ESR1 Activity Research Articles

Identification of novel microRNA regulatory pathways associated with heterogeneous prostate cancer

BackgroundMicroRNAs (miRNAs) are potential regulators that contribute to the pathogenesis of cancer. Microarray technologies have been widely used to characterize aberrant miRNA expression patterns in cancer. Nevertheless, the miRNAs expression signatures identified for a same cancer differs among laboratories due to the cancer heterogeneity. In addition, how the deregulated miRNAs coordinately contribute to the tumourigenic process of prostate cancer remains elusive.ResultsWe evaluated five outlier detection algorithms that take into account the heterogeneity of cancer samples. ORT was selected as the best method and applied to four prostate cancer associated microRNA expression datasets. After microRNA target prediction and pathway enrichment mapping, 38 Gene Ontology terms, 16 KEGG pathways and 99 GeneGO pathways are found putative prostate cancer associated. Comparison with our previous studies, we identified two putative novel pathways important in prostate cancer. The two novel pathways are 1) ligand-independent activation of ESR1 and ESR2 and 2) membrane-bound ESR1: interaction with growth factors signalling.ConclusionsWe proved that expression signatures of at the pathway level well address the cancer heterogeneity and are more consistent than at the miRNA/gene levels. Based on this observation, we identified putative novel microRNA regulatory pathways which will help us to elucidate the cooperative function of different microRNAs in prostate cancer.

Differential role of the estrogen receptors ESR1 and ESR2 on the regulation of proteins involved with proliferation and differentiation of Sertoli cells from 15-day-old rats

The aim of the present study was to investigate the role of each estrogen receptors on the regulation of proteins involved with proliferation and differentiation of Sertoli cells from 15-day-old rats. Activation of ESR1 by 17β-estradiol (E2) and ESR1-selective agonist PPT increased CCND1 expression, and this effect was dependent on NF-kB activation. E2 and the ESR2-selective agonist DPN, but not PPT, increased, in a PI3K and CREB-dependent manner, the expression of CDKN1B and the transcription factors GATA-1 and DMRT1. Analyzing the expression of ESR1 and ESR2 in different stages of development of Sertoli cells, we observed that the ESR1/ESR2 ratio decreased with age, and this ratio seems to be important to determine the end of cell proliferation and the start of cell differentiation. In Sertoli cells from 15-day-old rats, the ESR1/ESR2 ratio favors the effect of ESR1 and the activation of this receptor increased [Methyl-3H]thymidine incorporation. We propose that in Sertoli cells from 15-day-old rats E2 modulates Sertoli cell proliferation through ESR1/NF-kB-mediated increase of CCND1, and cell cycle exit and differentiation through ESR2/CREB-mediated increase of CDKN1B, GATA-1 and DMRT1. The present study reinforces the important role of estrogen for normal testis development.

Estrogen Receptor 1 Agonist PPT Stimulates Slc2a4 Gene Expression and Improves Insulin-Induced Glucose Uptake in Adipocytes

