Putative Estrogen Response Element Research Articles

EZH2 Mediates Proliferation, Migration, and Invasion Promoted by Estradiol in Human Glioblastoma Cells.

Glioblastomas (GBM) are the most frequent and aggressive brain tumors. 17β-estradiol (E2) increases proliferation, migration, and invasion of human GBM cells; however underlying mechanisms are no fully understood. Zeste 2 Enhancer Homologous enzyme (EZH2) is a methyltransferase part of Polycomb 2 repressor complex (PRC2). In GBM, EZH2 is overexpressed and involved in the cell cycle, migration, and invasion processes. We studied the role of EZH2 in the pro-oncogenic actions of E2 in human GBM cells. EZH2 gene silencing and pharmacological inhibition of EZH2 blocked proliferation, migration, and invasion of GBM cells induced by E2. We identified in silico additional putative estrogen response elements (EREs) at the EZH2 promoter, but E2 did not modify EZH2 expression. In silico analysis also revealed that among human GBM samples, EZH2 expression was homogeneous; in contrast, the heterogeneous expression of estrogen receptors (ERs) allowed the classification of the samples into groups. Even in the GBM cluster with high expression of ERs and those of their target genes, the expression of PCR2 target genes did not change. Overall, our data suggest that in GBM cells, pro-oncogenic actions of E2 are mediated by EZH2, without changes in EZH2 expression and by mechanisms that appear to be unrelated to the transcriptional activity of ERs.

Gene expression in oligodendrocytes during remyelination reveals cholesterol homeostasis as a therapeutic target in multiple sclerosis

Regional differences in neurons, astrocytes, oligodendrocytes, and microglia exist in the brain during health, and regional differences in the transcriptome may occur for each cell type during neurodegeneration. Multiple sclerosis (MS) is multifocal, and regional differences in the astrocyte transcriptome occur in experimental autoimmune encephalomyelitis (EAE), an MS model. MS and EAE are characterized by inflammation, demyelination, and axonal damage, with minimal remyelination. Here, RNA-sequencing analysis of MS tissues from six brain regions suggested a focus on oligodendrocyte lineage cells (OLCs) in corpus callosum. Olig1-RiboTag mice were used to determine the translatome of OLCs in vivo in corpus callosum during the remyelination phase of a chronic cuprizone model with axonal damage. Cholesterol-synthesis gene pathways dominated as the top up-regulated pathways in OLCs during remyelination. In EAE, remyelination was induced with estrogen receptor-β (ERβ) ligand treatment, and up-regulation of cholesterol-synthesis gene expression was again observed in OLCs. ERβ-ligand treatment in the cuprizone model further increased cholesterol synthesis gene expression and enhanced remyelination. Conditional KOs of ERβ in OLCs demonstrated that increased cholesterol-synthesis gene expression in OLCs was mediated by direct effects in both models. To address this direct effect, ChIP assays showed binding of ERβ to the putative estrogen-response element of a key cholesterol-synthesis gene (Fdps). As fetal OLCs are exposed in utero to high levels of estrogens in maternal blood, we discuss how remyelinating properties of estrogen treatment in adults during injury may recapitulate normal developmental myelination through targeting cholesterol homeostasis in OLCs.

Expression of Thyroid Hormone Responsive SPOT 14 Gene Is Regulated by Estrogen in Chicken (Gallus gallus)

Thyroid hormone responsive spot 14 (THRSP) is a small nuclear protein that responds rapidly to thyroid hormone. It has been shown that THRSP is abundant in lipogenic tissues such as liver, fat and the mammary gland in mammals. The THRSP gene acts as a key lipogenic activator and can be activated by thyroid hormone triiodothyronine (T3), glucose, carbohydrate and insulin. Here we report that chicken THRSP is also abundant in lipogenic tissues including the liver and the abdominal fat, and its expression levels increased with sex maturation and reached the highest level at the peak of egg production. Structure analysis of the THRSP gene indicates that there is a conscious estrogen response element (ERE) located in the −2390 – −2402 range of the gene promoter region. Further studies by ChIP-qPCR proved that the ERα interacts with the putative ERE site. In addition, THRSP was significantly upregulated (P < 0.05) when chickens or chicken primary hepatocytes were treated with 17β-estradiol in both the in vivo and in vitro conditions. We therefore conclude that THRSP is directly regulated by estrogen and is involved in the estrogen regulation network in chicken.

