Estrogen receptor α (ERα) is present in about 70 % of human breast cancers and, working in conjunction with extracellular signal-regulated kinase 2 (ERK2), this nuclear hormone receptor regulates the expression of many protein-encoding genes. Given the crucial roles of miRNAs in cancer biology, we investigated the regulation of miRNAs by estradiol (E2) through ERα and ERK2, and their impact on target gene expression and phenotypic properties of breast cancer cells. We identified miRNA-encoding genes harboring overlapping ERα and ERK chromatin binding sites in ERα-positive MCF-7 cells and showed ERα and ERK2 to bind to these sites and to be required for transcriptional induction of these miRNAs by E2. Hsa-miR-196a2*, the most highly estrogen up-regulated miRNA, markedly down-regulated tumor protein p63 (TP63), a member of the p53 family. In ERα-positive and ERα-negative breast cancer cells, proliferative and invasiveness properties were suppressed by hsa-miR-196a2* expression and enhanced by hsa-miR-196a2* antagonism or TP63 target protector oligonucleotides. Hsa-miR-196a2* and TP63 were inversely correlated in breast cancer cell lines and in a large cohort of human breast tumors, implying clinical relevance. The findings reveal a tumor suppressive role of hsa-miR-196a2* through regulation of TP63 by ERα and/or ERK2 signaling. Manipulating the hsa-miR-196a2*-TP63 axis might provide a potential tumor-suppressive strategy to alleviate the aggressive behavior and poor prognosis of some ERα-positive as well as many ERα-negative breast cancers. PMID: 23250869
Category Archives: Breast Cancer
Aryl hydrocarbon receptor modulation of estrogen receptor α-mediated gene regulation by a multimeric chromatin complex involving the two receptors and the coregulator RIP140.Madak-Erdogan Z, Katzenellenbogen B.S., Toxicological Sciences. 2012 Feb;125(2):401-11
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. PMID:22071320
Macrophage-elicited loss of estrogen receptor-α in breast cancer cells via involvement of MAPK and c-Jun at the ESR1 genomic locus.Stossi F, Madak-Erdoğan Z, Katzenellenbogen BS.Oncogene. 2012 Apr 5;31(14):1825-34
Estrogen receptor-α (ERα, ESR1) is a pivotal transcriptional regulator of breast cancer physiology and is targeted by endocrine therapies. Loss of ERα activity or expression is an indication of endocrine resistance and is associated with increased risk of tumor recurrence and worse prognosis. In this study, we sought to investigate whether elements of the tumor microenvironment, namely macrophages, would impact on ERα and we found that macrophage-derived factors caused loss of ERα expression in breast cancer cells. Conditioned media from macrophages caused activation of several intracellular pathways in breast cancer cells of which c-Src, protein kinase c and mitogen-activated protein kinase (MAPK) were essential for loss of ERα expression. Moreover, a prolonged hyperactivation of MAPK was observed. The activation of this kinase cascade resulted in recruitment of extracellular signal regulated kinase 2 (ERK2) directly to chromatin at the ESR1 gene locus in a process that was dependent upon activation and recruitment of the c-Jun transcription factor. Thus, we identify a novel mechanism for loss of ERα expression in breast cancer cells via macrophage activation of kinase cascades in the cancer cells causing transcriptional repression of the ESR1 gene by a direct chromatin action of a c-Jun/ERK2 complex. The findings in this study support an alternative mechanism, not intrinsic to the tumor cell but derived from the cross-talk with the tumor microenvironment, that could lead to endocrine resistance and might be targeted therapeutically to prevent loss of ERα expression in breast tumors.PMID:21860415
Genomic Collaboration of Estrogen Receptor alpha (ERα) and Extracellular Signal-Regulated Kinase 2 in Regulating Gene and Proliferation Programs, Madak-Erdogan Z, Lupien M, Stossi F., Brown M, Katzenellenbogen BS, Molecular and Cellular Biology. 2011, Jan;31(1):226-36
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. PMID: 20956553
Estrogen Receptor Alpha Represses Transcription of Early Target Genes via p300 and CtBP1, Stossi F, Madak-Erdogan Z, Katzenellenbogen BS, Molecular and Cellular Biology. 2009 Apr;29(7):1749-59
