Expression Of Estrogen-responsive Genes Research Articles

Inhibition of estrogen signaling through depletion of estrogen receptor alpha by ursolic acid and betulinic acid from Prunella vulgaris var. lilacina

Extracts of Prunella vulgaris have been shown to exert antiestrogenic effects. To identify the compounds responsible for these actions, we isolated the constituents of P. vulgaris and tested their individual antiestrogenic effects. Rosmarinic acid, caffeic acid, ursolic acid (UA), oleanolic acid, hyperoside, rutin and betulinic acid (BA) were isolated from the flower stalks of P. vulgaris var. lilacina Nakai (Labiatae). Among these constituents, UA and BA showed significant antiestrogenic effects, measured as a decrease in the mRNA level of GREB1, an estrogen-responsive protein; the effects of BA were stronger than those of UA. UA and BA were capable of suppressing estrogen response element (ERE)-dependent luciferase activity and expression of estrogen-responsive genes in response to exposure to estradiol, further supporting the suppressive role of these compounds in estrogen-induced signaling. However, neither UA nor BA was capable of suppressing estrogen signaling in cells ectopically overexpressing estrogen receptor α (ERα). Furthermore, both mRNA and protein levels of ERα were reduced by treatment with UA or BA, suggesting that UA and BA inhibit estrogen signaling by suppressing the expression of ERα. Interestingly, both compounds enhanced prostate-specific antigen promoter activity. Collectively, these findings demonstrate that UA and BA are responsible for the antiestrogenic effects of P. vulgaris and suggest their potential use as therapeutic agents against estrogen-dependent tumors.

Effects of elevated dissolved carbon dioxide and perfluorooctane sulfonic acid, given singly and in combination, on steroidogenic and biotransformation pathways of Atlantic cod

In the aquatic environments, the predicted changes in water temperature, pO2 and pCO2 could result in hypercapnic and hypoxic conditions for aquatic animals. These conditions are thought to affect several basic cellular and physiological mechanisms. Yet, possible adverse effects of elevated CO2 (hypercapnia) on teleost fish, as well as combined effects with emerging and legacy environmental contaminants are poorly investigated. In this study, juvenile Atlantic cod (Gadus morhua) were divided into groups and exposed to three different water bath PFOS exposure regimes (0 (control), 100 and 200μgL−1) for 5 days at 1h/day, followed by three different CO2-levels (normocapnia, moderate (0.3%) and high (0.9%)). The moderate CO2 level is the predicted near future (within year 2300) level, while 0.9% represent severe hypercapnia. Tissue samples were collected at 3, 6 and 9 days after initiated CO2 exposure. Effects on the endocrine and biotransformation systems were examined by analyzing levels of sex steroid hormones (E2, T, 11-KT) and transcript expression of estrogen responsive genes (ERα, Vtg-α, Vtg-β, ZP2 and ZP3). In addition, transcripts for genes encoding xenobiotic metabolizing enzymes (cyp1a and cyp3a) and hypoxia-inducible factor (HIF-1α) were analyzed. Hypercapnia alone produced increased levels of sex steroid hormones (E2, T, 11-KT) with concomitant mRNA level increase of estrogen responsive genes, while PFOS produced weak and time-dependent effects on E2-inducible gene transcription. Combined PFOS and hypercapnia exposure produced increased effects on sex steroid levels as compared to hypercapnia alone, with transcript expression patterns that are indicative of time-dependent interactive effects. Exposure to hypercapnia singly or in combination with PFOS produced modulations of the biotransformation and hypoxic responses that were apparently concentration- and time-dependent. Loading plots of principal component analysis (PCA) produced a significant grouping of individual scores according to the exposure scenarios at day 6 and 9. Overall, the PCA analysis produced a unique clustering of variables that signifies a positive correlation between exposure to high PFOS concentration and mRNA expression of E2 responsive genes. Notably, this pattern was not evident for individuals exposed to PFOS concentrations in combination with elevated CO2 scenarios. To our knowledge, the present study is the first of its kind, to evaluate such effects using combined exposure to a perfluoroalkyl sulfonate and elevated levels of CO2 saturation, representative of future oceanic climate change, in any fish species or lower vertebrate.

Combined effects of silver nanoparticles and 17α-ethinylestradiol on the freshwater mudsnail Potamopyrgus antipodarum.

