Hedgehog (Hh) is an oncogenic signaling pathway that regulates the activity of Gli transcription factors. Canonical Hh is a Smoothened- (Smo-) driven process that alters the post-translational processing of Gli2/Gli3 proteins. Though evidence supports a role for Gli action in prostate cancer (PCa) cell growth and progression, there is little indication that Smo is involved. Here we describe a non-canonical means for activation of Gli transcription in PCa cells mediated by the binding of transcriptionally-active androgen receptors (ARs) to Gli3. Androgens stimulated reporter expression from a Gli-dependent promoter in a variety of AR + PCa cells and this activity was suppressed by an anti-androgen, Enz, or by AR knockdown. Androgens also upregulated expression of endogenous Gli-dependent genes. This activity was associated with increased intranuclear binding of Gli3 to AR that was antagonized by Enz. Fine mapping of the AR binding domain on Gli2 showed that AR recognizes the Gli protein processing domain (PPD) in the C-terminus. Mutations in the arginine-/serine repeat elements of the Gli2 PPD involved in phosphorylation and ubiquitinylation blocked the binding to AR. β-TrCP, a ubiquitin ligase that recognizes the Gli PPD, competed with AR for binding to this site. AR binding to Gli3 suppressed its proteolytic processing to the Gli3 repressor form (Gli3R) whereas AR knockdown increased Gli3R. Both full-length and truncated ARs were able to activate Gli transcription. Finally, we found that an ARbinding decoy polypeptide derived from the Gli2 C-terminus can compete with Gli3 for binding to AR. Exogenous overexpression of this decoy suppressed Gli transcriptional activity in PCa cells. Collectively, this work identifies a novel pathway for non-canonical activation of Hh signaling in PCa cells and identifies a means for interference that may have clinical relevance for PCa patients.

中文翻译：

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转录活性雄激素受体蛋白与Gli3相互作用介导的前列腺癌细胞中刺猬的非规范激活。

刺猬（Hh）是调节Gli转录因子活性的致癌信号通路。Canonical Hh是一种平滑化（Smo-）驱动的过程，可改变Gli2 / Gli3蛋白的翻译后加工过程。尽管有证据支持Gli作用在前列腺癌（PCa）细胞生长和进展中起作用，但几乎没有迹象表明Smo参与其中。在这里，我们描述了PCa细胞中由转录活性雄激素受体（ARs）与Gli3结合介导的Gli转录激活的非规范方法。雄激素刺激了各种AR + PCa细胞中Gli依赖型启动子的报告基因表达，并且该活性被抗雄激素，Enz或AR敲低所抑制。雄激素也上调内源性Gli依赖性基因的表达。这种活性与被Enz拮抗的Gli3与AR的核内结合增加有关。AR结合域在Gli2上的精细定位表明，AR识别C末端的Gli蛋白加工域（PPD）。参与磷酸化和泛素化的Gli2 PPD的精氨酸/丝氨酸重复元件中的突变阻止了与AR的结合。β-TrCP是一种识别Gli PPD的泛素连接酶，它与AR竞争与该位点的结合。AR与Gli3的结合抑制了其对Gli3阻遏物形式（Gli3R）的蛋白水解过程，而AR敲低增加了Gli3R。全长AR和截短AR都能够激活Gli转录。最后，我们发现源自Gli2 C末端的AR结合诱饵多肽可以与Gli3竞争与AR的结合。该诱饵的外源过度表达抑制了PCa细胞中的Gli转录活性。总的来说，这项工作确定了PCa细胞中Hh信号非规范激活的新途径，并确定了可能对PCa患者具有临床意义的干扰手段。