Recruitment Of ERα Research Articles

The expression of cystathionine gamma-lyase is regulated by estrogen receptor alpha in human osteoblasts.

Hydrogen sulfide (H2S), generated in the osteoblasts predominantly via cystathionine-γ-lyase (CSE), is bone protective. Previous studies suggested that the onset of bone loss due to estrogen deficiency is associated to decreased levels of H2S and blunted gene expression of CSE. However, there are still a lot of unknowns on how H2S levels influence bone cells function. The present study aims to explore the mechanisms by which estrogen may regulate CSE expression, in particular the role of estrogen receptor alpha (ERα) in human osteoblasts (hOBs). Vertebral lamina derived hOBs were characterized and then assessed for CSE expression by western blot analysis in the presence or absence of ERα overexpression. Bioinformatic analysis, luciferase reporter assay and ChIP assay were performed to investigate ERα recruitment and activity on hCSE gene promoter.Three putative half Estrogen Responsive Elements (EREs) were identified in the hCSE core promoter and were found to participate in the ERα – mediated positive regulation of CSE expression. All osteoblast samples responded to ERα over-expression increasing the levels of CSE protein in a comparable manner. Notably, the ERα recruitment on the regulatory regions of the CSE promoter occurred predominantly in female hOBs than in male hOBs. The obtained results suggest that CSE/H2S system is in relation with estrogen signaling in bone in a gender specific manner.

ERβ compensates for the absence of ERα function to promote osteoblast viability by inhibition of SOST signaling.

Estrogen receptors α and β (ERα and ERβ) serve key functions in bone development and maintenance, and in the metabolism of bone mineral. ERβ and ERα form heterodimers, and ERβ negatively regulates the transactivation of ERα. ERβ also inhibits recruitment of ERα to the estrogen-responsive promoters. However, the relationship of ERα and ERβ in the regulation of osteoblast viability and differentiation remains unclear. The present study aimed to investigate whether ERβ plays a role in balancing ERα activity in osteoblast cells. Downregulation of ERα by short hairpin RNA (shRNA) was found to significantly increase cell cycle arrest at G1 phase (P<0.01). In addition, this effect was found to be significantly enhanced by downregulation of ERβ (P<0.05). Inversely, ERα-knocked down osteoblasts were treated with ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) to activate ERβ. It was found that activation of ERβ significantly rescued the arrest of cell cycle induced by the downregulation of ERα (P<0.05). Furthermore, downregulation of ERα was found to significantly inhibit cell viability (P<0.01), and knockdown of ERβ was found to have a significant synergic effect with ERα downregulation on the inhibition of cell viability (P<0.01). Treatment with ERβ agonist DPN significantly rescued the effects of downregulation of ERα on cell viability (P<0.01). It was also demonstrated that the synergic effects of ERα and ERβ deletion was via upregulation of SOST gene expression, and the subsequent inhibition of OPG and Runx2 gene expression. Thus, ERβ may serve a function in balancing osteoblast viability and differentiation induced by ERα.

BRCA1-mimetic compound NSC35446.HCl inhibits IKKB expression by reducing estrogen receptor-α occupancy in the IKKB promoter and inhibits NF-κB activity in antiestrogen-resistant human breast cancer cells.