Ecotoxicological studies have shown that nanosilver is among the most toxic nanomaterials to aquatic organisms. However, research has so far focused on the determination of acute effects. Combined effects of nanosilver with other substances have not yet been studied in aquatic organisms. The present study aimed to investigate the chronic toxicity of nanosilver as well as the potential of nanosilver to influence the effects of co-occurring substances on the freshwater mudsnail Potamopyrgus antipodarum. In 28-day chronic toxicity experiments, the effects of nanosilver on the reproduction of P. antipodarum were assessed. In order to evaluate the influence of nanosilver on other substances, 17α-ethinylestradiol (EE2) was chosen as model compound due to the well-characterized effects on P. antipodarum. In addition to effects on reproduction, exposure to nanosilver and EE2 was monitored by determining the expression of estrogen-responsive transcripts (estrogen receptor and vitellogenin encoding genes). Exposure to nanosilver decreased the reproduction of P. antipodarum (EC10: 5.57 μg l(-1); EC50: 15.0 μg l(-1)). Exposure to EE2 significantly stimulated the embryo production at 25 ng l(-1). The presence of nanosilver led to increased EE2 effects at EE2 concentrations that had no influence on reproduction when applied in absence of nanosilver. In contrast, combined exposure to nanosilver decreased EE2 effects at concentrations that stimulated reproduction and the expression of estrogen responsive genes when applied in the absence of nanosilver. This is the first study demonstrating an influence of nanosilver on the effects of co-contaminants on aquatic organisms. The study further highlights the need for chronic experiments to properly assess environmental risks of nanosilver and their effects on co-occurring contaminants.

Increased gene copy number of VAMP7 disrupts human male urogenital development through altered estrogen action.

Vesicle transport is intimately connected with key nuclear functions and transcriptional regulation. Here, children born with congenital genitourinary tract masculinization disorders were analyzed by array-Comparative Genomic Hybridization, which revealed the presence of de novo copy number gains on Xq28 encompassing the VAMP7 gene encoding a vesicle-trafficking protein. Humanized VAMP7 BAC transgenic mice displayed cryptorchidism, urethral defects, and hypospadias. Mutant mice exhibited reduced penile length, focal spermatogenic anomalies, diminished sperm motility, and subfertility. VAMP7 colocalized with estrogen receptor alpha (ESR1) in the presence of ligand. Elevated levels of VAMP7 markedly intensified ESR1 transcriptional activity by increasing ESR1 protein cellular content upon ligand stimulation and up-regulated the expression of estrogen-responsive genes including ATF3, CYR61, and CTGF, all of which are implicated in human hypospadias. Hence, increased gene dosage of the SNARE protein, VAMP7, enhances estrogen receptor action in male genitourinary tissues, affects the virilization of the reproductive tract, and results in genitourinary birth defects in humans.

SETD6 controls the expression of estrogen-responsive genes and proliferation of breast carcinoma cells

The lysine methyltransferase SETD6 modifies the histone variant H2AZ, a key component of nuclear receptor-dependent transcription. Herein, we report the identification of several factors that associate with SETD6 and are implicated in nuclear hormone receptor signaling. Specifically, SETD6 associates with the estrogen receptor α (ERα), histone deacetylase HDAC1, metastasis protein MTA2, and the transcriptional co-activator TRRAP. Luciferase reporter assays identify SETD6 as a transcriptional repressor, in agreement with its association with HDAC1 and MTA2. However, SETD6 behaves as a co-activator of several estrogen-responsive genes, such as PGR and TFF1. Consistent with these results, silencing of SETD6 in several breast carcinoma cell lines induced cellular proliferation defects accompanied by enhanced expression of the cell cycle inhibitor CDKN1A and induction of apoptosis. Herein, we have identified several chromatin proteins that associate with SETD6 and described SETD6 as an essential factor for nuclear receptor signaling and cellular proliferation.

Estrogen-like activity of Adenophora triphylla var. japonica water extract in MCF-7 cells

This study investigated the estrogen-like activity of Adenophora triphylla var. japonica, which has been shown to have pharmacological activities. Water extracts from A. triphylla (ATWE) could bind to estrogen receptors and displaced binding of E2 to ERα and ERβ. ATWE stimulated the proliferation of estrogen receptor-positive MCF-7 cells in a dose dependent manner (p<0.05). ATWE induced proliferation was blocked by the addition of the estrogen antagonist ICI 182,780 (p<0.05). Moreover, ATWE treatment caused a significant increase in mRNA expression of estrogen-responsive genes (pS2, PR, and cathepsin D; p<0.05). These results indicate that A. triphylla has estrogen-like activity and could be used to improve estrogen deficiency-related menopausal symptoms or diseases in postmenopausal women.

