ERα Gene Expression Research Articles

Effects of PCB 126 and PCB 153 on secretion of steroid hormones and mRNA expression of steroidogenic genes (STAR, HSD3B, CYP19A1) and estrogen receptors (ERα, ERβ) in prehierarchical chicken ovarian follicles

The objective of this study was to assess the in vitro effects of dioxin-like PCB 126 and non-dioxin-like PCB 153 on basal and ovine LH (oLH)-stimulated testosterone (T) and estradiol (E2) secretion and expression of steroidogenic genes (STAR, HSD3B and CYP19A1) and estrogen receptors α (ERα) and β (ERβ) in white (WF) and yellowish (YF) prehierarchical follicles of the hen ovary. Steroid concentrations in a medium and gene expression in follicles following 6h of exposition were determined by RIA and real-time qPCR, respectively. Both PCBs increased basal and oLH-stimulated T secretion by the WF follicles. PCB 126 reduced basal E2 secretion by the WF follicles. PCB 153 elevated but PCB 126 reduced oLH-stimulated E2 secretion by the prehierarchical follicles. PCB 126 increased basal STAR and HSD3B and reduced CYP19A1 mRNA expression in these follicles. PCB 153 increased basal expression of STAR and HSD3B in YF follicles, but diminished HSD3B mRNA levels in the WF. The studied PCBs had an opposite effect on basal and oLH-stimulated CYP19A1 mRNA expression in prehierarchical follicles. Both PCBs modulated basal and inhibited oLH-stimulated ERα and ERβ gene expression in the prehierarchical follicles. In conclusion, data of the current study demonstrate the congener-specific effects of PCBs on sex steroid secretion by prehierarchical follicles of the chicken ovary, which are at least partly related to STAR, HSD3B and CYP19A1 gene expression. It is suggested that PCBs, by influencing follicular steroidogenesis and expression of estrogen receptors, may impair development and selection of yellowish follicles to the preovulatory hierarchy.

Proximal and distal regulation of the HYAL1 gene cluster by the estrogen receptor α in breast cancer cells.

Chromosomal and genome abnormalities at the 3p21.3 locus are frequent events linked to epithelial cancers, including ovarian and breast cancers. Genes encoded in the 3p21.3 cluster include HYAL1, HYAL2 and HYAL3 members of hyaluronidases involved in the breakdown of hyaluronan, an abundant component of the vertebrate extracellular matrix. However, the transcriptional regulation of HYAL genes is poorly defined. Here, we identified the estrogen receptor ERα as a negative regulator of HYAL1 expression in breast cancer cells. Integrative data mining using METABRIC dataset revealed a significant inverse correlation between ERα and HYAL1 gene expression in human breast tumors. ChIP-Seq analysis identified several ERα binding sites within the 3p21.3 locus, supporting the role of estrogen as an upstream signal that diversely regulates the expression of 3p21.3 genes at both proximal and distal locations. Of these, HYAL1 was repressed by estrogen through ERα binding to a consensus estrogen response element (ERE) located in the proximal promoter of HYAL1 and flanked by an Sp1 binding site, required to achieve optimal estrogen repression. The repressive chromatin mark H3K27me3 was increased at the proximal HYAL1 ERE but not at other EREs contained in the cluster, providing a mechanism to selectively downregulate HYAL1. The HYAL1 repression was also specific to ERα and not to ERβ, whose expression did not correlate with HYAL1 in human breast tumors. This study identifies HYAL1 as an ERα target gene and provides a functional framework for the direct effect of estrogen on 3p21.3 genes in breast cancer cells.

Argonaute 2 Expression Correlates with a Luminal B Breast Cancer Subtype and Induces Estrogen Receptor Alpha Isoform Variation.

