Expression Of Estrogen Receptor Beta Research Articles

Expression and Prognostic Value of Oestrogen Receptor Beta in Colorectal Cancer.

Differences between men and women in the incidence and biological mechanisms of colorectal cancer (CRC) suggest that estrogens may play a role in the pathogenesis of this disease. The identification of the human estrogen receptor beta (ERβ) and its expression in the intestinal mucosa led to further studies that revealed that estrogens have a protective function against CRC mediated by the activation of ERβ. However, ERβ expression and its role in CRC is controversial. The purpose of this study was to determine the distribution and prognostic value of ERβ expression in the intestinal mucosa of patients diagnosed and surgically treated for CRC, and its association with other known prognostic factors. A total of 109 paraffin-embedded samples of the wild-type ERβ isoform were analyzed by immunohistochemical nuclear staining in patients with colorectal adenocarcinoma. Clinical/pathological and survival data were collected. Immunohistochemical quantification was performed using the category scoring system, which has been validated for assessing estrogen receptor alfa. The wild-type ERβ isoform -also called ERβ1- was positive in 101 patients (92.7%) and negative in nine patients (7.3%). Univariate analysis revealed that the absence of expression of the ERβ1 gene was correlated with mucinous adenocarcinoma (p<0.05). Also, a non-significant tendency was observed for ERβ expression to be down-regulated in advanced tumors. With a median follow-up of 47months, the overall survival and progression-free survival were not found to be associated with ERβ1 expression (p=0.2). Although the wild-type ERβ isoform was expressed in most study patients with colorectal cancer, it does not seem to have any prognostic value for the course of the disease. Further studies should be conducted to investigate whether the down-regulation of ERβ expression has any biological function in mucinous colorectal cancer.

Estrogen receptor alpha is required in GABAergic, but not glutamatergic, neurons to masculinize behavior

Masculinization of the altricial rodent brain is driven by estrogen signaling during a perinatal critical period. Genetic deletion of estrogen receptor alpha (Esr1/ERα) results in altered hypothalamic-pituitary-gonadal (HPG) axis signaling and a dramatic reduction of male sexual and territorial behaviors. However, the role of ERα in masculinizing distinct classes of neurons remains unexplored. We deleted ERα in excitatory or inhibitory neurons using either a Vglut2 or Vgat driver and assessed male behaviors. We find that Vglut2-Cre;Esr1lox/lox mutant males lack ERα in the ventrolateral region of the ventromedial hypothalamus (VMHvl) and posterior ventral portion of the medial amygdala (MePV). These mutants recapitulate the increased serum testosterone levels seen with constitutive ERα deletion, but have none of the behavioral deficits. In contrast, Vgat-Cre;Esr1lox/lox males with substantial ERα deletion in inhibitory neurons, including those of the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr), posterior dorsal MeA (MePD), and medial preoptic area (MPOA) have normal testosterone levels, but display alterations in mating and territorial behaviors. These mutants also show dysmasculinized expression of androgen receptor (AR) and estrogen receptor beta (Esr2). Our results demonstrate that ERα masculinizes GABAergic neurons that gate the display of male-typical behaviors.

Characterization of serum estradiol level and tissue estrogen receptor immunostaining with clinical response and reproductive factor changes in Chinese female patients with esophageal squamous cell carcinoma

Pre-menopausal female patients have a prolonged survival than post-menopausal patients, indicating that estrogen and/or estrogen receptor (ER) may have some biological effects on prognosis. ER expression in cancer tissue has been reported to be a significant prognostic marker in multiple human cancers. However, the prognostic value of estrogen and/or ER on female patients with esophageal squamous cell carcinoma (ESCC) is rarely reported. The present study was undertaken to elucidate the associations of serum estradiol level, tissue estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) expression with clinical response and reproductive factor changes in 387 female ESCC patients. Radioimmunoassay revealed that serum estradiol level was higher in pre-menopausal than those in peri-menopausal and post-menopausal patients. Furthermore, patients with higher serum estradiol level appeared to have a better survival. Immunostaining results suggested that ERα positive (+) expression was mainly located in cytoplasm of tumor cells with a positive rate of 69.9% and ERβ (+) was mainly located in nucleus of tumor cells with a positive rate of 64.9%. We did not find the relations of ER expression with tumor invasion (P>0.05), lymph node metastasis (P>0.05), TNM staging (P>0.05) and treatment method (P>0.05). Surprisingly, ERα (+) expression was higher in post-menopausal patients than those in pre-menopausal patients (P<0.05). Patients with number of pregnancy≥4 have a higher ERβ (+) expression than those patients with≤3 (P<0.05). Univariate and multivariate survival analysis showed that ERβ (+) expression in addition to ERα (−) expression are favorable prognostic markers in female ESCC patients (P<0.05). Further related study is needed to in-depth explore the potential mechanisms of ERα and ERβ in survival of female patients with ESCC.

