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1.  Hierarchical Modularity in ERα Transcriptional Network Is Associated with Distinct Functions and Implicates Clinical Outcomes 
Scientific Reports  2012;2:875.
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.
doi:10.1038/srep00875
PMCID: PMC3500769  PMID: 23166858
2.  Epigenetics meets estrogen receptor: regulation of estrogen receptor by direct lysine methylation 
Endocrine-related cancer  2009;16(2):319-323.
The nuclear hormone receptor estrogen receptor α (ERα) promotes cellular growth through ligand-dependent activation of specific target genes, a process which is targeted in the treatment of ERα-expressing breast cancers. ERα activity is regulated at the protein level by post-translational modifications including phosphorylation and acetylation. A study now shows that ERα can also be directly methylated at lysine 302 (K302) by SET7, a histone methyltransferase that is known to monomethylate H3K4 and is associated with transcriptional activation. It was shown that K302 methylation stabilizes ERα protein and is suggested to increase sensitivity of ERα to estrogens, enhancing transcription of estrogen response elements. Furthermore, SET7 methylation of K302 is enhanced by a breast cancer-associated mutation at K303 (K303R) in vitro. These findings provide an additional mechanism of SET7 mediated transcriptional activation, as well as potential insight into the complex regulation of ERα stability and ligand sensitivity.
doi:10.1677/ERC-08-0305
PMCID: PMC3901989  PMID: 19208734
3.  Aryl Hydrocarbon Receptor Modulation of Estrogen Receptor α-Mediated Gene Regulation by a Multimeric Chromatin Complex Involving the Two Receptors and the Coregulator RIP140 
Toxicological Sciences  2011;125(2):401-411.
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.
doi:10.1093/toxsci/kfr300
PMCID: PMC3262852  PMID: 22071320
estrogen receptor α; aryl hydrocarbon receptor; coregulator; gene regulation; breast cancer
4.  Genomic Collaboration of Estrogen Receptor α and Extracellular Signal-Regulated Kinase 2 in Regulating Gene and Proliferation Programs ▿  
Molecular and Cellular Biology  2010;31(1):226-236.
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.
doi:10.1128/MCB.00821-10
PMCID: PMC3019850  PMID: 20956553
5.  Inference of hierarchical regulatory network of estrogen-dependent breast cancer through ChIP-based data 
BMC Systems Biology  2010;4:170.
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.
doi:10.1186/1752-0509-4-170
PMCID: PMC3012048  PMID: 21167036
6.  Extranuclear Signaling by Estrogen: Role in Breast Cancer Progression and Metastasis 
Minerva ginecologica  2010;62(6):573-583.
The estrogen receptor (ERα) is implicated in the progression of breast cancer. Hormonal therapies which block ER functions or local and systemic estrogen production are currently used to treat ERα positive breast cancer. Hormonal therapy shows beneficial effects, however, initial or acquired resistance to endocrine therapies frequently occurs, and tumors recur as metastasis. Emerging evidence suggests in addition to exerting its well-studied nuclear functions, ERα also participates in extranuclear signaling that involve growth factor signaling components, adaptor molecules and stimulation of cytosolic kinases. ERα extranuclear pathways have the potential to activate gene transcription, modulate cytoskeleton, and promote tumor cell proliferation, survival, and metastasis. Cytoplasmic/membrane ERα is detected in a subset of breast tumors and expression of extranuclear components ERα is deregulated in tumors. The extranuclear actions of ER are emerging as important targets for tumorigenic and metastatic control. Inhibition of ERα extranuclear actions has the potential to prevent breast tumor progression and may be useful in preventing ERα positive metastasis. In this review, we summarize the results of recent research into the role of ERα mediated extranuclear actions in breast tumorigenesis and metastasis.
PMCID: PMC3729592  PMID: 21079578
7.  PES1 promotes breast cancer by differentially regulating ERα and ERβ 
The Journal of Clinical Investigation  2012;122(8):2857-2870.
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.
doi:10.1172/JCI62676
PMCID: PMC3408741  PMID: 22820289
8.  Epigenetics of Estrogen Receptor Signaling: Role in Hormonal Cancer Progression and Therapy 
Cancers  2011;3(3):1691-1707.
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.
doi:10.3390/cancers3021691
PMCID: PMC3147309  PMID: 21814622
estrogen receptor; histone modifications; epigenetics; breast cancer; hormonal cancers
9.  Epigenetics of Estrogen Receptor Signaling: Role in Hormonal Cancer Progression and Therapy 
Cancers  2011;3(2):1691-1707.
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.
doi:10.3390/cancers3021691
PMCID: PMC3147309  PMID: 21814622
estrogen receptor; histone modifications; epigenetics; breast cancer; hormonal cancers
10.  AKT Alters Genome-Wide Estrogen Receptor α Binding and Impacts Estrogen Signaling in Breast Cancer▿ † 
Molecular and Cellular Biology  2008;28(24):7487-7503.
