Repress Estrogen Receptor Research Articles

Serine starvation silences estrogen receptor signaling through histone hypoacetylation

Loss of estrogen receptor (ER) pathway activity promotes breast cancer progression, yet how this occurs remains poorly understood. Here, we show that serine starvation, a metabolic stress often found in breast cancer, represses estrogen receptor alpha (ERα) signaling by reprogramming glucose metabolism and epigenetics. Using isotope tracing and time-resolved metabolomic analyses, we demonstrate that serine is required to maintain glucose flux through glycolysis and the TCA cycle to support acetyl-CoA generation for histone acetylation. Consequently, limiting serine depletes histone H3 lysine 27 acetylation (H3K27ac), particularly at the promoter region of ER pathway genes including the gene encoding ERα, ESR1. Mechanistically, serine starvation impairs acetyl-CoA-dependent gene expression by inhibiting the entry of glycolytic carbon into the TCA cycle and down-regulating the mitochondrial citrate exporter SLC25A1, a critical enzyme in the production of nucleocytosolic acetyl-CoA from glucose. Consistent with this model, total H3K27ac and ERα expression are suppressed by SLC25A1 inhibition and restored by acetate, an alternate source of acetyl-CoA, in serine-free conditions. We thus uncover an unexpected role for serine in sustaining ER signaling through the regulation of acetyl-CoA metabolism.

Multi-walled carbon nanotubes inhibit estrogen receptor expression in vivo and in vitro through transforming growth factor beta1

Exposure to multi-walled carbon nanotubes (MWCNTs) is suspected to contribute to pulmonary fibrosis through modulation of transforming growth factor beta1 (TGF-β1). There is growing evidence that estrogen signaling is important in pulmonary function and modulates pro-fibrogenic signaling in multiple models of pulmonary fibrosis, however an interaction between MWCNT exposure and estrogen signaling in the lung is not known. The purpose of this work was to determine whether estrogen signaling in the lung is a target for MWCNTs and to identify potential signaling mechanisms mediating MWCNT-induced responses using a whole-body inhalation mouse model and an in vitro human lung cell model. Mice exposed to MWCNTs had reduced mRNA expression of estrogen receptor alpha and beta (Esr1 and Esr2, respectively) in lung tissue at multiple time-points post exposure, whereas expression of G-protein coupled estrogen receptor 1 (Gper1) was more variable. We localized ESR1 protein expression as primarily associated with bronchioles and within inflammatory macrophages. The reduction in estrogen receptor expression was concomitant to an increase in TGF-β1 levels in the bronchoalveolar lavage fluid (BALF) of MWCNT-exposed animals. We confirmed a role for TGF-β1 in mediating MWCNT-induced repression of ESR1 mRNA expression using a TGF-β type-I receptor inhibitor in bronchial epithelial cells in vitro. Overall these results highlight a novel mechanism of MWCNT-induced signaling where MWCNT-induced regulation of TGF-β1 represses estrogen receptor expression. Dysregulated estrogen signaling through altered receptor expression may have potential consequences on lung function.

Z-ligustilide restores tamoxifen sensitivity of ERa negative breast cancer cells by reversing MTA1/IFI16/HDACs complex mediated epigenetic repression of ERa.

Emerging evidence indicates epigenetic modification represses estrogen receptor α (ERα) and contributes to the resistance to tamoxifen in aggressive ERα-negative (ERα−) breast cancer. Z-ligustilide is a major compound in Radix Angelica sinensis, an herb from traditional Chinese medicine (TCM) most frequently prescribed for breast cancer. However, the role of Z-ligustilide in ERα− breast cancer and epigenetic modification remains largely unknown. Herein we showed, for the first time, that Z-ligustilide restored the growth inhibition of tamoxifen on ERα− breast cancer cells. Apoptosis and S and G2/M phases cell cycle arrest were induced by combinatorial Z-ligustilide and tamoxifen. Importantly, Z-ligustilide reactivated the ERα expression and transcriptional activity, which is proved to be indispensable for restoring the sensitivity to tamoxifen. Interestingly, Z-ligustilide increased Ace-H3 (lys9/14) enrichment in the ERα promoter. Moreover, Z-ligustilide dramatically reduced the enrichment of metastasis-associated protein 1 (MTA1) as well as IFN-γ-inducible protein 16 (IFI16) and histone deacetylases (HDACs) onto the ERα promoter. Meanwhile, Z-ligustilide downregulated MTA1, IFI16 and HDACs, which caused destabilization of the corepressor complex. Collectively, our study not only highlights Z-ligustilide as a novel epigenetic modulator, but also opens new possibilities from TCM for treating aggressive tamoxifen-resistant breast cancer.