Abstract 5531: Significance and functions of estrogen receptor beta (ESR2) isoforms in glioblastoma

Abstract Background: Glioblastoma (GBM) are the deadliest form of primary brain neoplasms. Several lines of evidence suggest tumor suppressive role of female sex hormones on brain tumors. However, mechanisms by which estrogens mediate protection against the GBM remains unknown. GBM preferentially express estrogen receptor beta (ESR2). Emerging evidence suggests that ESR2 is expressed as multiple isoforms, (ESR2-1 to 5, with variation at the C-terminal domain); however, much of the published information is focused primarily on ESR2-1. Little is known about the expression and functions of other ESR2 isoforms in GBM. The objective of this study is to examine the expression and determine the functions of ESR2 isoforms in GBM cells. Methods: Expression of ESR2 isoforms was profiled using 10 different patients derived and 5 established GBM cells. To study the functions of individual ESR2 isoforms in GBM cells, we have generated ESR2 knockout (ESR2-KO) cells using CRISPR/Cas9 system and then established GBM model cells expressing individual ESR2-1, 2, and 5 using lentiviral transduction in the ESR2-KO background GBM cells. IPMS analysis was used to identify binding proteins of each ESR2 isoforms. Effect of each isoform on the growth, apoptosis, cell cycle progression, migration and invasion was analyzed using established methods. Mechanistic studies were conducted using reporter gene assays, RT-qPCR and signaling analysis. Results: RT-qPCR results demonstrated that ESR2-5 is highly expressed in majority of primary and established GBM cells compared to ESR2-1 and ESR2-2, with ESR2-4 is the least expressed. Expression of ESR2-1 but not ESR2-5 significantly reduced proliferation of GBM cells. Further, ESR2-KO cells exhibited high migratory and invasive potential compared to parental GBM cells and re-expression of ESR2-1, but not ESR2-5 resulted in reduction in migratory and invasive potential of GBM cells. ESR2-KO GBM cells exhibited decreased levels of cell cycle arrest and apoptosis proteins p27, p21 and PUMA compared to parental GBM cells and re-expression of ESR2-1 or ESR2-5 in ESR2-KO cells rescued the phenotype. IPMS studies identified several common and unique proteins that bind to each of the isoforms. Both ESR2-1 and ESR2-5 interacted with mTOR, while ESR2-5 uniquely interacted with several proteins related to DNA repair, immunomodulatory and apoptosis pathways. Overexpression of ESR2-1 reduced the activation of mTOR signaling molecules including p-mTOR, p-S6K and p-S6 in GBM cells compared to ESR2-KO cells, while ESR2-5 enhanced mTOR downstream signaling. Conclusion: Using ESR2KO cells, we have provided genetic evidence for the role of ESR2-1 in GBM tumor suppression. Our results also discovered that ESR2-5 is highly expressed in GBMs. Unlike ESR2-1, ESR2-5 has lesser tumor suppression ability and its interactions with mTOR, DNA repair, and apoptotic pathways may have important implications in GBM progression. Citation Format: Jinyou Liu, Gangadhara Reddy Sareddy, Mei Zhou, Suryavathi Viswanadhapalli, Andrew Brenner, Rajeshwar R. Tekmal, Ratna K. Vadlamudi. Significance and functions of estrogen receptor beta (ESR2) isoforms in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5531. doi:10.1158/1538-7445.AM2017-5531

Estrogen-dependent efficacy of limb ischemic preconditioning in female rats.