Type 2 diabetes mellitus is characterized by disruption in glycemic homeostasis, involving impaired insulin-induced glucose disposal. For that, reduced glucose transporter GLUT4, encoded by Slc2a4 gene, plays a fundamental role. Conversely, increase in Slc2a4/GLUT4 expression improves glycemic homeostasis. Recent studies have proposed that estradiol is able to modulate Slc2a4 expression, according to distinct effects upon estrogen receptors ESR1/ESR2. We hypothesize that ESR1-agonist effect could stimulate Slc2a4 expression; thus, increasing cellular glucose disposal, which could be beneficial to glycemic control. Differentiated 3T3-L1 adipocytes were treated (24 hours) with selective ESR1- agonist PPT 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, selective ESR1-antagonist MPP 1,3-Bis(4- hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride, and selective ESR2 agonist DPN 2,3-bis(4-Hydroxyphenyl)-propionitrile, with/without 17β-estradiol (E2). We analyzed Slc2a4 mRNA (real time PCR) and GLUT4 protein (Western blotting) expression, transcriptional activity of the Slc2a4 repressor Nuclear Factor- κB (NF-κB) (electrophoretic mobility shift assay), and cellular glucose disposal (2-deoxi-D-[(3)H]glucose uptake, 2-DG). ESR1-agonist PPT enhanced Slc2a4/GLUT4 expression (~30%) in the absence or presence of 0.1 and 10 nmol/L E2, and decreased the NF-κB binding activity (~50%). Conversely, ESR1-antagonist MPP, together with E2, decreased Slc2a4/GLUT4 expression (20-40%) and increased NF-κB binding activity (~30%). Furthermore, treatment with ESR2- agonist DPN decreased Slc2a4/GLUT4 expression (20-50%). 2-DG uptake was modulated in parallel to that observed in GLUT4 protein. The present results reveal that ESR1 activity enhances, whereas ESR2 activity represses, Slc2a4/GLUT4 expression. These effects are partially mediated by NF-κB, and allow parallel changes in adipocyte glucose disposal. Furthermore, the data provide evidences that ESR1-agonist PPT, as a Slc2a4/GLUT4 enhancer, can be a promising coadjuvant drug for diabetes mellitus therapy.

Estrogen receptor (ESR) 2 partially offsets the absence of ESR1 in gonadotropes of pituitary-specific Esr1 knockout female mice

Estrogen receptor 1 and 2 (ESR1 and 2) mediate estrogen (E) action on gonadotrope function. While much is known about the effects of ESR1 on the gonadotrope, there is still some controversy regarding the effects of ESR2. To investigate the role of ESR2 in the gonadotrope, 45-day-old female mice of two different genotypes were used: wild type (WT) and pituitary (gonadotropes and thyrotropes)-specific Esr1 knockout (KO). All mice were ovariectomized (OVX) and 15 days later injected over 3 days with 2.5 μg 17β-estradiol (E(2)), 0.2 mg of the selective ESR1 or 2 agonists, propylpyrazole triol and diarylpropionitrile, respectively, or 0.1 ml oil. The day after treatment, anterior pituitary glands were dissected out for evaluation of gonadotrope ultrastructural morphology and pituitary immunohistochemical expression of progesterone receptor (Pgr (Pr)). Blood was collected and serum LH levels were assessed. Activation of ESR1 in WT mice resulted in the following: i) uterine ballooning and vaginal cornification, ii) negative feedback on LH secretion, iii) increased number of homogeneous (functional) gonadotropes, and iv) pituitary Pgr expression (35.9±2.0% of pituitary cells). Activation of ESR1 in KO mice induced normal uterine, vaginal, and LH secretion responses, but failed to increase the number of functional gonadotropes, and induced significantly lower Pgr expression (21.0±3.0% of pituitary cells) than in WT mice. Whilst activation of ESR2 had no significant effects in WT mice, it doubled the number of functional gonadotropes exhibited by KO mice injected with oil. It is concluded that E(2) exerted its action in KO mouse gonadotropes via ESR2.

In Vivo Treatments with Fulvestrant and Anastrozole Differentially Affect Gene Expression in the Rat Efferent Ductules1