We previously identified small molecules that fit into a BRCA1-binding pocket within estrogen receptor-alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity ("BRCA1-mimetics"), and overcome antiestrogen resistance. One such compound, the hydrochloride salt of NSC35446 ("NSC35446.HCl"), also inhibited the growth of antiestrogen-resistant LCC9 tumor xenografts. The purpose of this study was to investigate the down-stream effects of NSC35446.HCl and its mechanism of action. Here, we studied antiestrogen-resistant (LCC9, T47DCO, MCF-7/RR, LY2), ERα-negative (MDA-MB-231, HCC1806, MDA-MB-468), and antiestrogen-sensitive (MCF-7) cell lines. Techniques utilized include RNA-seq, qRT-PCR, cell growth analysis, cell-cycle analysis, Western blotting, luciferase reporter assays, TUNEL assays, in silico analysis of the IKKB gene, and ChIP assays. SC35446.HCl inhibited proliferation and induced apoptosis in antiestrogen-resistant LCC9, T47DCO, MCF-7/RR, and LY2 cells but not in ERα-negative breast cancer cell lines. IKKB (IKKβ, IKBKB), an upstream activator of NF-κB, was identified as a BRCA1-mimetic-regulated gene based on an RNA-seq analysis. NSC35446.HCl inhibited IKKB, IKKA, and IKKG/NEMO mRNA and protein expression in LCC9 cells. NSC35446.HCl also inhibited NF-κB activity and expression of NF-κB target genes. In silico analysis of the IKKB promoter identified nine estrogen response element (ERE) half-sites and one ERE-like full-site. ChIP assays revealed that ERα was recruited to the ERE-like full-site and five of the nine half-sites and that ERα recruitment was inhibited by NSC35446.HCl in LCC9 and T47DCO cells. These studies identify functional EREs in the IKKB promoter and identify IKKB as an ERα and NSC35446.HCl-regulated gene, and they suggest that NF-κB and IKKB, which were previously linked to antiestrogen resistance, are targets for NSC35446.HCl in reversing antiestrogen resistance.

Daidzein down-regulates ubiquitin-specific protease 19 expression through estrogen receptor β and increases skeletal muscle mass in young female mice

Ubiquitin-specific protease 19 (USP19) is a key player in the negative regulation of muscle mass during muscle atrophy. Loss-of-function approaches demonstrate that 17β-estradiol (E2) increases USP19 expression through estrogen receptor (ER) α and consequently decreases soleus muscle mass in young female mice under physiological conditions. Daidzein is one of the main isoflavones in soy, and activates ERβ-dependent transcription. Here, we investigated the effects of daidzein on E2-increased USP19 expression and E2-decreased soleus muscle mass in young female mice. Daidzein stimulated the transcriptional activity of ERβ in murine C2C12 cells and down-regulated USP19 expression. Consistently, daidzein inhibited E2-induced USP19 expression in a reporter activity using a functional half-estrogen response element (hERE) from Usp19. Daidzein inhibited E2-induced recruitment of ERα and promoted recruitment of ERβ to the Usp19 hERE. Dietary daidzein down-regulated the expression of USP19 at the mRNA and protein levels and increased soleus muscle mass in female mice, but not in males. In soleus muscle from ovariectomized (OVX) female mice, dietary daidzein inhibited E2-increased USP19 mRNA expression and E2-decreased muscle mass. Furthermore, E2 induced the recruitment of ERα and ERβ to the hERE, whereas daidzein inhibited E2-induced recruitment of ERα, and enhanced E2-increased recruitment of ERβ, to the Usp19 hERE. These results demonstrate that dietary daidzein decreases USP19 mRNA expression through ERβ and increases soleus muscle mass in young female mice, but not in male mice, under physiological conditions.

Abstract 5504: Regulation of the receptor for advanced glycation end products by estrogen receptor ligands in endometrial cancer

Abstract Endometrial cancer has numerous established risk factors including excessive unopposed estrogen exposure, tamoxifen, obesity and polycystic ovary syndrome (PCOS). These risk factors may promote inflammation, an important hallmark of cancer. Since the endometrium is a site of regular and repeated inflammation during the menstrual cycle, the regulation of inflammation may be involved in endometrial cancer. The receptor for advanced glycation end products (RAGE) stimulates numerous inflammatory signalling pathways and its expression is elevated in a number of inflammatory pathologies including diabetes and more recently cancer. This work aims to investigate the regulation of RAGE by estrogen receptor ligands in endometrial cancer. RAGE and estogen receptor isoform protein expression were examined in a patient cohort by immunohistochemistry. RAGE and ER isoform expression was assessed following estrogen receptor ligand exposure in both type I and type II endometrial cancer cell line models. ERα, ERβ, p65 and ER co-regulator recruitment to the RAGE promoter following ER ligand treatment was determined in vitro by chromatin immunoprecipitation. Data revealed (i) RAGE and ER isoform endometrial expression is altered in endometrial cancer patients and patients receiving tamoxifen compared to postmenopausal women, (ii) RAGE is expressed in type I and II endometrial cancer cell lines and its expression can be modulated by estrogen and tamoxifen exposure, and (iii) by chromatin immunoprecipitation, estrogen receptor ligands were shown to alter ERα, ERβ and ER co-regulator binding at the RAGE promoter. To conclude, results from this work suggest RAGE is associated with endometrial cancer development. Once clinically validated, RAGE and associated targets investigated in this study could be potentially used as a panel of endometrial cancer biomarkers and may also be used to indicate if patients are at risk of the adverse agonist action of tamoxifen in the endometrium. Citation Format: Julia Davies, Deyarina Gonzalez, R.Steven Conlan. Regulation of the receptor for advanced glycation end products by estrogen receptor ligands in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5504. doi:10.1158/1538-7445.AM2017-5504