Mediator of ERBB2-driven Cell Motility (MEMO) Promotes Extranuclear Estrogen Receptor Signaling Involving the Growth Factor Receptors IGF1R and ERBB2*

In addition to nuclear estrogen receptor (ER) acting as a transcription factor, extranuclear ER also plays an important role in cancer cell growth regulation through activation of kinase cascades. However, the molecular mechanisms by which extranuclear ER exerts its function are still poorly understood. Here, we report that mediator of ERBB2-driven cell motility (MEMO) regulates extranuclear functions of ER. MEMO physically and functionally interacted with ER. Through its interaction with the growth factor receptors IGF1R and ERBB2, MEMO mediated extranuclear functions of ER, including activation of mitogen-activated protein kinase (MAPK) and protein kinase B/AKT, two important growth regulatory protein kinases, and integration of function with nuclear ER. Activation of MAPK and AKT was responsible for MEMO modulation of ER phosphorylation and estrogen-responsive gene expression. Moreover, MEMO increased anchorage-dependent and -independent growth of ER-positive breast cancer cells in vitro and was required for estrogen-induced breast tumor growth in nude mice. Together, our studies identified MEMO as a new component of extranuclear ER signalosome and suggest an essential role for MEMO in the regulation of ER-positive breast cancer cell growth.

Estrogen receptor splice variants as a potential source of false-positive estrogen receptor status in breast cancer diagnostics

It is well established that only estrogen receptor (ER)-positive tumors benefit from hormonal therapies. We hypothesized that a subgroup of breast cancer patients expresses estrogen receptor α (ERα), but fails to respond to hormonal therapy due to the expression of a non-functional receptor. We analyzed a series of 2,658 ERα-positive HER2-negative breast tumors for ERα and progesterone receptor (PR) status as determined by mRNA expression and for their molecular subtypes (Luminal type vs Basal type, assessed by BluePrint™ molecular subtyping assay). In addition, we assessed the recurrence risk (low vs high) using the 70-gene MammaPrint™ signature. We found that 55 out of 2,658 (2.1 %) tumors that are ERα positive by mRNA analysis also demonstrate a Basal molecular subtype, indicating that they lack expression of estrogen-responsive genes. These ERα-positive Basal-type tumors express significantly lower levels of both ERα and PR mRNA as compared to Luminal-type tumors (P < 0.0001) and almost invariably (94.5 %) have a high-risk MammaPrint™ profile. Twelve of the MammaPrint™ genes are directly ERα responsive, indicating that MammaPrint™ assesses ERα function in breast cancer without considering ERα mRNA levels. We find a relatively high expression of the dominant negative ERα splice variant ERΔ7 in ERα-positive Basal-type tumors as compared to ERα-positive Luminal-type tumors (P < 0.0001). Expression of the dominant negative ERα variant ERΔ7 provides a rationale as to why tumors are of the Basal molecular subtype while staining ERα positive by immunohistochemistry. These tumors may lack a functional response to estrogen and consequently may not respond to hormonal therapy. Our data indicate that such patients are of MammaPrint™ high recurrence risk and might benefit from adjuvant chemotherapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2648-1) contains supplementary material, which is available to authorized users.

An estrogen receptor dependent mechanism of Oroxylin A in the repression of inflammatory response.

Oroxylin A, a natural flavonoid, is one of the main bioactive compounds that underlie the anti-inflammatory effect of the medicinal herb Scutellaria baicalensis Georgi widely used in southeastern Asia; however, the molecular mechanisms for the therapeutic benefits remain largely unclear. In this study, we found that Oroxylin A induces estrogen-responsive gene expression and promoter activity. In macrophages, Oroxylin A treatment significantly attenuates lipopolysaccharide (LPS)-induced but not basal inflammatory response, including nitric oxide (NO) production and the expression of inflammatory mediators (i.e., iNOS and COX-2) and cytokines (i.e., TNF-α, IL-1β, and IL-6), in an estrogen receptor (ER)-dependent manner. Oroxylin A treatment also dramatically decreases LPS-induced secretion of pro-inflammatory cytokines. Furthermore, the downregulation of all these inflammatory parameters by Oroxylin A was abolished when cells were pretreated with specific ER antagonist. Thus, Oroxylin A is a novel phytoestrogen and exhibits anti-inflammatory effects that are mediated by ER activity.