Abstract B108: Cancer-associated fibroblasts contribute to establishment of ER-negative breast cancer phenotype through secreted factors and miRNAs

Abstract The tumor microenvironment (TME) has been shown to play a vital role in tumor development and progression. Carcinoma associated fibroblasts (CAFs) form a very essential component of the TME. In fact, the TME in different types of carcinomas, particularly carcinomas of the breast, is comprised mainly of fibroblasts. CAFs in breast cancer TME secrete cytokines and growth factors that are known to activate a multitude of signaling pathways in breast cancer cells. One of these pathways is the MAPK pathway, a signaling pathway activated downstream of ERBB receptor tyrosine kinase family members. We have previously demonstrated that activation of the MAPK pathway represses estrogen receptor (ER) expression leading to an ER-negative phenotype in breast cancer. We have generated dissociated CAF cell cultures from ER+, ER-/Her2+, and triple negative (TN) primary breast tumors. We have characterized these CAFs on the basis of fibroblast markers, epithelial markers, microarray analysis, soft agar assay and in vivo tumorigenecity studies. Our results show that these CAF populations from primary breast tumors are pure populations of CAF cell cultures. These CAFs provide a model that allows us to examine the role of CAF interaction with ER+ breast cancer cells regarding activation of MAPK and subsequent repression of ER expression. We demonstrate that treatment of the ER+ MCF-7 cell line with conditioned media (CM) from the CAFs results in transient activation of MAPK signaling and subsequent repression of ER. Continuous exposure of ER+ breast cancer cells to soluble factors from CAFs also results in activation of MAPK and down-regulation of ER expression. Importantly, CM from human mammary fibroblasts (HMFs) and CAFs generated from an ER+ tumor do not down-regulate ER expression. Gene expression analysis and cytokine arrays indicate key differences in gene and cytokine expression between HMFs and the ER+ tumor CAFs compared to the two CAFs generated from ER- tumors. We have previously identified a miRNA signature associated with hyperactivation of MAPK signaling (hMAPK). This signature identifies the majority of ER- breast cancers as well as a population of ER+ breast cancers that express lower levels of ER. Like the ER- breast cancers identified by this signature, ER+ cancers identified by this signature exhibit gene expression patterns indicative of activated MAPK signaling, and exhibit significantly increased disease recurrence and significantly reduced disease survival. Here we show that breast tumors cancers bearing this hMAPK-miRNA signature have elevated expression of numerous stromal markers associated with poor clinical outcome, as well as a number of microRNAs that are differentially expressed between normal human mammary fibroblasts (HMFs) and mammary tumor carcinoma CAFs. Some of these miRNAs have been established as targeting ER. Using reporter constructs to investigate microRNA regulation of ER, we have observed that treatment of ER+ breast cancer cells with CM from ER- CAFs results in enhanced microRNA-dependent repression of ER. These data are suggestive of a role for CAF secreted factors in activating MAPK in breast cancer cells leading to repression of ER. In addition, CAFs are able to increase the expression of hMAPK-miRNAs that can down-regulate ER expression either via the activation of MAPK in the cancer cell or secreted directly from the CAFs. We hypothesize that CAFs may contribute to an ER-negative phenotype in breast cancer cells not only by activating MAPK signaling in the tumor cells via secretion of growth factors, but also potentially by transfer of MAPK-regulated miRNAs to the breast cancer cells as well. Citation Format: Sanket H. Shah, Philip Miller, Katherine Drews-Elger, Joeli Brinkman, Stefania Lairet, Alana Steinberg, Dorraya El-Ashry. Cancer-associated fibroblasts contribute to establishment of ER-negative breast cancer phenotype through secreted factors and miRNAs. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B108.

