E2-target Genes Research Articles

Abstract P6-12-02: Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers

Abstract Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. Estrogen receptor-alpha, ERɑ, functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the predominant naturally-occurring estrogen. This results in dimerization, DNA binding to cis-regulatory elements called enhancers, and gene regulatory function of ERɑ. In vivo, E2 production and action fluctuates during reproductive cycles and possibly even in a circadian manner. In tissues and cells, E2-regulated gene expression is dynamic, with rapid induction of enhancer formation and target gene expression (or repression), followed by enhancer “decommissioning” and cessation of the gene regulatory effects. The effects of sustained E2 production and signaling, fluctuations in E2 levels, and removal of E2 on estrogen-regulated gene expression are unknown. Classical experiments exploring the gene regulatory effects of E2 involve stimulation of cultured cells throughout the duration of the experiment with a continuous and typically high dose of hormone, with the cells harvested at the end of the stimulation period. While this has allowed us to understand the molecular underpinnings of signal-dependent transcription, it has not allowed analysis of physiological fluctuations in estrogen signaling involving periods of stimulation (i.e., the presence of hormone) and non-stimulation (i.e., the absence of hormone). In our studies, we are applying a combination of next generation sequencing techniques and fluorescence microscopy to investigate signal-dependent responses to fluctuating and short duration E2 exposures using ERɑ-positive MCF-7 human breast cancer cells after E2 treatment and removal. Our initial results suggest that different E2 target genes respond differently to low dose E2 treatment and E2 withdrawal. Moreover, for some E2 target genes, sustained E2 treatment may not be required for a complete E2 transcriptional response. With these studies, we hope to connect physiological aspects of E2 production, release, and action with the molecular mechanisms of E2-dependent gene expression - an aspect of our understanding of estrogen signaling that has been lacking. Citation Format: Hyung Bum Kim, William L. Kraus. Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-02.

RF03 | PMON297 Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers

Abstract Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. Estrogen receptor-alpha, ERɑ, functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the predominant naturally-occurring estrogen. This results in dimerization, DNA binding to cis-regulatory elements called enhancers, and gene regulatory function of ERɑ. In vivo, E2 production and action fluctuates during reproductive cycles and possibly even in a circadian manner. In tissues and cells, E2-regulated gene expression is dynamic, with rapid induction of enhancer formation and target gene expression (or repression), followed by enhancer "decommissioning" and cessation of the gene regulatory effects. The effects of sustained E2 production and signaling, fluctuations in E2 levels, and removal of E2 on estrogen-regulated gene expression are unknown. Classical experiments exploring the gene regulatory effects of E2 involve stimulation of cultured cells throughout the duration of the experiment with a continuous dose of hormone, with the cells harvested at the end of the stimulation period. While this has allowed us to understand the molecular underpinnings of signal-dependent transcription, it has not allowed analysis of physiological fluctuations in estrogen signaling involving periods of stimulation and non-stimulation. In our studies, we are applying a combination of next generation sequencing techniques and fluorescence microscopy to investigate signal-dependent responses to fluctuating E2 levels using ERɑ-positive MCF-7 human breast cancer cells after E2 treatment and removal. Our initial results suggest that different E2 target genes respond differently to E2 withdrawal. Moreover, for some E2 traget genes, sustained E2 treatment may not be required for a complete E2 transcriptional response. With these studies, we hope to connect physiological aspects of E2 production, release, and action with the molecular mechanisms of E2-dependent gene expression - an aspect of our understanding of estrogen signaling that has been lacking. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K. Presentation: Saturday, June 11, 2022 1:12 p.m. - 1:17 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells.

Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call 'progestin control regions' (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.

Ligand dependent gene regulation by transient ERα clustered enhancers.