Abstract 3602: Novel BRCA1 mimetic 35446HCl inhibits growth of anti estrogen resistant breast cancer cells through the NF-κB pathway

Abstract Nearly a third of breast cancer patients develop resistance to antiestrogen treatments although the estrogen receptor ER-α continues to affect cell survival and proliferation. Thus there is a need for new interventions to inhibit ER-α function. Based on high-resolution mapping, we proposed interaction sites on both BRCA1 and ER-α in a three-dimensional model of BRCA1. Utilizing this model, we identified a class of compounds that fits into a predicted BRCA1-binding cavity within ER-α, mimics the ability of BRCA1 to repress ER-α activity, and functions differently than other antiestrogens. Previous publication has shown that the 1st generation BRCA1 mimetic compound (A7) reduced proliferation and inhibited ER-α activity in antiestrogen-sensitive and resistant breast cancer cells with negligible non-specific toxicity. More importantly we have shown that the 2nd generation compounds (35446, 4631) partially restored sensitivity to Tam/ICI-resistant cells (LCC9), caused dissociation of ER-α from a model estrogen response element (ERE) in MCF-7 cells, and inhibited growth of LCC9 tumor xenografts at nontoxic concentrations utilizing compound 35446-HCl (35446 prepared as a hydrochloride salt). The focus of this study is the mechanism of action of 35446-HCl compound’s inhibition of ER-α, which includes inhibition of a major signaling pathway and mode of cell death. Eight genes identified from RNA-seq in LCC9 cells treated with BRCA1 mimetic A7 were verified by qPCR using 35446-HCl. One verified gene is the NF-κB activator IKKB, which is linked to therapy resistance. Treatment with 35446-HCl inhibited IKKB expression and proliferation in LCC9 cells compared to untreated Control at 48 and 72 hrs (p&amp;lt;0.0001). Transcriptional activity from the p65 promoter as measured with a luciferase assay was decreased in the presence of BRCA1 mimetic treatment (p&amp;lt;0.0001). Expression of downstream targets of NF-κB, including genes known to inhibit apoptosis, was reduced (p value ranging from &amp;lt; 0.05- 0.001). Apoptosis due to 35446-HCl treatment was verified by caspase-7 cleavage and increased TUNEL staining at both 48 and 72 hrs. In silico analysis of the IKBKB promoter within 10kb upstream of the transcription start site identified one putative full ERE and five putative half ERE sites. Of these, 35446-HCl inhibited the interaction of ER-α with the full ERE (p&amp;lt;0.0001) and five half ERE (p value ranging from &amp;lt; 0.05- 0.001) sites upstream of the IKKB start site in LCC9 cells as well as Tam resistant/ER-α mutated T47Dco cells. These results identify a mechanism of a novel compound that can overcome antiestrogen resistance. Citation Format: Shyam Nathan, Eliot M. Rosen. Novel BRCA1 mimetic 35446HCl inhibits growth of anti estrogen resistant breast cancer cells through the NF-κB pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3602. doi:10.1158/1538-7445.AM2017-3602

The constitutively active estrogen receptor (ER) binds and activates the promoter of the vitellogenin (Vtg) gene in the Sydney rock oyster, Saccostrea glomerata

Vitellogenin (Vtg) is a well-established biomarker of estrogenic exposure in aquatic animals. In vertebrates, Vtg gene transcription is controlled by the estrogen receptors (ERs). Although an ER ortholog is present in molluscs, its role as a transcriptional regulator remains elusive. Here, we tested the hypothesis that in the Sydney rock oyster, Saccostrea glomerata, the ER ortholog activates Vtg gene transcription through specific interaction with its promoter. Luciferase reporter assays indicated that sgER activated both a minimal promoter containing the consensus estrogen-responsive elements (EREs) and the sgVtg promoter in an estrogen-independent manner. The sgVtg promoter-luciferase activation was significantly reduced when any of three putative ERE half sites (½EREs) in the promoter were mutated. Electrophoretic mobility shift assay (EMSA) confirmed that sgER binds specifically to a 68-bp promoter sequence where these ½EREs reside. Overall, the results suggest that sgER is a constitutively active transcription factor that binds and activates the sgVtg promoter.

