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Recent genome-wide profiling reveals highly complex regulation networks among ERα and its targets. We integrated estrogen (E2)-stimulated time-series ERα ChIP-seq and gene expression data to identify the ERα-centered transcription factor (TF) hubs and their target genes, and inferred the time-variant hierarchical network structures using a Bayesian multivariate modeling approach. With its recurrent motif patterns, we determined three embedded regulatory modules from the ERα core transcriptional network. The GO analyses revealed the distinct biological function associated with each of three embedded modules. The survival analysis showed the genes in each module were able to render a significant survival correlation in breast cancer patient cohorts. In summary, our Bayesian statistical modeling and modularity analysis not only reveals the dynamic properties of the ERα-centered regulatory network and associated distinct biological functions, but also provides a reliable and effective genomic analytical approach for the analysis of dynamic regulatory network for any given TF.

The nuclear hormone receptor, estrogen receptor α (ERα), and mitogen-activated protein kinases (MAPKs) play key roles in hormone-dependent cancers, and yet their interplay and the integration of their signaling inputs remain poorly understood. In these studies, we document that estrogen-occupied ERα activates and interacts with extracellular signal-regulated kinase 2 (ERK2), a downstream effector in the MAPK pathway, resulting in ERK2 and ERα colocalization at chromatin binding sites across the genome of breast cancer cells. This genomic colocalization, predominantly at conserved distal enhancer sites, requires the activation of both ERα and ERK2 and enables ERK2 modulation of estrogen-dependent gene expression and proliferation programs. The ERK2 substrate CREB1 was also activated and recruited to ERK2-bound chromatin following estrogen treatment and found to cooperate with ERα/ERK2 in regulating gene transcription and cell cycle progression. Our study reveals a novel paradigm with convergence of ERK2 and ERα at the chromatin level that positions this kinase to support nuclear receptor activities in crucial and direct ways, a mode of collaboration likely to underlie MAPK regulation of gene expression by other nuclear receptors as well.

Although crosstalk between aryl hydrocarbon receptor (AhR) and estrogen receptor α (ERα) is well established, the mechanistic basis and involvement of other proteins in this process are not known. Because we observed an enrichment of AhR-binding motifs in ERα-binding sites of many estradiol (E2)-regulated genes, we investigated how AhR might modulate ERα-mediated gene transcription in breast cancer cells. Gene regulations were categorized based on their pattern of stimulation by E2 and/or dioxin and were denoted E2-responsive, dioxin-responsive, or responsive to either ligand. ERα, AhR, aryl hydrocarbon receptor translocator, and receptor interacting protein 140 (RIP140) were recruited to gene regulatory regions in a gene-specific and E2/dioxin ligand-specific manner. Knockdown of AhR markedly increased the expression of ERα-mediated genes upon E2 treatment. This was not attributable to a change in ERα level, or recruitment of ERα, phosphoSer5-RNA Pol II, or several coregulators but rather was associated with greatly diminished recruitment of the coregulator RIP140 to gene regulatory sites. Changing the cellular level of RIP140 revealed coactivator or corepressor roles for this coregulator in E2- and dioxin-mediated gene regulation, the choice of which was determined by the presence or absence of ERα at gene regulatory sites. Coimmunoprecipitation and chromatin immunoprecipitation (ChIP)-reChIP studies documented that E2- or dioxin-promoted formation of a multimeric complex of ERα, AhR, and RIP140 at ERα-binding sites of genes regulated by either E2 or dioxin. Our findings highlight the importance of cross-regulation between AhR and ERα and a novel mechanism by which AhR controls, through modulating the recruitment of RIP140 to ERα-binding sites, the kinetics and magnitude of ERα-mediated gene stimulation.

Background

Global profiling of in vivo protein-DNA interactions using ChIP-based technologies has evolved rapidly in recent years. Although many genome-wide studies have identified thousands of ERα binding sites and have revealed the associated transcription factor (TF) partners, such as AP1, FOXA1 and CEBP, little is known about ERα associated hierarchical transcriptional regulatory networks.

Results

In this study, we applied computational approaches to analyze three public available ChIP-based datasets: ChIP-seq, ChIP-PET and ChIP-chip, and to investigate the hierarchical regulatory network for ERα and ERα partner TFs regulation in estrogen-dependent breast cancer MCF7 cells. 16 common TFs and two common new TF partners (RORA and PITX2) were found among ChIP-seq, ChIP-chip and ChIP-PET datasets. The regulatory networks were constructed by scanning the ChIP-peak region with TF specific position weight matrix (PWM). A permutation test was performed to test the reliability of each connection of the network. We then used DREM software to perform gene ontology function analysis on the common genes. We found that FOS, PITX2, RORA and FOXA1 were involved in the up-regulated genes.

