Abstract 1294: Identification of the binding domains that allow ligand-independent co-activation of androgen receptor by Gli2.

Abstract

Abstract Gli proteins are transcription factors that mediate the cellular response to Hedgehog signaling. Gli activity guides cell fate decisions during development and is required for the proper morphogenesis of embryonic tissues, including the prostate gland. Previously, we showed that exogenous overexpression of Gli1 or Gli2 in androgen-deprived LNCaP cells increased the expressions of androgen-dependent genes in prostate cancer (PCa) cells and conferred androgen-independent growth behavior to them (Chen et al., Mol Cancer, 9: 89-101, 2010). We also showed that Gli1 or Gli2 proteins co-immunoprecipitate with AR, suggesting that AR interacts with Gli proteins to mediate these effects. Based upon this data, we proposed that Gli1/2 proteins are ligand-independent co-activators of AR in PCa. To better understand the Gli2-AR interaction and its effects on PCa responsiveness to androgen ablation, we fine mapped the specific interaction domains between Gli2 and AR. Complete or partial recombinant Gli2 cDNAs were tagged with GST and the GST-tagged proteins were purified on glutathione beads prior to incubation with in vitro translated human AR protein fragments. GST pulldown assays showed that GLI2 specifically recognizes and binds the Tau5 region within the AR N-terminal domain (AA 391-555). Likewise, AR binds to Gli2 within an extended C-terminal domain (AA 628-1091) that may involve two distinct AR binding sites. AR co-activation studies in 293T cells shows that the presence of binding site(s) and the Gli2 activation domain are required for Gli2 co-activation of AR. Gli2 was tested for its ability to co-activate natural truncated variants of the AR. The interaction of Gli2 with the Tau4 domain of AR was consistent with the ability of Gli2 to co-activate truncated variants of the AR that lack the DNA binding domain. Our work demonstrates that Gli2 co-activation of AR requires direct interaction between the tau5 domain in the AR N-terminus and the AR binding domains in the Gli2 C-terminus as well as the Gli2 activation domain. Gli2 binding at tau5 supports co-activation of the wildtype AR (in both the presence and absence of androgen) as well as co-activation of truncated receptors that do not bind androgens. As such, the evidence that Gli2 expression is increased in PCa by androgen ablation suggests that it may be a factor in progression to castration resistant PCa. Supported by the DOD PCRP (W81XH-10-1-0493, to RB). Citation Format: Na Li, Mengqian Chen, Ralph Buttyan. Identification of the binding domains that allow ligand-independent co-activation of androgen receptor by Gli2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1294. doi:10.1158/1538-7445.AM2013-1294