E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma

E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

Stress-related disorders, pituitary adenylate cyclase—activating peptide (PACAP)ergic system, and sex differences

Trauma-related disorders, such as posttraumatic stress disorder (PTSD) are remarkably common and debilitating, and are often characterized by dysregulated threat responses. Across numerous epidemiological studies, females have been found to have an approximately twofold increased risk for PTSD and other stress-related disorders. Understanding the biological mechanisms of this differential risk is of critical importance. Recent data suggest that the pituitary adenylate cyclase-activating polypeptide (PACAP) pathway is a critical regulator of the stress response across species. Moreover, increasing evidence suggests that this pathway is regulated by both stress and estrogen modulation and may provide an important window into understanding mechanisms of sex differences in the stress response. We have recently shown that PACAP and its receptor (PAC1R) are critical mediators of abnormal processes after psychological trauma. Notably, in heavily traumatized human subjects, there appears to be a robust sex-specific association of PACAP blood levels and PAC1R gene variants with fear physiology, PTSD diagnosis, and symptoms, specifically in females. The sex-specific association occurs within a single-nucleotide polymorphism (rs2267735) that resides in a putative estrogen response element involved in PAC1R gene regulation. Complementing these human data, the PAC1R messenger RNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP-PAC1R pathway are regulated by estrogen and are involved in abnormal fear responses underlying PTSD.

Potential mechanisms underlying estrogen-induced expression of the molluscan estrogen receptor (ER) gene

In vertebrates, estrogens and estrogen mimicking chemicals modulate gene expression mainly through a genomic pathway mediated by the estrogen receptors (ERs). Although the existence of an ER orthologue in the mollusc genome has been known for some time, its role in estrogen signalling has yet to be deciphered. This is largely due to its constitutive (ligand-independent) activation and a limited mechanistic understanding of its regulation. To fill this knowledge gap, we cloned and characterised an ER cDNA (sgER) and the 5⿲-flanking region of the gene from the Sydney rock oyster Saccostrea glomerata. The sgER cDNA is predicted to encode a 477-amino acid protein that contains a DNA-binding domain (DBD) and a ligand-binding domain (LBD) typically conserved among both vertebrate and invertebrate ERs. A comparison of the sgER LBD sequence with those of other ligand-dependent ERs revealed that the sgER LBD is variable at several conserved residues known to be critical for ligand binding and receptor activation. Ligand binding assays using fluorescent-labelled E2 and purified sgER protein confirmed that sgER is devoid of estrogen binding. In silico analysis of the sgER 5⿲-flanking sequence indicated the presence of three putative estrogen responsive element (ERE) half-sites and several putative sites for ER-interacting transcription factors, suggesting that the sgER promoter may be autoregulated by its own gene product. sgER mRNA is ubiquitously expressed in adult oyster tissues, with the highest expression found in the ovary. Ovarian expression of sgER mRNA was significantly upregulated following in vitro and in vivo exposure to 17β-estradiol (E2). Notably, the activation of sgER expression by E2 in vitro was abolished by the specific ER antagonist ICI 182, 780. To determine whether sgER expression is epigenetically regulated, the in vivo DNA methylation status of the putative proximal promoter in ovarian tissues was assessed using bisulfite genomic sequencing. The results showed that the promoter is predominantly hypomethylated (with 0⿿3.3% methylcytosines) regardless of sgER mRNA levels. Overall, our investigations suggest that the estrogen responsiveness of sgER is regulated by a novel ligand-dependent receptor, presumably via a non-genomic pathway(s) of estrogen signalling.