17β-Estradiol up-regulates miR-155 expression and reduces TP53INP1 expression in MCF-7 breast cancer cells

In estrogen responsive breast cancer cells, estradiol (E2) is a key regulator of cell proliferation and survival. MiR-155 has emerged as an "oncomiR", which is the most significantly up-regulated miRNA in breast cancer. Moreover, miR-155 is higher in ERα (+) breast tumors than ERα (-), but no one has examined whether E2 regulates miR-155 expression in MCF-7 cells. In this study, the aim was to explore whether miR-155 involved in E2 regulated expression of estrogen responsive genes. We evaluated miR-155 expression in human breast cancer cells by real-time PCR, finding out miR-155 was overexpressed in MCF-7 cells compared with MDA-MB-231 cells. Treatment with E2 in MCF-7 cells increased miR-155 expression, promoting proliferation and decreasing apoptosis, similarly, transfection of miR-155m to MCF-7 cells gave the similar results. In contrast, inhibited miR-155 expression by transfection with miR-155 inhibitors reduced proliferation and promoted apoptosis of MCF-7 cells. Moreover, TP53INP1 is one of the targets of miR-155. E2 negatively regulated TP53INP1 mRNA expression and the protein expression of TP53INP1, cleaved-caspase-3, -8, -9, and p21, whereas transfection with miR-155 inhibitors increased TP53INP1, cleaved-caspase-3, -8, -9, and p21 protein level. These results demonstrated that E2 promoted breast cancer development and progression possibly through increasing the expression of miR-155, which was overexpressed in MCF-7 cells, contributes to proliferation of MCF-7 cells possibly through down-regulating TP53INP1.

Effects of triclocarban on the transcription of estrogen, androgen and aryl hydrocarbon receptor responsive genes in human breast cancer cells

Triclocarban (TCC) is an antimicrobial agent that is used in detergents, soaps and other personal hygiene products. Similarly to triclosan the widespread use of TCC has raised concerns about its endocrine potential. In luciferase-based reporter assays TCC has been shown to enhance estrogenic and androgenic activities following cellular coexposure with estrogen or dihydrotestosterone, respectively. The present study demonstrates that although coexposure with TCC enhances the estrogenic and androgenic readout of luciferase-based reporter cell lines such as HeLa9908 and MDA-kb2, it fails to act as a xenoandrogen on transcriptional level, nor does it induce cell proliferation in the estrogen sensitive E-screen. In addition TCC did not alter the expression of estrogen responsive genes in human mammary carcinoma MCF-7 cells exposed to 17β-estradiol, bisphenol A, butylparaben or genistein.However, TCC was shown to interfere with the regulon of the aryl hydrocarbon receptor (AhR) as TCC showed a costimulatory effect on transcription of CYP1A1 and CYP1B1, effectively lowering the transcriptional threshold for both genes in the presence of estrogens. It thus seems, that while the induction of the respective luciferase reporter assays by TCC is an unspecific false positive signal caused by luciferase stabilisation, TCC has the potential to interfere with the regulatory crosstalk of the estrogen receptor (ER) and the AhR regulon.

Abstract PL01-03: Developmental reprogramming of cancer susceptibility

Abstract PL01-03: Developmental reprogramming of cancer susceptibility

Effects of the conjugated equine estrogen/bazedoxifene tissue-selective estrogen complex (TSEC) on mammary gland and breast cancer in mice.

