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Non-canonical activation of Hedgehog signaling in prostate cancer cells is mediated by the interaction of Gli proteins and transcriptionally active androgen receptor

University of British Columbia

The Hedgehog (Hh) pathway is an embryonic development pathway, driven by peptide ligands called hedgehogs. During progression of prostate cancer (PCa), Hh signaling is increased, with especially high activation in castration resistant disease (CRPC). Evidence is lacking for canonical Hh signaling in PCa, indicating the likelihood that non-canonical pathways are involved. Our work shows that transcriptionally active androgen receptor (AR) binding to Gli proteins drives non-canonical Hh signaling in PCa. Androgen-sensitive LNCaP and androgen-independent LNCaP-AI and LN95 cells were transfected with a Gli-promoter driven luciferase reporter and treated with R1881, enzalutamide, or both, and luciferase activity was measured. Androgen treatment (R1881) induced Gli transcriptional activity while enzalutamide reversed this effect. Similarly, siRNA knockdown of full-length AR (AR-FL) suppressed R1881-induced Gli transcription. Western blot and qPCR confirmed increased expression of endogenous Gli target genes Gli1 and Ptch1 with androgen treatment. Androgen treatment stabilized expression of full-length active Gli3 in a dose-dependent manner but this was reversed by AR knockdown using siRNA. AR binds to Gli3 at the protein processing domain (PPD), which, given the above data, suggests that AR binding to Gli stabilizes full-length Gli3 by preventing phosphorylation and ubiquitination of the PPD, thereby stopping proteolytic cleavage and proteosomal degradation from occurring. Finally, we found that an AR-binding decoy peptide derived from the Gli2 C-terminus can compete with Gli3 for binding to AR, suppressing Gli transcriptional activity in PCa cells. Our data supports the idea that transcriptionally active AR binding to Gli proteins provides a means for Hh signaling to occur. Not only does AR co-activate Gli transcriptional activity, but it also alters the proteolytic processing of Gli proteins by preventing phosphorylation and ubiquitination of the Gli PPD by competing with β-TrCP for binding to Gli. Collectively, our findings show the importance of AR-Gli interaction in PCa progression.

Text

2017-12-20

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/64120

Interdisciplinary Oncology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/