Unliganded Estrogen receptor alpha (ERα) has been implicated in ligand-dependent gene regulation. Upon ligand exposure, ERα binds to several EREs relatively proximal to the pre-marked, unliganded ERα-bound sites and affects transient but robust gene expression. However, the underlying mechanisms are not fully understood. Here we demonstrate that upon ligand stimulation, persistent sites interact extensively, via chromatin looping, with the proximal transiently ERα-bound sites, forming Ligand Dependent ERα Enhancer Cluster in 3D (LDEC). The E2-target genes are regulated by these clustered enhancers but not by the H3K27Ac super-enhancers. Further, CRISPR-based deletion of TFF1 persistent site disrupts the formation of its LDEC resulting in the loss of E2-dependent expression of TFF1 and its neighboring genes within the same TAD. The LDEC overlap with nuclear ERα condensates that coalesce in a ligand and persistent site dependent manner. Furthermore, formation of clustered enhancers, as well as condensates, coincide with the active phase of signaling and their later disappearance results in the loss of gene expression even though persistent sites remain bound by ERα. Our results establish, at TFF1 and NRIP1 locus, a direct link between ERα condensates, ERα enhancer clusters, and transient, but robust, gene expression in a ligand-dependent fashion.

Growth inhibitory efficacy and anti-aromatase activity of Tabebuia avellanedae in a model for post-menopausal Luminal A breast cancer.

Aromatase inhibitors (AIs) represent a treatment option for post-menopausal estrogen receptor-positive (ER+) breast cancer as monotherapy, or in combination with cyclin-dependent kinase 4/6 or mTOR inhibitors. Long-term treatment with these agents leads to dose-limiting toxicity and drug resistance. Natural substances provide testable alternatives to current therapy. Tabebuia avellanedae (TA) tree is indigenous to the Amazon rainforest. The inner bark of TA represents a medicinal dietary supplement known as Taheebo. Non-fractionated aqueous extract from TA is an effective growth inhibitor in the Luminal A and triple negative breast cancer models. The quinone derivative naphthofurandione (NFD) is a major bioactive agent in TA. The present study examined the efficacy of finely ground powder from the inner bark of TA, available under the name of Taheebo-NFD-Marugoto (TNM). The ER+ MCF-7 cells stably transfected with the aromatase gene MCF-7AROM represented a model for aromatase-expressing post-menopausal breast cancer. Anchorage-independent colony formation, cell cycle progression, pro-apoptotic caspase 3/7 activity, apoptosis-specific gene expression, aromatase activity and select estradiol (E2) target gene expression represented the mechanistic end points. Treatment of MCF-7AROM cells with TNM induced a dose-dependent reduction in E2-promoted anchorage-independent colony number. Mechanistic assays on TNM-treated MCF-7AROM cells demonstrated that TNM at a concentration of 10 µg (NFD content: 2 ng), induced S-phase arrest, increased pro-apoptotic caspase 3/7 activity, increased pro-apoptotic BAX and decreased anti-apoptotic BCL-2 gene expression, and inhibited aromatase activity. Additionally, TNM treatment downregulated ESR-1 (gene for ER-α), aromatase and progesterone gene expression and reduced mRNA levels of E2 target genes pS2, GRB2 and cyclin D1. Inhibition of aromatase activity, based on the NFD content of TNM was superior to the clinical AIs Letrozole and Exemestane. These data demonstrated the potential efficacy of TNM as a nutritional alternative for current therapy of aromatase positive, post-menopausal breast cancer.

Alterations of estradiol-induced histone H3 acetylation in the preoptic area and anteroventral periventricular nucleus of middle-aged female rats