A tissue-selective estrogen complex (TSEC), combining a selective estrogen receptor modulator, bazedoxifene (BZA), with conjugated equine estrogen (CEE), represents a novel strategy of menopausal hormone therapy without involving a progestin. We hypothesized that the antiestrogenic properties of BZA can also block the estrogenic effects of CEE on breast tissue and thereby prevent breast cancer in women. To test our hypothesis, the effects of estradiol (E(2)), CEE, and BZA on mammary gland and breast cancer xenografts were assessed in mouse models. In immature castrate mice, BZA completely blocked CEE- or E(2)-stimulated ductal and terminal end bud growth of mammary gland as well as estrogen-responsive gene expression. As a positive control, E(2) stimulated tumor growth in nude mice bearing MCF-7 xenografts. This effect was completely blocked by BZA as were E(2)-stimulated expression of PR, pS2 (trefoil factor 1), cMyc, and AREG; the enhancement of Ki67 and proliferating cell nuclear antigen (PCNA); and the antiapoptotic effect. CEE was much less potent than E(2) in stimulating Ki67, reducing apoptosis, and stimulating gene expression, but all effects were blocked by BZA. Unexpectedly, CEE alone, even at high doses, did not stimulate tumor growth. As confirmation of its absorption and deconjugation, CEE caused a 6-fold increase in uterine weight and stimulation of gene expression. These data support our hypothesis that the net effect of the CEE/BZA TSEC is to block estrogen action in benign and malignant breast tissue. These findings provide a rationale for a clinical study to determine whether this TSEC prevents breast cancer in women.

Identification of Estrogen Receptor β as a SUMO-1 Target Reveals a Novel Phosphorylated Sumoylation Motif and Regulation by Glycogen Synthase Kinase 3β

SUMO conjugation has emerged as a dynamic process in regulating protein function. Here we identify estrogen receptor β (ERβ) to be a new target of SUMO-1. ERβ SUMO-1 modification occurs on a unique nonconsensus sumoylation motif which becomes fully competent upon phosphorylation of its contained serine residue, which provides the essential negative charge for sumoylation. This process is further regulated by phosphorylation of additional adjacent serine residues by glycogen synthase kinase 3β (GSK3β), which maximizes ERβ sumoylation in response to hormone. SUMO-1 attachment prevents ERβ degradation by competing with ubiquitin at the same acceptor site and dictates ERβ transcriptional inhibition by altering estrogen-responsive target promoter occupancy and gene expression in breast cancer cells. These findings uncovered a novel phosphorylated sumoylation motif (pSuM), which consists of the sequence ψKXS (where ψ represents a large hydrophobic residue) and which is connected to a GSK3-activated extension that functions as a SUMO enhancer. This extended pSuM offers a valuable signature to predict SUMO substrates under protein kinase regulation.

17beta-hydroxysteroid dehydrogenase type 1 modulates breast cancer protein profile and impacts cell migration

IntroductionHuman 17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a steroid-converting enzyme that has long been known to play critical roles in estradiol synthesis and more recently in dihydrotestosterone (DHT) inactivation, showing a dual function that promotes breast cancer cell proliferation. Previously, we reported the first observation of the influence of the enzyme on endogenous estrogen-responsive gene expression. Here, we demonstrate the impact of 17β-HSD1 expression on the breast cancer cell proteome and investigate its role in cell migration.Methods17β-HSD1 was stably transfected in MCF7 cells and the proteome of the generated cells overexpressing 17β-HSD1 (MCF7-17βHSD1 cells) was compared to that of the wild type MCF7 cells. Proteomics study was performed using two-dimensional gel electrophoresis followed by mass spectrometry analysis of differentially expressed protein spots. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the transcription of individual gene. The effect of 17β-HSD1 on MCF7 cell migration was verified by a wound-healing assay.ResultsProteomic data demonstrate that the expression of more than 59 proteins is modulated following 17β-HSD1 overexpression. 17β-HSD1 regulates the expression of important genes and proteins that are relevant to cell growth control, such as BRCA2 and CDKN1A interacting protein (BCCIP) and proliferating cell nuclear antigen (PCNA) which are down- and upregulated in MCF7-17βHSD1 cells, respectively. RT-qPCR data reveal that 17β-HSD1 increases the mRNA levels of estrogen receptors (ER) alpha and beta by 171 and 120%, respectively, while decreasing that of the androgen receptor by 64%. Interestingly, 17β-HSD1 increases the mRNA transcript (by 3.6 times) and the protein expression of the metastasis suppressor gene nm23-H1 and the expression of the two enzymes are closely correlated. We have further shown that 17β-HSD1 expression is associated with an increase of MCF7 cell migration.ConclusionsIn addition to the regulation of important genes, we have demonstrated for the first time that 17β-HSD1 increases breast cancer cell migration, in spite of its positive regulation of the antimetastatic gene NM23. This is also correlated to its stimulation of breast cancer cell growth, further confirming its targeting in ER positive breast cancer. The novel findings in this study suggest several directions for future research on the contribution of 17β-HSD1 to breast cancer progression and related treatment.