Essential role of endogenous prolactin and CDK7 in estrogen-induced upregulation of the prolactin receptor in breast cancer cells.

Our early studies have shown that Estradiol (E2)/Estrogen Receptor α (ER) in a non-DNA dependent manner through complex formation with C/EBPβ/SP1 induced transcriptional activation of the generic hPIII promoter and expression of the Prolactin Receptor (PRLR) receptor in MCF-7 cells. Subsequent studies demonstrated effects of unliganded ERα with requisite participation of endogenous PRL on the activation of PRLR transcription. Also, EGF/ERBB1 in the absence of PRL and E2 effectively induced upregulation of the PRLR. In this study we have delineated the transcriptional mechanism of upregulation of PRLR receptor induced by E2 incorporating knowledge of the various transcriptional upregulation modalities from our previous studies. Here, we demonstrate an essential requirement of STAT5a induced by PRL via PRLR receptor which associates at the promoter and its interaction with phoshoERα S118. Knock-down of PRL by siRNA significantly reduced E2-induced PRLR promoter activity, mRNA and protein expression, recruitment of ERα to the complex at promoter, C/EBPβ association to its DNA site and productive complex formation at hPIII promoter. The specific CDK7 inhibitor (THZ1) that attenuates E2-induced ERα phosphorylation at S118 abrogated E2-induced PRLR promoter activation. Further studies demonstrated that E2 induced cell migration was inhibited by PRL siRNA and THZ1 indicating its dependence on PRL/PRLR and CDK7, respectively. Our studies have demonstrated the essential role of endogenous PRL and CDK7 in the upregulation of PRLR by E2 and provide insights for therapeutic approaches that will mitigate the transcription/expression of PRLR and its participation in breast cancer progression fueled by E2 and PRL via their cognate receptors.

Progesterone induces progesterone receptor gene (PGR) expression via rapid activation of protein kinase pathways required for cooperative estrogen receptor alpha (ER) and progesterone receptor (PR) genomic action at ER/PR target genes

Progesterone Receptors (PRs) are critical effectors of estrogen receptor (ER) signaling required for mammary gland development and reproductive proficiency. In breast and reproductive tract malignancies, PR expression is a clinical prognostic marker of ER action. While estrogens primarily regulate PR expression, other factors likely contribute to a dynamic range of receptor expression across diverse tissues. In this study, we identified estrogen-independent but progestin (R5020)-dependent regulation of ER target genes including PGR in ER+/PR+ cancer cell lines. R5020 (10nM–10μM range) induced dose-dependent PR mRNA and protein expression in the absence of estrogen but required both PR and ERα. Antagonists of either PR (RU486, onapristone) or ERα (ICI 182,780) attenuated R5020 induction of TFF1, CTSD, and PGR. Chromatin immunoprecipitation (ChIP) assays performed on ER+/PR+ cells demonstrated that both ERα and PR were recruited to the same ERE/Sp1 site-containing region of the PGR proximal promoter in response to high dose progestin (10μM). Recruitment of ERα and PR to chromatin and subsequent PR mRNA induction were dependent upon rapid activation of MAPK/ERK and AKT; inhibition of these kinase pathways via U0126 or LY294002 blocked these events. Overall, we have identified a novel mechanism of ERα activation initiated by rapid PR-dependent kinase pathway activation and associated with phosphorylation of ERα Ser118 for estrogen-independent but progestin-dependent ER/PR cross talk. These studies may provide insight into mechanisms of persistent ER-target gene expression during periods of hormone (i.e. estrogen) ablation and suggest caution following prolonged treatment with aromatase or CYP17 inhibitors (i.e. contexts when progesterone levels may be abnormally elevated).

