Abstract 4396: Gli, not Androgen Receptor, is the primary driver of prostate cell growth

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Abstract Introduction Hedgehog (Hh) signaling regulates the activity of Gli transcription factors. Gli controls genes needed for development, cell growth and cell motility. Canonical Hh signaling through Smoothened [Smo] suppresses the proteolysis of Gli2 and Gli3, maintaining them in high molecular weight active forms. Previously, we showed that active androgen receptors ([AR] liganded AR full length and AR-V7) recognize and binds Gli3 its Protein Processing Domain. This binding overrides the need for Smo action and provides a means for stabilization and activation of Gli3 in prostate cancer (PCa) cells without Hh. Studies on the effects of AR binding to Gli2 are more challenging because it is expressed at much lower levels than Gli3. Here, we report that AR also affects Gli2 protein stability and show that Gli2, like Gli3, is a driver of PCa cell growth that is overexpressed in castration resistant disease. Method Immunohistochemistry (IHC) using Gli2- and Gli3-specific antibodies was used to assess expression of Gli2 and Gli3 in human PCa tumor microarrays. Western blot were used to identify and quantify levels of active Gli2 and Gli3 in androgen-treated PCa cells (LNCaP, LNCaP-AI, LAPC4). Proximity ligation assays (PLA) was used to visualize in situ and quantify AR-Gli2 complexes in PCa cells. Gli2- and Gli3-specific siRNAs were used to knock down Gli expression in AR+ (LNCaP-AI, LAPC4, 22Rv1) and AR- (PC3) PCa, cells followed by the Gli reporter assay and the cell growth assay. Results IHC outcomes showed that nuclear Gli2 and Gli3 protein expression was significantly higher in castration resistant tumors compared to primary disease. Androgens increased expression of active Gli2 and Gli3 protein expression in PCa cells and stimulated Gli reporter activity. PLA showed the presence of intranuclear complexes of Gli2/Gli3-AR-Full-length in androgen-treated LNCaP and Gli2-AR-V7 complexes in CWR22rv1 cells. Knockdown of Gli2 or Gli3 present significant reduction in Gli transcriptional activity as well as inhibition on growth of both AR+ and AR- PCa cells that were tested. Conclusion AR activity affects Gli2 processing, as it does for Gli3. We can visualize and quantify AR-Gli2/Gli3 complexes in situ in PCa cells. Gli2 suppression, as well as Gli3 suppression, has a profound effect on PCa cell growth but combined suppression has the strongest effects in certain PCa cells. Finally, Gli2 and Gli3 protein expression is elevated in castration resistant disease. Citation Format: Jane Foo, Na Li, Shabnam Massah, Mannan Nouri, Mike Wang, Ralph Buttyan. Gli, not Androgen Receptor, is the primary driver of prostate cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4396.