Abstract 89: 17beta-Estradiol-activated Estrogen Receptors Attenuate Cardiac Fibrosis Through Inhibition of Collagen I and III Expression in Female Hearts

Objectives: Female pressure-overloaded hearts show less fibrosis compared with males. 17β-Estradiol (E2) attenuates cardiac fibrosis in female mice. Whether this is mediated by direct E2-effects on collagen synthesis is still unknown. Therefore, we investigated the role of E2 and estrogen receptors (ER) on collagen I and III expression and analyzed involved mechanisms. Methods: Female C57BL/6J mice (7 weeks) underwent sham operation, ovariectomy (OVX), OVX with E2-supplementation (390mg E2-containing pellets) or placebo. After 2 weeks, animals underwent transverse aortic constriction (TAC) or sham surgery. Mice were sacrificed after 9 weeks. Collagen amount, collagen I and III protein in left ventricular tissue were detected by Sirius Red and antibody staining, respectively. Gene and protein expression were determined by quantitative Real-Time PCR and Western blot. Adult female rat cardiac fibroblasts were treated with E2 (10 -8 M), vehicle, ERα- and β-agonists (10 -7 M) for 24h or pre-treated with PD98059 for 1h. ER binding to the collagen I and III promoter was analyzed by chromatin immunoprecipitation assays. Findings: In female OVX mice, undergoing TAC surgery, E2-supplementation significantly reduced collagen deposition, collagen I and III mRNA and protein levels in comparison with mice without E2. In female rat cardiac fibroblasts, E2 significantly down-regulated collagen I and III mRNA and protein level. Specific ER-agonist-treatment showed that E2-mediated regulation of collagen I and III expression was mediated via activation of ERα, but not ERβ. Further, upon E2-treatment, ERα was phosphorylated at Ser118, which occurred by E2-induced activation of ERK1/2 signaling. Furthermore, we could show that ERα and ERβ bind to two putative half-palindromic estrogen response elements within the collagen I and III promoter in female cardiac fibroblasts. Conclusion: E2 inhibits cardiac collagen I and III mRNA and protein in female mice under pressure overload. Data from rat female cardiac fibroblasts suggest that this is mediated via E2-activated ERK1/2 signaling and ERα, which binds with ERβ to the collagen I and III promoter. Understanding of how E2/ER attenuate collagen I and III expression in pathological hypertrophy may improve therapy.

Transcriptional regulation of 5′-flanking regions of stanniocalcin genes by estrogen receptor and estrogen receptor-related receptor

Stanniocalcin (STC), a glycoprotein hormone originally identified in bony fish, is a regulator of calcium and phosphate homeostasis. Two related STC genes, STC1 and STC2, are expressed in various tissues in vertebrates, but their transcriptional regulation and differences between the two genes are poorly understood. The aim of this study was to determine and compare estrogen-dependent transcriptional activities of the 5′-flanking regions of the mouse STC1 (mStc1) and STC2 (mStc2) genes. Using HEK293 cells and a transient transfection method, the transcriptional activity of the mStc1 and mStc2 genes was measured by a luciferase reporter assay. In this study, we have identified the existence of putative estrogen response elements (EREs) and estrogen receptor (ER)-related receptor (ERR) response elements (ERREs) in the 5′-flanking regions of the mStc1 gene, but the putative ERRE sites only within the mStc2. Luciferase reporter assays showed that ligand bound ERα and ERRγ increase the transcription of mStc1 but not mStc2. In addition, the ERα- and ERRγ-mediated transactivation of the 5′-flanking region of mStc1 was further enhanced by the coregulators, steroid receptor coactivator 1 and peroxisome proliferator-activated receptor γ coactivator 1 α. Taken together, our results suggest that the putative promoter region of mStc1, but not mStc2, is transcriptionally enhanced by ligand-activated ERα and ERRγ.

Estradiol stimulates apolipoprotein A-IV gene expression in the nucleus of the solitary tract through estrogen receptor-α.

