Hedgehog/Gli supports androgen signaling in androgen deprived and androgen independent prostate cancer cells

Abstract

BackgroundCastration resistant prostate cancer (CRPC) develops as a consequence of hormone therapies used to deplete androgens in advanced prostate cancer patients. CRPC cells are able to grow in a low androgen environment and this is associated with anomalous activity of their endogenous androgen receptor (AR) despite the low systemic androgen levels in the patients. Therefore, the reactivated tumor cell androgen signaling pathway is thought to provide a target for control of CRPC. Previously, we reported that Hedgehog (Hh) signaling was conditionally activated by androgen deprivation in androgen sensitive prostate cancer cells and here we studied the potential for cross-talk between Hh and androgen signaling activities in androgen deprived and androgen independent (AI) prostate cancer cells.ResultsTreatment of a variety of androgen-deprived or AI prostate cancer cells with the Hh inhibitor, cyclopamine, resulted in dose-dependent modulation of the expression of genes that are regulated by androgen. The effect of cyclopamine on endogenous androgen-regulated gene expression in androgen deprived and AI prostate cancer cells was consistent with the suppressive effects of cyclopamine on the expression of a reporter gene (luciferase) from two different androgen-dependent promoters. Similarly, reduction of smoothened (Smo) expression with siRNA co-suppressed expression of androgen-inducible KLK2 and KLK3 in androgen deprived cells without affecting the expression of androgen receptor (AR) mRNA or protein. Cyclopamine also prevented the outgrowth of AI cells from androgen growth-dependent parental LNCaP cells and suppressed the growth of an overt AI-LNCaP variant whereas supplemental androgen (R1881) restored growth to the AI cells in the presence of cyclopamine. Conversely, overexpression of Gli1 or Gli2 in LNCaP cells enhanced AR-specific gene expression in the absence of androgen. Overexpressed Gli1/Gli2 also enabled parental LNCaP cells to grow in androgen depleted medium. AR protein co-immunoprecipitates with Gli2 protein from transfected 293T cell lysates.ConclusionsCollectively, our results indicate that Hh/Gli signaling supports androgen signaling and AI growth in prostate cancer cells in a low androgen environment. The finding that Gli2 co-immunoprecipitates with AR protein suggests that an interaction between these proteins might be the basis for Hedgehog/Gli support of androgen signaling under this condition.

Gli Transcription Factors Mediate the Oncogenic Transformation of Prostate Basal Cells Induced by a Kras-Androgen Receptor Axis

“…This is further supported by evidence showing that Gli1 or Gli2 is associated with AR through direct protein-protein interactions and can regulate AR-controlled gene expression in prostate cancer (39,40). Additionally, co-activation of AR with the C-terminal domain of Gli2 regulates androgen-responsive genes in prostate cancer cells (41).…”

Emerging data on androgen receptor splice variants in prostate cancer

“…Gli2, which binds to the Tau5/AF5 region in the N-terminal domain of AR to enhance AR-FL activity (Chen, et al 2010), can also coactivate AR-V7 and AR v567es (Li, et al 2014b). However, there are also cofactors that interact with AR-FL and AR-Vs through different interfaces and with differing affinity.…”

Targeting the CB2 cannabinoid receptor in osteoporosis

“…The notion that hedgehog/Gli also affects androgen signaling originated from observations of a dose-dependent effect of cyclopamine on the expression of androgen-regulated genes [111] in LNCaP and other prostate cancer cells. Here, cyclopamine treatment was shown to specifically suppress expression of kallikrein-related peptidase (KLK)2, KLK3 and PGC in androgen-deprived, but not androgen-supplemented, LNCaP cells, whereas it further induced expression of Shh, which represents an androgen-repressed gene.…”

“…Here, it is notable that the GANT drugs did not significantly affect expression of Ptch1. Finally, in the reverse paradigm, exogenous expression of Gli1 or Gli2 in androgen-deprived prostate cancer cells not only increased the expression of androgen-dependent genes but also enabled these cells to grow in an androgen-deficient medium [111]. Collectively, the outcomes of these studies support the presence of a Smo-dependent signaling process, at least in androgen-deprived prostate cancer cells, which cross-talks with the androgen signaling pathway through Gli to affect androgen-regulated gene expression.…”

“…The involvement of Gli in the regulation of androgen-dependent genes suggests that the effect might be mediated by some form of Gli/AR interaction. Indeed, coimmunoprecipitation or two-hybrid analysis shows that Gli1 or Gli2 can directly bind to the AR protein [111,112]. Based on these reports, the Gli proteins may have AR coactivation functions that contribute to androgen signaling, especially in the androgen-deprived state.…”