Estrogen receptor alpha (ERα) signaling pathways are frequently disrupted in breast cancer and contribute to disease progression. ERα signaling is multifaceted and many ERα regulators have been identified including transcription factors and growth factor pathways. More recently, microRNAs (miRNAs) are shown to deregulate ERα activity in breast carcinomas, with alterations in both ERα and miRNA expression correlating to cancer progression. In this study, we show that a high expression of Argonaute 2 (AGO2), a translation regulatory protein and mediator of miRNA function, correlates with the luminal B breast cancer subtype. We further demonstrate that a high expression of AGO2 in ERα+ tumors correlates with a poor clinical outcome. MCF-7 breast cancer cells overexpressing AGO2 (MCF7-AGO2) altered ERα downstream signaling and selective ERα variant expression. Enhanced ERα-36, a 36 kDa ERα isoform, protein and gene expression was observed in vitro. Through quantitative polymerase chain reaction (qPCR), we demonstrate decreased basal expression of the full-length ERα and progesterone receptor genes, in addition to loss of estrogen stimulated gene expression in vitro. Despite the loss, MCF-7-AGO2 cells demonstrated increased estrogen stimulated tumorigenesis in vivo. Together with our clinical findings on AGO2 expression and the luminal B subtype, we suggest that AGO2 is a regulator of altered ERα signaling in breast tumors.

Estrogen receptor expression and gene promoter methylation in non-small cell lung cancer - a short report.

In the past, anomalous estrogen receptor (ER) regulation has been associated with various lung pathologies, but so far its involvement in lung cancer initiation and/or progression has remained unclear. Here, we aimed at assessing in vivo and in vitro ER expression and its possible epigenetic regulation in non-small cell lung cancer (NSCLC) samples and their corresponding normal tissues and cells. ERα and ERβ gene expression levels were assessed using real time quantitative PCR (RT-qPCR), whereas ERα and ERβ gene promoter methylation levels were assessed using DNA bisulfite conversion followed by pyrosequencing. We included NSCLC (n=87) and adjacent histologically normal lung tissue samples from lung cancer patients (n=184), primary normal bronchial epithelial-derived cell cultures (n=11), immortalized bronchial epithelial-derived cell lines (n=3) and NSCLC derived cell lines (n=9). Using RT-qPCR we found significantly lower ERα and ERβ expression levels in the NSCLC tissue samples compared to their normal adjacent tissue samples. These lower ER expression levels were confirmed in vitro using primary normal bronchial epithelial-derived cell cultures, immortalized bronchial epithelial-derived cell lines and NSCLC-derived cell lines. By using this latter panelof cells, we found that ER gene promoter hypermethylation was associated with decreased ER expression. In addition we found that in tumor and normal lung tissues, smoking was associated with decreased ER expression and that normal lung tissues with a low ERβ expression level exhibited increased smoking-related DNA adducts. Taken together, our results indicate that decreased ER expression mediated by DNA methylation may play a role in NSCLC development.

Daidzein increases OPG/RANKL ratio and suppresses IL-6 in MG-63 osteoblast cells

Daidzein is a major dietary source of isoflavones found in Leguminosae, and belongs to the family of diphenolic compounds. The estrogenic effects of daidzein to prompt bone formation and prevent bone resorption have been observed in animal models and cultured cells. In our study, we studied the effects of daidzein, raloxifene and E2 on expression of the osteoblast-produced bone regulatory factors OPG, RANKL and IL-6 in human osteoblastic MG-63 cells. Results suggest that treatment with daidzein, raloxifene and E2 increased the levels of OPG and decreased those of RANKL and IL-6. The effects of daidzein on OPG and RANKL expression are mediated by both ERα and ERβ but those on IL-6 production primarily by ERα. Moreover, daidzein may promote activation of the classic estrogen response element (ERE) pathway through increasing ERα, ERβ and steroid hormone receptor coactivator (SRC)-1 expression. E2 was also able to enhance transcription derived from the ERE, while raloxifene has no effect on it. Raloxifene increased ERα protein and gene expression levels but had no effect on ERβ protein and gene expression at 0.1μM. E2 was found significantly increased the protein and mRNA levels of SRC-1, while raloxifene has no effect on it compared with control. This ability of daidzein to affect osteoblastic cells makes it a good candidate for the treatment of bone loss in postmenopausal women.