Germacrone Inhibits Estrogen Receptor α-Mediated Transcription in MCF-7 Breast Cancer Cells.

Estrogen receptor (ER)α-positive breast cancer cells regulate the expression of estrogen-responsive genes, which are involved in cell proliferation, differentiation, and cell cycle progression. Clinically, the inhibition of ERα-mediated gene expression in breast cancer cells has long been considered an effective way to prevent the development and progression of cancer. Germacrone, a terpenoid compound isolated from Rhizoma curcuma, has been known to have antitumor activity in various human cancer cell lines. However, the mechanism by which germacrone inhibits the proliferation of breast cancer cells is still unclear. Here, we demonstrated that germacrone inhibits ERα-mediated gene expression at the transcriptional level in MCF-7 cells. Germacrone inhibits the recruitment of ERα to the estrogen response element on chromatin and consequently compromises the binding of switch/sucrose non-fermentable chromatin remodeling complex and RNA polymerase II to target gene promoter, thereby inhibiting the estrogen-induced chromatin accessibility. In addition, germacrone efficiently potentiates the antitumor activity of methotrexate and 5-fluorouracil. Our results not only provide substantial molecular mechanism of germacrone on ERα-mediated signaling in breast cancer cells but also demonstrate the benefits of germacrone as a combination therapy with other drugs for the treatment of breast cancer. Copyright © 2016 John Wiley & Sons, Ltd.

Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors.

DNA-protein interactions are vital to fundamental cellular events including transcription, replication, DNA repair, and recombination. Thus, their study holds the key to our understanding of mechanisms underlying normal development and homeostasis as well as disease. Transcriptional regulation is a highly complex process that involves recruitment of numerous factors resulting in formation of multi-protein complexes at gene promoters to regulate gene expression. The studied proteins can be, for example, transcription factors, epigenetic regulators, co-activators, co-repressors, or ligand-activated nuclear receptors as estrogen receptor-α (ERα) bound either directly to the DNA or indirectly by interaction with other DNA-bound factors. Chromatin immunoprecipitation (ChIP) assay is a powerful method to study interactions of proteins and a specific genomic DNA region. Recruitment of ERα to promoters of estrogen-dependent genes is a common mechanism to activate or enhance gene transcription in breast cancer thus promoting tumor progression. In this chapter, we demonstrate a stepwise protocol for ChIP assay using binding of ERα to its genomic targets after stimulation with 17β-estradiol (E2) in breast cancer cells as an example.

Ctr9, a key subunit of PAFc, affects global estrogen signaling and drives ERα-positive breast tumorigenesis

The human RNA polymerase II (RNAPII)-associated factor complex (hPAFc) and its individual subunits have been implicated in human diseases, including cancer. However, its involvement in breast cancer awaits investigation. Using data mining and human breast cancer tissue microarrays, we found that Ctr9, the key scaffold subunit in hPAFc, is highly expressed in estrogen receptor α-positive (ERα(+)) luminal breast cancer, and the high expression of Ctr9 correlates with poor prognosis. Knockdown of Ctr9 in ERα(+) breast cancer cells almost completely erased estrogen-regulated transcriptional response. At the molecular level, Ctr9 enhances ERα protein stability, promotes recruitment of ERα and RNAPII, and stimulates transcription elongation and transcription-coupled histone modifications. Knockdown of Ctr9, but not other hPAFc subunits, alters the morphology, proliferative capacity, and tamoxifen sensitivity of ERα(+) breast cancer cells. Together, our study reveals that Ctr9, a key subunit of hPAFc, is a central regulator of estrogen signaling that drives ERα(+) breast tumorigenesis, rendering it a potential target for the treatment of ERα(+) breast cancer.