Our aim was to examine the effects of ischemic preconditioning (IPC) on the local periosteal and systemic inflammatory consequences of hindlimb ischemia-reperfusion (IR) in Sprague-Dawley rats with chronic estrogen deficiency (13 weeks after ovariectomy, OVX) in the presence and absence of chronic 17beta-estradiol supplementation (E2, 20 µg kg-1 , 5 days/week for 5 weeks); sham-operated (non-OVX) animals served as controls. As assessed by intravital fluorescence microscopy, rolling and the firm adhesion of polymorphonuclear neutrophil leukocytes (PMNs) gave similar results in the Sham + IR and OVX + IR groups in the tibial periosteal microcirculation during the 3-h reperfusion period after a 60-min tourniquet ischemia. Postischemic increases in periosteal PMN adhesion and PMN-derived adhesion molecule CD11b expressions, however, were significantly reduced by IPC (two cycles of 10'/10') in Sham animals, but not in OVX animals; neither plasma free radical levels (as measured by chemiluminescence), nor TNF-alpha release was affected by IPC. E2 supplementation in OVX animals restored the IPC-related microcirculatory integrity and PMN-derived CD11b levels, and TNF-alpha and free radical levels were reduced by IPC only with E2. An enhanced estrogen receptor beta expression could also be demonstrated after E2 in the periosteum. Overall, the beneficial periosteal microcirculatory effects of limb IPC are lost in chronic estrogen deficiency, but they can be restored by E2 supplementation. This suggests that the presence of endogenous estrogen is a necessary facilitating factor of the anti-inflammatory protection provided by limb IPC in females. The IPC-independent effects of E2 on inflammatory reactions should also be taken into account in this model. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:97-105, 2018.

Effect of simvastatin on baboon endometriosis.

Endometriosis, a common disorder affecting women of reproductive age, is characterized by ectopic growth of the endometrial tissues, altered steroid hormone response, and inflammation. Previous studies revealed that statins, selective inhibitors of the key step of mevalonate pathway, inhibit growth of endometrial stromal cells in vitro and reduce endometriotic lesions in murine models of endometriosis. This study evaluated the effects of simvastatin on the development of endometriosis in a baboon model of this disease. Sixteen baboons were randomly assigned to the treatment group (simvastatin, 20 mg daily) or to the control group. Endometriotic lesions were evaluated by laparoscopy after 3 months. The volume of red, orange-red, and white endometriotic lesions was significantly reduced by 78% in animals treated with simvastatin. The expression of a marker of proliferation, proliferating cell nuclear antigen (PCNA), was significantly reduced in animals receiving simvastatin in red lesions, white lesions, black lesions, and in adhesions. Simvastatin was also associated with an increase in the expression of estrogen receptor alpha in red lesions, and a decrease in the expression of estrogen receptor beta in black lesions, in adhesions, and in eutopic endometrium. Furthermore, simvastatin significantly reduced the expression of neopterin, a marker of inflammation, oxidative stress, and immune system activation. Collectively, the present findings indicate that the inhibition of the mevalonate pathway by simvastatin reduces the risk of developing endometriosis in the primate model of this disease by decreasing the growth of endometrial lesions, by modulating the expression of genes encoding for estrogen receptors, and by reducing inflammation.

Estradiol via estrogen receptor beta influences ROS levels through the transcriptional regulation of SIRT3 in human seminoma TCam-2 cells.

Human testis, gonocytes, and adult germ cells mainly express estrogen receptor beta, and estrogen receptor beta loss is associated with advanced tumor stage; however, the molecular mechanisms of estrogen receptor beta-protective effects are still to be defined. Herein, we provide evidence that in human seminoma TCam-2 cells, E2 through estrogen receptor beta upregulates the mitochondrial deacetylase sirtuin-3 at protein and messenger RNA levels. Specifically, E2 increases sirtuin-3 expression through a transcriptional mechanism due to the occupancy of sirtuin-3 promoter by estrogen receptor beta, together with the transcription factor Sp1 as evidenced by Chip reChIp assay. This complex binds to a GC cluster located between -128 bp/+1 bp and is fundamental for E2 effects, as demonstrated by Sp1 small interfering RNA studies. Beside, after 24 h, E2 stimulus significantly increased activities of superoxide dismutase and catalase to scavenge reactive oxygen species produced by 30 min of E2 stimulus. In summary, this article indicates a novel functional interplay between estrogen receptor beta and sirtuin-3 counteracting reactive oxygen species production in TCam-2 cells. Our findings thus show that an important tumor-suppressive pathway through estrogen receptor beta is target of E2, actually proposing a distinctive protecting action against seminoma. Future studies may lead to additional strategies for the current therapy of seminoma.