Distinct Roles for Aryl Hydrocarbon Receptor Nuclear Translocator and Ah Receptor in Estrogen-Mediated Signaling in Human Cancer Cell Lines

The activated AHR/ARNT complex (AHRC) regulates the expression of target genes upon exposure to environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Importantly, evidence has shown that TCDD represses estrogen receptor (ER) target gene activation through the AHRC. Our data indicates that AHR and ARNT act independently from each other at non-dioxin response element sites. Therefore, we sought to determine the specific functions of AHR and ARNT in estrogen-dependent signaling in human MCF7 breast cancer and human ECC-1 endometrial carcinoma cells. Knockdown of AHR with siRNA abrogates dioxin-inducible repression of estrogen-dependent gene transcription. Intriguingly, knockdown of ARNT does not effect TCDD-mediated repression of estrogen-regulated transcription, suggesting that AHR represses ER function independently of ARNT. This theory is supported by the ability of the selective AHR modulator 3′,4′-dimethoxy-α-naphthoflavone (DiMNF) to repress estrogen-inducible transcription. Furthermore, basal and estrogen-activated transcription of the genes encoding cathepsin-D and pS2 are down-regulated in MCF7 cells but up-regulated in ECC-1 cells in response to loss of ARNT. These responses are mirrored at the protein level with cathepsin-D. Furthermore, knock-down of ARNT led to opposite but corresponding changes in estrogen-stimulated proliferation in both MCF7 and ECC-1 cells. We have obtained experimental evidence demonstrating a dioxin-dependent repressor function for AHR and a dioxin-independent co-activator/co-repressor function for ARNT in estrogen signalling. These results provide us with further insight into the mechanisms of transcription factor crosstalk and putative therapeutic targets in estrogen-positive cancers.

Nuclear Orphan Receptor, COUP-TFII, in Reproduction.

COUP-TFII is an orphan nuclear receptor of the nuclear receptor superfamily. COUP-TFII is highly expressed in the uterine stroma and its expression is controlled by progesterone-Ihh-Ptch signaling from the epithelium to the stroma. Mutants with uterine-specific deletion of COUP-TFII are defective in embryo attachment and decidualization, phenotypes similar to mutant mice with the conditional ablation of Ihh or PR. Our results indicate that COUP-TFII is a downstream target of Indian hedgehog that mediates PR/hedgehog signaling to regulate embryo attachment and decidualization. Interestingly, the expression of estrogen receptor target genes in the epithelium is enhanced in the absence of COUP-TFII, suggesting COUP-TFII may mediate progesterone signaling to repress estrogen receptor activity. To assess this, the mutants were treated with ICI 182,780 to determine if receptivity could be restored by the inhibition of estrogen activity. Intriguingly, the implantation sites are partially restored upon ICI 182,780 treatment. Also, the rescued mice exhibited decidualization by expressing markers BMP2 and Wnt4. Our findings reveal that COUP-TFII mediates progesterone signaling to inhibit estrogen receptor activity and establish the requirement for epithelial-stroma interactions during the implantation process. In addition to COUP-TFII's role in the stroma, we found that COUP-TFII is also expressed in the myometrium. Deletion of COUP-TFII in the smooth muscle results in leiomyoma-like ectopic tissues. The ectopic tissues express smooth muscle actin and collagen, characteristic markers of leiomyoma. The development of leiomyoma may be associated with improper wound repair processes in the COUP-TFII deficient mice. Since leiomyoma is one of the most common benign tumors in the female reproductive tract, our findings provide a new perspective on women's health care associated with dysfunction of COUP-TFII. This work is supported by U54 HD 07495, Project 4, HL076448 and DK 62434. (platform)