Abstract 285: Disulfiram, a dual MGMT and aldehyde dehydrogenase inhibitor, sensitizes ER-positive breast cancer cells to temozolomide and cyclophosphamide

Abstract Background: O6 methylguanine DNA methyltransferase (MGMT) is overexpressed in a majority of cancers, including breast cancer. MGMT is a DNA repair protein leading to chemo resistance. MGMT expression directly correlates with ER expression and tamoxifen resistance. Aldehyde Dehydrogenase (ALDH) activity, as a progenitor/stem cell marker, while it has been reported to inversely correlate with MGMT expression in other cancers, has also been linked to chemotherapy and radiation resistance. Methods: We have tested the effect of Antabuse (disulfiram, DSF), as a dual MGMT and ALDH inhibitor, at nontoxic doses, in combination with Temozolomide (TMZ) or/and Cyclophosphamide (CP) on ER+ breast cancer cells. Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases ER+ breast cancer cell growth (MCF7, T47D and ZR75) in a dose-dependent manner. DSF further sensitizes breast cancer cells to TMZ or/and CP and significantly inhibits breast cancer growth without causing unwanted side effects on the normal breast epithelial cells. Dose effect and isobologram studies confirm synergistic activity of DSF + CP and moderate synergism for DSF + TMZ. DSF, alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP), significantly inhibits expression of MGMT, aldehyde dehydrogenase, ERα, E2F and BIRC5 gene (survivin) - all involved in breast cancer tumorigenesis. DSF, alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP) caused significant apoptosis in breast cancer cells. In a dose dependent manner, DSF inhibited colony formation, effect which was further enhanced by addition of TMZ/CP (DSF ± TMZ/CP). Similarly, DSF alone or in combination with TMZ (DSF ± TMZ) and/or CP (DSF ± CP) decreased the metastatic potential of breast cancer cells. Conclusions: Our findings suggest that DSF as dual inhibitor of MGMT and ALDH significantly enhances the antitumor effect of alkylators as CP and TMZ in ER+ breast cancer Citation Format: George C. Bobustuc, Deborah Donohoe, Alisher Holmuhamedov, Srivenugopal Kalkunte, Santhi D. Konduri. Disulfiram, a dual MGMT and aldehyde dehydrogenase inhibitor, sensitizes ER-positive breast cancer cells to temozolomide and cyclophosphamide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 285.

Sex differences in obesity development in pair-fed neuronal lipoprotein lipase deficient mice

ObjectiveCompared to men, postmenopausal women suffer from a disproportionate burden of many co-morbidities associated with obesity, e.g. cardiovascular disease, cancer, and dementia. The underlying mechanism for this sex difference is not well understood but is believed to relate to absence of the protective effect of estrogen through the action of estrogen receptor alpha (ERα) in the central nervous system. With the recently developed neuron-specific lipoprotein lipase deficient mice (NEXLPL−/−) (Wang et al., Cell Metabolism, 2011 [15]), we set to explore the possible role of lipid sensing in sex differences in obesity development. MethodsBoth male and female NEXLPL−/− mice and littermate WT controls were subjected to pair feeding (pf) where daily food amount given was adjusted according to body weight to match the food intake of ad libitum (ad) fed control WT mice. Food intake and body weight were measured daily, and pair feeding was maintained to 42 wk in male mice and to 38 wk in female mice. Various brain regions of the mice were harvested, and ERα gene expression was examined in both male and female NEXLPL−/− and WT control mice under both ad- and pf-fed conditions. ResultsAlthough both male and female NEXLPL−/− mice developed obesity similarly on standard chow, male NEXLPL−/− mice still developed obesity under with pair feeding, but on a much delayed time course, while female NEXLPL−/− mice were protected from extra body weight and fat mass gain compared to pair-fed WT control mice. Pair feeding alone induced extra fat mass gain in both male and female WT mice, and this was mostly driven by the reduction in physical activity. LPL deficiency resulted in an increase in ERα mRNA in the hypothalamus of ad-fed female mice, while pair feeding alone also resulted in an increase of ERα in both female WT control and NEXLPL−/− mice. The effect on increasing ERα by pair feeding and LPL deficiency was additive in pair-fed female NEXLPL−/− mice. ERα mRNA levels were not significantly modified in other brain regions examined, nor in the hypothalamus of male NEXLPL−/− mice compared to control mice. ConclusionsThese results suggest that the mechanism underlying ERα regulation of body weight interacts with the LPL-dependent lipid processing in the hypothalamus in a sex specific way. ERα could provide the link between brain lipid sensing and sex differences in obesity development. This study has the potential important clinical implication to provide better management for women who suffer from obesity and obesity-related co-morbidities.