We also conducted the ERα and Pol-II ChIP-seq experiments in tamoxifen resistance MCF7 cells (denoted as MCF7-T in this study) and compared the difference between MCF7 and MCF7-T cells. The result showed very little overlap between these two cells in terms of targeted genes (21.2% of common genes) and targeted TFs (25% of common TFs). The significant dissimilarity may indicate totally different transcriptional regulatory mechanisms between these two cancer cells.

Conclusions

Our study uncovers new estrogen-mediated regulatory networks by mining three ChIP-based data in MCF7 cells and ChIP-seq data in MCF7-T cells. We compared the different ChIP-based technologies as well as different breast cancer cells. Our computational analytical approach may guide biologists to further study the underlying mechanisms in breast cancer cells or other human diseases.

The initiation of breast cancer is associated with increased expression of tumor-promoting estrogen receptor α (ERα) protein and decreased expression of tumor-suppressive ERβ protein. However, the mechanism underlying this process is unknown. Here we show that PES1 (also known as Pescadillo), an estrogen-inducible protein that is overexpressed in breast cancer, can regulate the balance between ERα and ERβ. We found that PES1 modulated many estrogen-responsive genes by enhancing the transcriptional activity of ERα while inhibiting transcriptional activity of ERβ. Consistent with this regulation of ERα and ERβ transcriptional activity, PES1 increased the stability of the ERα protein and decreased that of ERβ through the ubiquitin-proteasome pathway, mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). Moreover, PES1 transformed normal human mammary epithelial cells and was required for estrogen-induced breast tumor growth in nude mice. Further analysis of clinical samples showed that expression of PES1 correlated positively with ERα expression and negatively with ERβ expression and predicted good clinical outcome in breast cancer. Our data demonstrate that PES1 contributes to breast tumor growth through regulating the balance between ERα and ERβ and may be a better target for the development of drugs that selectively regulate ERα and ERβ activities.

Estrogen receptor (ERα) signaling plays a key role in hormonal cancer progression. ERα is a ligand-dependent transcription factor that modulates gene transcription via recruitment to the target gene chromatin. Emerging evidence suggests that ERα signaling has the potential to contribute to epigenetic changes. Estrogen stimulation is shown to induce several histone modifications at the ERα target gene promoters including acetylation, phosphorylation and methylation via dynamic interactions with histone modifying enzymes. Deregulation of enzymes involved in the ERα-mediated epigenetic pathway could play a vital role in ERα driven neoplastic processes. Unlike genetic alterations, epigenetic changes are reversible, and hence offer novel therapeutic opportunities to reverse ERα driven epigenetic changes. In this review, we summarize current knowledge on mechanisms by which ERα signaling potentiates epigenetic changes in cancer cells via histone modifications.

Estrogen regulates several biological processes through estrogen receptor α (ERα) and ERβ. ERα-estrogen signaling is additionally controlled by extracellular signal activated kinases such as AKT. In this study, we analyzed the effect of AKT on genome-wide ERα binding in MCF-7 breast cancer cells. Parental and AKT-overexpressing cells displayed 4,349 and 4,359 ERα binding sites, respectively, with ∼60% overlap. In both cell types, ∼40% of estrogen-regulated genes associate with ERα binding sites; a similar percentage of estrogen-regulated genes are differentially expressed in two cell types. Based on pathway analysis, these differentially estrogen-regulated genes are linked to transforming growth factor β (TGF-β), NF-κB, and E2F pathways. Consistent with this, the two cell types responded differently to TGF-β treatment: parental cells, but not AKT-overexpressing cells, required estrogen to overcome growth inhibition. Combining the ERα DNA-binding pattern with gene expression data from primary tumors revealed specific effects of AKT on ERα binding and estrogen-regulated expression of genes that define prognostic subgroups and tamoxifen sensitivity of ERα-positive breast cancer. These results suggest a unique role of AKT in modulating estrogen signaling in ERα-positive breast cancers and highlights how extracellular signal activated kinases can change the landscape of transcription factor binding to the genome.

