Abstract 4492: Androgen receptor affects GLI activity by promoting transcriptionally active states of GLI upon androgen stimulation

Abstract

Abstract Introduction: While “castration” therapies benefit metastatic prostate cancer (PCa) patients, their effects are transient and progression to castration resistant disease (CRPC) remains a vexing clinical problem. Despite low levels of circulating androgens in treated patients, CRPC typically remains dependent on androgen/androgen receptor (AR) signaling for further growth. Previously we showed that Gli protein binding to AR co-activates AR transcriptional activity and supports androgen-independent growth. Here we show evidence that the interaction of AR with Gli proteins, particularly Gli3, allows for a “non-canonical” activation of Hedgehog signaling that contributes to PCa cell growth and progression to CRPC. Methods: Androgen growth-dependent (LNCaP) or -independent (LNCaP-AI, LN95) PCa cells were transfected with a Gli-luciferase (Gli-luc) reporter and were treated with R1881, enzalutamide (Enz), or a combination. Luciferase activity was measured. R1-D567 (harboring a genomic deletion of AR) served as a negative control. Knockdown of AR-FL with siRNA or overexpression of a small decoy peptide from the Gli2 AR-binding domain (Gli-DP) was also performed in above cells. Western blot and real-time PCR confirmed both AR and Gli target gene/protein expressions in LNCaP and LNCaP-AI cells (-/+ R1881). Sonic hedgehog (Shh) ligand-responsive 3T3-E1 cells were transiently expressed with AR-FL to confirm the involvement of canonical Shh-Gli activation pathway in AR co-activation of Gli transcription. Co-immunoprecipitation (co-IP) was performed to verify the blocking efficiency of Gli-DP in AR-Gli3 interaction in LN95 cells and AR-overexpressing 293FT. Results: Androgen (R1881) treatment strongly induced Gli transcription activity in AR+ PCa cells while Enz reversed this effect; AR-FL-specific knockdown or exogenous Gli-DP expression suppressed androgen-induced Gli reporter expression as well as expression of endogenous Gli-responsive genes. R1881 treatment promoted Gli2/3 protein processing to transcriptionally active states (Gli3-FullLength and Gli2-Active) and enhanced the expression of Gli-target genes, Gli1/Ptch1, at protein and/or mRNA levels. Co-IP revealed that exogenous Gli-DP blocked the interaction between AR and transcriptionally active Gli3-FL in PCa cells. Overexpression of AR in 3T3-E1 cells confirmed that AR interaction with Gli stabilizes the active Gli3-FL form by suppressing degradation of Gli3 in a manner independent of the canonical Shh-Gli activation pathway. Conclusions: Our findings further identify the importance of AR-Gli interaction in progression of PCa to CRPC and shows that cooperative binding of AR to Gli proteins enhances both AR and Hedgehog signaling in PCa cells. Interference with the AR-Gli interaction using a decoy peptide provides a means to suppress AR activation of Hedgehog in PCa cells. Citation Format: Sarah Truong, Na Li, Mannan Nouri, Ralph Buttyan. Androgen receptor affects GLI activity by promoting transcriptionally active states of GLI upon androgen stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2017-4492