Neoadjuvant tamoxifen synchronizes ERα binding and gene expression profiles related to outcome and proliferation.

Estrogen receptor alpha (ERα)-positive breast cancers are frequently treated with tamoxifen, but resistance is common. It remains elusive how tamoxifen resistance occurs and predictive biomarkers for treatment outcome are needed. Because most biomarker discovery studies are performed using pre-treatment surgical resections, the effects of tamoxifen therapy directly on the tumor cell in vivo remain unexamined. In this study, we assessed DNA copy number, gene expression profiles and ERα/chromatin binding landscapes on breast tumor specimens, both before and after neoadjuvant tamoxifen treatment. We observed neoadjuvant tamoxifen treatment synchronized ERα/chromatin interactions and downstream gene expression, indicating that hormonal therapy reduces inter-tumor molecular variability. ERα-synchronized sites are associated with dynamic FOXA1 action at these sites, which is under control of growth factor signaling. Genes associated with tamoxifen-synchronized sites are capable of differentiating patients for tamoxifen benefit. Due to the direct effects of therapeutics on ERα behavior and transcriptional output, our study highlights the added value of biomarker discovery studies after neoadjuvant drug exposure.

Maternal folic acid supplementation modulates DNA methylation and gene expression in the rat offspring in a gestation period-dependent and organ-specific manner

Maternal folic acid supplementation can alter DNA methylation and gene expression in the developing fetus, which may confer disease susceptibility later in life. We determined which gestation period and organ were most sensitive to the modifying effect of folic acid supplementation during pregnancy on DNA methylation and gene expression in the offspring. Pregnant rats were randomized to a control diet throughout pregnancy; folic acid supplementation at 2.5× the control during the 1st, 2nd or 3rd week of gestation only; or folic acid supplementation throughout pregnancy. The brain, liver, kidney and colon from newborn pups were analyzed for folate concentrations, global DNA methylation and gene expression of the Igf2, Er-α, Gr, Ppar-α and Ppar-γ genes. Folic acid supplementation during the 2nd or 3rd week gestation or throughout pregnancy significantly increased brain folate concentrations (P<.001), while only folic acid supplementation throughout pregnancy significantly increased liver folate concentrations (P=.005), in newborn pups. Brain global DNA methylation incrementally decreased from early to late gestational folic acid supplementation and was the lowest with folic acid supplementation throughout pregnancy (P=.026). Folic acid supplementation in late gestation or throughout pregnancy significantly decreased Er-α, Gr and Ppar-α gene expression in the liver (P<.05). The kidney and colon were resistant to the effect of folic acid supplementation. Maternal folic acid supplementation affects tissue folate concentrations, DNA methylation and gene expression in the offspring in a gestation-period-dependent and organ-specific manner.

Diethylstilbestrol at environmental levels affects the development of early life stage and target gene expression in Japanese Medaka (Oryzias latipes).

In this study, the biologic effects of DES on the early life and adult life stages of Japanese medaka (Oryzias latipes) were evaluated. At the early life stage, the fertilized eggs were exposed to 1-1000 ng/L diethylstilbestrol (DES) for 15 days and the hatched larvae were continually exposed to the same concentrations for an additional 25 days. Significant adverse effects on hatchability, time to hatching and mortality rate occurred at DES concentrations of 100 and 1000 ng/L, while the abnormality (scoliosis and abdominal swelling) rate was significantly increased at 10 ng/L and above. After exposure, the fish were maintained in charcoal-dechlorinated tap water for a further 30 days. Only the male gonadosomatic index (GSI) at 1000 ng/L was significantly increased. At concentrations greater than 1 ng/L, estrogen receptor α (ERα) mRNA in both sexes and vitellogenin-I (Vtg-I) mRNA in males were significantly down-regulated; while Vtg-I mRNA in females was significantly up-regulated. When sexually mature medaka were exposed to 10 and 1000 ng/L DES for 21 days, only the GSI in females was significantly decreased at 1000 ng/L. At 10 and 1000 ng/L, ERα mRNA in both sexes was significantly down-regulated, while Vtg-I mRNA in males was significantly up-regulated. These findings showed that DES at the environmental concentration of 10 ng/L can affect the early life stage development of medaka and alter liver ERα and Vtg-I gene expression. Therefore, if we only focused on these sensitive toxicity endpoints such as ERα and Vtg-I mRNA expression, DES has a strong estrogenic effect on Japanese medaka.