The way in which estrogen receptor α (ERα) mediates gene transcription and hormone-dependent cancer cell proliferation is now being largely reconsidered in view of several recent discoveries. ERα-mediated transcription appears to be a cyclic and transient process where the proteasome - and thus receptor degradation - plays a pivotal role. In view of our recent investigations, which demonstrate the estrogenic activity of a synthetic peptide corresponding to a regulatory motif of the receptor (ERα17p), we propose that ERα proteasomal degradation could induce the emergence of regulatory peptide(s). The latter would function as a signal and contribute to the ERα activation process, amplifying the initial hormonal stimulation and giving rise to sustained estrogenic response.

The regulation of gene expression by nuclear receptors controls the phenotypic properties and diverse biologies of target cells. In breast cancer cells, estrogen receptor alpha (ERα) is a master regulator of transcriptional stimulation and repression, yet the mechanisms by which agonist-bound ERα elicits repression are poorly understood. We analyzed early estrogen-repressed genes and found that ERα is recruited to ERα binding sites of these genes, albeit more transiently and less efficiently than for estrogen-stimulated genes. Of multiple cofactors studied, only p300 was recruited to ERα binding sites of repressed genes, and its knockdown prevented estrogen-mediated gene repression. Because p300 is involved in transcription initiation, we tested whether ERα might be trying to stimulate transcription at repressed genes, with ultimately failure and a shift to a repressive program. We found that estrogen increases transcription in a rapid but transient manner at early estrogen-repressed genes but that this is followed by recruitment of the corepressor CtBP1, a p300-interacting partner that plays an essential role in the repressive process. Thus, at early estrogen-repressed genes, ERα initiates transient stimulation of transcription but fails to maintain the transcriptional process observed at estrogen-stimulated genes; rather, it uses p300 to recruit CtBP1-containing complexes, eliciting chromatin modifications that lead to transcriptional repression.

ERα is a ligand-dependent nuclear receptor that is important in breast cancer genesis, behavior and response to hormone-based therapies. A T7 phage display screen against full-length human ERα, coupled with genome-wide exon arrays, was used to identify RAC3 as a putative ERα co-regulator. RAC3 is a rho family small GTPase that is associated with cytoskeletal rearrangement. We demonstrate a novel role for nuclear RAC3 as an ERα transcriptional activator, with prognostic implications for metastatic disease. Through in vitro and cell-based studies, RAC3 was shown to exist in a GTP-bound state and act as a ligand specific ERα co-activator of E2-induced transcription. Over expression of RAC3 induced pro-growth and pro-migratory genes that resulted in increased migration of ERα-positive breast cancer cells. Chemical inhibition and genetic knockdown of RAC3 antagonized E2-induced cell proliferation, cell migration, and ERα mediated gene expression, indicating that RAC3 is necessary for full ERα transcriptional activity. In agreement with the molecular and cellular data, RAC3 over expression in ERα-positive breast cancers correlated with a significant decrease in recurrence free survival and a significant increase in the odds ratio of metastasis. In conclusion, RAC3 is novel ERα co-activator that promotes cell migration and has prognostic value for ERα-positive breast cancer metastasis. RAC3 may also be a useful therapeutic target for ERα-positive breast cancers.

Estrogen receptor α (ERα), FOXA1, and GATA3 form a functional enhanceosome in MCF-7 breast carcinoma cell that is significantly associated with active transcriptional features such as enhanced p300 co-activator and RNA Pol II recruitment as well as chromatin opening.The enhanceosome exerts significant impact and optimal transcriptional control in the regulation of E2-responsive genes.The presence of FOXA1 and GATA3 is indispensable in restoring the ERα growth-response machinery in the ERα-negative cells and recapitulating the appropriate expression cassette.

Estrogen receptor α (ERα) is a ligand-inducible hormone nuclear receptor that has important physiology and pathology roles in reproduction, cancer, and cardiovascular biology. The regulation of ERα involves its binding to the DNA recognition sequence also known as estrogen-response elements (EREs) and recruits a variety of co-activators, corepressors, and chromatin remodeling enzymes to initiate transcription machinery. In our previous (Lin et al, 2007) and recent (Joseph et al, 2010) studies, we have identified high confidence ERα binding sites in MCF-7 human mammary carcinoma cells. With known motif scanning and de novo motif detection, we identified that FOXA1 and GATA3 motifs were commonly enriched around ERα binding sites. Moreover, numerous microarray studies have documented the co-expression of ERα, FOXA1, and GATA3 in primary breast tumors (Badve et al, 2007; Wilson and Giguere, 2008). This evidence suggests that these three transcription factors (TFs) may cluster on DNA binding sites and contribute to the breast cancer phenotype. However, there is little understanding as to the nature of their coordinated interaction at the genome level or the biological consequences of their detailed interaction.