Although estrogens have been implicated in the regulation of apolipoprotein A-IV (apo A-IV) gene expression in the nucleus tractus solitarius, previous studies have not defined the molecular mechanism. The aim of this study was to examine the transcriptional mechanisms involved in regulation of apo A-IV gene expression. Using cultured primary neuronal cells from rat embryonic brainstems, we found that treatment with 10nM 17β-estradiol-3-benzoate (E2) or 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (an estrogen receptor [ER]α agonist), but not 2,3-bis(4-hydroxyphenyl)-propionitrile (an ERβ agonist), significantly increased apo A-IV gene expression, compared with vehicle treatment. This effect of E2 was abolished when the cells were incubated with E2 linked to BSA, which prevents E2 from entering cells, implying that a nongenomic mechanism of E2 is not involved. Two putative estrogen response elements were identified at the 5'-upstream region of the apo A-IV gene promoter, but only 1 of them was able to recruit ERα, leading to increased apo A-IV gene expression, as determined by chromatin immunoprecipitation assay and luciferase activity analysis. A cyclic regimen of E2 or 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol treatment for 8 cycles (4 d/cycle, mimicking the ovarian cycle of female rats) in ovariectomized female rats significantly reduced food intake and body weight gain and increased apo A-IV gene expression in the nucleus tractus solitarius, relative to vehicle. These data collectively demonstrate that nuclear ERα is the primary mediator of E2's action on apo A-IV gene expression and suggest that increased signaling of endogenous apo A-IV may at least partially mediate E2-induced inhibitory effect on feeding.

THU0481 Aromatase Inhibitor Treatment and Musculoskeletal Adverse Events: SNP Modulated, Estrogen-Dependent Variation in Ccr6/Ccl20 Expression

BackgroundJoint pain and stiffness are major side effects associated with aromatase inhibitor (AI) therapy. In a recent genome-wide association study, we identified 4 SNPs near the T-cell leukemia 1A (TCL1A)...

Mechanisms of transcriptional activation of the mouse claudin-5 promoter by estrogen receptor alpha and beta

Claudin-5 is an integral membrane protein and a critical component of endothelial tight junctions that control paracellular permeability. Claudin-5 is expressed at high levels in the brain vascular endothelium. Estrogens have multiple effects on vascular physiology and function. The biological actions of estrogens are mediated by two different estrogen receptor (ER) subtypes, ER alpha and ER beta. Estrogens have beneficial effects in several vascular disorders. Recently we have cloned and characterized a murine claudin-5 promoter and demonstrated 17beta-estradiol (E2)-mediated regulation of claudin-5 in brain and heart microvascular endothelium on promoter, mRNA and protein level. Sequence analysis revealed a putative estrogen response element (ERE) and a putative Sp1 transcription factor binding site in the claudin-5 promoter. The aim of the present study was to further characterize the estrogen-responsive elements of claudin-5 promoter. First, we introduced point mutations in ERE or Sp1 site in −500/+111 or in Sp1 site of −268/+111 claudin-5 promoter construct, respectively. Basal and E2-mediated transcriptional activation of mutated constructs was abrogated in the luciferase reporter gene assay. Next, we examined whether estrogen receptor subtypes bind to the claudin-5 promoter region. For this purpose we performed chromatin immunoprecipitation assays using anti-estrogen receptor antibodies and cellular lysates of E2-treated endothelial cells followed by quantitative PCR analysis. We show enrichment of claudin-5 promoter fragments containing the ERE- and Sp1-binding site in immunoprecipitates after E2 treatment. Finally, in a gel mobility shift assay, we demonstrated DNA–protein interaction of both ER subtypes at ERE. In summary, this study provides evidence that both a non-consensus ERE and a Sp1 site in the claudin-5 promoter are functional and necessary for the basal and E2-mediated activation of the promoter.

Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway.