Abstract P6-08-06: Neoadjuvant tamoxifen therapy synchronizes ERα binding and gene expression profiles

Abstract Background: The majority of breast cancer patients are diagnosed with ERα-positive breast cancer. Most ERα-positive patients are treated with adjuvant endocrine therapy — typically tamoxifen or aromatase inhibitors — to block cellular proliferation. Although these treatments are considered successful, resistance is common. Notably, cross-resistance between the two types of therapies is not always observed suggesting molecular heterogeneity and underlining the need for development of personalized treatments. The Anastrozole, Fulvestrant or Tamoxifen Exposure — Response in molecular profile study (AFTER study, NCT00738777) aims to investigate prospectively whether short-term treatment can induce molecular changes indicative of pre-operative therapy response. Study Design: ERα-positive breast cancer patients are included in this open-label multicenter study. Post-menopausal patients are randomized between tamoxifen, anastrozole and fulvestrant and pre-menopausal and male patients receive tamoxifen. Treatment occurs during the pre-operative window between diagnosis and surgery (4±2 weeks). Clinical characteristics collected are ERα/PR and HER2 status as well as lymph-node status. The primary endpoint is the decrease in tumor cell proliferation, as assessed by Ki67 gene expression and published cell proliferation gene expression signatures. All data are collected from both pre- and post-treatment samples. Additionally, we will compare the changes induced by treatment in gene expression, ERα/DNA binding interactions, DNA copy number, endoxifen and estradiol levels. Results: Among 67 patients currently enrolled, we examined the data from the subset of 28 tamoxifen treated patients. ERα and PR levels did not differ significantly between pre- and post-treatment. All tumors were HER2-negative. Proliferation examined by Ki67 (IHC and gene expression, MKI67) was significantly lower in post-treatment samples (P &amp;lt; 0.01). A significant association was identified with the change in gene expression proliferation signature score and change in MKI67 (rho = 0.7, P &amp;lt; 0.001). We identified two samples, which changed from MammaPrint (MP) low-risk to high-risk among 17 pairs with data. One sample's score was on the cutoff for high-risk definition. Interestingly, the second sample also had an increase in Ki67 gene expression and proliferation gene signature score in the post-treatment sample. Overall, ERα/DNA binding interaction regions overlapped significantly more among post-treatment samples as compared to pre-treatment samples (P &amp;lt;0.001). There were 3 samples that increased in MKI67 gene expression after drug exposure. Among these, only the MP low- to high-risk sample had an increase in proliferation gene signature and decrease in ERα/DNA binding interactions. Conclusions: Pre-treatment samples were more variable for both proliferation gene expression signatures and ERα/DNA binding interactions indicating the underlying molecular heterogeneity of the group prior to therapy. This inter-tumor heterogeneity appears to have been lowered by exposure to tamoxifen. Interestingly, not all samples were uniform in their response to tamoxifen exposure as measured by Ki67 and MP scores suggesting samples taken after treatment exposure may be useful for predictive biomarker discovery. Citation Format: Linn SC, Severson TM, Nevedomskaya E, Peeters J, van Rossum A, Kuilman T, Krijgsman O, Goossens I, Glas A, Koornstra R, Peeper D, Wesseling J, Simon I, Wessels L, Zwart W. Neoadjuvant tamoxifen therapy synchronizes ERα binding and gene expression profiles. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-06.

Moving Toward Integrating Gene Expression Profiling Into High-Throughput Testing: A Gene Expression Biomarker Accurately Predicts Estrogen Receptor α Modulation in a Microarray Compendium.