Abstract 4053: A label-free spectroscopic signature associated with hormone sensitivity in 3D co-culture models of breast cancer

Abstract Treatment of estrogen-receptor positive breast cancers relies on endocrine therapies; however, up to 30% of patients become resistant to this treatment. It is beneficial to identify patients who will remain sensitive to endocrine therapy using markers inherent in tissue at the time of diagnosis. Using a three-dimensional (3D) co-culture, we developed a method to determine the chemical signature of hormone sensitivity based on Fourier Transform Infrared (FT-IR) spectroscopic imaging, a highly sensitive label-free method that can detect chemical changes in tissue samples. We used a 3D co-culture model comprised of MCF7 breast cancer cells and MCF7 Tam-resistant cells grown as spheroids on Matrigel™ and different densities of human mammary fibroblasts (HMFs) embedded within collagen. Upon treatment with 17β-estradiol (E2) or tamoxifen (Tam), samples were paraffin embedded for imaging and, in parallel, RNA was isolated to determine hormone sensitivity. MCF7 grown in 3D showed comparable E2 and Tam sensitivity compared to 2D culture as determined through expression of estrogen-responsive genes. Co-culture led to a decrease in the ability of E2 and Tam to regulate known response genes, and this was dependent on fibroblast density. Furthermore, EMT markers were up-regulated after co-culture, showing that this model more closely mimics an early, more invasive tumor stage. FT-IR detection of spectroscopic features identified changes occurring after treatment with Tam in MCF7 grown alone or in co-culture. A significant decrease in a peak associated with nucleic acids (1080 cm-1) was seen compared to untreated or Tam-resistant cells, emphasizing the inhibitory effects of Tam on proliferation of MCF7. Moreover, Tam induced a significant decrease in peaks associated with fatty acid synthesis (2930 cm-1, 2850 cm-1) that have been related to apoptosis. When cells were exposed to E2 in the presence of HMFs, there was a distinct increase in the peak associated with glycosylation (1030 cm-1), suggesting a synergistic metabolic effect between E2 and the surrounding microenvironment. Based on the application of FT-IR, we identified a novel spectroscopic signature that can distinguish between hormone-responsive and non-responsive tumor cell lines. Although there is already considerable insight into molecular signatures of endocrine response in cell culture and in vivo models of breast cancer, FT-IR spectroscopic imaging provides a label-free and global chemical signature that could be used in addition to immunohistochemical analysis of the initial biopsy as a way to predict endocrine sensitivity. (Supported by grants from The Breast Cancer Research Foundation to BSK, DOD postdoctoral fellowship (W81XWH-09-1-0398) to AB, NIH NCI Alliance for Nanotechnology in Cancer ‘Midwest Cancer Nanotechnology Training Center’ (R25 CA154015A) to SEH, and National Institutes of Health (RO1CA138882) to RB.) 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 4053. doi:1538-7445.AM2012-4053

Environmental Estrogens Differentially Engage the Histone Methyltransferase EZH2 to Increase Risk of Uterine Tumorigenesis

Environmental exposures during sensitive windows of development can reprogram normal physiologic responses and alter disease susceptibility later in life in a process known as developmental reprogramming. For example, exposure to the xenoestrogen diethylstilbestrol during reproductive tract development can reprogram estrogen-responsive gene expression in the myometrium, resulting in hyperresponsiveness to hormone in the adult uterus and promotion of hormone-dependent uterine leiomyoma. We show here that the environmental estrogens genistein, a soy phytoestrogen, and the plasticizer bisphenol A, differ in their pattern of developmental reprogramming and promotion of tumorigenesis (leiomyomas) in the uterus. Whereas both genistein and bisphenol A induce genomic estrogen receptor (ER) signaling in the developing uterus, only genistein induced phosphoinositide 3-kinase (PI3K)/AKT nongenomic ER signaling to the histone methyltransferase enhancer of zeste homolog 2 (EZH2). As a result, this pregenomic signaling phosphorylates and represses EZH2 and reduces levels of H3K27me3 repressive mark in chromatin. Furthermore, only genistein caused estrogen-responsive genes in the adult myometrium to become hyperresponsive to hormone; estrogen-responsive genes were repressed in bisphenol A-exposed uteri. Importantly, this pattern of EZH2 engagement to decrease versus increase H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by genistein promoted development of uterine leiomyomas, increasing tumor incidence and multiplicity, whereas bisphenol A did not. These data show that environmental estrogens have distinct nongenomic effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark in chromatin during developmental reprogramming, and promote uterine tumorigenesis.

