Abstract
Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor (AR)-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and dihydrotestosterone stimulate expression of a versican promoter-driven luciferase reporter vector (versican-Luc). Further, both basal and androgen-stimulated versican-Luc activities were significantly diminished in LNCaP cells, when AR gene expression was knocked down using a short hairpin RNA. Methylation-protection footprinting analysis revealed an AR-protected element between positions +75 and +102 of the proximal versican promoter, which strongly resembled a consensus steroid receptor element. Electrophoretic mobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated versican-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce versican promoter activity and mRNA levels in AR-positive prostate stromal fibroblasts. Interestingly, overexpression of beta-catenin in the presence of androgen augmented versican promoter activity 10- and 30-fold and enhanced versican mRNA levels 2.8-fold in fibroblasts. In conclusion, we demonstrate that AR transactivates versican expression, which may augment tumor-stromal interactions and may contribute to prostate cancer progression.
Highlights
Prostatic stroma is necessary in the maintenance of adult prostatic secretory epithelium [2, 3]
Versican Proximal Promoter Luciferase Reporter Vector Responds to Steroid Stimulation—The versican-Luc vector, in which expression of the luciferase gene is under the control of the versican proximal promoter (Ϫ634 to ϩ118 relative to the transcriptional start site), was used in transient transfection assays to determine the effects of steroid stimulation on versican promoter activity [36]
We have found that androgens dose-dependently enhance human versican promoter activity in LNCaP androgen receptor (AR)-positive epithelial human prostate cancer cells (Fig. 1A)
Summary
Genes that are preferentially expressed in human prostate tissues are often regulated by androgens at the transcriptional level. AI progression is a multifactorial process by which cells acquire the ability to both survive in the absence of androgens and proliferate using non-androgenic stimuli for mitogenesis It probably involves variable combinations of several processes, including adaptive up-regulation of antiapoptotic genes, ligand-independent activation of the AR, and alternative signaling pathways. Using transient transfection of synthetic reporter and endogenous gene reporter constructs in prostate stromal fibroblasts, we demonstrated that -catenin is required for AR-mediated transcription in both ligand-dependent and ligand-independent manner in prostate stromal fibroblast cells. These data identify a novel role for -catenin in nuclear hormone receptor-mediated transcription in prostate stromal cells
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