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 (ERalpha) is a master regulator of transcriptional stimulation and repression, yet the mechanisms by which agonist-bound ERalpha elicits repression are poorly understood. We analyzed early estrogen-repressed genes and found that ERalpha is recruited to ERalpha 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 ERalpha binding sites of repressed genes, and its knockdown prevented estrogen-mediated gene repression. Because p300 is involved in transcription initiation, we tested whether ERalpha 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, ERalpha 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. PMID:19188451
Nuclear and Extranuclear Pathway Inputs in the Regulation of Global Gene Expression by Estrogen Receptors, Madak-Erdogan Z, Kieser KJ, Kim SH, Komm B, Katzenellenbogen JA, Katzenellenbogen BS,Molecular Endocrinology. 2008; 22 (9): 2116-2127
Whereas estrogens exert their effects by binding to nuclear estrogen receptors (ERs) and directly altering target gene transcription, they can also initiate extranuclear signaling through activation of kinase cascades. We have investigated the impact of estrogen-mediated extranuclear-initiated pathways on global gene expression by using estrogen-dendrimer conjugates (EDCs), which because of their charge and size remain outside the nucleus and can only initiate extranuclear signaling. Genome-wide cDNA microarray analysis of MCF-7 breast cancer cells identified a subset of 17beta-estradiol (E2)-regulated genes ( approximately 25%) as EDC responsive. The EDC and E2-elicited increases in gene expression were due to increases in gene transcription, as observed in nuclear run-on assays and RNA polymerase II recruitment and phosphorylation. Treatment with antiestrogen or ERalpha knockdown using small interfering RNA abolished EDC-mediated gene stimulation, whereas GPR30 knockdown or treatment with a GPR30-selective ligand was without effect, indicating ER as the mediator of these gene regulations. Inhibitors of MAPK kinase and c-Src suppressed both E2 and EDC stimulated gene expression. Of note, in chromatin immunoprecipitation assays, EDC was unable to recruit ERalpha to estrogen-responsive regions of regulated genes, whereas ERalpha recruitment by E2 was very effective. These findings suggest that other transcription factors or kinases that are downstream effectors of EDC-initiated extranuclear signaling cascades are recruited to regulatory regions of EDC-responsive genes in order to elicit gene stimulation. This study thus highlights the importance of inputs from both nuclear and extranuclear ER signaling pathways in regulating patterns of gene expression in breast cancer cells. PMID:18617595
Estrogen dendrimer conjugates that preferentially activate extranuclear, nongenomic versus genomic pathways of estrogen action, Harrington WR, Kim SH, Funk CC, Madak-Erdogan Z, Schiff R, Katzenellenbogen JA, Katzenellenbogen BS, Molecular Endocrinology. 2006; 20(3):491-502.
Estrogenic hormones are classically thought to exert their effects by binding to nuclear estrogen receptors and altering target gene transcription, but estrogens can also have nongenomic effects through rapid activation of membrane-initiated kinase cascades. The development of ligands that selectively activate only the nongenomic pathways would provide useful tools to investigate the significance of these pathways. We have prepared large, abiotic, nondegradable poly(amido)amine dendrimer macromolecules that are conjugated to multiple estrogen molecules through chemically robust linkages. Because of their charge and size, these estrogen-dendrimer conjugates (EDCs) remain outside the nucleus. They stimulate ERK, Shc, and Src phosphorylation in MCF-7 breast cancer cells at low concentrations, yet they are very ineffective in stimulating transcription of endogenous estrogen target genes, being approximately 10,000-fold less potent than estradiol in genomic actions. In contrast to estradiol, EDC was not effective in stimulating breast cancer cell proliferation. Because these EDC ligands activate nongenomic activity at concentrations at which they do not alter the transcription of estrogen target genes, they should be useful in studying extranuclear initiated pathways of estrogen action in a variety of target cells. PMID:16306086