We mapped the genome-wide binding profiles of ERα, FOXA1, and GATA3 using the massive parallel chromatin immunoprecipitation-sequencing (ChIP-seq) approach. We observed that ERα, FOXA1, and GATA3 colocalized in a coordinated manner where ∼30% of all ERα binding sites were overlapped with FOXA1 and GATA3 bindings upon estrogen (E2) stimulation. Moreover, we found that the ERα+FOXA1+GATA3 conjoint sites were associated with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Such results indicate that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. And such enhanceosome binding sites appear to regulate the genes driving core ERα function.

To further validate that ERα+FOXA1+GATA3 co-binding represents an optimal configuration for E2-mediated transcriptional activation, we have performed luciferase reporter assays on GREB1 locus that actively engages ERα enhanceosome sites in gene regulation (Figure 5C). The presence of ERα induced the GREB1 luciferase activity to ∼246% (as compared with the control construct). The individual presence of FOXA1 and GATA3 or combination of both only produced subtle changes to the GREB1 luciferase activity. The combination of ERα+FOXA1 and ERα+GATA3 has increased the luciferase activity to ∼330%. Interestingly, the assemblage of ERα+FOXA1+GATA3 provided the optimal ER responsiveness to 370%. This suggests that ERα provides the fundamental gene regulatory module but that FOXA1 and GATA3 incrementally improve ERα-regulated transcriptional induction.

It is known that ERα is a ligand-activated TF that mediates the proliferative effects of E2 in breast cancer cells. Garcia et al (1992) showed inhibited growth in MDA-MB-231 cells with forced expression of ERα upon E2 treatment. The rationale for these different outcomes has remained elusive. We posited that these higher order regulatory mechanisms of ERα function such as the formation and composition of enhanceosomes may explain the establishment of transcriptional regulatory cassettes favoring either growth enhancement or growth repression.

To test this hypothesis, we stably transfected the MDA-MB-231 cells with individual ERα, FOXA1, GATA3, or in combinations (Figure 6A). We observed inhibited growth in cells with enforced expression of ERα or FOXA1. There was unaltered growth in cells with expression of GATA3. Co-expression of ERα+FOXA1 or ERα+GATA3 exhibited inhibition of cell proliferation as compared with control cells. However, the co-expression of ERα together with FOXA1 and GATA3 resulted in marked induction of cell proliferation under E2 stimulation. We have recapitulated this cellular reprogramming in another ERα-negative breast cancer cell line, BT-549 and observed similar E2-responsive growth induction in the ERα+FOXA1+GATA3-expressing BT-549 cells. This suggests that only with the full activation of conjoint binding sites by the three TFs will the proliferative phenotype associated with ligand induced ERα be manifest.

To assess the nature of this transcriptional reprogramming, we asked the question if the reprogrammed MDA-MB-231 cells display any similarity in the expression profile of the ERα-positive breast cancer cell line, MCF-7 (Figure 6C). We combined the E2-regulated genes from these differently transfected MDA-MB-231 cells, and compared their expressions in these MDA-MB-231-transfected cells and MCF-7 cells. Strikingly, we found that the expression profiles of ERα+FOXA1+GATA3-expressing MDA-MB-231 cells display a good correlation (R=0.42) with the E2-induced expression profile of MCF-7. We did not observe such correlation between the expression profiles of MDA-MB-231 transfected with ERα only (R=−0.21). Furthermore, we observed that there is marginal induced expression of luminal marker genes and reduced expression of basal genes in the ERα+FOXA1+GATA3-expressing MDA-MB-231 as compared with the vector control cells. This suggests that the enhanceosome component is competent to partially reprogramme the basal cells to resemble the luminal cells.

Taken together, we have uncovered the genomics impact as well as the functional importance of an enhanceosome comprising ERα, FOXA1, and GATA3 in the estrogen responsiveness of ERα-positive breast cancer cells. This enhanceosome exerts significant combinatorial control of the transcriptional network regulating growth and proliferation of ERα-positive breast cancer cells. Most importantly, we show that the transfection of the enhanceosome component was necessary to reprogramme the ERα-negative cells to restore the estrogen-responsive growth and to transcriptionally induce a basal to luminal transition.

