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Estrogen receptor beta as a novel target of androgen receptor action in breast cancer cell lines.

ABSTRACT: 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.

SUBMITTER: Rizza P

PROVIDER: S-EPMC3978907 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Estrogen receptor beta as a novel target of androgen receptor action in breast cancer cell lines.

Rizza Pietro P Barone Ines I Zito Domenico D Giordano Francesca F Lanzino Marilena M De Amicis Francesca F Mauro Loredana L Sisci Diego D Catalano Stefania S Dahlman Wright Karin K Gustafsson Jan-Ake JA Andò Sebastiano S

Breast cancer research : BCR 20140219 1

<h4>Introduction</h4>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 ...[more]

PMID: 24552459

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Project description:BACKGROUND: Estrogen receptors alpha (ER?) and beta (ER?) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ER? being able to modulate the effects of ER? on gene transcription and cell proliferation. ER? is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ER? in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. RESULTS: Expression of full-length ER? in hormone-responsive, ER?-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ER? and 6024 ER? binding sites in estrogen-stimulated cells, comprising sites occupied by either ER?, ER? or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ER?+ vs ER?- cells, 424 showed one or more ER? site within 10 kb. These putative primary ER? target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ER? binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions. CONCLUSIONS: Results indicate that the vast majority of the genomic targets of ER? can bind also ER?, suggesting that the overall action of ER? on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell.

Project description:BackgroundTumors are heterogeneous in nature, composed of different cell populations with various mutations and/or phenotypes. Using a single drug to encounter cancer progression is generally ineffective. To improve the treatment outcome, multiple drugs of distinctive mechanisms but complementary anticancer activities (combination therapy) are often used to enhance antitumor efficacy and minimize the risk of acquiring drug resistance. We report here the synergistic effects of salinomycin (a polyether antibiotic) and dasatinib (a Src kinase inhibitor).MethodsFunctionally, both drugs induce cell cycle arrest, intracellular reactive oxygen species (iROS) production, and apoptosis. We rationalized that an overlapping of the drug activities should offer an enhanced anticancer effect, either through vertical inhibition of the Src-STAT3 axis or horizontal suppression of multiple pathways. We determined the toxicity induced by the drug combination and studied the kinetics of iROS production by fluorescence imaging and flow cytometry. Using genomic and proteomic techniques, including RNA-sequencing (RNA-seq), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western Blot, we subsequently identified the responsible pathways that contributed to the synergistic effects of the drug combination.ResultsCompared to either drug alone, the drug combination showed enhanced potency against MDA-MB-468, MDA-MB-231, and MCF-7 human breast cancer (BC) cell lines and tumor spheroids. The drug combination induces both iROS generation and apoptosis in a time-dependent manner, following a 2-step kinetic profile. RNA-seq data revealed that the drug combination exhibited synergism through horizontal suppression of multiple pathways, possibly through a promotion of cell cycle arrest at the G1/S phase via the estrogen-mediated S-phase entry pathway, and partially via the BRCA1 and DNA damage response pathway.ConclusionTranscriptomic analyses revealed for the first time, that the estrogen-mediated S-phase entry pathway partially contributed to the synergistic effect of the drug combination. More importantly, our studies led to the discoveries of new potential therapeutic targets, such as E2F2, as well as a novel drug-induced targeting of estrogen receptor β (ESR2) approach for triple-negative breast cancer treatment, currently lacking of targeted therapies.

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Estrogen receptor beta as a novel target of androgen receptor action in breast cancer cell lines.

Publications

Estrogen receptor beta as a novel target of androgen receptor action in breast cancer cell lines.

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