Estrogen deficiency is the main reason of bone loss, leading to postmenopausal osteoporosis, and estrogen replacement therapy (ERT) has been demonstrated to protect bone loss efficiently. Notch signaling controls proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Moreover, imperfect estrogen-responsive elements (EREs) were found in the 5′-untranslated region of Notch1 and Jagged1. Thus, we examined the molecular and biological links between estrogen and the Notch signaling in postmenopausal osteoporosis in vitro. hBMSCs were obtained from healthy women and patients with postmenopausal osteoporosis. Notch signaling molecules were quantified using real-time polymerase chain reaction (real-time PCR) and Western Blot. Luciferase reporter constructs with putative EREs were transfected into hBMSCs and analyzed. hBMSCs were transduced with lentiviral vectors containing human Notch1 intracellular domain (NICD1). We also used N-[N-(3, 5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester, a γ-secretase inhibitor, to suppress the Notch signaling. We found that estrogen enhanced the Notch signaling in hBMSCs by promoting the expression of Jagged1. hBMSCs cultured with estrogen resulted in the up-regulation of Notch signaling and increased proliferation and differentiation. Enhanced Notch signaling could enhance the proliferation and differentiation of hBMSCs from patients with postmenopausal osteoporosis (OP-hBMSCs). Our results demonstrated that estrogen preserved bone mass partly by activating the Notch signaling. Because long-term ERT has been associated with several side effects, the Notch signaling could be a potential target for treating postmenopausal osteoporosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11010-014-2021-7) contains supplementary material, which is available to authorized users.

A distal estrogen responsive element upstream the cap site of human transthyretin gene is an enhancer-like element upon ERα and/or ERβ transactivation

Previous studies reported that 17 beta-estradiol (E2) is responsible for the up-regulation of transthyretin (TTR) expression via an estrogen receptor (ER)-dependent pathway in rat choroid plexus (CP) and liver. A computer-assisted homology search identified a putative estrogen-responsive element (ERE) in the 5′ flanking region of the human TTR (hTTR) gene (ERETTR), with the sequence aAGTCAAAGTGACCa, between −3406 and −3392bp. Luciferase reporter assays and electrophoretic mobility shift (EMSA) and supershift analysis were carried out to investigate if E2 regulates TTR transcription via this putative ERE. Luciferase reporter assays in COS-7 cells were carried out with a plasmid construction where the TTR fragment containing the putative ERETTR was cloned in pGL2-promoter vector (pGL2-P) (pGL2-P/TTR), co-transfected with estrogen receptor α (ERα) and/or estrogen receptor β (ERβ) expression vectors. These assays demonstrated that, upon incubation with E2, one or both ERs (α and/or β) transactivate the reporter gene. The pGL2-P/TTR showed a significant transactivation of up to 6.8-fold, by E2, when co-transfected with ERβ, and up to 4-fold with ERα. Specific binding of ER (α and/or β) to ERETTR was demonstrated by EMSA and supershift assays confirmed the binding to ERα and/or ERβ. Our findings further suggest a mechanism underlying the regulation of TTR expression through the identification of a novel ERE in the TTR gene, which functions as an E2-dependent enhancer-like element.

Estradiol Represses the GD3 Synthase Gene ST8SIA1 Expression in Human Breast Cancer Cells by Preventing NFκB Binding to ST8SIA1 Promoter

Recent data have underlined a possible role of GD3 synthase (GD3S) and complex gangliosides in Estrogen Receptor (ER) negative breast cancer progression. Here, we describe the main transcript of the GD3S coding gene ST8SIA1 expressed in breast tumors. We characterized the corresponding core promoter in Hs578T breast cancer cells and showed that estradiol decreases ST8SIA1 mRNA expression in ER-positive MCF-7 cells and ERα-transfected ER-negative Hs578T cells. The activity of the core promoter sequence of ST8SIA1 is also repressed by estradiol. The core promoter of ST8SIA1 contains two putative Estrogen Response Elements (ERE) that were not found to be involved in the promoter activity pathway. However, NFκB was shown to be involved in ST8SIA1 transcriptional activation and we demonstrated that estradiol prevents NFκB to bind to ST8SIA1 core promoter in ERα expressing breast cancer cells by inhibiting p65 and p50 nucleus localization. The activation of NFκB pathway in ER-negative tumors, due to the absence of estradiol signaling, might explain the overexpression of GD3 synthase in this tumor subtype.

Abstract 4765: The role of estrogen receptor signalling in serous ovarian cancer.