Despite the role of the estrogen receptor α (ERα) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERα into ERα-negative cells paradoxically has been growth inhibition. We mapped the binding profiles of ERα and its interacting transcription factors (TFs), FOXA1 and GATA3 in MCF-7 breast carcinoma cells, and observed that these three TFs form a functional enhanceosome that regulates the genes driving core ERα function and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. We demonstrate that these enhanceosome occupied sites are associated with optimal enhancer characteristics with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Most importantly, we show that the transfection of all three TFs was necessary to reprogramme the ERα-negative MDA-MB-231 and BT-549 cells to restore the estrogen-responsive growth resembling estrogen-treated ERα-positive MCF-7 cells. Cumulatively, these results suggest that all the enhanceosome components comprising ERα, FOXA1, and GATA3 are necessary for the full repertoire of cancer-associated effects of the ERα.

Estrogen receptor α (ERα) is a ligand-regulated transcription factor with a broad range of physiological functions and one of the most important classifiers in breast cancer. MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as important regulators of gene expression in a plethora of physiological and pathological processes. Upon binding the 3′ untranslated region (UTR) of target mRNAs, miRNAs typically reduce their stability and/or translation. The ERα mRNA has a long 3′ UTR of about 4.3 kb which has been reported to reduce mRNA stability and which bears evolutionarily conserved miRNA target sites, suggesting that it might be regulated by miRNAs. We have performed a comprehensive and systematic assessment of the regulatory role of all miRNAs that are predicted to target the 3′ UTR of the ERα mRNA. We found that miR-22 represses ERα expression most strongly and by directly targeting the ERα mRNA 3′ UTR. Of the three predicted miR-22 target sites in the 3′ UTR, the evolutionarily conserved one is the primary target. miR-22 overexpression leads to a reduction of ERα levels, at least in part by inducing mRNA degradation, and compromises estrogen signaling, as exemplified by its inhibitory impact on the ERα-dependent proliferation of breast cancer cells.

Estrogen drives both transcriptional activation and proteolysis of estrogen receptor α (ERα; encoded by ESR1). Here we observed variable and overlapping ESR1 mRNA levels in 200 ERα-negative and 50 ERα-positive primary breast cancers examined, which suggests important posttranscriptional ERα regulation. Our results indicate that Src cooperates with estrogen to activate ERα proteolysis. Inducible Src stimulated ligand-activated ERα transcriptional activity and reduced ERα t1/2. Src and ERα levels were inversely correlated in primary breast cancers. ERα-negative primary breast cancers and cell lines showed increased Src levels and/or activity compared with ERα-positive cancers and cells. ERα t1/2 was reduced in ERα-negative cell lines. In both ERα-positive and -negative cell lines, both proteasome and Src inhibitors increased ERα levels. Src inhibition impaired ligand-activated ERα ubiquitylation and increased ERα levels. Src siRNA impaired ligand-activated ERα loss in BT-20 cells. Pretreatment with Src increased ERα ubiquitylation and degradation in vitro. These findings provide what we believe to be a novel link between Src activation and ERα proteolysis and support a model whereby crosstalk between liganded ERα and Src drives ERα transcriptional activity and targets ERα for ubiquitin-dependent proteolysis. Oncogenic Src activation may promote not only proliferation, but also estrogen-activated ERα loss in a subset of ERα-negative breast cancers, altering prognosis and response to therapy.

Estrogen receptor α (ERα) is a marker predictive for response of breast cancers to endocrine therapy. About 30% of breast cancers, however, are hormone- independent because of lack of ERα expression. New strategies are needed for re-expression of ERα and sensitization of ER-negative breast cancer cells to selective ER modulators. The present report shows that arsenic trioxide induces reactivated ERα, providing a target for therapy with ER antagonists. Exposure of ER-negative breast cancer cells to arsenic trioxide leads to re-expression of ERα mRNA and functional ERα protein in in vitro and in vivo. Luciferase reporter gene assays and 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assays show that, upon exposure to arsenic trioxide, formerly unresponsive, ER-negative MDA-MB-231 breast cancer cells become responsive to ER antagonists, 4-hydroxytamoxifen and ICI 182,780. Furthermore, methylation- specific PCR and bisulfite-sequencing PCR assays show that arsenic trioxide induces partial demethylation of the ERα promoter. A methyl donor, S-adenosylmethionine (SAM), reduces the degree of arsenic trioxide-induced re-expression of ERα and demethylation. Moreover, Western blot and ChIP assays show that arsenic trioxide represses expression of DNMT1 and DNMT3a along with partial dissociation of DNMT1 from the ERα promoter. Thus, arsenic trioxide exhibits a previously undefined function which induces re-expression ERα in ER-negative breast cancer cells through demethylation of the ERα promoter. These findings could provide important information regarding the application of therapeutic agents targeting epigenetic changes in breast cancers and potential implication of arsenic trioxide as a new drug for the treatment of ER–negative human breast cancer.