SUMOylation Modulates the Transcription Repressor Function of RIP140

RIP140/NRIP1 (receptor-interacting protein 140) functions as a corepressor of nuclear receptors. It plays an important role in the transcriptional control of energy metabolism and female fertility. RIP140 contains four distinct repression domains (RD1-RD4), and the repressive activity of RIP140 involves complex mechanisms. The function of both RD1 and RD2 is linked to recruitment of histone deacetylases and C-terminal binding protein, respectively, but the mechanism of repression for RD3 and RD4 has remained elusive. Because covalent modification by small ubiquitin-like modifiers (SUMO-1, -2, and -3; SUMOylation) is often associated with transcriptional repression, we studied whether SUMOylation is involved in the repressive activity of RIP140. We show that two conserved lysines, Lys(756) and Lys(1154), located in RD3 and RD4, respectively, are subject to reversible SUMOylation, with SUMO-1 being more efficiently conjugated than SUMO-2. Interestingly, mutations of the RIP140 SUMOylation sites compromised the transcription repressor function of RIP140 and blunted its capacity to repress estrogen receptor alpha-dependent transcription. Conjugation of SUMO-1 also influenced the subnuclear distribution pattern of RIP140. In sum, our demonstration that the function of RIP140 repression domains 3 and 4 can be modulated by reversible SUMO modification thus adds a novel level to the regulation of RIP140 activity, which may have ramifications in the control of gene networks exerted by RIP140.

Different Modulation of ER‐Mediated Transactivation by Xenobiotic Nuclear Receptors Depending on the Estrogen Response Elements and Estrogen Target Cell Types

Recent studies demonstrated that constitutive androstane receptor (CAR) inhibits ER-mediated transactivation of both endogenous and synthetic estrogen responsive promotor in Hep G2. Whereas steroid and xenobiotic receptor (SXR) but not peroxisome proliferator-activated receptor-gamma (PPAR-gamma) was also reported to repress estrogen receptor (ER) transactivation of the synthetic 4ERE in Hep G2, the effects of these xenobiotic nuclear receptors (XNRs) on the endogenous estrogen responsive promotor remain to be determined. Effects of CAR, SXR, and PPAR-gamma on ER transactivation were also examined in three different kinds of breast cancer cell lines. However, except in MCF-7, studies were limited either in single dose response (MDA-MB-231) or with CAR only (MCF-7-K3). And there is presently no report on the effects of CAR, SXR, and PPAR-gamma on ER-mediated transactivation in ovarian-derived CHO-S cells. Accordingly, this article further examined the effects of the endogenous vitellogenin B1 estrogen responsive promotor on the SXR- and PPAR-gamma-modulated ER transactivation in Hep G2, and either dose-dependent or single dose effects of SXR, PPAR-gamma, and CAR in two different breast cancer cell lines and the ovarian-derived cell line respectively, on the ER-mediated transactivation of the synthetic (4ERE)-tk-luciferase reporter. Consistent with the previous report, CAR significantly repressed ER-mediated transactivation of the endogenous vitellogenin B1 promotor in Hep G2 cells. However, contrary to the effects on the synthetic promotor, PPAR-gamma potentiated whereas SXR did not have any effects on the ER transactivation of the vitellogenin promotor in Hep G2. In the breast cancer cell line of MDA-MB-231 in which endogenous ER is known not to be expressed, CAR modestly stimulated ER transactivation of the synthetic 4ERE in a low dose whereas both SXR and PPAR-gamma did not have any effects in all doses examined (20-500 ng). And in both CHO-S and estrogen-independent breast cancer cell line, MCF-7-K3, none of the three xenobiotic receptors significantly influenced the ER-mediated 4ERE transactivation in all doses examined. XNRs modulate ER-mediated transactivation depending on the estrogen response elements (EREs) and estrogen target cell types.

Activation of p21-activated Kinase 6 by MAP Kinase Kinase 6 and p38 MAP Kinase

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.