Induction of cell proliferation and survival genes by estradiol-repressed microRNAs in breast cancer cells

BackgroundIn estrogen responsive MCF-7 cells, estradiol (E2) binding to ERα leads to transcriptional regulation of genes involved in the control of cell proliferation and survival. MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression. The aim of this study was to explore whether miRNAs were involved in hormonally regulated expression of estrogen responsive genes.MethodsWestern blot and QPCR were used to determine the expression of estrogen responsive genes and miRNAs respectively. Target gene expression regulated by miRNAs was validated by luciferase reporter assays and transfection of miRNA mimics or inhibitors. Cell proliferation was evaluated by MTS assay.ResultsE2 significantly induced bcl-2, cyclin D1 and survivin expression by suppressing the levels of a panel of miRNAs (miR-16, miR-143, miR-203) in MCF-7 cells. MiRNA transfection and luciferase assay confirmed that bcl-2 was regulated by miR-16 and miR-143, cyclinD1 was modulated by miR-16. Importantly, survivin was found to be targeted by miR-16, miR-143, miR-203. The regulatory effect of E2 can be either abrogated by anti-estrogen ICI 182, 780 and raloxifene pretreatment, or impaired by ERα siRNA, indicating the regulation is dependent on ERα. In order to investigate the functional significance of these miRNAs in estrogen responsive cells, miRNAs mimics were transfected into MCF-7 cells. It revealed that overexpression of these miRNAs significantly inhibited E2-induced cell proliferation. Further study of the expression of the miRNAs indicated that miR-16, miR-143 and miR-203 were highly expressed in triple positive breast cancer tissues, suggesting a potential tumor suppressing effect of these miRNAs in ER positive breast cancer.ConclusionsThese results demonstrate that E2 induces bcl-2, cyclin D1 and survivin by orchestrating the coordinate downregulation of a panel of miRNAs. In turn, the miRNAs manifest growth suppressive effects and control cell proliferation in response to E2. This sheds a new insight into the integral post-transcriptional regulation of cell proliferation and survival genes by miRNAs, a potential therapeutic option for breast cancer.

Cannibalism, cell survival, and endocrine resistance in breast cancer

Breast cancer cells often respond to an endocrine therapy by altering expression of specific estrogen-responsive genes and inducing autophagy, a cannibalistic lysosomal pathway. Autophagy eliminates damaged or other organelles, allowing the recovery of the energy stored in their macromolecules to attempt restoration of metabolic homeostasis. Induction of autophagy can result from activation of the unfolded protein response following metabolic stress, the final cell fate often being determined by the extent and duration of autophagy. A study by Gonzalez-Malerva and colleagues builds upon this extensive knowledge, adding HSPB8 to the list of altered genes associated with endocrine resistance in breast cancer and describing the ability of HSPB8 to regulate autophagy and confer tamoxifen resistance.

Suppression of Estrogen Receptor Transcriptional Activity by Connective Tissue Growth Factor

Secreted growth factors have been shown to stimulate the transcriptional activity of estrogen receptors (ER) that are responsible for many biological processes. However, whether these growth factors physically interact with ER remains unclear. Here, we show for the first time that connective tissue growth factor (CTGF) physically and functionally associates with ER. CTGF interacted with ER both in vitro and in vivo. CTGF interacted with ER DNA-binding domain. ER interaction region in CTGF was mapped to the thrombospondin type I repeat, a cell attachment motif. Overexpression of CTGF inhibited ER transcriptional activity as well as the expression of estrogen-responsive genes, including pS2 and cathepsin D. Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity. The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF. Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels. The results suggested the transcriptional regulation of estrogen signaling through interaction between CTGF and ER, and thus may provide a novel mechanism by which cross-talk between secreted growth factor and ER signaling pathways occurs.