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.
doi:10.1128/MCB.00799-08
PMCID: PMC2593438  PMID: 18838536
11.  Integrative genomics of gene and metabolic regulation by estrogen receptors α and β, and their coregulators 
To define how the estrogen receptors α and β control specific responses in breast cancer cells, genome-wide patterns of chromatin binding of the ERα and ERβ receptors and their coregulators, SRC3 and RIP140, were determined and integrated with gene expression data and functional analyses.
The closely related transcription factors, estrogen receptors ERα and ERβ, can elicit differential cellular responses.To understand the basis of this specificity, chromatin binding of ERs and key coregulators, and gene expression, were analyzed genome wide in human breast cancer cells containing ERα only, ERα+ERβ, and ERβ only.A clustering-based combinatorial analysis of ChIP-Seq and gene expression data was used to parse genes into groups, specifying their mode of functional regulation in a particular cell background.Through this analysis, RIP140 was identified as an ERβ-preferential cofactor regulating cell proliferation, apoptosis, and adipogenesis programs.A 20-gene ERβ and RIP140 signature was developed, which predicted outcome and disease-free survival in breast cancer patients.
The closely related transcription factors (TFs), estrogen receptors ERα and ERβ, regulate divergent gene expression programs and proliferative outcomes in breast cancer. Utilizing breast cancer cells with ERα, ERβ, or both receptors as a model system to define the basis for differing response specification by related TFs, we show that these TFs and their key coregulators, SRC3 and RIP140, generate overlapping as well as unique chromatin-binding and transcription-regulating modules. Cistrome and transcriptome analyses and the use of clustering algorithms delineated 11 clusters representing different chromatin-bound receptor and coregulator assemblies that could be functionally associated through enrichment analysis with distinct patterns of gene regulation and preferential coregulator usage, RIP140 with ERβ and SRC3 with ERα. The receptors modified each other's transcriptional effect, and ERβ countered the proliferative drive of ERα through several novel mechanisms associated with specific binding-site clusters. Our findings delineate distinct TF-coregulator assemblies that function as control nodes, specifying precise patterns of gene regulation, proliferation, and metabolism, as exemplified by two of the most important nuclear hormone receptors in human breast cancer.
doi:10.1038/msb.2013.28
PMCID: PMC3964312  PMID: 23774759
coregulator usage; estrogen receptors α and β; gene regulation; metabolism; proliferation
12.  Regulatory function of the P295-T311 motif of the estrogen receptor α - does proteasomal degradation of the receptor induce emergence of peptides implicated in estrogenic responses?  
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.
doi:10.1621/nrs.06007
PMCID: PMC2329824  PMID: 18432312
13.  Estrogen Receptor Alpha Represses Transcription of Early Target Genes via p300 and CtBP1▿  
Molecular and Cellular Biology  2009;29(7):1749-1759.
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.
doi:10.1128/MCB.01476-08
PMCID: PMC2655624  PMID: 19188451
14.  RAC3 is a promigratory co-activator of ERα 
Oncogene  2011;30(17):1984-1994.
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.
doi:10.1038/onc.2010.583
PMCID: PMC3084334  PMID: 21217774
15.  miR-22 Inhibits Estrogen Signaling by Directly Targeting the Estrogen Receptor α mRNA▿ † 
Molecular and Cellular Biology  2009;29(13):3783-3790.
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.
doi:10.1128/MCB.01875-08
PMCID: PMC2698751  PMID: 19414598
16.  Cellular reprogramming by the conjoint action of ERα, FOXA1, and GATA3 to a ligand-inducible growth state 
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.
doi:10.1038/msb.2011.59
PMCID: PMC3202798  PMID: 21878914
enhanceosome; estrogen receptor α; FOXA1; GATA3; synthetic phenotypes
17.  SUMOylation of ZFP282 potentiates its positive effect on estrogen signaling in breast tumorigenesis 
Oncogene  2012;32(35):10.1038/onc.2012.420.
Estrogen receptor α (ERα) plays critical roles in development and progression of breast cancer, and the coiled-coil co-activator (CoCoA) is an important ERα co-activator for estrogen-induced gene expression. The small ubiquitin-like modifier (SUMO) pathway is hyperactivated in breast cancer, but the mechanism by which SUMOylation regulates ERα-mediated transcription remains poorly understood. Here, we identified ZFP282 as a CoCoA binding protein. ZFP282 associates directly with ERα and cooperates synergistically with CoCoA to enhance ERα function. ZFP282 is required for estrogen-induced expression of ERα target genes and estrogen-dependent breast cancer cell growth and tumorigenesis. In addition, we found that ZFP282 is SUMOylated and that SUMOylation positively regulates the co-activator activity of ZFP282 by increasing its binding affinity to ERα and CoCoA and consequently increasing recruitment of ZFP282-CoCoA complex to the promoter of ERα target genes. These findings reveal essential roles for ZFP282 and its SUMOylation in estrogen signaling and breast tumorigenesis.
doi:10.1038/onc.2012.420
PMCID: PMC3874449  PMID: 22986521
breast cancer; co-activator; CoCoA; SUMOylation; ZFP282
18.  Src promotes estrogen-dependent estrogen receptor α proteolysis in human breast cancer 
Journal of Clinical Investigation  2007;117(8):2205-2215.