In this study we investigated the characteristics of histone H3 acetylation in the anterior hypothalamus under E2 positive feedback to gain a better understanding of the mechanism underlying reduced GnRH neuron activation and altered gene expression in female reproductive aging. Young and middle-aged female rats were ovariectomized (OVX) and treated with estradiol (E2) or oil. C-Fos expression, the number of GnRH neurons co-localized with c-Fos in the preoptic area (POA), and the number of acetylated histone H3 cells in the POA and anteroventral periventricular nucleus (AVPV) were quantified at the time of the expected GnRH neuron activation. We used real-time PCR to evaluate the expression of Esr1 target genes including Kiss1 and VGluT2 and genes known as Esr1 coregulators in the anterior hypothalamus. Our results show that in the young females, E2 markedly increased histone H3 acetylation in the POA and AVPV, coincident with increased c-Fos and GnRH neuron activation in the POA. In middle-aged females, E2-induced histone H3 acetylation was reduced in the POA but was not significantly altered in the AVPV. This occurred in association with a reduction of c-Fos expression and the number of GnRH cells expressing c-Fos in the POA as well as a down-regulation of Kiss1 and VGluT2 mRNA expression in the anterior hypothalamus of the animals. E2 caused significant decreases in Ncoa2 and Crebbp mRNA expression in the anterior hypothalamus of young, but not middle-aged females. Taken together, these data suggest that alterations of histone H3 acetylation in the POA and AVPV and the inability of Ncoa2 and Crebbp to respond to E2 in the middle-aged anterior hypothalamus partially contribute to the decline of GnRH neuron activation and E2 target gene expression changes that occur in female along with reproductive aging.

RNA Stabilizes Transcription-Dependent Chromatin Loops Induced By Nuclear Hormones

We show that transcription induced by nuclear receptors for estrogen (E2) or retinoic acid (RA) is associated with formation of chromatin loops that juxtapose the 5’ end (containing the promoter) with the enhancer and the 3′ polyA addition site of the target gene. We find three loop configurations which change as a function of time after induction: 1. RA or E2-induced loops which connect the 5′ end, the enhancer and the 3′ end of the gene, and are stabilized by RNA early after induction; 2. E2-independent loops whose stability does not require RNA; 3. Loops detected only by treatment of chromatin with RNAse H1 prior to hormonal induction. RNAse H1 digests RNA that occludes the relevant restriction sites, thus preventing detection of these loops. R-loops at the 5′ and 3′ ends of the RA or E2-target genes were demonstrated by immunoprecipitation with anti-DNA-RNA hybrid antibodies as well as by sensitivity to RNAse H1. The cohesin RAD21 subunit is preferentially recruited to the target sites upon RA or E2 induction of transcription. RAD21 binding to chromatin is eliminated by RNAse H1. We identified E2-induced and RNase H1-sensitive antisense RNAs located at the 5′ and 3′ ends of the E2-induced transcription unit which stabilize the loops and RAD21 binding to chromatin. This is the first report of chromatin loops that form after gene induction that are maintained by RNA:DNA hybrids.

Abstract P5-05-16: Role of estrogen receptor alpha acetylation in estrogen-dependent gene regulation in breast cancers

Abstract Estrogens, such as 17β-estradiol (E2), act through estrogen receptor alpha (ERα), a ligand-regulated transcription factor that binds across the genome to promote enhancer formation and regulate gene expression. ERα is expressed in approximately 70% of breast cancers, where it regulates the transcription of genes involved in mitogenic and inflammatory pathways. We are exploring the the acetylation of ERα on lysines 266 and 268, a modification that enhances the DNA binding and transcriptional activities of ERα. ERα acetylation is catalyzed by the lysine acetyltransferases p300 and CBP in an E2- and steroid receptor coregulator (SRC)-dependent manner. Acetylation-dependent activation of ERα has potential implications in breast cancers associated with enhanced coregulator interactions, such as SRC-3/Amplified in Breast (AIB1) gene amplifications and gain-of-function ERα mutations in endocrine resistant metastatic tumors, such as Y537S and D538G. Increased association of ERα with SRCs and p300/CBP leading to enhanced ERα acetylation may promote gain-of-function ERα activity. Our hypothesis is that acetylation of ERα alters its function by increasing E2-responsive gene transcription and signaling in breast cancers. Our current efforts are focused on investigating the role of ERα acetylation on chromatin binding and accessibility, enhancer activity, and target gene transcription. We are using the ER-positive MCF-7 breast cancer cell line with a knockdown/re-expression strategy with biochemical mimics of acetylated (K266/268Q) or unacetylated (K266/268R) ERα. Genomic ERα binding profiles using chromatin immunoprecipitation-sequencing (ChIP-seq) has defined overlapping, but unique, sets of transcriptional targets and recruitment kinetics for the ERα mutants. Current efforts are focused on defining global transcriptional responses associated with the unique binding activities using precision run-on sequencing (PRO-seq). Ultimately, our goal is to define how acetylation affects the binding of ERα to chromatin and its effect on the transcriptional activity of E2-target genes in breast cancers in the context of gain-of-function ERα mutations and coregulator amplifications. Supported in part by a postdoctoral fellowship from the Lalor Foundation to YMV, and grants from the NIDDK, NICHD, and CPRIT to WLK, as well as support from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment. Citation Format: Vasquez YM, Setlem R, Murakami S, Kraus WL. Role of estrogen receptor alpha acetylation in estrogen-dependent gene regulation in breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-16.