Estrogen receptor-alpha (ERα) is a major therapeutic target of hormonal therapies in breast cancer and its expression in tumors is predictive of clinical response. Protein levels of ERα are tightly controlled by the 26S proteasome, yet how the clinical proteasome inhibitor, bortezomib, impacts ERα regulation has not been studied. Bortezomib selectively inhibits the chymotrypsin-like activity of the proteasome. Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERα protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity. Unexpectedly, however, chronic bortezomib exposure caused a reduction of ERα levels in multiple ER+ breast cancer cell lines. This response can be explained by the fact that bortezomib induced a dramatic decrease in ERα mRNA due to direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERα gene promoter. Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related to occupancy of ERα and RNA PolII on endogenous promoters. In addition, bortezomib inhibited estrogen-dependent growth in soft agar. These results reveal a novel link between proteasome activity and expression of ERα in breast cancer and uncover distinct roles of the chymotrypsin-like activity of the proteasome in the regulation of the ERα pathway.

Breast cancers expressing estrogen receptor α (ERα) are often more differentiated histologically than ERα-negative tumors, but the reasons for this difference are poorly understood. One possible explanation is that transcriptional co-factors associated with ERα determine the expression of genes which promote a more differentiated phenotype. In this study, we identify one such cofactor as coactivator associated arginine methyltransferase 1 (CARM1), a unique co-activator of ERα that can simultaneously block cell proliferation and induce differentiation through global regulation of ERα-regulated genes. CARM1 was evidenced as an ERα co-activator in cell-based assays, gene expression microarrays, and mouse xenograft models. In human breast tumors, CARM1 expression positively correlated with ERα levels in ER+ tumors but was inversely correlated with tumor grade. Our findings suggest that co-expression of CARM1 and ERα may provide a better biomarker of well-differentiated breast cancer. Further, our findings define an important functional role of this histone arginine methyltransferase in re-programming ERα-regulated cellular processes, implicating CARM1 as a putative epigenetic target in ER-positive breast cancers.

The differential recruitment of coregulatory proteins to the DNA-bound estrogen receptor α (ERα) plays a critical role in mediating estrogen-responsive gene expression. We previously isolated and identified retinoblastoma-associated proteins 46 (RbAp46) and 48 (RbAp48), which are associated with chromatin remodeling, histone deacetylation, and transcription repression, as proteins associated with the DNA-bound ERα. We now demonstrate that RbAp46 and RbAp48 interact with ERα in vitro and in vivo, associate with ERα at endogenous, estrogen-responsive genes, and alter expression of endogenous, ERα-activated and -repressed genes in MCF-7 breast cancer cells. Our findings reveal that RbAp48 limits expression of estrogen-responsive genes and that RbAp46 modulates estrogen responsiveness in a gene-specific manner. The ability of RbAp46 and RbAp48 to interact with ERα and influence its activity reveals yet another role for these multifunctional proteins in regulating gene expression.

Estrogen receptor alpha (ERα)-positive breast cancers that co-express trans cription factors GATA-3 and FOXA1 have a favorable prognosis. These transcription factors form an autoregulatory hormonal network that influences estrogen responsiveness and sensitivity to hormonal therapy. Disruption of this network may be a mechanism whereby ERα positive breast cancers become resistant to therapy. The transcription factor T-bet is a negative regulator of GATA-3 in the immune system. In this study, we report that insulin increases the expression of T-bet in breast cancer cells, which correlates with reduced expression of GATA-3, FOXA1 and the ERα:FOXA1:GATA-3 target gene GREB-1. The effects of insulin on GATA-3 and FOXA1 could be recapitulated through overexpression of T-bet in MCF-7 cells (MCF-7-T-bet). Chromatin immunoprecipitation assays revealed reduced ERα binding to GREB-1 enhancer regions in MCF-7-T-bet cells and in insulin treated MCF-7 cells. MCF-7-T-bet cells were resistant to tamoxifen in the presence of insulin and displayed prolonged ERK and AKT activation in response to epidermal growth factor treatment. ERα-positive cells with intrinsic tamoxifen resistance as well as MCF-7 cells with acquired tamoxifen and fulvestrant resistance expressed elevated levels of T-bet and/or reduced levels of FOXA1 and GATA-3. Analysis of publicly available databases revealed ERα-positive/T-bet-positive breast cancers expressing lower levels of FOXA1 (p=0.0137) and GATA-3 (p=0.0063) compared to ERα-positive/T-bet-negative breast cancers. Thus, T-bet expression in primary tumors and circulating insulin levels may serve as surrogate biomarkers to identify ERα-positive breast cancers with a dysfunctional hormonal network, enhanced growth factor signaling, and resistance to hormonal therapy.