Expression of unusual immunohistochemical markers in mucinous breast carcinoma

BackgroundMucinous breast carcinoma is characterized by the production of variable amounts of mucin. Some studies have addressed immunohistochemical characterization of mucinous breast carcinoma using a limited set of antibodies. However, the purpose of the present study was to investigate a larger panel of markers not widely used in daily practice and to determine their pathological implications. MethodsForty patients diagnosed with mucinous breast carcinoma were enrolled. An immunohistochemical study was performed on whole sections of paraffin embedded tissue, using antibodies for the following markers: estrogen receptor alpha and beta, progesterone receptor, androgen receptor, HER2, EGFR, Ki-67, E-cadherin, β-catenin, p53, chromogranin, synaptophysin, GCDFP15, mammaglobin, and CDX2. ResultsThe pure mucinous type was more prevalent in older patients and more frequently expressed GCDFP15. Capella type B presented more frequently with a high Ki-67 index and neuroendocrine differentiation. Although there was a lower frequency of vascular invasion and lymph node metastases in the pure type, the difference was not statistically significant. No case expressed CDX2 (a marker for gastrointestinal tumors), while 85% of the cases expressed at least one of the two typical breast markers (GCDFP15 and mammaglobin), suggesting that these markers may be reliably used for differential diagnosis. Expression of estrogen receptor beta was related to the presence of mucin cell producing lymph node metastasis, with potential prognostic and predictive value. Conclusionour findings support the immunohistochemical homogeneity of mucinous breast carcinomas because only minor differences were found when subgrouping them into Capella types A and B or into types pure and mixed.

Abstract P5-04-04: Activation of ERβ in triple negative breast cancer results in cell cycle arrest

Abstract Background: Triple negative breast cancer (TNBC), which comprises approximately 20% of breast cancer diagnoses, lacks estrogen receptor alpha, progesterone receptor and Her-2 expression. However, we have identified that 30% of TNBC patients express estrogen receptor beta (ERβ), a nuclear hormone receptor and potential therapeutic target. Here we examine the effects of ERβ in triple negative breast cancer cell lines. Methods: Cell lines that stably express ERβ were used to perform microarray analyses following five days of estrogen treatment. Ingenuity pathway analysis was conducted on differentially expressed genes to determine alterations in biological pathways. The effects of ERβ on cell cycle progression and apoptosis was determined. Cell cycle-related expression changes were confirmed with RT-qPCR and western blotting. The impact of siRNA mediated gene silencing of CDK1 on TNBC cell proliferation was assessed as was the ability of ERβ to elicit anti-proliferative effects in the setting of CDK1 knockdown. Results: We have shown that estrogen or ERβ-specific agonist treatment causes decreased proliferation of ERβ+ TNBC cells. This inhibitory effect is not due to programmed cell death but rather a G1/S phase cell cycle arrest as indicated by flow cytometry experiments. Microarray data and ingenuity pathway analysis revealed a number of down regulated genes involved in cell-cycle progression. Specifically, estrogen treatment of ERβ positive TNBC cells was shown to result in suppression of cyclin-dependent kinase 1 (CDK1) and Cyclin B, effects that were confirmed following ERβ-specific agonist treatment at both the mRNA and protein levels via RT-qPCR and western blotting, respectively. Knockdown of CDK1 in ERβ+ TNBC cells using siRNA resulted in decreased proliferation and diminished the anti-proliferative effects observed following estrogen or ERβ-specific agonist treatment. Conclusions: Our data demonstrate that estrogen and ERβ-specific agonists cause cell cycle arrest in ERβ positive TNBC. These effects are due to ERβ-mediated suppression of multiple genes involved in cell cycle progression including CDK1 and Cyclin B. Following knockdown of CDK1, estrogen or ERβ-specific agonist treatment displayed minimal impact on cell proliferation. Therefore, ERβ's effects on proliferation may primarily be mediated by blockade of CDK1 and Cyclin B. Regardless of ERβ, our data suggest that inhibition of CDK1 activity may have therapeutic benefit in a subset of TNBC patients, an area of study that has yet to be explored. Citation Format: Reese JM, Bruinsma ES, Monroe DG, Goetz MP, Hawse JR. Activation of ERβ in triple negative breast cancer results in cell cycle arrest [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-04-04.