Abstract 5042: Dynamic transcriptional regulation of ERα targets in prostate cancer

Abstract Introduction: Prostate cancer is the second most common cause of cancer death in American men. Prostate cancer is mainly an androgen receptor regulated disease, however estrogen signaling is also important in prostate physiology and development particularly in aging men as decreasing testosterone is accompanied by increasing estrogen levels. Further, estrogen signaling through estrogen receptor alpha (ERα) has been implicated in prostate cancer progression, and ERα expression increases in high grade tumors, however its precise role remains unclear. Thus far there have been no systematic approaches to assay ERα transcriptional regulation in prostate cancer, and in this study we aim to determine the role of ERα in prostate cancer by identifying direct ERα target genes. Methods: ERα recruitment profile was assayed by ChIP-Seq in both NCI-H660 and ERα overexpressing VCaP cells in response to estrogen treatment. ERα transcriptional regulation of target genes was examined utilizing RNA-Seq in VCaP cells with and without ERα overexpression. Candidate gene expression was assayed via qPCR in prostate cancer cell lines, and by RNA-Seq in a 126 sample prostate cancer patient cohort. Results: In order to delineate the role of ERα in prostate cancer we integrated RNA- and ChIP-Seq datasets from two different cell lines in response to ERα overexpression and estrogen treatment. In doing so we identified an ERα signature comprising multiple genes that were significantly up or down-regulated (&amp;gt; 5-fold) and had an ERα binding site near the gene promoter. Many of these ERα target genes have been previously identified as oncogenes or tumor suppressors, and they include members several key pathways including PI3 kinase signaling and cytoskeleton maintenance. Expression of these target genes was queried in multiple prostate cancer cell lines, and interestingly ERα binding sites and estrogen regulation were cell line specific in both androgen receptor positive and negative lines. Further, significant differences in target gene expression were demonstrated between benign prostate and prostate adenocarcinoma (PCa), and/or between PCa and castration-resistant prostate cancer (CRPC) or neuroendocrine prostate cancer (NEPC). Combinatorial targeting of both ERα and PI3K signaling proved beneficial for prostate cancer. Conclusion: These results demonstrate that estrogen regulates a distinct set of genes in prostate cancer in a direct genomic fashion, including several components of the PI3K pathway. They also support the hypothesis that this ERα signature is dynamic and changes during the course of prostate cancer progression to CRPC and NEPC. Further validation of the pathways involved is necessary, however these findings may lead to the discovery of novel co-targeting treatment strategies and/or prostate cancer biomarkers, and assessment of ERα signaling may help predict a subclass of prostate cancer patients that are likely to respond to PI3K inhibition. Citation Format: Kellie Cotter, Dimple Chakravarty, Andrea Sboner, Mark A. Rubin. Dynamic transcriptional regulation of ERα targets in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5042. doi:10.1158/1538-7445.AM2015-5042

17β-Estradiol Ameliorates Tight Junction Disruption via Repression of MMP Transcription.

The blood-brain barrier (BBB) or blood-spinal cord barrier (BSCB) formed by capillary endothelial cells provides a physical wall between the central nervous system (CNS) and circulating blood with highly selective permeability. BBB/BSCB disruption by activation of matrix metalloproteinases (MMPs) has been shown to result in further neurological damage after CNS injury. Recently it has been discovered that estrogen attenuates BBB/BSCB disruption in in vitro and in vivo models. However, the molecular mechanism underlying the estrogen-mediated attenuation of BBB/BSCB disruption has not been elucidated fully. In the present study, we found that 17β-estradiol (E2) suppresses nuclear factor-κB-dependent MMP-1b, MMP-2, MMP-3, MMP-9, MMP-10, and MMP-13 gene activation in microvessel endothelial bEnd.3 cells subjected to oxygen and glucose deprivation/reperfusion injury. E2 induced the recruitment of ERα and nuclear receptor corepressor to the nuclear factor-κB binding site on the MMPs' gene promoters. Consistently, ER antagonist ICI 182.780 showed opposite effects of E2. We further found that E2 attenuates tight junction disruption through the decreased degradation of tight junction proteins in bEnd.3 cells subjected to oxygen and glucose deprivation-reperfusion injury. In addition, E2 suppressed the up-regulation of MMP expression, leading to a decreased BSCB disruption in the injured spinal cord. In conclusion, we discovered the molecular mechanism underlying the protective role of estrogenin BBB/BSCB disruption using an in vitro and in vivo model. Our study suggests that estrogens may provide a potential therapeutic intervention for preserving BBB/BSCB integrity after CNS injury.