Estrogen receptor alpha (ERα) degradation is regulated by ubiquitination, but the signaling pathways that modulate ERα turnover are unknown. We found that extracellular signal-regulated kinase 7 (ERK7) preferentially enhances the destruction of ERα but not the related androgen receptor. Loss of ERK7 was correlated with breast cancer progression, and all ERα-positive breast tumors had decreased ERK7 expression compared to that found in normal breast tissue. In human breast cells, a dominant-negative ERK7 mutant decreased the rate of endogenous ERα degradation >4-fold in the presence of hormone and potentiated estrogen responsiveness. ERK7 targets the ERα ligand-binding domain for destruction by enhancing its ubiquitination. Thus, ERK7 is a novel regulator of estrogen responsiveness through its control of ERα turnover.

Serine protease PRSS23 is a newly discovered protein that has been associated with tumor progression in various types of cancers. Interestingly, PRSS23 is coexpressed with estrogen receptor α (ERα), which is a prominent biomarker and therapeutic target for human breast cancer. Estrogen signaling through ERα is also known to affect cell proliferation, apoptosis, and survival, which promotes tumorigenesis by regulating the production of numerous downstream effector proteins.

In the present study, we aimed to clarify the correlation between and functional implication of ERα and PRSS23 in breast cancer. Analysis of published breast cancer microarray datasets revealed that the gene expression correlation between ERα and PRSS23 is highly significant among all ERα-associated proteases in breast cancer. We then assessed PRSS23 expression in 56 primary breast cancer biopsies and 8 cancer cell lines. The results further confirmed the coexpression of PRSS23 and ERα and provided clinicopathological significance. In vitro assays in MCF-7 breast cancer cells demonstrated that PRSS23 expression is induced by 17β-estradiol-activated ERα through an interaction with an upstream promoter region of PRSS23 gene. In addition, PRSS23 knockdown may suppress estrogen-driven cell proliferation of MCF-7 cells.

Our findings imply that PRSS23 might be a critical component of estrogen-mediated cell proliferation of ERα-positive breast cancer cells. In conclusion, the present study highlights the potential for PRSS23 to be a novel therapeutic target in breast cancer research.

Background

The status of estrogen receptor-α (ERα) is critical to the clinical prognosis and therapeutic approach in breast cancer. ERα-negative breast cancer is clinically aggressive and has a poor prognosis because of the lack of hormone target-directed therapies. Previous studies have shown that epigenetic regulation plays a major role in ERα silencing in human breast cancer cells. Dietary green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), is believed to be an anticancer agent in part through its regulation of epigenetic processes.

Results

In our current studies, we found that EGCG can reactivate ERα expression in ERα-negative MDA-MB-231 breast cancer cells. Combination studies using EGCG with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), revealed a synergistic effect of reactivation of ERα expression in ERα-negative breast cancer cells. Reactivation of ERα expression by EGCG and TSA treatment was found to sensitize ERα-dependent cellular responses to activator 17β-estradiol (E2) and antagonist tamoxifen in ERα-negative breast cancer cells. We also found that EGCG can lead to remodeling of the chromatin structure of the ERα promoter by altering histone acetylation and methylation status thereby resulting in ERα reactivation. A decreased binding of the transcription repressor complex, Rb/p130-E2F4/5-HDAC1-SUV39H1-DNMT1, in the regulatory region of the ERα promoter also contributes to ERα transcriptional activation through treatment with EGCG and/or TSA.

Conclusions

Collectively, these studies show that green tea EGCG can restore ERα expression by regulating epigenetic mechanisms, and this effect is enhanced when combined with an HDAC inhibitor. This study will facilitate more effective uses of combination approaches in breast cancer therapy and will help to explore more effective chemotherapeutic strategies toward hormone-resistant breast cancer.