Estrogen plays a key role in maintaining the morphology and function of the efferent ductules. We previously demonstrated that the antiestrogen fulvestrant markedly affected gene expression in the rat efferent ductules. The mechanism of fulvestrant action to modulate gene expression may involve not only the blockade of ESR1 and ESR2 estrogen receptors, but also the activation of ESR1 and ESR2 when the receptors are tethered to AP-1 or SP1 transcription factors, or the activation of the G protein-coupled estrogen receptor 1. We therefore compared the effects of two strategies to interfere with estrogen action in the rat efferent ductules: treatment with fulvestrant or with the aromatase inhibitor anastrozole. Whereas fulvestrant markedly increased Mmp7 and Spp1, and reduced Nptx1 mRNA levels, no changes were observed with anastrozole. Fulvestrant caused changes in epithelial morphology that were not seen with anastrozole. Fulvestrant shifted MMP7 immunolocalization in the epithelial cells from the supranuclear to the apical region; this effect was less pronounced with anastrozole. In vitro studies of (35)S-methionine incorporation showed that protein release was increased, whereas tissue protein content in the efferent ductules of fulvestrant-treated rats was decreased. Although fulvestrant markedly affected gene expression, no changes were observed on AP-1 and SP1 DNA-binding activity. The blockade of ESRs seems to be the major reason explaining the differences between both treatments. At least some of the effects of fulvestrant appear to result from compensatory mechanisms activated by the dramatic changes caused by ESR1 blockade.

Abstract 4220: Identification of novel developmental and differentiation markers in the breast

Abstract Background: The majority of cell growth pathways implicated in breast cancer have dual physiologic roles in normal mammary gland development. Similarly, founder stem cells in breast cancer may share important phenotypic characteristics with progenitor cells in early mammary gland development. The goal of this project was to investigate transcriptional profiles in the early pubertal primate mammary gland in order to identify novel markers and pathways involved in development, differentiation, and potentially breast cancer risk. Materials and Methods: Mammary gland biopsies from 31 female cynomolgus macaque monkeys were evaluated. Animals ranged in age from 1.5 to 21.8 years. Reproductive stage was determined based on ovarian hormones and breast histology as developmental (pre/peripubertal, n=8) or adult (premenopausal, n=7; pregnant, n=6; lactating, n=5; and postmenopausal, n=5). Transcriptional profiles were generated using Affymetrix cDNA rhesus macaque gene microarrays and analyzed using Genesifter software. All statistical comparisons were adjusted for multiple comparisons. Results: Predetermined developmental stem cell markers included CD44, CD29, CD24, OSF-2, and ALDH1; no significant changes were noted between developmental and adult profiles for any of these markers at a fold-change >2. Marked upregulation of estrogen receptor alpha (ESR1) and several markers of ESR1 activity were highly upregulated in the developing breast samples (e.g. PGR at 22-fold, TFF1 at 108-fold, and IGFBP1 at 54-fold) (adjusted P<0.0001 for all), while genes markedly upregulated in all adult samples included markers of lipid metabolism / milk production (e.g. lactalbumin at 33-fold, casein alpha s1 at 29-fold) and immunoglobulin production (e.g. IGHG1 at 36-fold, IGHM at 33-fold) (adjusted P<0.0001 for all). Novel markers highly upregulated in the developing breast included POSTN at 22-fold, KLK11 at 34-fold, KLK12 at 18-fold, PENK at 22-fold, IL13RA2 at 14-fold, CST1 at 13-fold, and NEK10 at 15-fold (adjusted P<0.0001 for all). Pathways significantly overrepresented (z-score > 2) among genes increased in developmental samples included extracellular matrix remodeling, focal adhesion, cytokine receptor interactions, and transforming growth factor-beta signaling. Discussion: Mammary morphogenesis during puberty is characterized by dramatic bursts of proliferation, differentiation, migration, and remodeling orchestrated by a complex but poorly understood network of molecular signals, many of which may also have more ominous dual roles in breast carcinogenesis. In this study we identify a novel group of markers that are highly expressed during this critical window of development. These findings should prove useful in the future study of breast cancer progenitor / stem cells and the role of aberrant developmental signals in early breast carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4220.