Abstract P3-07-20: Biological functions of ERβ in triple negative breast cancer and its utility as a novel therapeutic drug target

Abstract Background: Triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancer diagnoses. It is the most aggressive form of breast cancer and clinical management is problematic due to lack of available targeted therapies. We have shown that approximately 30% of all TNBCs express estrogen receptor beta (ERβ), a ligand binding transcription factor, and a potential drug target for patients with this form of the disease. Methods: Using novel ERβ-expressing TN cell lines developed in our laboratory, we assessed the impacts of ERβ on proliferation, invasion, migration, and alterations in cell cycle progression following estrogen and ERβ-specific agonist treatment. We also characterized the ERβ transcriptome and cistrome in these models through microarray and ChIP-Seq, respectively. Finally, we determined the tumoral response of cell line xenografts and PDXs treated with 17β-estradiol. Results: We found that both estrogen and multiple ERβ-specific agonists elicit significant anti-tumor effects in ERβ+ TNBC cell lines and tumor xenografts. Activation of ERβ with estrogen and ERβ-specific agonists resulted in inhibition of cell proliferation primarily through a G1/S phase cell cycle arrest. Substantial reductions in cell migration and invasion were also observed following treatment. Microarray studies revealed that ERβ differentially regulated the expression of approximately 1000 genes following estrogen treatment. Of these genes, the most striking effects were observed in a family of small secreted cysteine protease inhibitors known as cystatins, which were highly induced following ERβ activation. ChIP-Seq and ChIP-PCR identified ERβ binding sites in the promoter region of each cystatin and demonstrated ERβ-mediated alterations in chromatin marks and recruitment of PolII around these promoters. We found that cystatins directly interact with TGFβ receptor 2 (TGFβR2) and block downstream TGFβ ligand-mediated activation of the canonical signaling pathway. Depletion of cystatins from conditioned media or through siRNA-mediated silencing reduced the ability of ERβ to elicit these anti-tumor effects. In vivo, estrogen treatment of mice harboring ERβ+ TNBC cell line xenografts or PDXs resulted in increased tumoral expression and serum levels of cystatins, and suppressed tumor growth. Conclusions: Our data demonstrated that estrogen and ERβ-specific agonists elicit anti-cancer effects in ERβ+ TNBC, both in vitro and in vivo. These effects are partially mediated by cystatins which can interact with, and inhibit, canonical TGFβ signaling, a pathway known to drive TNBC progression. Given the lack of targeted therapies for TNBC patients, the present data suggests that estrogen or ERβ-specific agonists offer a novel approach to manage this subset of patients. Citation Format: Reese JM, Bruinsma ES, Suman VJ, Nelson AW, Chernukhin I, Carroll JS, Ingle JN, Goetz MP, Hawse JR. Biological functions of ERβ in triple negative breast cancer and its utility as a novel therapeutic drug target [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-20.

Serotonin receptor 4 (5-hydroxytryptamine receptor Type 4) regulates expression of estrogen receptor beta and cell migration in hormone-naive prostate cancer.

Estrogens are considered to potentially play some roles in the development and progression of prostate cancer through estrogen receptor beta (ERβ). However, additional factors which could influence the clinical outcome of the patients through modulating these steroid signalings have also been proposed. Among these, increased expression of serotonin receptor especially that of 5-hydroxytryptamine receptor Type 4 (5-HTR4) has been recently proposed to be involved in autocrine/paracrine mechanisms of castration-resistant prostate cancer, but the presence and clinical significance of 5-HTR4 in hormone-naive prostate cancer (HNPC) and its interaction with hormonal signaling pathways have remained virtually unknown. We evaluated the status of 5-HTR4 in 112 human HNPC cases (acinar adenocarcinoma) using immunohistochemistry and correlated the findings with clinicopathological features of individual patients and the status of androgen receptor (AR) and ERβ. To further elucidate its underlying mechanisms, androgen-dependent human prostate carcinoma cell line, LNCaP, expressing 5-HTR4, was treated by 5-HTR4 agonist. 5-HTR4 immunoreactivity was detected in 34% of prostate cancer cases examined (38/112) and was significantly correlated with the status of ERβ but not with that of AR and other clinicopathological factors of the patients. Results of in vitro studies demonstrated that 24 h incubation with 5-HTR4 agonist (10 nM) increased the expression level of ERβ messenger RNA compared to controls. 5-HTR4 agonist (100 nM) significantly inhibited LNCaP carcinoma cell migration (P < 0.05). Results of our present study indicated that 5-HTR4 signaling upregulated ERβ expression in HNPCs and could impact on biological processes in HNPC.