Female-specific regulation of skeletal muscle mass by USP19 in young mice.

17β-Estradiol (E₂) is thought to be responsible for sex-specific differences in skeletal muscle mass. The biological function of E₂ is exerted through its binding to estrogen receptor α (ERα). The expression of ubiquitin-specific peptidase 19 (USP19) is upregulated during muscle atrophy and by E₂-activated ERα. Here, we investigated the involvement of USP19 in sex difference in muscle mass in young mice. Knockdown of USP19 in hindlimb muscles increased the mass and fiber size in soleus muscle in females but not males. Using Usp19 promoter reporter constructs, a functional half-estrogen response element (hERE) was identified in intron 1 of Usp19. ERα bound to hERE in an E₂-dependent manner in C2C12 myoblasts and in soleus muscle in ovariectomized (OVX) female mice. Furthermore, under normal physiological conditions, ERα bound to hERE in soleus muscle only in females. In contrast, administration of E₂ resulted in increased Usp19 mRNA expression, decreased muscle mass, and recruitment of ERα to hERE in soleus muscle in males. Knockdown of ERα in hindlimb muscles decreased Usp19 mRNA expression and increased the mass of soleus muscle only in females. Knockdown of USP19 resulted in increased levels of ubiquitin conjugates in soleus muscle in females. OVX increased the levels of ubiquitin conjugates and administration of E₂ decreased OVX-induced levels of ubiquitin conjugates. These results demonstrate that in soleus muscle in young female mice under physiological conditions, E₂ upregulates USP19 expression through ERα and consequently leads to decreases in ubiquitin conjugates and muscle mass.

A functional interplay between ZNF217 and Estrogen Receptor alpha exists in luminal breast cancers

We aimed at highlighting the role of ZNF217, a Krüppel-like finger protein, in Estrogen Receptor-α (ERα)-positive (ER+) and luminal breast cancers. Here we report for the first time that ZNF217 and ERα proteins bind to each other in both breast cancer cells and breast tumour samples, via the ERα hinge domain and the ZNF217 C-terminal domain. ZNF217 enhances the recruitment of ERα to its estrogen response elements (ERE) and the ERα-dependent transcription of the GREB1 estrogen-regulated gene. The prognostic power of ZNF217 mRNA expression levels is most discriminatory in breast cancers classified with a “good prognosis”, particularly the Luminal-A subclass. A new immunohistochemistry ZNF217 index, based on nuclear and cytoplasmic ZNF217 staining, also allowed the identification of intermediate/poor relapse-free survivors in the Luminal-A subgroup. ZNF217 confers tamoxifen resistance in ER+ breast cancer cells and is a predictor of relapse under endocrine therapy in patients with ER+ breast cancer. ZNF217 thus allows the re-stratification of patients with ER+ breast cancers considered as cancers with good prognosis where no other biomarkers are currently available and widely used. Here we propose a model in ER+ breast cancer where ZNF217-driven aggressiveness incorporates ZNF217 as a positive enhancer of ERα direct genomic activity and where ZNF217 possesses its highest discriminatory prognostic value.

Effects of Estetrol on Migration and Invasion in T47-D Breast Cancer Cells through the Actin Cytoskeleton.

