Functional localization and competition between the androgen receptor and T-cell factor for nuclear beta-catenin: a means for inhibition of the Tcf signaling axis.
Recent reports suggest that the beta-catenin-T-cell factor (Tcf) (BCT) signaling pathway is important in the progression of prostate cancer. Evidence suggests that the androgen receptor (AR) can repress BCT-mediated transcription both in prostate cancer and colon cancer cells (Chesire and Isaacs, 2002). In this study, we validate such findings and show that repression of BCT signaling is facilitated by competition between the AR and Tcf. Measurements of the Tcf transcriptional reporter (TOPFLASH) indicated that AR+DHT-mediated repression can inhibit BCT transcription in the presence of WT and exogenous activating beta-catenin (Delta1-130 bp). Transient transfections in SW480 cells (APC(mut/mut)) showed that this mode of repression is functionally independent of APC-mediated beta-catenin ubiquitination. Using a recently developed red flourescent protein (HcRed), we demonstrate novel observations about the nuclear distribution of Tcf. Furthermore, with the use of red (HcRed-AR and HcRed-Tcf) and green fusion proteins (beta-catenin-EGFP), we provide morphological evidence of a reciprocal balance of nuclear beta-catenin-EGFP (BC-EGFP). By cotransfecting in LNCaP prostate tumor cells and using quantitative imaging software, we demonstrated a 62.0% colocalization of HcRed-AR and BC-EGFP in the presence of DHT and 63.3% colocalization of HcRed-Tcf/BC-EGFP in the absence of DHT. Costaining for activated RNA Pol II (phosphoserine 2) and HcRed-Tcf suggested that Tcf foci contain transcriptional 'hotspots' validating that these sites have the capacity for transcriptional activity. Given this apparent androgen-dependent competition for nuclear BC-EGFP, we chose to assess our hypothesis by in vivo and in vitro binding assays. SW480 cells transiently transfected with an AR expression construct, treated with DHT and immunoprecipitated for Tcf showed less associated beta-catenin when compared to Tcf precipitates from untreated cells. Furthermore, by treating cells with DHT+Casodex, we were able to abrogate the androgen-sensitive AR/beta-catenin interaction, in addition to relieving transcriptional repression of the TOPFLASH reporter. In vitro binding assays, with increasing amounts of AR(S35), resulted in decreased Tcf(S35) association with immunoprecipitated recombinant beta-catenin-HIS. These data suggest that in steady-state conditions, AR has the ability to compete out Tcf binding for beta-catenin. Finally, using SW480 cells, we show that AR-mediated repression of the BCT pathway has implications for cell cycle progression and in vitro growth. Using FACs analysis, we observed a 26.1% increase in accumulation of cells in the G1 phase of the cell cycle, while in vitro growth assays showed a 35% reduction in viable cells transfected with AR+DHT treatment. Together, our data strongly suggest that a reciprocal balance of nuclear beta-catenin facilitates AR-mediated repression of BCT-driven transcription and cell growth.
Mulholland
David J
DJ
The Prostate Centre, Jack Bell Research, Vancouver, BC, Canada V6H 3Z6. djm@interchange.ubc.ca
Read
Jason T
JT
Rennie
Paul S
PS
Cox
Michael E
ME
Nelson
Colleen C
CC
D000813:Anilides / Q000494:pharmacology; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D013196:Dihydrotestosterone / Q000494:pharmacology; D006801:Humans; D008297:Male; D009570:Nitriles; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D011944:Receptors, Androgen / Q000378:metabolism*; D051820:TCF Transcription Factors; D014105:Tosyl Compounds; D015534:Trans-Activators / Q000378:metabolism*; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000502:physiology*; D014407:Tumor Cells, Cultured; D051176:beta Catenin
D000813:Anilides; C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D009570:Nitriles; D011944:Receptors, Androgen; D051820:TCF Transcription Factors; C544894:TCF7L2 protein, human; D014105:Tosyl Compounds; D015534:Trans-Activators; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D051176:beta Catenin; D013196:Dihydrotestosterone; C053541:bicalutamide; D012321:DNA-Directed RNA Polymerases