Expression of estrogen receptor beta isoforms in pancreatic adenocarcinoma.

337 Background: There have been several limited trials in which tamoxifen efficacy was tested in patients with pancreatic adenocarcinoma (PAC). Most of these studies were small series of patients with unresectable PAC and reported mixed results. In these studies, patients were not stratified by estrogen receptor status because estrogen receptor beta (ER-b) had not yet been identified, and PAC did not express the traditional ER-alpha. Recent studies showed that the effects of estrogens, phytoestrogens and tamoxifen on PAC cell lines depended on ER-b expression. The aim of this study was to investigate ER-b expression in human PAC and whether such expression correlates with any clinicopathologic parameters. Methods: Sections of tissue microarray containing 18 formalin fixed and paraffin embedded human PAC were stained by immunohistochemistry (IHC) using monoclonal antibodies to ER-b isoforms 1, 2, and 5 (ER-b1, ER-b2, and ER-b5, respectively), and for Cyclin A. The levels of ER-b isoform expression in tumor cells and the S-phase fraction (SPF) were determined using a quantitative digital image analysis solution (OTMIAS). Results: All isoforms were expressed in PAC, although at different levels. Higher mean ER-b2 levels correlated with male sex (p = 0.057), older age (p = 0.005), and lower pT stage (p = 0.008), but not with grade, pN stage, or SPF. Mean ER-b5 levels correlated negatively with SPF (p = 0.021), but not with sex, age, grade, pT or pN. Mean ER-b1 expression did not correlate with any of the above mentioned clinicopathologic factors. Conclusions: ER-b1, ER-b2, and ER-b5 are expressed in PAC. Higher ER-b2 and ER-b5 levels of expression are significantly correlated with lower tumor pT stage and with lower SPF, respectively, suggesting that they may play a tumor suppressive role in PAC. The association between ER-b2 levels and patient sex and age suggest that it could be influenced by endogenous/exogenous hormonal exposure.

Expression of Estrogen Receptor Alpha in Malignant Melanoma.

Background:Features of malignant melanoma (MM) vary in the different geographic regions of the world. This may be attributable to environmental, ethnic, and genetic factors. The aim of this study was to determine the expression of estrogen receptor alpha (ER-α) in MM in Isfahan, Iran.Materials and Methods:This study was planned as a descriptive, analytical, cross-sectional investigation. During this study, paraffin-embedded tissue blocks of patients with a histopathologic diagnosis of MM was studied for ER-α using immunohistochemistry (IHC).Results:In this study, 38 patients (female/male; 20/18) with a definite diagnosis of malignant cutaneous melanoma and mean age of 52.4 ± 11.2 years were investigated. Using envision IHC staining, there were not any cases with ER-α expression.Conclusion:In confirmation to the most previous studies, expression of ER-α was negative in MM. It is recommended to investigate the expression of estrogen receptor beta and other markers in MM.

The presence and impact of estrogen metabolism on the biology of triple-negative breast cancer.