Estetrol (E4) is a natural human estrogen present at high concentrations during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications. E4 acts as a selective estrogen receptor (ER) modulator, exerting estrogenic actions on the endometrium or the central nervous system, while antagonizing the actions of estradiol in the breast. We tested the effects of E4 on its own or in the presence of 17β-estradiol (E2) on T47-D ER+ breast cancer cell migration and invasion of three-dimensional matrices. E4 administration to T47-D cells weakly stimulated migration and invasion. However, E4 decreased the extent of movement and invasion induced by E2. Breast cancer cell movement requires a remodeling of the actin cytoskeleton. During exposure to E4, a weak, concentration-dependent, re-distribution of actin fibers toward the cell membrane was observed. However, when E4 was added to E2, an inhibition of actin remodeling induced by E2 was seen. Estrogens stimulate ER+ breast cancer cell movement through the ezrin–radixin–moesin family of actin regulatory proteins, inducing actin and cell membrane remodeling. E4 was a weak inducer of moesin phosphorylation on Thr558, which accounts for its functional activation. In co-treatment with E2, E4 blocked the activation of this actin controller in a concentration-related fashion. These effects were obtained through recruitment of estrogen receptor-α. In conclusion, E4 acted as a weak estrogen on breast cancer cell cytoskeleton remodeling and movement. However, when E2 was present, E4 counteracted the stimulatory actions of E2. This contributes to the emerging hypothesis that E4 may be a naturally occurring ER modulator in the breast.

Estrogen-dependent sushi domain containing 3 regulates cytoskeleton organization and migration in breast cancer cells.

Aromatase inhibitors (AI) are the standard endocrine therapy for postmenopausal breast cancer; however, currently used biomarkers, i.e., estrogen receptor-alpha/progesterone receptor (ERα/PR), predict only slightly more than half of the potential responders to AI treatment. To identify novel markers of AI responsiveness, a genome-wide microarray analysis was performed using primary breast tumor samples from 50 postmenopausal women (PMW) who later developed metastatic breast cancer. Sushi domain containing 3 (SUSD3) was significantly differentially expressed gene, with 3.38-fold higher mRNA levels in AI-responsive breast tumors versus non-responders (p<0.001). SUSD3 was highly expressed in ERα-positive breast tumors and treatment with estradiol increased SUSD3 expression in ERα-positive breast cancer cells. Treatment with an antiestrogen or ERα knockdown abolished basal and estradiol-dependent SUSD3 expression. Recruitment of ERα upstream of the transcription start site of SUSD3 was demonstrated by chromatin immunoprecipitation (ChIP)-PCR. Flow cytometric analysis of SUSD3 knockdown cells revealed blunted estradiol effects on progression into S and M phases. SUSD3 was localized to the plasma membrane of breast cancer cells. SUSD3 knockdown decreased the appearance of actin-rich protrusions, stress fibers and large basal focal adhesions, while increasing the presence of cortical actin concomitant with a decrease in Rho and FAK activity. SUSD3-deficient cells demonstrated diminished cell spreading, cell-cell adhesion, and motility. In conclusion, SUSD3 is a novel promoter of estrogen-dependent cell proliferation and regulator of cell-cell and cell-substrate interactions and migration in breast cancer. It may serve as a novel predictor of response to endocrine therapy and potential therapeutic target.

Zinc Finger Nuclease–Mediated Knockout of AHR or ARNT in Human Breast Cancer Cells Abolishes Basal and Ligand-Dependent Regulation of CYP1B1 and Differentially Affects Estrogen Receptor α Transactivation

In this study, we used zinc finger nuclease-mediated knockout of the aryl hydrocarbon receptor (AHR) or AHR nuclear translocator (ARNT) in MCF7 and AHR knockout in MDA-MB-231 human breast cancer cells to investigate cross talk among AHR, ARNT, and estrogen receptor α (ERα). Knockout of AHR or ARNT prevented the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent induction of all AHR target genes examined. Knockout of AHR or ARNT also significantly reduced basal cytochrome P4501B1 (CYP1B1) expression levels, which were restored with overexpression of either protein but not with a DNA binding-deficient AHR mutant. Basal and TCDD-, 17β-estradiol (E2)-, or TCDD + E2-dependent recruitment of AHR, ARNT, ERα, NCoA3, and RNA polymerase II to CYP1B1 as well as CYP1B1 mRNA levels were abolished in MCF7-AHR((ko)) and MDA-MB-231 AHR(ko) cells. However, reduced but significant E2-dependent recruitment of ERα, NCoA3, and RNA polymerase II to CYP1B1 and weak increases in CYP1B1 mRNA levels were observed in MCF7 ARNT((ko)) cells. Interestingly, E2-dependent increases in trefoil factor 1, but not growth regulation by estrogen in breast cancer 1 (GREB1) mRNA levels, were dependent on ARNT expression. Moreover, the TCDD-dependent increases in the proteolytic degradation of ERα were prevented by the loss of AHR or ARNT. Our data show that AHR and ARNT play critical roles in the basal, TCDD, and E2-induced regulation of CYP1B1 but also reveal distinct roles for both proteins in ERα transactivation.