Corepressor subnuclear organization is regulated by estrogen receptor via a mechanism that requires the DNA-binding domain

The restriction of transcription factors to certain domains within the cell nucleus must serve an important regulatory function. The silencing mediator of retinoic acid and thyroid hormone (SMRT) and other members of the corepressor complex are enriched in spherical intranuclear foci, and repress estrogen receptor alpha (ERα)-dependent transcriptional activity. When fluorescent protein (FP)-labeled SMRT and ERα were co-expressed, the proteins co-localized. The subnuclear organization and positioning of the complexes, however, depended on the ligand state of the receptor. Automated image analysis was used to quantify the ERα-dependent change in SMRT organization in randomly selected living cell populations. The results demonstrate that the subnuclear positioning of SMRT is influenced by the ligand-bound ERα, and this activity is dependent on the ratio of the co-expressed ERα and SMRT. A deletion mutant of ERα showed that the receptor DNA-binding domain was necessary for the ligand-dependent positioning of SMRT. These results define important organizational mechanisms that underlie nuclear receptor regulation of gene expression.

Intermittent Hypoxia Induces Proteasome-Dependent Down-Regulation of Estrogen Receptor α in Human Breast Carcinoma

Hypoxia may influence gene expression to promote malignancy, and acute hypoxia has been shown to transiently repress estrogen receptor (ER)-alpha expression in breast cell lines. However, the effect of intermittent hypoxia, which is likely more prevalent in breast cancers, remains to be determined. ER-alpha expression was assessed by Western blot and immunohistochemistry in a selected cohort of 51 ER-alpha-positive breast carcinomas, in relation to markers of hypoxia. The effect of acute and intermittent hypoxia on ER-alpha expression was also determined in MCF7 and ZR-75 breast cell lines, together with the role of proteasome function with the proteasome inhibitor bortezomib. Regional loss of ER-alpha expression occurs in breast tumors and is consistently present in hypoxic regions defined by the proximity of necrosis and induction of hypoxia-induced genes carbonic anhydrase IX (CA-IX) and glucose transporter 1 (Glut-1), in both in situ (n = 29; P < 0.0001) and invasive (n = 20; P = 0.0001) carcinomas. In MCF7 and ZR-75 cells, ER-alpha is transiently down-regulated by acute hypoxia and rapidly restored by reoxygenation. However, intermittent, acute hypoxia can cause a similar down-regulation of ER-alpha that is not attributable to decreased mRNA and persists in MCF7 cells despite reoxygenation for up to 14 days. This effect occurs with no change in cell viability but a corresponding reduction in growth response to estradiol. However, ER-alpha expression can be restored by bortezomib. Intermittent hypoxia can cause persistent changes in proteasome function that may contribute to reduced ER-alpha expression in breast tumors and consequently to diminished response and development of resistance to endocrine therapy.

Metastasis-Associated Protein 1 Interacts with NRIF3, an Estrogen-Inducible Nuclear Receptor Coregulator

The transcriptional activity of estrogen receptor alpha (ER-alpha) is modified by regulatory action and interactions of coactivators and corepressors. Recent studies have shown that the metastasis-associated protein 1 (MTA1) represses estrogen receptor element (ERE)-driven transcription in breast cancer cells. With a yeast two-hybrid screen to clone MTA1-interacting proteins, we identified a known nuclear receptor coregulator (NRIF3) as an MTA1-binding protein. NRIF3 interacted with MTA1 both in vitro and in vivo. NRIF3 bound to the C-terminal region of MTA1, while MTA1 bound to the N-terminal region of NRIF3, containing one nuclear receptor interaction LXXLL motif. We showed that NRIF3 is an ER coactivator, hyperstimulated ER transactivation functions, and associated with the endogenous ER and its target gene promoter. MTA1 repressed NRIF3-mediated stimulation of ERE-driven transcription and interfered with NRIF3's association with the ER target gene chromatin. In addition, NRIF3 deregulation enhanced the responsiveness of breast cancer cells to estrogen-induced stimulation of growth and anchorage independence. Furthermore, we found that NRIF3 is an estrogen-inducible gene and activated ER associated with the ER response element in the NRIF3 gene promoter. These findings suggest that NRIF3, an MTA1-interacting protein, is an estrogen-inducible gene and that regulatory interactions between MTA1 and NRIF3 might be important in modulating the sensitivity of breast cancer cells to estrogen.