Microarray profiling of chemical-induced effects is being increasingly used in medium- and high-throughput formats. Computational methods are described here to identify molecular targets from whole-genome microarray data using as an example the estrogen receptor α (ERα), often modulated by potential endocrine disrupting chemicals. ERα biomarker genes were identified by their consistent expression after exposure to 7 structurally diverse ERα agonists and 3 ERα antagonists in ERα-positive MCF-7 cells. Most of the biomarker genes were shown to be directly regulated by ERα as determined by ESR1 gene knockdown using siRNA as well as through chromatin immunoprecipitation coupled with DNA sequencing analysis of ERα-DNA interactions. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression datasets from experiments using MCF-7 cells, including those evaluating the transcriptional effects of hormones and chemicals. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% and 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) ER reference chemicals including "very weak" agonists. Importantly, the biomarker predictions accurately replicated predictions based on 18 in vitro high-throughput screening assays that queried different steps in ERα signaling. For 114 chemicals, the balanced accuracies were 95% and 98% for activation or suppression, respectively. These results demonstrate that the ERα gene expression biomarker can accurately identify ERα modulators in large collections of microarray data derived from MCF-7 cells.

CTCF May Not Directly Regulate ERα mRNA Expression in the ER+ MCF7 Breast Cancer Cell Line

Introduction: CTCF is an evolutionally conserved 11-zinc finger protein factor involved in an array of processes whose deregulation could lead to cellular transformation. Through interactions with ERα binding regions and ERregulated genes, CTCF was shown to compartmentalize the cellular genome into domains. It also colocalized with ERα in MCF7 cells and had interactions with ERα during histone deacetylase recruitment and fork-head activity. A fast-running isoform was previously shown to be expressed in breast cancer tissue but not in normal breast tissue. It is not clear whether there is a regulatory relationship between CTCF and ERα in breast cancer. Aim: To determine whether CTCF expression regulated ERα expression in the ER+ MCF7 breast cancer cell line. Methods: MCF7 breast cancer cells were transfected with either CTCF expression vectors or siRNA against CTCF. Following CTCF over-expression and knock-down, changes in endogenous expression of ERα gene and protein expression were monitored by quantitative polymerase chain reaction (using MIQE guidelines) and western blot analysis respectively. Results: CTCF plasmid overexpression and siRNA knockdown was associated with cell rounding but with 96.4% and 95.7% cell viability respectively. Increase in CTCF mRNA on over-expression was associated with a rise in CTCF protein expression. siRNA knockdown of CTCF mRNA was accompanied by a corresponding decrease in CTCF protein expression. CTCF over-expression and knockdown appeared to inhibit the ability to detect ERα protein expression by western blotting. Neither the over-expression nor knockdown of CTCF altered ERα mRNA expression as detected by QPCR. Conclusion: Alterations in CTCF mRNA expression did not affect ERα gene expression in MCF7 cells suggesting that CTCF interactions with the estrogen receptor in breast cancer may not be mediated via direct regulation of ERα mRNA expression.

Endocrine control of canine mammary neoplasms: serum reproductive hormone levels and tissue expression of steroid hormone, prolactin and growth hormone receptors.

BackgroundNeoplasms of the mammary gland are among the most common diseases in female domestic dogs (Canis familiaris). It is assumed that reproductive hormones influence tumorigenesis in this species, although the precise role of the endocrine milieu and reproductive state is subject to continuing discussion. In line with this, a recent systematic review of available data on the development of mammary neoplasms revealed weak evidence for risk reduction after neutering and an effect of age at neutering. Investigation of several hormone receptors has revealed decreased expression of estrogen receptor-alpha (ERα, ESR1), progesterone (P4) receptor (PGR), prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) associated with neoplastic differentiation of mammary tissues. In other studies, increased levels of estrogens, progesterone and prolactin were found in serum and/or tissue homogenates of dogs with malignant neoplasms. However, the association between these entities within one animal population was never previously examined. Therefore, this study investigated the association between circulating serum concentrations of estradiol-17β, progesterone and prolactin, and gene expression of ERα (ESR1), ERβ (ESR2), PGR, PRLR, PRL and GHR, with respect to reproductive state (spayed vs. intact) and cycle stage (anestrus vs. diestrus). Additionally, the expression of E-cadherin (CDH-1) was evaluated as a possible indicator of metastatic potential.ResultsFor all receptors, the lowest gene expression was found in malignant tumors compared to normal tissues of affected dogs. Steroid levels were not influenced by their corresponding receptor expression in mammary neoplasms, but increased PRL levels were negatively associated with low PRLR gene expression in malignant tumors. The expression of CDH-1 was influenced by tumor malignancy and cycle stage, i.e., the highest gene expression was found in benign mammary tumors in diestrous dogs compared to normal and malignant mammary tissues of anestrous and spayed dogs.ConclusionsHerein, it has been confirmed that transformation towards malignant neoplasms is associated with significant reduction of gene expression of particular hormone receptors. Only PRLR in malignant tumors seems to be influenced by circulating PRL levels. In dogs, CDH-1 can be used as a prognostic factor; its expression, however, in benign tumors is influenced by cycle stage.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0546-y) contains supplementary material, which is available to authorized users.

