Abstract: PD33-01
Sources of Funding: Canadian Institutes of Health Research and the Terry Fox Research Institute

Introduction

Canonical (smoothened-driven) Hedgehog signaling regulates the activities of Gli transcription factors and their ability to induce cell proliferation, motility and invasion. While there are numerous reports of constitutive Hedgehog/Gli activity in prostate cancer (PCa) cells, there is little evidence for a role of smoothened in this activity. Previously we showed that Gli proteins bind to full length- (FL-) and truncated- (t-) androgen receptors (ARs) at the N-terminal tau5 transactivation domain and that this binding co-activates AR transcriptional activity in PCa cells. Here we show, conversely, that transcriptionally active AR binds to a critical protein processing domain on Gli2/Gli3, alters their post-translational proteolytic processing and drives non-canonical Hedgehog signaling that facilitates androgen-independent growth of PCa cells.

Methods

Site-specific mutations were introduced into the Gli2 C-terminal domain to identify the peptides involved in AR recognition. GST-pulldown was used to test binding of the mutated peptide to AR-tau5 peptide. Western blotting was used to quantify AR expression and the proteolytic states of Gli2 and 3 in LNCaP, LNCaP-AI, LN95 and VCaP cells. AR or Gli3 protein was knocked down with siRNAs. A Gli-luciferase reporter was used to measure functional Gli activity in cells and qPCR was used to determine how AR or Gli expression manipulation affected expressions of endogenous Gli-responsive genes. Proximity ligation assays with antibodies against AR and Gli3 was used to quantify the relative binding of these proteins in androgen-dependent and androgen-independent cell lines.

Results

Mutations in a repeat serine/arginine-rich peptide in the C-terminal domain of Gli2 (aa820-836) blocked Gli2 binding to AR. This peptide encompasses the Gli protein processing domain that regulates a site-specific proteolysis that renders transcriptionally active Gli proteins into transcriptional repressors. Binding of liganded AR to this site blocked Gli proteolysis and maintained Gli in transcriptionally active states. Knockdown of AR in androgen growth-independent cell lines induced proteolytic inactivation of Gli in PCa cells. Knockdown of Gli3 in these cells induced a loss of AR protein. Proximity ligation assays showed that binding between Gli3 and AR was higher in androgen-independent cell lines.

Conclusions

Transcriptionally active AR protein binding to Gli proteins protects both from a natural degradation process and provides a non-canonical means to activate Hedgehog signaling in PCa cells. Since Gli proteins regulate the expressions of growth-promoting genes, our findings suggest that the growth effects of androgens on PCa cells are the results of a non-canonical Hedgehog activation process.

Funding

Canadian Institutes of Health Research and the Terry Fox Research Institute