Abstract P6-08-02: Disruption of the estradiol-regulated NTN1-UNC5A dependence receptor signaling axis causes a hybrid basal/luminal molecular phenotype in estrogen receptor-positive breast cancer cells

Abstract Luminal subtype of breast cancers that express the estrogen receptor alpha (ERα) represents approximately two-thirds of all breast cancer cases. ER+ tumors tend to have the most favorable prognoses when treated with endocrine therapy. However, a relapse or endocrine therapy resistance is often seen in ER+ breast tumors. UNC5A belongs to the dependence receptor family which can mediate two different intracellular signals: cell survival, differentiation or migration when engaged with its ligand (such as Netrin-1; NTN1) or cell death/apoptosis in the absence of the ligand. Here we demonstrate that, depending upon the cell type, UNC5A and NTN1 are estradiol (E2)-inducible genes. Using shRNA or CRISPR knockdown strategies, we show that the disruption of the NTN1-UNC5A signaling axis in ER+ (MCF7 and T-47D) cells generates a mixed basal-like/luminal phenotype with stem cell-like characteristics. RNA-seq of UNC5A knockdown cells showed deregulated expression of several E2-target genes in both cell lines. Moreover, knockdown of UNC5A resulted in increased cell proliferation, and elevated expression of the E2-inducible anti-apoptotic, BCL2. Furthermore, the expression of ΔNp63 was enhanced in UNC5A knockdown cells. ΔNp63 is a TP53 family transcription factor that promotes breast epithelial stem cell maintenance and basal-like breast cancer. Accordingly, UNC5A knockdown cells displayed cancer stem cell phenotype as evident from ~3-fold increase in the number of CD44+/CD24+, CD44+/EPCAM+ and ITGA6+/EPCAM+ subpopulation compared with control cells. In addition, the expression of NTN4, a pro-angiogenic and lymphangiogenic factor, was increased upon UNC5A knockdown. In vivo, UNC5A knockdown cells implanted in nude mice were able to form tumors in the mammary fat pad independent of E2 supplementation and were able to colonize and develop into overt metastasis in multiple organs such as lungs, ovaries and adrenal glands. Consequently, analysis of mammary fat pad tumors from animals that received UNC5A knockdown cells revealed an increased expression of PECAM1 (CD31), a marker for endothelial cells used to evaluate tumor angiogenesis. In contrast to UNC5A, knocking down NTN1, decreased the expression of BCL2 and TP63 in both cell lines. Thus, knockdown of UNC5A resulted in deregulated expression of E2-regulated genes, E2-independent and anti-estrogen-resistant growth in vitro, and E2-independent tumor formation in xenograft models. Consistent with results of in vitro studies, analysis of tissue samples from breast cancer patients (n=196) revealed that lower expression of UNC5A is associated with lower overall survival (P < 0.05). Thus, loss or mutational inactivation of UNC5A could lead to unrestricted E2:ERα signaling and anti-estrogen resistant growth while simultaneously enabling ERα-positive luminal breast cancer cells to acquire basal-like and cancer stem cell-like features. Citation Format: Padua MB, Bhat-Nakshatri P, Anjanappa M, Hao Y, Liu Y, McElyea K, Sandusky G, Althouse S, Perkins S, Nakshatri H. Disruption of the estradiol-regulated NTN1-UNC5A dependence receptor signaling axis causes a hybrid basal/luminal molecular phenotype in estrogen receptor-positive breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-02.