Response of adult mouse uterus to early disruption of estrogen receptor-α signaling is influenced by Krüppel-like factor 9

Inappropriate early exposure of the hormone-responsive uterus to estrogenic compounds is associated with increased risk for adult reproductive diseases including endometrial cancers. While the dysregulation of estrogen receptor-alpha (ESR1) signaling is well acknowledged to mediate early events in tumor initiation, mechanisms contributing to sustained ESR1 activity later in life and leading to induction of oncogenic pathways remain poorly understood. We had shown previously that the transcription factor Krüppel-like factor 9 (KLF9) represses ESR1 expression and activity in Ishikawa endometrial glandular epithelial cells. We hypothesized that KLF9 functions as a tumor suppressor, and that loss of its expression enhances ESR1 signaling. Here, we evaluated the contribution of KLF9 to early perturbations in uterine ESR1 signaling pathways elicited by the administration of synthetic estrogen diethylstilbestrol (DES) to wild-type (WT) and Klf9 null (KO) mice on postnatal days (PNDs) 1-5. Uterine tissues collected at PND84 were subjected to histological, immunological, and molecular analyses. Compared with WT mice, KO mice demonstrated larger endometrial glands and lower endometrial gland numbers; DES exposure exacerbated these differences. Loss of KLF9 expression resulted in increased glandular ESR1 immunoreactivity with DES, without effects on serum estradiol levels. Quantitative RT-PCR analyses indicated altered expression of uterine genes commonly dysregulated in endometrial cancers (Akt1, Mmp9, Slpi, and Tgfbeta1) and of those involved in growth regulation (Fos, Myc, Tert, and Syk), with loss of Klf9, alone or in concert with DES. Our data support a molecular network between KLF9 and ESR1 in the uterus, and suggest that silencing of KLF9 may contribute to endometrial dysfunctions initiated by aberrant estrogen action.

ERBB2 influences the subcellular localization of the estrogen receptor in tamoxifen-resistant MCF-7 cells leading to the activation of AKT and RPS6KA2

Acquired resistance to endocrine therapies remains a major clinical obstacle in hormone-sensitive breast tumors. We used an MCF-7 breast tumor cell line (Tam(R)-1) resistant to tamoxifen to investigate this mechanism. We demonstrate that Tam(R)-1 express elevated levels of phosphorylated AKT and MAPK3/1-activated RPS6KA2 compared with the parental MCF-7 cell line (MCF-7). There was no change in the level of total ESR between the two cell lines; however, the Tam(R)-1 cells had increased phosphorylation of ESR1 ser(167). SiRNA blockade of AKT or MAPK3/1 had little effect on ESR1 ser(167) phosphorylation, but a combination of the two siRNAs abrogated this. Co-localization studies revealed an association between ERBB2 and ESR1 in the Tam(R)-1 but not MCF-7 cells. ESR1 was redistributed to extranuclear sites in Tam(R)-1 and was less transcriptionally competent compared with MCF-7 suggesting that nuclear ESR1 activity was suppressed in Tam(R)-1. Tamoxifen resistance in the Tam(R)-1 cells could be partially overcome by the ERBB2 inhibitor AG825 in combination with tamoxifen, and this was associated with re-localization of ESR1 to the nucleus. These data demonstrate that tamoxifen-resistant cells have the ability to switch between ERBB2 or ESR1 pathways promoting cell growth and that pharmacological inhibition of ERBB2 may be a therapeutic strategy for overcoming tamoxifen resistance.

17Beta-Estradiol Induces the Translocation of the Estrogen Receptors ESR1 and ESR2 to the Cell Membrane, MAPK3/1 Phosphorylation and Proliferation of Cultured Immature Rat Sertoli Cells1