Background

Estrogen receptors alpha (ERα) and beta (ERβ) 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 ERβ being able to modulate the effects of ERα on gene transcription and cell proliferation. ERβ is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERβ 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 ERβ in hormone-responsive, ERα-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 ERβ and 6024 ERα binding sites in estrogen-stimulated cells, comprising sites occupied by either ERβ, ERα 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 ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb. These putative primary ERβ 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. ERβ 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 ERβ can bind also ERα, suggesting that the overall action of ERβ on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell.

The nuclear hormone receptor estrogen receptor α (ERα) mediates the actions of estrogens in target cells and is a master regulator of the gene expression and proliferative programs of breast cancer cells. The presence of ERα in breast cancer cells is crucial for the effectiveness of endocrine therapies, and its loss is a hallmark of endocrine-insensitive breast tumors. However, the molecular mechanisms underlying the regulation of the cellular levels of ERα are not fully understood. Our findings reveal a unique cellular pathway involving the p38 mitogen-activated protein kinase (p38MAPK)-mediated phosphorylation of ERα at Ser-294 that specifies its turnover by the SCFSkp2 proteasome complex. Consistently, we observed an inverse relationship between ERα and Skp2 or active p38MAPK in breast cancer cell lines and human tumors. ERα regulation by Skp2 was cell cycle stage dependent and critical for promoting the mitogenic effects of estradiol via ERα. Interestingly, by the knockdown of Skp2 or the inhibition of p38MAPK, we restored functional ERα protein levels and the control of gene expression and proliferation by estrogen and antiestrogen in ERα-negative breast cancer cells. Our findings highlight a novel pathway with therapeutic potential for restoring ERα and the responsiveness to endocrine therapy in some endocrine-insensitive ERα-negative breast cancers.

Background

The estrogen receptor α (ERα) is a ligand-regulated transcription factor. However, a wide variety of other extracellular signals can activate ERα in the absence of estrogen. The impact of these alternate modes of activation on gene expression profiles has not been characterized.

Methodology/Principal Findings

We show that estrogen, growth factors and cAMP elicit surprisingly distinct ERα-dependent transcriptional responses in human MCF7 breast cancer cells. In response to growth factors and cAMP, ERα primarily activates and represses genes, respectively. The combined treatments with the anti-estrogen tamoxifen and cAMP or growth factors regulate yet other sets of genes. In many cases, tamoxifen is perverted to an agonist, potentially mimicking what is happening in certain tamoxifen-resistant breast tumors and emphasizing the importance of the cellular signaling environment. Using a computational analysis, we predicted that a Hox protein might be involved in mediating such combinatorial effects, and then confirmed it experimentally. Although both tamoxifen and cAMP block the proliferation of MCF7 cells, their combined application stimulates it, and this can be blocked with a dominant-negative Hox mutant.

Conclusions/Significance

The activating signal dictates both target gene selection and regulation by ERα, and this has consequences on global gene expression patterns that may be relevant to understanding the progression of ERα-dependent carcinomas.

Multiple factors influence estrogen receptor α (ERα) transcriptional activity. Current models suggest that the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor functions within a histone deactylase-containing protein complex that binds to antiestrogen-bound ERα and contributes to negative regulation of gene expression. In this report, we demonstrate that SMRT is required for full agonist-dependent ERα activation. Chromatin immunoprecipitation assays demonstrate that SMRT, like ERα and the SRC-3 coactivator, is recruited to an estrogen-responsive promoter in estrogen-treated MCF-7 cells. Depletion of SMRT, but not histone deacetylases 1 or 3, negatively impacts estradiol-stimulated ERα transcriptional activity, while exogenous expression of SMRT's receptor interaction domains blocks ERα activity, indicating a functional interaction between this corepressor and agonist-bound ERα. Stimulation of estradiol-induced ERα activity by SMRT overexpression occurred in HeLa and MCF-7 cells, but not HepG2 cells, indicating that these positive effects are cell type specific. Similarly, the ability of SMRT depletion to promote the agonist activity of tamoxifen was observed for HeLa but not MCF-7 cells. Furthermore, impairment of agonist-stimulated activity by SMRT depletion is specific to ERα and not observed for receptors for vitamin D, androgen, or thyroid hormone. Nuclear receptor corepressor (N-CoR) depletion increased the transcriptional activity of all four tested receptors. SMRT is required for full expression of the ERα target genes cyclin D1, BCL-2, and progesterone receptor but not pS2, and its depletion significantly attenuated estrogen-dependent proliferation of MCF-7 cells. Taken together, these data indicate that SMRT, in conjunction with gene-specific and cell-dependent factors, is required for positively regulating agonist-dependent ERα transcriptional activity.