Abstract
IntroductionThe 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.MethodsER-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.ResultsIn 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.ConclusionsCollectively, 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.
Highlights
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
Mibolerone increases ER beta expression in breast cancer cells We have previously demonstrated that the non-aromatizable androgen DHT decreased cell proliferation in a dosedependent manner in ER-positive MCF-7 breast cancer cells [21,26]
MCF-7 and ZR75 breast cancer cells were treated with mibolerone for 24 and 48 hours and ER beta mRNA and protein levels were evaluated by real time reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting analysis
Summary
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. In vitro studies have demonstrated that androgen signaling may counteract the proliferative effect of estrogens in AR-positive breast cancer cells [19], while over-expression of the AR markedly decreases ER alpha transcriptional activity in ERpositive breast cancer cells [20,21]. In these latter models, basal as well as estradiol-induced proliferation are inhibited by the non-aromatizable androgen 5a-dihydrotestosterone (DHT) [21,22,23], through an increase in AR protein cell content [21], along with a block in G1 to S transition of the cell cycle [21,22]. We have shown a direct down-regulation of the cyclin D1 gene expression by AR activation via interaction with the orphan nuclear receptor DAX1, as an additional mechanism involved in the androgen-dependent inhibition of ER-positive breast cancer cell growth [25]
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