: implications for AR and ARΔLBD-signalling in human prostate cancer cells

Abstract

Background: Advanced castration resistant prostate cancer (CRPC) is often characterized by an increase of C-terminally truncated, constitutively active androgen receptor (AR) variants. Due to the absence of a ligand binding domain located in the AR-C-terminus, these receptor variants (also termed ARΔLBD) are unable to respond to all classical forms of endocrine treatments like surgical/chemical castration and/or application of anti-androgens.

Methodology: In this study we tested the effects of the naturally occurring stilbene resveratrol (RSV) and (E)-4-(2, 6-Difluorostyryl)-N, N-dimethylaniline, a fluorinated dialkylaminostilbene (FIDAS) on AR- and ARΔLBD in prostate cancer cells. The ability of the compounds to modulate transcriptional activity of AR and the ARΔLBD-variant Q640X was shown by reporter gene assays. Expression of endogenous AR and ARΔLBD mRNA and protein levels were determined by qRT-PCR and Western Blot. Nuclear translocation of AR-molecules was analyzed by fluorescence microscopy. AR and ARΔLBD/Q640X homo-/heterodimer formation was assessed by mammalian two hybrid assays. Biological activity of both compounds in vivo was demonstrated using a chick chorioallantoic membrane xenograft assay.

Results: The stilbenes RSV and FIDAS were able to significantly diminish AR and Q640X-signalling. Successful inhibition of the Q640X suggests that RSV and FIDAS are not interfering with the AR-ligand binding domain like all currently available anti-hormonal drugs. Repression of AR and Q640X-signalling by RSV and FIDAS in prostate cancer cells was caused by an inhibition of the AR and/or Q640X-dimerization. Although systemic bioavailability of both stilbenes is very low, both compounds were also able to downregulate tumor growth and AR-signalling in vivo.

Conclusion: RSV and FIDAS are able to inhibit the dimerization of AR and ARΔLBD molecules suggesting that stilbenes might serve as lead compounds for a novel generation of AR-inhibitors.

Figure 1. Regulation of AR- and ARΔLBD-protein by RSV and FIDAS in human PC cell lines.

Prostate cancer cells were grown in presence/absence of DHT and incubated with RSV or FIDAS for 24 hours. Subsequently, intracellular AR or ARΔLBD-levels (AR-V7, Q640X) were determined by Western Blotting as described in Material and Methods: (A) LNCaP, (B) 22Rv1, (C) PC-3 transfected with full length AR, (D) PC-3 transfected with Q640X.

Figure 2. Effects of FIDAS and RSV on AR and Q640X signalling.

PC-3 cells were transiently co-transfected with AR or Q640X, the pARE(2x)-luc reporter and the pRL-TK control plasmid as described in Material and Methods. (A) PC-3 cells transfected with AR were grown with/without 5 nM DHT in the presence/absence of RSV or FIDAS for 24 hours. Data are expressed as % transactivation of DHT stimulated AR ( =  % activity AR_DHT/AR_basal) which was set at 100%; *p<0.05. (B) PC-3 cells transfected with constitutively active Q640X were treated with RSV or FIDAS for 24 hours. Data are expressed as % transactivation of Q640X which was set at 100%, *p<0.05).

Figure 3. Effects of RSV and FIDAS on AR/Q640X-signalling under androgen deprived conditions.

PC-3 cells were transiently transfected with AR, Q640X or AR+Q640X, together with pPSA-61luc and pRL-TK reporter plasmids as described in Material and Methods. Cells expressing Q640X and/or AR were grown for 24 hours in presence/absence of RSV (100 µM) or FIDAS (50 µM). Data are expressed in fold AR basal activity which was set to 1. As depicted, RSV and FIDAS significantly decreased PSA-promoter mediated reporter gene activity in Q640X and AR co-expressing cells, *p>0.05.

Figure 4. RSV and FIDAS do not inhibit nuclear translocation of AR and Q640X.

PC-3 cells were transiently transfected with expression plasmids coding for green fluorescent AR-EosFP or Q640X-EGFP fusion proteins as described in Material and Methods. Subsequently cells were treated with/without 5 nM DHT in presence/absence of 100 µM RSV for 120 minutes. Nuclear localization of fluorescent AR or Q640X proteins was analyzed by fluorescence microscopy.

Figure 5. Effects of RSV and FIDAS on the dimerization of AR and Q640X.

Formation of AR and Q640X homo/hetero-dimers was analyzed 24 hours after RSV or FIDAS treatment using a M2H as described in Material and Methods. Within this time frame RSV as well as FIDAS did not exhibit a significant in vitro toxicicity (see Table S1). AR/AR dimers: Formation of AR/AR homodimers was analyzed in PC-3 cells grown under androgenic stimuli (5 nM DHT) in presence/absence of RSV or FIDAS (FIDAS/RSV untreated + DHT  = 100%). AR/Q640X and Q640X/Q640X dimers: Formation of AR/Q640X heterodimers (AR-VP16/ACT and Q640X-GAL4/BIND) or Q640X/Q640X homodimers was analyzed in the absence of DHT in RSV/FIDAS treated/untreated PC-3 cells (FIDAS/RSV untreated was set at 100%), *p<0.05.

Figure 6. Effects FIDAS on prostate cancer micro-tumors growing on the CAM.

CAM assays were performed with PC-3 (AR negative) and LNCaP (AR-positive) as described in Material and Methods. Proliferation of PC cells was determined by nuclear staining of KI67. AR-activity was analyzed by PSA-staining.