Abstract Epithelial ovarian cancer is a heterogeneous group of diseases with multiple histotypes which can be broadly divided into a dualistic model based on morphological, molecular genetic, and clinical features. The most common and aggressive of all EOC, accounting for 90% of deaths, is High Grade Serous Carcinoma (HGSC). It is characterized by a genetically unstable, rapidly growing phenotype, is diagnosed at advanced stage with poorly defined cancer precursors compared to the more indolent Low Grade Serous Carcinoma (LGSC). Reproductive hormone receptor status (estrogen/ progesterone receptor) may be an important indicator of response to anti-hormonal therapy in ovarian cancer. There have been few reports detailing the expression of ER in HGSC, LGSC and the normal fallopian tube epithelium, the likely cell of origin of both histotypes. We hypothesize that some ER mediated signaling is maintained despite the loss of progesterone receptor in HGSC compared to the LGSC. Methodology: Snap-frozen tissues (43 HGSC and 18 LGSC) were selected from the UHN Biobank. We used our previously published gene expression profiles to generate a candidate gene list, which was chosen based on the presence of known estrogen responsive elements. They were validated by qPCR and immunohistochemistry on HGSC and LGSC tissue microarrays. Human fallopian tube epithelial cell lines were treated with estradiol at 50nm to determine ‘normal’ ER response. An ER positive cancer cell line (SKOV3) was used to determine the cellular response to candidate genes. IHC was scored using automated image analysis. Statistical analysis was performed using ANOVA and Fisher's Exact Test. Results: 35/43 (81%) of HGSC were ER+/PR- whilst 9/27 of LGSC were ER+/PR- and 18/27 ER+/PR+. Gene expression analysis of ER+ HGSC and ER- HGSC revealed 881 genes with more than a 2-FC in gene expression including 202 genes with known or putative estrogen responsive elements. They are involved in immune response, locomotion, metanephrous development and cellular adhesion. We compared genes that were differentially expressed between normal fallopian tube cells obtained during pre- or post-ovulation (93 genes). 42 ‘FTE-normal’ genes were differentially expressed between ER- versus ER+ HGSC. We selected 5 genes to validate by qPCR and IHC, based on gene ontology and known associations to cancer pathways. Conclusions: These results show that HGSC is predominantly an ER+/ PR- cancer (80%) while LGSC is predominantly an ER positive/ PR positive cancer (66%). Our data also indicate that some ER signalling is maintained in the absence of the progesterone receptor and ER+ HGSC are transcriptionally different from ER- HGSC although, there is no distinct clinical benefit between these two groups. ER is still able to transcriptionally activate a subset of known ER target genes and this information should provide further insight into the use of hormone receptors as indicators of anti-hormone therapy in ovarian cancer. Citation Format: Sophia Hl George, Anca Milea, Ramlogan Sowamber, Danielle Toccalino, Patricia A. Shaw. The role of estrogen receptor signalling in serous ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2013-4765

Novel estrogen target gene ZAS3 is overexpressed in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a prototypic, inflammatory autoimmune disease characterized by significant gender bias. Previous studies have established a role for hormones in SLE pathogenesis, including the sex hormone estrogen. Estrogen regulates gene expression by translocating estrogen receptors (ER) α and β into the nucleus where they induce transcription by binding to estrogen response elements (EREs) of target genes. The ZAS3 locus encodes a signaling and transcriptional molecule involved in regulating inflammatory responses. We show that ZAS3 is significantly up-regulated in SLE patients at both the protein and mRNA levels in peripheral blood mononuclear cells (PBMCs). Furthermore, estrogen stimulates the expression of ZAS3 in vitro in several leukocyte and breast cancer cell lines of both human and murine origin. In vivo estrogen treatment mediates induction of tissue specific ZAS3 expression in several lymphoid organs in mice. Estrogen stimulation also significantly up-regulates ZAS3 expression in primary PBMCs, while treatment with testosterone has no effect. Mechanistically, estrogen induces differential ERα binding to putative EREs within the ZAS3 gene and ERα knockdown with siRNA prevents estrogen induced ZAS3 up-regulation. In contrast, siRNA targeting IFNα has no effect. These data demonstrate that ZAS3 expression is directly regulated by estrogen and that ZAS3 is overexpressed in lupus. Since ZAS3 has been shown to regulate inflammatory pathways, its up-regulation by estrogen could play a critical role in female-biased autoimmune disorders.

