Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells.

The androgen receptor (AR) is activated in patients with castration resistant prostate cancer (CRPC) despite low circulating levels of androgen, suggesting that intracellular signaling pathways and non-androgenic factors may contribute to AR activation. Many G-protein coupled receptors (GPCR) and their ligands are also activated in these cells indicating that they may play a role in development of Prostate Cancer (PCa) and CRPC. Although a cross talk has been suggested between the two pathways, yet, the identity of GPCRs which may play a role in androgen signaling, is not established yet. By using blast analysis of 826 GPCRs, we identified a GPCR, GPCR 205, which exhibited maximum similarity with the ligand binding domain of the AR. We demonstrate that adhesion GPCR 205, also known as GPR56, can be activated by androgens to stimulate the Rho signaling pathway, a pathway that plays an important role in prostate tumor cell metastasis. Testosterone stimulation of GPR56 also activates the cAMP/ Protein kinase A (PKA) pathway, that is necessary for AR signaling. Knocking down the expression of GPR56 using siRNA, disrupts nuclear translocation of AR and transcription of prototypic AR target genes such as PSA. GPR56 expression is higher in all twenty-five prostate tumor patient's samples tested and cells expressing GPR56 exhibit increased proliferation. These findings provide new insights about androgen signaling and identify GPR56 as a possible therapeutic target in advanced prostate cancer patients.

Phosphorylation of HSP90 by protein kinase A is essential for the nuclear translocation of androgen receptor.

The androgen receptor (AR) is often activated in prostate cancer patients undergoing androgen-ablative therapy because of the activation of cellular pathways that stimulate the AR despite low androgen levels. In many of these tumors, the cAMP-dependent protein kinase A (PKA) pathway is activated. Previous studies have shown that PKA can synergize with low levels of androgen to enhance androgen signaling and consequent cell proliferation, leading to castration-resistant prostate cancer. However, the mechanism by which PKA causes AR stimulation in the presence of low/no androgen is not established yet. Here, using immunofluorescence immunoblotting assays, co-immunoprecipitation, siRNA-mediated gene silencing, and reporter gene assays, we demonstrate that PKA activation is necessary for the phosphorylation of heat shock protein (HSP90) that binds to unliganded AR in the cytoplasm, restricting its entry into the nucleus. We also found that PKA-mediated phosphorylation of the Thr89 residue in HSP90 releases AR from HSP90, enabling AR binding to HSP27 and its migration into the nucleus. Substitution of the Thr89 in HSP90 prevented its phosphorylation by PKA and significantly reduced AR transactivation and cellular proliferation. We further observed that the transcription of AR target genes, such as prostate-specific antigen (PSA), is also lowered in the HSP90 Thr89 variant. These results suggest that using a small-molecule inhibitor against the HSP90 Thr89 residue in conjunction with existing androgen-ablative therapy may be more effective than androgen-ablative therapy alone in the treatment of prostate cancer patients.