Establishment of active chromatin structure at enhancer elements by mixed-lineage leukemia 1 to initiate estrogen-dependent gene expression

A number of genome-wide analyses have revealed that estrogen receptor α binding to and regulation of its target genes correlate with binding of FOXA1, a pioneer factor, to nearby DNA sites in MCF-7 breast cancer cells. The enhancer element-specific histone H3K4me1/2 mark is enriched at the specific FOXA1/ERα recruitment sites in chromatin, but the mechanism by which these enhancer marks are established in chromatin before hormone treatment is unclear. Here, we show that mixed-lineage leukemia 1 (MLL1) protein is a key determinant that maintains permissive chromatin structure of the TFF1 enhancer region. MLL1 occupies the TFF1 enhancer region and methylates H3K4 before hormone stimulation. In vitro, MLL1 binds directly to the CpG-rich region of the TFF1 enhancer, and its binding is dependent on hypomethylation of DNA. Furthermore, the depletion of MLL1 in MCF-7 cells results in a dramatic decrease of chromatin accessibility and recruitment of FOXA1 and ERα to the enhancer element. Our study defines the mechanism by which MLL1 nucleates histone H3K4 methylation marks in CpG-enriched regions to maintain permissive chromatin architecture and allow FOXA1 and estrogen receptor α binding to transcriptional regulatory sites in breast cancer cells.

RhoA and RhoC differentially modulate estrogen receptor α recruitment, transcriptional activities, and expression in breast cancer cells (MCF-7)

RhoA and RhoC are closely related, small GTPases that are clearly involved in breast cancer tumorigenesis. Nonetheless, their specific roles in the control of estrogen receptor alpha (ERα) activities have not been elucidated. We used siRNA sequences to specifically down-regulate RhoA and RhoC expression in ERα-positive breast adenocarcinoma MCF-7 cells. We then analyzed the consequences of down-regulation on ERα expression, ERα recruitment to the promoters of four target genes, and the mRNA levels of those genes. We demonstrated that RhoA and RhoC clearly and similarly modulated ERα recruitment to the vitellogenin estrogen responsive element (ERE) present in a luciferase reporter gene and to the promoters of progesterone receptor (PR), cathepsin D, and pS2 genes. Besides, RhoA up-regulated the ERE-luciferase reporter gene activity and PR mRNA expression and tended to down-regulate cathepsin D and pS2 mRNA expression. Conversely, RhoC inhibition had no significant effect at the mRNA level. Furthermore, RhoA inhibition, and to a lesser extent RhoC inhibition, increased ERα expression. No alteration in ERα mRNA levels was observed, suggesting potential post-translational control. Taken together, our results strongly suggest that RhoA and RhoC play different, but clear, roles in ERα signaling. These GTPases are definitely involved, along with RhoB, in ERα recruitment and, to some extent, ERα cofactor balance. We hypothesize a differential role of RhoA in breast cancer tumors that depend on hormone status.

Co-regulated gene expression by oestrogen receptor α and liver receptor homolog-1 is a feature of the oestrogen response in breast cancer cells

Oestrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, regulates breast cancer cell proliferation and promotes motility and invasion. To determine the mechanisms of LRH-1 action in breast cancer, we performed gene expression microarray analysis following RNA interference for LRH-1. Interestingly, gene ontology (GO) category enrichment analysis of LRH-1–regulated genes identified oestrogen-responsive genes as the most highly enriched GO categories. Remarkably, chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1 showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα−regulated genes by LRH-1 through binding to oestrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 overexpression stimulated ERα recruitment, while LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at oestrogen response elements controls the expression of oestrogen-responsive genes.