Implications of Sex Hormone Receptor Gene Expression in the Predominance of Hepatocellular Carcinoma in Males: Role of Natural Products.

The present study was planned to investigate the role of sex hormone receptor gene expression in the pathogenesis of hepatocellular carcinoma (HCC). Adult male Wistar rats were divided into seven groups. Group (1) was negative control. Groups (2), (5), (6), and (7) were orally administered with N-nitrosodiethylamine for the induction of HCC, then group (2) was left untreated, group (5) was orally treated with curcumin, group (6) was orally treated with carvacrol, and group (7) was intraperitoneally injected with doxorubicin, whereas groups (3) and (4) were orally administered only curcumin and carvacrol, respectively. The HCC group showed significant upregulation in the androgen receptor (AR) and the estrogen receptor-alpha (ERα) gene expression levels in the liver tissue. On the contrary, HCC groups treated with either curcumin or carvacrol showed significant downregulation in AR and ERα gene expression levels in the liver tissue. In conclusion, the obtained data highlight that both AR and ERα but not estrogen receptor-beta (ERβ) gene expression may contribute to the male prevalence of HCC induced in male rats. Interestingly, both curcumin and carvacrol were found to have a promising potency in alleviating the male predominating HCC.

Adipocyte hypoxia promotes epithelial-mesenchymal transition-related gene expression and estrogen receptor-negative phenotype in breast cancer cells.

The development of breast cancer is linked to the loss of estrogen receptor (ER) during the course of tumor progression, resulting in loss of responsiveness to hormonal treatment. The mechanisms underlying dynamic ERα gene expression change in breast cancer remain unclear. A range of physiological and biological changes, including increased adipose tissue hypoxia, accompanies obesity. Hypoxia in adipocytes can establish a pro-malignancy environment in breast tissues. Epidemiological studies have linked obesity with basal-like breast cancer risk and poor disease outcome, suggesting that obesity may affect the tumor phenotype by skewing the microenvironment toward support of more aggressive tumor phenotypes. In the present study, human SGBS adipocytes were co-cultured with ER-positive MCF7 cells for 24 h. After co-culture, HIF1α, TGF-β, and lectin-type oxidized LDL receptor 1 (LOX1) mRNA levels in the SGBS cells were increased. Expression levels of the epithelial-mesenchymal transition (EMT)-inducing transcription factors FOXC2 and TWIST1 were increased in the co-cultured MCF7 cells. In addition, the E-cadherin mRNA level was decreased, while the N-cadherin mRNA level was increased in the co-cultured MCF7 cells. ERα mRNA levels were significantly repressed in the co-cultured MCF7 cells. ERα gene expression in the MCF7 cells was decreased due to increased HIF1α in the SGBS cells. These results suggest that adipocytes can modify breast cancer cell ER gene expression through hypoxia and also can promote EMT processes in breast cancer cells, supporting an important role of obesity in aggressive breast cancer development.

Estrogen induces global reorganization of chromatin structure in human breast cancer cells.

In the cell nucleus, each chromosome is confined to a chromosome territory. This spatial organization of chromosomes plays a crucial role in gene regulation and genome stability. An additional level of organization has been discovered at the chromosome scale: the spatial segregation into open and closed chromatins to form two genome-wide compartments. Although considerable progress has been made in our knowledge of chromatin organization, a fundamental issue remains the understanding of its dynamics, especially in cancer. To address this issue, we performed genome-wide mapping of chromatin interactions (Hi-C) over the time after estrogen stimulation of breast cancer cells. To biologically interpret these interactions, we integrated with estrogen receptor (ERα) binding events, gene expression and epigenetic marks. We show that gene-rich chromosomes as well as areas of open and highly transcribed chromatins are rearranged to greater spatial proximity, thus enabling genes to share transcriptional machinery and regulatory elements. At a smaller scale, differentially interacting loci are enriched for cancer proliferation and estrogen-related genes. Moreover, these loci are correlated with higher ERα binding events and gene expression. Taken together these results reveal the role of a hormone - estrogen - on genome organization, and its effect on gene regulation in cancer.