UPF1 regulates myeloid cell functions and S100A9 expression by the hnRNP E2/miRNA-328 balance.

UPF1 is a key player in nonsense mediated mRNA decay (NMD) but also involved in posttranscriptional gene regulation. In this study we found that UPF1 regulates the expression of genes with functions in inflammation and myeloid cell differentiation via hnRNP E2. The majority of the UPF1-regulated genes identified in monocytic cells contain a binding site for hnRNP E2 within 5′ UTR located introns with hnRNP E2 acting here as splicing regulator. We found that miRNA-328 which is significantly induced during monocytic cell differentiation acts independently from its gene silencing function as RNA decoy for hnRNP E2. One representative gene controlled by the hnRNP E2/miRNA-328 balance is S100A9 which plays an important role in cell differentiation and oxidative stress response of monocytes. Induction of miRNA-328 expression during cell differentiation antagonizes the blockade by hnRNP E2 which results in the upregulation of CD11b expression and ROS production in monocytic cells. Taken together, our data indicate that upregulation of miR-328 is responsible for the induction of hnRNP E2 target genes during myeloid cell differentiation.

A deregulated expression of estrogen-target genes is associated with an altered response to estradiol in aged rats perinatally exposed to bisphenol A

Here we assessed the effects of perinatal exposure to bisphenol A (BPA) on the uterine response to 17β-estradiol (E2) in aged rats. Pregnant rats were orally exposed to 0.5 or 50 μg BPA/kg/day from gestational day 9 until weaning. On postnatal day (PND) 360, the rats were ovariectomized and treated with E2 for three months. The uterine tissue of BPA50 and BPA0.5 rats showed increased density of glands with squamous metaplasia (GSM) and glands with daughter glands respectively. Wnt7a expression was lower in GSM of BPA50 rats than in controls. The expression of estrogen receptor 1 (ESR1) and its 5′- untranslated exons ESR1-O and ESR1-OT was lower in BPA50 rats. Both doses of BPA modified the expression of coactivator proteins and epigenetic regulatory enzymes. Thus, perinatal BPA-exposed rats showed different glandular abnormalities associated with deregulated expression of E2-target genes. Different mechanisms would be involved depending on the BPA dose administered.

Shugan Liangxue Decoction () Down-Regulates Estrogen Receptor α Expression in Breast Cancer Cells.

To observe the effect of Shugan Liangxue Decoction (, SGLXD) on estrogen receptor α (ERα) in human breast cancer cells. The effect of SGLXD (0.85-5.10 mg/mL) on the proliferation of breast cancer cells were evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The nuclear ERα protein levels in MCF-7, T47D and ZR-75-1 cells which treated by SGLXD for 24 h were examined by western blot and immunofluorescence assay. MCF-7 and MDA-MB-231 cells were treated by 17β-estradiol (E2) with or without SGLXD, for 24 h, and the E2 targeted genes c-myc and bcl-2 protein product was evaluated by western blot. SGLXD showed dose-dependent inhibition on the proliferation of MCF-7, T47D and ZR-75-1 cells, but did not inhibit the proliferation of MDA-MB-231 cells. Furthermore, the promotive effect on cell growth induced by E2 was also significantly inhibited by SGLXD treatment. With the treatment of 1.70, 3.40, 5.10 mg/mL SGLXD, the nuclear ERα protein level was reduced to 88.1%, 70.4% and 60.9% in MCF-7 cells, and was decreased to 43.0%, 38.4% and 5.9% in ZR-75-1 cells as compared with the control group. In T47D cells, the nuclear ERα protein was down-regulated to 51.3% and 4.3% by 3.40 and 5.10 mg/mL SGLXD treatment. The down-regulative effect of SGLXD on nuclear ERα was confirmed by immunofluorescence assay. SGLXD decreased the protein product of c-myc and bcl-2. SGLXD may exhibit selective inhibition effect on the proliferation of ER positive breast cancer cells. SGLXD reduced the nuclear ERα expression and the protein product of E2 target gene c-myc and bcl-2.

Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

Estrogenic Potency of Benzophenone UV Filters in Breast Cancer Cells: Proliferative and Transcriptional Activity Substantiated by Docking Analysis

The results from recent studies show that some benzophenones (BPs) and their hydroxylated metabolites can function as weak estrogens (E2) in the environment. However, little is known about the structure-activity relationship of these molecules. We have examined the effects of exposure to ten different BPs on the proliferation of estrogen receptor (ER)-positive breast cancer cells and on the transcriptional activity of E2-target genes. We analyzed two genes that are tightly linked with estrogen-mediated proliferation, the CXCL12 and amphiregulin genes and two classical estrogen-responsive genes, the pS2 and progesterone receptor. Significant differences in the BPs efficiency to induce cell proliferation and endogenous E2-target gene expressions were observed. Using ERE-, Sp1-, AP1- and C3-reporter genes that contain different ER-binding sites in their promoter, we also showed significant differences in the BPs efficiency in activation of the ER transactivation. Together, our analyzes showed that the most active molecule is 4-hydroxy-BP. Docking analysis of the interaction of BPs in the ligand-binding pocket of ERα suggests that the minimum structural requirement for the estrogenic activity of BPs is a hydroxyl (OH) group in the phenyl A-ring that allows interaction with Glu-353, Arg-394 or Phe-404, which enhances the stability between BPs and ERα. Our modeling also indicates a loss of interaction between the OH groups of the phenyl B-ring and His-524. In addition, the presence of some OH groups in the phenyl B-ring can create repulsion forces, which may constrain helix 12 in an unfavorable position, explaining the differential estrogenic effects of BPs. These results, together with our analysis of BPs for their potency in activation of cell proliferation and ER-mediated transcription, report an improved understanding of the mechanism and structure–activity relationship of BPs.

Role of estrogen receptor α in estradiol‐induced cardiac growth

Contrary to its effects in the diseased heart, we recently discovered that estradiol (E2) unexpectedly exerts pro‐hypertrophic effects in the healthy heart of ovariectomized (OVX) mice. Our aim was to investigate the mechanism(s) involved in the regulation of cardiac growth by E2 testing the hypothesis that estrogen receptor α (ERα) mediates these E2 effects. Female wild‐type (WT) C57Bl/6J and estrogen receptor α knockout (ERKO) mice were ovariectomized at two months old and were fed an E2‐containing or soy‐free (Ctrl) diet for three months (n = 5–6/group). E2 supplementation in OVX mice led to a lower body weight (15% decrease; P < 0.05) and a higher uterus‐to‐body‐weight ratio (5‐fold increase; P < 0.001) compared with Ctrl mice, but these E2 effects were abrogated in ERKO mice. E2 led to a higher heart‐to‐body‐weight ratio compared with WT Ctrl mice (20% increase; P < 0.05). Deletion of ERα abolished this cardiac effect of E2. The expression of three E2 target genes in the heart was significantly induced by E2 in WT mice compared with Ctrl, whereas in ERKO mice E2 had no effect on Act2, Igf1 and Myocd expression. In conclusion, our study demonstrates that E2 regulates body weight and exerts significant effects on cardiac growth and gene expression via an ERα‐dependent mechanism. Funding: EUGeneHeart (LSHM‐CT‐2005–018833)

17β-Estradiol-induced interaction of ERα with NPPA regulates gene expression in cardiomyocytes

17β-Oestradiol (E2) and its receptors (ERα and ERβ) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.