While triple-negative breast cancer (TNBC) is negative for estrogen receptor alpha, a substantial proportion of carcinomas express estrogen receptor beta (ERβ); consequently, estrogen actions and metabolism may be relevant in this cancer subtype. A cohort of 81 TNBC patients from Tohoku University Hospital, Japan were characterised with regard to the expression of estrogen receptor beta and enzymes known to modulate levels of estrogens in breast and other tissues (Aromatase, 17-beta- Hydroxysteroid dehydrogenases 1, 2 and 6). This was done at the protein level by means of immunohistochemistry. As this cohort has been previously characterised for androgens, this also allows for comparison between the expressions of estrogen-related proteins and of androgen-related proteins. Preliminary mechanistic studies in cell culture were also undertaken. 17βHSD2 was detected in the highest number of cases followed by 17βHSD1, 17βHSD6 and aromatase. When comparing the expression of ERβ with that of the enzymes, it was positively correlated with the expression of 17βHSD6 (p<0.05) and trended towards correlation with dual expression of 17βHSD1 and 2 (p<0.07). 17βHSD1 was associated with significantly reduced tumour volume (p=0.0025), while ERβ was associated with a trend towards reduced lymphovascular invasion, (p<0.061). Interestingly, in survival analysis, 17βHSD6 expression was the only one of these five factors that influenced survival, with positive samples being associated with longer disease-free survival compared to those that were negative for 17βHSD6 (p<0.05). In assessing associations with expression of proteins in the androgenic pathway, expression of aromatase appeared to be associated with androgenic pathways in TNBC patients (p<0.05). Due to this association and the potential relevance to androgen-directed therapies in TNBC, we evaluated this interaction in vitro. We observed androgen-dependent upregulation of aromatase and ERβ in a subset of AR expressing TNBC cell lines (MDA-MB-453, SUM-185-PE and MFM-223). Overall this study suggests the presence of, and a potential protective effect of estrogens in TNBC.

Insulin-like growth factor-I stimulates estrogen receptor beta and aromatase expression via PI3K/AKT-mediated transcriptional activation in endometriosis

Insulin-like growth factor-I stimulates estrogen receptor beta and aromatase expression via PI3K/AKT-mediated transcriptional activation in endometriosis

Developmental Toxicant Exposure Is Associated with Transgenerational Adenomyosis in a Murine Model.

The common environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or, commonly, dioxin) is a known endocrine disruptor that has been linked to the development of endometriosis in experimental models. Using a murine model, we previously demonstrated that in utero TCDD exposure promotes the transgenerational development of an “endometriosis-like” uterine phenotype consisting of reduced responsiveness to progesterone, as well as subfertility and an increased risk of preterm birth. Because adenomyosis is frequently observed as a comorbidity in women with endometriosis, herein we sought to determine the incidence of adenomyosis in nonpregnant mice with a history of direct or indirect TCDD exposure. Using histologic assessment and immunohistochemical staining, we analyzed murine uteri for adenomyosis, microvessel density, and expression of estrogen receptors alpha and beta (ESR1 and ESR2). Our studies revealed that unexposed control mice did not exhibit adenomyosis, whereas this disease was frequently observed in mice with a history of early-life TCDD exposure. A transgenerational impact of developmental TCDD exposure was demonstrated, because a subset of mice with only an indirect exposure (F3) also exhibited adenomyosis. Microvessel density within the uterus was significantly higher in all groups of TCDD-exposed mice compared with control animals, with density correlated to the severity of disease. Both ESR1 and ESR2 proteins exhibited alterations in expression in experimental mice compared with controls. Similar to women with endometriosis, we observed a significant reduction in the ratio of Esr1:Esr2 mRNA in all F1 mice compared with controls. Although this retrospective study was not designed to specifically address mechanisms associated with the development of adenomyosis, our data suggest that developmental TCDD exposure permanently alters adult steroid responses, which may contribute to the transgenerational development of adenomyosis.

Abstract 3263: Dietary supplement 3, 3’-diindolylmethane (DIM) as an antiangiogenic agent in breast cancer