Structure-Function Analysis of the Estrogen Receptor α Corepressor Scaffold Attachment Factor-B1: IDENTIFICATION OF A POTENT TRANSCRIPTIONAL REPRESSION DOMAIN

Scaffold attachment factor-B1 (SAFB1) is a nuclear matrix protein that has been proposed to couple chromatin structure, transcription, and RNA processing. We have previously shown that SAFB1 can repress estrogen receptor (ERalpha)-mediated transactivation. Here we present a structure-function study showing that transactivation is mediated via an intrinsic and transferable C-terminal repression domain (RD). A similar C-terminal RD was found in the family member SAFB2. Removal of the RD from SAFB1 resulted in a dominant-negative SAFB1 protein that increased ligand-dependent and -independent ERalpha activity. SAFB1RD-mediated repression was partly blocked by histone deacetylase inhibitors; however, no histone deacetylase inhibitors were identified in a yeast two-hybrid screen using the RD as bait. Instead, SAFB1RD was found to interact with TAFII68, a member of the basal transcription machinery. We propose a model in which SAFB1 represses ERalpha activity via indirect association with histone deacetylation and interaction with the basal transcription machinery.

ω‐6 Polyunsaturated fatty acid γ‐linolenic acid (18:3n‐6) is a selective estrogen‐response modulator in human breast cancer cells: γ‐Linolenic acid antagonizes estrogen receptor‐dependent transcriptional activity, transcriptionally represses estrogen receptor expression and synergistically enhances tamoxifen and ICI 182,780 (Faslodex) efficacy in human breast cancer cells

ω‐6 Polyunsaturated fatty acid γ‐linolenic acid (18:3n‐6) is a selective estrogen‐response modulator in human breast cancer cells: γ‐Linolenic acid antagonizes estrogen receptor‐dependent transcriptional activity, transcriptionally represses estrogen receptor expression and synergistically enhances tamoxifen and ICI 182,780 (Faslodex) efficacy in human breast cancer cells

Antisense blocking of BRCA1 enhances sensitivity to plumbagin but not tamoxifen in BG-1 ovarian cancer cells.

Previous studies have shown that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG-1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally act as a growth inhibitor in these cells. Also, there are other reports that suggest that wild-type BRCA1 protein may repress estrogen receptor (ER) function either directly or indirectly. However, response to antiestrogen drugs in BRCA1-blocked ER-positive ovarian cancer cells has not been reported, and this served as the rationale for this study. We analyzed the effect of tamoxifen, emodin, and plumbagin in BRCA1-blocked ER-positive BG-1 ovarian cancer cells. For all three drugs, BRCA1-blocked cells were more sensitive than the corresponding control cells as assessed by MTT assay; however, only plumbagin showed a statistically significant difference in mean viability (P < 0.05). All three drugs induced loss of mitochondrial membrane potential (DeltaPsi(m)), nuclear condensation, DNA fragmentation, and morphological changes, as observed after 6 h of drug treatment, suggesting apoptosis induction in both BRCA1-blocked and control cells. However, apoptosis induction was greater in BRCA1-blocked cells, the efficacy being in the order of plumbagin > tamoxifen > emodin. The dose of plumbagin needed to kill 50% was 5 microM in the control cells and 2.68 microM for the BRCA1-blocked cells, indicating that the latter was about twofold more sensitive to plumbagin than the wild-type cells. This throws light on the fact that plumbagin may have chemotherapeutic potential as an anticancer agent in BRCA1-mutated ovarian cancer patients.

Histone H1 represses estrogen receptor alpha transcriptional activity by selectively inhibiting receptor-mediated transcription initiation.

Chromatin is the physiological template for many nuclear processes in eukaryotes, including transcription by RNA polymerase II. In vivo, chromatin is assembled from genomic DNA, core histones, linker histones such as histone H1, and nonhistone chromatin-associated proteins. Histone H1 is thought to act as a general repressor of transcription by promoting the compaction of chromatin into higher-order structures. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the effects of histone H1 on estrogen receptor alpha (ER alpha)-mediated transcription with chromatin templates. We show that histone H1 acts as a potent repressor of ligand- and coactivator-regulated transcription by ER alpha. Histone H1 exerts its repressive effect without inhibiting the sequence-specific binding of ER alpha to chromatin or the overall extent of targeted acetylation of nucleosomal histones by the coactivator p300. Instead, histone H1 acts by blocking a specific step in the ER alpha-dependent transcription process, namely, transcription initiation, without affecting transcription reinitiation. Together, our data indicate that histone H1 acts selectively to reduce the overall level of productive transcription initiation by restricting promoter accessibility and preventing the ER alpha-dependent formation of a stable transcription pre-initiation complex.

