PD33-01 NON-CANONICAL ACTIVATION OF HEDGEHOG SIGNALING IN PROSTATE CANCER CELLS MEDIATED BY THE BINDING OF TRANSCRIPTIONALLY ACTIVE ANDROGEN RECEPTORS TO GLI TRANSCRIPTION FACTORS

Abstract

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2017PD33-01 NON-CANONICAL ACTIVATION OF HEDGEHOG SIGNALING IN PROSTATE CANCER CELLS MEDIATED BY THE BINDING OF TRANSCRIPTIONALLY ACTIVE ANDROGEN RECEPTORS TO GLI TRANSCRIPTION FACTORS Na Li, Sarah Truong, Mannan Nouri, Amy Anne Lubik, and Ralph Buttyan Na LiNa Li More articles by this author , Sarah TruongSarah Truong More articles by this author , Mannan NouriMannan Nouri More articles by this author , Amy Anne LubikAmy Anne Lubik More articles by this author , and Ralph ButtyanRalph Buttyan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3315AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES 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. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e593 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Na Li More articles by this author Sarah Truong More articles by this author Mannan Nouri More articles by this author Amy Anne Lubik More articles by this author Ralph Buttyan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...