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.
doi:10.1172/JCI21739
PMCID: PMC1906730  PMID: 17627304
19.  Arsenic Induces Functional Re-Expression of Estrogen Receptor α by Demethylation of DNA in Estrogen Receptor-Negative Human Breast Cancer 
PLoS ONE  2012;7(4):e35957.
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.
doi:10.1371/journal.pone.0035957
PMCID: PMC3338760  PMID: 22558281
20.  Proteasome inhibition represses ERα gene expression in ER+ cells- a new link between proteasome activity and estrogen signaling in breast cancer 
Oncogene  2009;29(10):1509-1518.
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.
doi:10.1038/onc.2009.434
PMCID: PMC2837136  PMID: 19946334
nuclear receptor; proteolysis; hormone-dependent cancer; bortezomib; transcription
21.  CARM1 is an important determinant of ERα-dependent breast cancer cell differentiation and proliferation in breast cancer cells 
Cancer research  2011;71(6):2118-2128.
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.
doi:10.1158/0008-5472.CAN-10-2426
PMCID: PMC3076802  PMID: 21282336
CARM1; histone methylation; breast cancer; differentiation; epigenetics
22.  Structure-Function Relationships of the Raloxifene-Estrogen Receptor-α Complex for Regulating Transforming Growth Factor-α Expression in Breast Cancer Cells* 
The Journal of biological chemistry  2001;277(11):9189-9198.
Amino acid Asp-351 in the ligand binding domain of estrogen receptor α (ERα) plays an important role in regulating the estrogen-like activity of selective estrogen receptor modulator-ERα complexes. 4-Hydroxyta-moxifen is a full agonist at a transforming growth factor a target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with the wild-type ERα. In contrast, raloxifene (Ral), which is also a selective estrogen receptor modulator, is a complete antiestrogen in this system. Because D351G ERα allosterically silences activation function-1 activity in the 4-hydroxytamox-ifen-ERα complex with the complete loss of estrogenlike activity, we examined the converse interaction of amino acid 351 and the piperidine ring of the antiestrogen side chain of raloxifene to enhance estrogen-like action. MDA-MB-231 cells were either transiently or stably transfected with Asp-351 (the wild type), D351E, D351Y, or D351F ERα expression vectors. Profound differences in the agonist and antagonist actions of Ral-ERα complexes were noted only in stable transfec-tants. The agonist activity of the Ral-ERα complex was enhanced with D351E and D351Y ERα, but raloxifene lost its agonist activity with D351F ERα. The distance between the piperidine nitrogen of raloxifene and the negative charge of amino acid 351 was critical for estrogen-like actions. The role of the piperidine ring in neutralizing Asp-351 was addressed using compound R1h, a raloxifene derivative replacing the nitrogen on its piperidine ring with a carbon to form cyclohexane. The derivative was a potent agonist with wild type ERα. These results support the concept that the side chain of raloxifene shields and neutralizes the Asp-351 to produce an antiestrogenic ERα complex. Alteration of either the side chain or its relationship with the negative charge at amino acid 351 controls the estrogen-like action at activating function 2b of the selective estrogen receptor modulator ERα complex.
doi:10.1074/jbc.M108335200
PMCID: PMC3696956  PMID: 11751902
23.  Prognosis of hormone-dependent breast cancers: implications of the presence of dysfunctional transcriptional networks activated by insulin via the immune transcription factor T-bet 
Cancer research  2010;70(2):685-696.
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.
doi:10.1158/0008-5472.CAN-09-1530
PMCID: PMC2807987  PMID: 20068169
estrogen receptor; GATA-3; FOXA1; T-bet; breast cancer
24.  The Role of Retinoblastoma Associated Proteins 46 and 48 in Estrogen Receptor α Mediated Gene Expression 
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.
doi:10.1016/j.mce.2008.05.016
PMCID: PMC2642675  PMID: 18577416
estrogen receptor; RbAp46; RbAp48; retinoblastoma-associated protein; transcription
25.  Extracellular Signal-Regulated Kinase 7, a Regulator of Hormone-Dependent Estrogen Receptor Destruction 
Molecular and Cellular Biology  2003;23(17):5979-5988.
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.
doi:10.1128/MCB.23.17.5979-5988.2003
PMCID: PMC180983  PMID: 12917323

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