Abstract Tumor angiogenesis refers to the sprouting and cooption of proliferating endothelial cells (EC’s) from adjacent pre-existing host vasculature, and is a key target of cancer therapy. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to promote and support angiogenesis. Within this complex tumor microenvironment, we and others have shown that tumors can recruit bone marrow derived endothelial progenitor cells that differentiate into mature bone marrow-derived endothelial cells and incorporate into sprouting tumor neovessels. Under pathological circumstances, such as breast cancer, a clear association between estrogen receptor expression by EC’s, angiogenic activity, and/or tumor invasiveness has been made. Approximately, 80% of breast cancers are hormone-receptor-positive cancers, thus enabling tamoxifen as the mainstay of breast cancer therapy. The roles of the anti-estrogens fulvestrant (ICI) and the dietary supplement 3, 3’-diindolylmethane (DIM) on cell-cell interaction and angiogenesis have not been fully elucidated. This study is designed to evaluate and compare the effect of these antiestrogens on angiogenesis at the cellular and molecular levels using tube formation of human umbilical vein endothelial cells (HUVEC) as an in vitro angiogenesis model. HUVEC cells were treated with serial dilutions of either DIM or ICI in presence and absence of (3nM) estrogen, and subjected to in vitro tube formation, proliferation, migration, and angiogenesis antibody array assays. We report that HUVEC cells are more sensitive to DIM than ICI. At 25 μM concentration, DIM significantly inhibited the crucial steps of angiogenesis including HUVEC cells proliferation, migration, cytokine release, and tube formation in an estrogen independent manner. On the other hand, at 1 μM concentration, ICI significantly exerted an antiangiogenic effects inhibiting HUVEC cells proliferation, migration, and tube formation, but this effect was totally dependent on the presence of estrogen. These results are validated by our observation that HUVEC cells express estrogen receptor beta (ER-β) and not estrogen receptor alpha (ER-α). A correlative effect between the antiangiogenic activity of DIM and ER-β upregulation was noted. We believe that the anti-estrogenic activity of DIM is mediated through the genomic and non-genomic activity of ER-β in endothelial cells predicting a new target for DIM to manifest its antiangiogenic effect. Citation Format: Ghada M. Ben Rahoma, Neha Y. Tuli, Robert B. Bednarczyk, Rachana R. Maniyar, Abraham Mittelman, Jan Geliebter, Raj Tiwari. Dietary supplement 3, 3’-diindolylmethane (DIM) as an antiangiogenic agent in breast cancer. [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 3263.

353 - Quantitative analysis of estrogen receptors alpha and beta expression in ovarian cancer patients by flow cytometry

353 - Quantitative analysis of estrogen receptors alpha and beta expression in ovarian cancer patients by flow cytometry

Hindbrain estrogen receptor-beta antagonism normalizes reproductive and counter-regulatory hormone secretion in hypoglycemic steroid-primed ovariectomized female rats

Hindbrain dorsal vagal complex A2 noradrenergic signaling represses the pre-ovulatory luteinizing hormone (LH) surge in response to energy deficiency. Insulin-induced hypoglycemia augments A2 neuron adenosine 5′-monophosphate-activated protein kinase (AMPK) activity and estrogen receptor-beta (ERβ) expression, coincident with LH surge suppression. We hypothesized that ERβ is critical for hypoglycemia-associated patterns of LH secretion and norepinephrine (NE) activity in key reproduction-relevant forebrain structures. The neural mechanisms responsible for tight coupling of systemic energy balance and procreation remain unclear; here, we investigated whether ERβ-dependent hindbrain signals also control glucose counter-regulatory responses to hypoglycemia. Gonadal steroid-primed ovariectomized female rats were pretreated by caudal fourth ventricular administration of the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) or vehicle before insulin injection at LH surge onset. Western blot analysis of laser-microdissected A2 neurons revealed hypoglycemic intensification of AMPK activity and dopamine-β-hydroxylase protein expression; the latter response was attenuated by PHTPP pretreatment. PHTPP regularized LH release, but not preoptic GnRH-I precursor protein expression in insulin-injected rats, and reversed hypoglycemic stimulation of glucagon and corticosterone secretion. Hypoglycemia caused PHTPP-reversible changes in NE and prepro-kisspeptin protein content in the hypothalamic arcuate (ARH), but not anteroventral periventricular nucleus. Results provide novel evidence for ERβ-dependent caudal hindbrain regulation of LH and counter-regulatory hormone secretion during hypoglycemia. Observed inhibition of LH likely involves mechanisms at the axon terminal that impede GnRH neurotransmission. Data also show that caudal hindbrain ERβ exerts site-specific control of NE activity in forebrain projection sites during hypoglycemia, including the ARH where prepro-kisspeptin may be a target of that signaling.

Reversal of fortune: estrogen receptor-β in endometriosis.

Enhanced inflammation and reduced apoptosis sustain the growth of endometriotic lesions. Alterations in the expression of estrogen receptor-alpha (ERα) and estrogen receptor-beta (ERβ) accompany the conversion of resident endometrial cells within the normal uterine environment to ectopic lesions located in extrauterine sites. Recent studies highlighted in this focused review linked ERβ to dysregulation of apoptotic and inflammatory networks involving novel interacting partners in endometriosis. The elucidation of these nongenomic actions of ERβ using human cells and mouse models is an important step in understanding key regulatory pathways that are disrupted leading to disease establishment and progression.