Modulation of Estrogen Receptor-mediated Transactivation by Orphan Receptor TR4 in MCF-7 Cells

The human testicular orphan receptor 4 (TR4) is a member of the nuclear receptor superfamily that shows a broad tissue distribution with higher expression in the nervous system and male reproductive tract. TR4 functions as a transcriptional modulator that controls various target genes via binding to the DNA hormone response elements. Here we report that instead of direct binding to hormone response elements for gene regulation, TR4 can also go through direct protein-protein interaction to repress estrogen receptor (ER)-mediated transactivation. Electrophoretic mobility shift and glutathione S-transferase pull-down assays clearly demonstrate that the direct interaction between TR4 and ER will inhibit the homodimerization of ER and interrupt/prevent ER binding to the estrogen response element. The consequence of these events may then result in the suppression of ER target genes, such as cyclin D1 and pS2 and inhibition of ER-mediated cell proliferation in the MCF-7 cells stably transfected with TR4. Together, our results showing that TR4 can suppress ER function via protein-protein interaction not only represent a unique cross-talk signaling pathway in the nuclear receptor superfamily, it may also provide us with a new strategy to modulate ER function in the breast cancer cells.

Isothiocyanates repress estrogen receptor α expression in breast cancer cells

The isothiocyanates (ITCs) have long been known to possess chemopreventive activities for a variety of neoplasms including breast cancer, but the molecular mechanism by which ITCs prevent breast cancer development has not been established. In this study, we investigated the effects of benzyl and phenethyl isothiocyanate (BITC and PEITC) on the estrogen-stimulated growth of estrogen receptor alpha (ERalpha)-positive breast cancer MCF7 and T-47D cells. BITC and PEITC inhibited estrogen-stimulated cell growth and reduced the expression levels of ERalpha in MCF7 and T-47D cells in a dose- and time-dependent and reversible manner. In addition, BITC and PEITC also abrogated the transcriptional activity of ERalpha and hence inhibited estrogen-stimulated expression of the estrogen responsive gene, pS2. These results demonstrated that BITC and PEITC function as potent ERalpha disruptors to abrogate mitogenic estrogen signaling in ER-positive breast cancer cells, which provides a molecular explanation for the growth inhibitory function of ITCs in breast cancer development, and a rational for further exploration of ITCs as chemopreventive agents for human mammary carcinogenesis.

Thyroid hormone receptors repress estrogen receptor activation of a TRE.

The identification of hormone response elements in the promoter regions of hormonally regulated genes has revealed a striking similarity between the estrogen response element (ERE) and a palindromic thyroid hormone response element (TRE) derived from the GH gene promoter. In addition, this TRE was described as a strong retinoic acid receptor response element for all three subtypes: alpha, beta, and gamma. We show here that the TRE in the absence of thyroid hormone receptor (TR) behaves similarly to imperfect EREs, which can synergize to mediate a strong estrogen-dependent activation of transcription. However, in the presence of TR, but the absence of T3, activation of the TRE constructs by estrogen receptor (ER) is inhibited. In vitro, ER and TR were found to bind to the TRE in the absence and presence of their respective ligands; however, TRs form a more stable complex with the TRE than does ER. To examine whether repression of ER activity on the TRE constructs by TR was due to heterodimer formation, we employed truncated TR mutants (tTR) that lacked the DNA-binding domain, but contained the ligand-binding/dimerization domain. The tTRs were shown to be efficient inhibitors of TR, but not of ER. Thus, inhibition of ER activity on TREs by TRs does not result from heterodimer formation. We discuss a mechanism in which TRs, in the absence of thyroid hormone, control TRE activation by related receptors by preventing their access to the TRE. This mechanism can greatly enhance the fidelity of the ligand-specific response from a TRE.