The aim of the present study was to determine the mechanisms involved in estrogen actions in cultured rat Sertoli cells. RT-PCR detected transcripts for the estrogen receptors ESR1 and ESR2 in cultured immature Sertoli cells and in the testis of 15-, 28-, and 120-day-old rats. The expression of ESR1 and ESR2 was confirmed in Sertoli cells by immunofluorescence and Western blot. Immunohistochemistry with cryosections of testes from immature and adult rats revealed that ESR1 is present in Sertoli, Leydig, and some peritubular myoid cells, and ESR2 is present in multiple cell types, including germ cells. Treatment of Sertoli cells with 17beta-estradiol (E(2)) induced a translocation of ESR1 and ESR2 to the plasma membrane and a concomitant phosphorylation of MAPK3/1. Both effects reached a maximum after 10 min and were blocked by PP2, an inhibitor of the SRC family of protein tyrosine kinases, and by the antiestrogen ICI 182,780 (ICI). MAPK3/1 phosphorylation was also decreased in the presence of AG 1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase, and in the presence of MAP2K1/2 inhibitor UO126. Treatment with E(2) for 24 h increased the incorporation of [methyl-(3)H]thymidine, which was blocked by ICI. These results indicate that E(2) activates an SRC-mediated translocation of estrogen receptors to the plasma membrane, which results in the activation of EGFR and the mitogen-activated protein kinase signaling pathway. In addition, activation of ESR1 and/or ESR2 by E(2) is involved in proliferation of immature Sertoli cells. The estrogen actions in Sertoli cells might be a key step mediating cellular events important for spermatogenesis and fertility.

Estrogen and Different Aspects of Vascular Disease in Women and Men

See related article, pages 477–484 Considerable evidence indicates that sex hormones have an important influence on cardiovascular physiology and pathology.1,2 Recent work suggests that a mechanism based on estrogen receptor-alpha (ESR1) contributes to a range of structural and functional responses that relate to vascular disease. Most of this work focuses on ESR1 activity within the endothelium and yields variable findings due, at least in part, to differences in the vascular bed or species studied, as well as other experimental conditions such as age, estrogen status, degree of preconstriction, etc. But many findings in conduit arteries (eg, more frequent plaque erosion, increased wall stiffness, spasm, etc) and microvessels (eg, reduced coronary flow reserve, hot flashes, etc) implicate smooth muscle cells (SMCs) in gender differences in vascular disease. These cells are directly responsible for extracellular matrix production and assure the integrity of the artery wall. They may be decreased, apoptotic, or dysfunctional in terms of synthesis and repair of extracellular matrix, which is destroyed in vulnerable plaque by macrophages. In this issue of Circulation Research Montague and colleagues3 report that activation of ESR1 decreases human aorta SMC differentiation (Figure). Importantly, they found that SMCs of men, compared with women, had reduced ESR1 expression associated with increased differentiation markers. Their work suggests that ESR1 activation switches SMC to a form that may promote plaque vulnerability. Could different activational states of ESR1 be responsible for shifting the functional characteristics of SMCs toward a phenotype that explains some complexities of gender-related differences in vascular disease? Hypothetical construct based on Montague et al suggests that the activational state …

Novel role of the RET finger protein in estrogen receptor-mediated transcription in MCF-7 cells

The Scaffold attachment factor B1 (SAFB1) is an estrogen receptor (ESR1) repressor that has been proposed to inhibit breast tumorigenesis. To obtain insight into the functions of SAFB1 we utilized a yeast two-hybrid screen and identified the Ret finger protein (RFP) as interacting with the SAFB1 C-terminus. RFP is a member of the trimotif (TRIM) family of proteins, which we found widely expressed in a series of breast cancer cell lines. We confirmed the interaction between SAFB1 and RFP through in vitro (GST-pull-down) and in vivo (coimmunoprecipitations) assays. We hypothesized that SAFB1 functions as a scaffolding protein to recruit proteins such as RFP into proximity with ESR1. Consequently, we asked whether RFP would modulate ESR1 activity and we discovered that RFP was important for the ESR1-dependent expression of cyclin D1 (CCND1) and the progesterone receptor (PR), but not IRS1 or MYC. Although RFP did not interact with ESR1 directly, it does coimmunoprecipitate with ESR1, demonstrating that RFP is found within the same protein complex. Chromatin immunoprecipitation assays (ChIP) located RFP to the TFF1 promoter, a known ESR1-regulated gene. Taken together, our study provides further evidence that coactivation and corepression are integrally linked processes and that RFP is a component of an ESR1 regulatory complex.