Abstract 4704: Effects of the HDAC inhibitor S78454/PCI-24781 on ER signalling in ERα-positive antiestrogen-sensitive and -resistant breast cancer cells

Abstract Background: Estrogen receptors (ER) drive breast cancer development through the regulation of gene transcription and the inhibition of ER signalling is the main therapeutic strategy for hormone-dependent breast cancers. Selective ER modulators (such as tamoxifen) or aromatase inhibitors are commonly administered to patients. However, de novo and acquired resistance are frequently observed and combinatorial treatments are required to bypass this phenomenon. Acetylation plays a key role in estrogen signalling by regulating ERα and ERα expression and activity (for a recent review see Linares et al, J. Biomed. Biotech. 2011). This post-translational mark is removed by histone deacetylases (HDACs) whose enzymatic activity could be blocked by specific HDAC inhibitors (HDIs). Interestingly, it has been shown that HDIs are able to regulate ER signalling and to potentiate the anti-tumor effects of anti-estrogens in ER-positive breast cancer cells. In this study, we analyzed the effects of a new hydroxamic acid HDI S78454 on several cellular parameters regulated by estrogens in ERα expressing MCF-7 breast cancer cells. Method: The following parameters were investigated both in sensitive and in antiestrogen-resistant MCF7 cells, that we developed in our laboratory: 1) ERα expression at the mRNA and protein levels by quantitative RT-PCR and western blot, respectively 2) transcriptional activity of ERα by quantifying the expression of several estrogen-regulated mRNAs 3) HDAC expression at the mRNA level and 4) 2D cell proliferation using MTS assay. Results: In MCF-7 breast cancer cells, S78454 decreased ERα expression, modulated gene expression (E2 target genes, HDACs and HDI-regulated genes) and inhibited cell proliferation. S78454 globally behaved as the reference HDIs yielding very similar amplitudes on the parameters that we have analyzed with EC50 being comprised between those of Vorinostat and Panobinostat. Interestingly, the IC50 of S78454 on MCF-7 cell proliferation was lower in cells treated with antiestrogens than in control cells or in cells treated with E2. Moreover, in antiestrogen-resistant MCF-7 cells that we developed in our laboratory, S78454 was able to significantly restore the sensitivity to the pure antiestrogen Fulvestrant. Conclusions and perspectives: Our findings suggest that in breast tumor cells, S78454 exhibits antiproliferative effects, can exert synergistic effects with antiestrogens and can restore antiestrogen-sensitivity in a model of antiestrogen-resistant MCF7 cell line. These results suggest that the combination of S78454 and antiestrogens could be valuable in the treatment of breast cancers resistant to hormonal therapies. This effect should be validated in vivo on tumor xenografts. In addition, it would be interesting to investigate whether S78454 also reinforces the anti-growth factor activity of antiestrogens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4704. doi:1538-7445.AM2012-4704

Conditional Ablation of the Progesterone Receptor Specifically in the Uterine Epithelium Demonstrates Its Necessary Role in the Regulation of Uterine Function.

Uterine progesterone receptor (PR), expressed in both the epithelium and stroma, mediates epithelial-stromal crosstalk during embryo implantation and pregnancy. Ex vivo tissue reconstitution studies have pointed to stromal PR as being critical for the regulation of uterine epithelial gene expression (i.e., Indian hedgehog (Ihh)) and inhibition of mitogenic estrogen (E2) action whereas the role of epithelial PR has been largely unexplored. Here, we investigated the vivo role of epithelial PR during pregnancy and in the regulation of epithelial gene expression using compartment-specific ablation of PR. Using the Wnt7a-Cre mouse model, PR was ablated specifically in the uterine epithelium. This ablation rendered female mice infertile, demonstrating that epithelial PR is critical for female fertility. We examined uterine function by assessing the ability of these mice to undergo the decidual response. The epithelial PR conditional knockout mice failed to display a decidual response indicating that, despite the presence of PR in the stroma, decidualization is unable to occur without epithelial PR. We next wanted to determine if epithelial PR may regulate the expression of progesterone (P4)-target genes. Epithelial PR is critical for P4-induction of epithelial P4-target genes (Ihh, Areg, Gata2, Cyp26a1), but not stromal P4-target genes (Il13ra2). Transfection analysis revealed that epithelial PR can transactivate Ihh gene expression in Hec1a cells further confirming its critical role in P4-target gene expression. Therefore, epithelial PR plays a critical role in epithelial P4-target gene expression which differs from previous observations using tissue reconstitution experiments. In the uterus, P4 inhibits E2 actions such as uterine weight gain, epithelial proliferation, and E2-target gene expression. Normal P4-inhibition of estrogen-induced weight gain was observed in these mice. However, examination of P4-inhibition of specific E2-target genes demonstrated that epithelial PR plays a gene-specific role in P4-inhibition exhibiting inhibition of Ltf and Clca3 expression, but not Lif expression. We are currently studying its potential role in P4-inhibition of E2-induced epithelial proliferation. In summary, using an in vivo mouse model, we have shown that epithelial PR is necessary for female fertility as a mediator of decidualization, P4-target gene expression, and the P4-inhibition of E2 action. This work was supported by NIH Grant RO1CA077530 (to J.P.L.) and R01HD042311 (to F.J.D.). (platform)