Identification of estrogen responsive genes using esophageal squamous cell carcinoma (ESCC) as a model

BackgroundEstrogen therapy has positively impact the treatment of several cancers, such as prostate, lung and breast cancers. Moreover, several groups have reported the importance of estrogen induced gene regulation in esophageal cancer (EC). This suggests that there could be a potential for estrogen therapy for EC. The efficient design of estrogen therapies requires as complete as possible list of genes responsive to estrogen. Our study develops a systems biology methodology using esophageal squamous cell carcinoma (ESCC) as a model to identify estrogen responsive genes. These genes, on the other hand, could be affected by estrogen therapy in ESCC.ResultsBased on different sources of information we identified 418 genes implicated in ESCC. Putative estrogen responsive elements (EREs) mapped to the promoter region of the ESCC genes were used to initially identify candidate estrogen responsive genes. EREs mapped to the promoter sequence of 30.62% (128/418) of ESCC genes of which 43.75% (56/128) are known to be estrogen responsive, while 56.25% (72/128) are new candidate estrogen responsive genes. EREs did not map to 290 ESCC genes. Of these 290 genes, 50.34% (146/290) are known to be estrogen responsive. By analyzing transcription factor binding sites (TFBSs) in the promoters of the 202 (56+146) known estrogen responsive ESCC genes under study, we found that their regulatory potential may be characterized by 44 significantly over-represented co-localized TFBSs (cTFBSs). We were able to map these cTFBSs to promoters of 32 of the 72 new candidate estrogen responsive ESCC genes, thereby increasing confidence that these 32 ESCC genes are responsive to estrogen since their promoters contain both: a/mapped EREs, and b/at least four cTFBSs characteristic of ESCC genes that are responsive to estrogen. Recent publications confirm that 47% (15/32) of these 32 predicted genes are indeed responsive to estrogen.ConclusionTo the best of our knowledge our study is the first to use a cancer disease model as the framework to identify hormone responsive genes. Although we used ESCC as the disease model and estrogen as the hormone, the methodology can be extended analogously to other diseases as the model and other hormones. We believe that our results provide useful information for those interested in genes responsive to hormones and in the design of hormone-based therapies.

Molecular Basis for Involvement of CYP1B1 in MYOC Upregulation and Its Potential Implication in Glaucoma Pathogenesis

CYP1B1 has been implicated in primary congenital glaucoma with autosomal recessive mode of inheritance. Mutations in CYP1B1 have also been reported in primary open angle glaucoma (POAG) cases and suggested to act as a modifier of the disease along with Myocilin (MYOC). Earlier reports suggest that over-expression of myocilin leads to POAG pathogenesis. Taken together, we propose a functional interaction between CYP1B1 and myocilin where 17β estradiol acts as a mediator. Therefore, we hypothesize that 17β estradiol can induce MYOC expression through the putative estrogen responsive elements (EREs) located in its promoter and CYP1B1 could manipulate MYOC expression by metabolizing 17β estradiol to 4-hydroxy estradiol, thus preventing it from binding to MYOC promoter. Hence any mutation in CYP1B1 that reduces its 17β estradiol metabolizing activity might lead to MYOC upregulation, which in turn might play a role in glaucoma pathogenesis. It was observed that 17β estradiol is present in Human Trabecular Meshwork cells (HTM) and Retinal Pigment Epithelial cells (RPE) by immunoflouresence and ELISA. Also, the expression of enzymes related to estrogen biosynthesis pathway was observed in both cell lines by RT-PCR. Subsequent evaluation of the EREs in the MYOC promoter by luciferase assay, with dose and time dependent treatment of 17β estradiol, showed that the EREs are indeed active. This observation was further validated by direct binding of estrogen receptors (ER) on EREs in MYOC promoter and subsequent upregulation in MYOC level in HTM cells on 17β estradiol treatment. Interestingly, CYP1B1 mutants with less than 10% enzymatic activity were found to increase the level of endogenous myocilin in HTM cells. Thus the experimental observations are consistent with our proposed hypothesis that mutant CYP1B1, lacking the 17β estradiol metabolizing activity, can cause MYOC upregulation, which might have a potential implication in glaucoma pathogenesis.