Breast cancer research : BCR · 2014
Estrogen receptor beta as a novel target of androgen receptor action in breast cancer cell lines.
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Rizza Pietro, Barone Ines, Zito Domenico, Giordano Francesca, Lanzino Marilena, De Amicis Francesca, Mauro Loredana, Sisci Diego, Catalano Stefania, Dahlman Wright Karin, Gustafsson Jan-Ake, Andò Sebastiano
Аннотация
INTRODUCTION: The two isoforms of estrogen receptor (ER) alpha and beta play opposite roles in regulating proliferation and differentiation of breast cancers, with ER-alpha mediating mitogenic effects and ER-beta acting as a tumor suppressor. Emerging data have reported that androgen receptor (AR) activation inhibits ER-positive breast cancer progression mainly by antagonizing ER-alpha signaling. However, to date no studies have specifically evaluated a potential involvement of ER-beta in the inhibitory effects of androgens.
METHODS: ER-beta expression was examined in human breast cancer cell lines using real-time PCR, Western blotting and small interfering RNA (siRNA) assays. Mutagenesis studies, electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were performed to assess the effects of mibolerone/AR on ER-beta promoter activity and binding.
RESULTS: In this study, we demonstrate that mibolerone, a synthetic androgen ligand, up-regulates ER-beta mRNA and protein levels in ER-positive breast cancer cells. Transient transfection experiments, using a vector containing the human ER-beta promoter region, show that mibolerone increases basal ER-beta promoter activity. Site-directed mutagenesis and deletion analysis reveal that an androgen response element (ARE), TGTTCT motif located at positions -383 and -377, is critical for mibolerone-induced ER-beta up-regulation in breast cancer cells. This occurs through an increased recruitment of AR to the ARE site within the ER-beta promoter region, along with an enhanced occupancy of RNA polymerase II. Finally, silencing of ER-beta gene expression by RNA interference is able to partially reverse the effects of mibolerone on cell proliferation, p21 and cyclin D1 expression.
CONCLUSIONS: Collectively, these data provide evidence for a novel mechanism by which activated AR, through an up-regulation of ER-beta gene expression, inhibits breast cancer cell growth.
METHODS: ER-beta expression was examined in human breast cancer cell lines using real-time PCR, Western blotting and small interfering RNA (siRNA) assays. Mutagenesis studies, electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were performed to assess the effects of mibolerone/AR on ER-beta promoter activity and binding.
RESULTS: In this study, we demonstrate that mibolerone, a synthetic androgen ligand, up-regulates ER-beta mRNA and protein levels in ER-positive breast cancer cells. Transient transfection experiments, using a vector containing the human ER-beta promoter region, show that mibolerone increases basal ER-beta promoter activity. Site-directed mutagenesis and deletion analysis reveal that an androgen response element (ARE), TGTTCT motif located at positions -383 and -377, is critical for mibolerone-induced ER-beta up-regulation in breast cancer cells. This occurs through an increased recruitment of AR to the ARE site within the ER-beta promoter region, along with an enhanced occupancy of RNA polymerase II. Finally, silencing of ER-beta gene expression by RNA interference is able to partially reverse the effects of mibolerone on cell proliferation, p21 and cyclin D1 expression.
CONCLUSIONS: Collectively, these data provide evidence for a novel mechanism by which activated AR, through an up-regulation of ER-beta gene expression, inhibits breast cancer cell growth.
Предметные рубрики MeSH
Breast NeoplasmsCell Line, TumorCell ProliferationCyclin D1Enzyme ActivationEstrogen Receptor alphaEstrogen Receptor betaFemaleHumansMCF-7 CellsMutagenesis, Site-DirectedNandrolonePromoter Regions, GeneticProtein BindingRNA InterferenceRNA Polymerase IIRNA, MessengerRNA, Small InterferingReceptors, AndrogenTestosterone CongenersUp-Regulationrho GTP-Binding Proteins