Synthesis and biological evaluation of phenolic 4,5-dihydroisoxazoles and 3-hydroxy ketones as estrogen receptor α and β agonists

In this work, 52 diphenyl-4,5-dihydroisoxazoles and -3-hydroxy ketones were prepared and their estrogen receptor α (ERα) and estrogen receptor β (ERβ) activities were explored in order to systematize and maximize their biological activity. The biological activity was firstly screened by using ERE reporter assay to find out how aromatic hydroxylation and methylation of the chiral centers of the compounds affect the ability of ER to mediate biological responses. For selected 19 compounds, the relative binding affinities (RBA, relative to 3,17β-estradiol) and ability to induce transcription of primary E2 target gene pS2 in human MCF-7 breast cancer cells were determined. In the reporter assay, many compounds showed even stronger activity than E2 and some of them showed RBA larger than 1%. The highest RBAs were determined for the enantiomers of 1-hydroxy-6-(4-hydroxy-phenyl)-1-phenyl-hexan-3-one ( 50a and 50b). Isomer 50a showed high binding affinity both to ERα (with RBA ∼200%) and ERβ (with RBA ∼60%), while the RBAs of 50b were ca. 40% of those. Some of the other compounds (with RBA ∼1–16%) showed also notable ERα binding selectivity. When four most promising ligands ( 50a, 50b, 45a, and 45b) were studied with respect to their ability to induce the transcription of primary E2 target gene pS2, the compounds acted as agonists or partial agonists. Computer modeling was used to predict receptor binding conformations and to rationalize the RBA differences of the compounds.

Estradiol-induced proliferation of papillary and follicular thyroid cancer cells is mediated by estrogen receptors α and β

Premenopausal women are at highest risk for papillary and follicular thyroid carcinoma, implicating a role for estrogens in thyroid cancer. The expression of estrogen receptors alpha and beta (ER), the effects of estradiol (E2), selective estrogen receptor modulators (SERMs) 4-hydroxytamoxifen and raloxifene, and ER subtype selective agonists were examined in NPA87 and KAT5 papillary and WRO follicular thyroid carcinoma cell lines. All three thyroid cancer cell lines expressed full-length ERalpha and ERbeta proteins with cytoplasmic localization that was unaffected by E2. ICI 182,780 (Fulvestrant, an ER antagonist), and inhibitors of non-genomic E2-activated MAPK and PI3K signaling blocked E2-induced cell proliferation. SERMs acted in a cell line-specific manner. No E2-induced estrogen response element (ERE)-driven reporter activity was observed in transiently transfected thyroid cancer cells. However, E2 increased transcription of established endogenous E2-target genes, i.e., cathepsin D in WRO and cyclin D1 in both KAT5 and WRO cells in an ER-dependent manner as validated by inhibitor and siRNA experiments. In contrast, E2 did not increase progesterone receptor expression in the thyroid cancer cell lines. E2 stimulated phosphorylation of ERK1/2 in KAT5 and WRO cells and siERalpha or siERbeta inhibited E2-induced ERK phosphorylation. Expression of the putative membrane estrogen receptor GPR30 was detected in WRO, but not NPA87 or KAT5 cells. GPR30 expression was lower in WRO than MCF-7 human breast cancer cells. Overall, these findings suggest E2-mediated thyroid cancer cell proliferation involves ERalpha and ERbeta transcriptional and non-genomic signaling events.