Early warning signs of endocrine disruption in adult fish from the ingestion of polyethylene with and without sorbed chemical pollutants from the marine environment

Plastic debris is associated with several chemical pollutants known to disrupt the functioning of the endocrine system. To determine if the exposure to plastic debris and associated chemicals promotes endocrine-disrupting effects in fish, we conducted a chronic two-month dietary exposure using Japanese medaka (Oryzias latipes) and environmentally relevant concentrations of microplastic (<1mm) and associated chemicals. We exposed fish to three treatments: a no-plastic (i.e. negative control), virgin-plastic (i.e. virgin polyethylene pre-production pellets) and marine-plastic treatment (i.e. polyethylene pellets deployed in San Diego Bay, CA for 3months). Altered gene expression was observed in male fish exposed to the marine-plastic treatment, whereas altered gene expression was observed in female fish exposed to both the marine- and virgin-plastic treatment. Significant down-regulation of choriogenin (Chg H) gene expression was observed in males and significant down-regulation of vitellogenin (Vtg I), Chg H and the estrogen receptor (ERα) gene expression was observed in females. In addition, histological observation revealed abnormal proliferation of germ cells in one male fish from the marine-plastic treatment. Overall, our study suggests that the ingestion of plastic debris at environmentally relevant concentrations may alter endocrine system function in adult fish and warrants further research.

Different action of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolites on ERα and ERβ gene and protein expression

In our previously published data we showed that PBDEs act as endocrine disruptors in ovarian follicles by altering steroid secretion.In this study we try to answer a question if BDE-47 and its hydroxylated metabolites (5-OH-BDE-47 and 6-OH-BDE-47) can act as endocrine disruptors in the ovary by changing the expression of the steroid nuclear receptors, estrogen receptor alpha (ERα) and beta (ERβ), androgen receptor (AR), and receptors associated with the metabolism of xenobiotics and steroid hormones, constitutive androstane receptor (CAR) and pregnane X-receptor (PXR), in porcine ovarian follicles. Expression of mRNA was evaluated by real-time PCR, whereas protein level by western blotting.CAR and PXR mRNAs were not expressed in porcine ovarian follicular cells. BDE-47 and its hydroxylated metabolites had no effect on the expression of AR mRNA and protein. Decreased expression of ERβ mRNA and protein under BDE-47 influence and increase both ERα and ERβ gene and protein expression in cells exposed to hydroxylated metabolites was noted. These findings indicate that BDE-47, by altering the ratio of ERα to ERβ toward ERα, and the hydroxylated metabolites of BDE-47, by increase estrogen receptors expression, may result in excessive ovarian exposure to estrogens.

An epigenomic approach to therapy for tamoxifen-resistant breast cancer.

Tamoxifen has been a frontline treatment for estrogen receptor alpha (ERα)-positive breast tumors in premenopausal women. However, resistance to tamoxifen occurs in many patients. ER still plays a critical role in the growth of breast cancer cells with acquired tamoxifen resistance, suggesting that ERα remains a valid target for treatment of tamoxifen-resistant (Tam-R) breast cancer. In an effort to identify novel regulators of ERα signaling, through a small-scale siRNA screen against histone methyl modifiers, we found WHSC1, a histone H3K36 methyltransferase, as a positive regulator of ERα signaling in breast cancer cells. We demonstrated that WHSC1 is recruited to the ERα gene by the BET protein BRD3/4, and facilitates ERα gene expression. The small-molecule BET protein inhibitor JQ1 potently suppressed the classic ERα signaling pathway and the growth of Tam-R breast cancer cells in culture. Using a Tam-R breast cancer xenograft mouse model, we demonstrated in vivo anti-breast cancer activity by JQ1 and a strong long-lasting effect of combination therapy with JQ1 and the ER degrader fulvestrant. Taken together, we provide evidence that the epigenomic proteins BRD3/4 and WHSC1 are essential regulators of estrogen receptor signaling and are novel therapeutic targets for treatment of Tam-R breast cancer.