Estrogen Receptor Subtypes Research Articles

Prognostically Relevant Subtypes and Survival Prediction for Breast Cancer Based on Multimodal Genomics Data

Cancer is one of the deadliest diseases caused by abnormal behaviors of genes that control the cell division and growth. Genomics data and clinical outcomes from multiplatform and heterogeneous sources are used to make clinical decisions for the cancer patients, where both multimodality and heterogeneity impose significant challenges to bioinformatics tools and algorithms. Numerous works have been proposed to overcome these challenges by using sophisticated bioinformatics and machine learning algorithms as either primary or supporting tools. In this paper, we propose a new approach to analyze genomics data from The Cancer Genome Atlas (TCGA) to classify breast cancer patients based on their subtypes and survival rates. Since multiple factors such as estrogen receptor (ER), progesterone receptor (PGR), and human epidermal growth factor receptor 2 (HER2) statuses are involved in breast cancer diagnosis, we used DNA methylation, gene expression (GE), and miRNA expression data by creating a multiplatform network called Multimodal Autoencoders (MAE) classifier to support each data type. Experiment results demonstrate that our approach is promising with high confidence for predicting both breast cancer subtypes and survival rates. In particular, we achieved state-of-the-art results with accuracies of 91% and 86%, respectively for the ER and PGR-based subtype prediction and moderately low accuracy for the HER2-based subtype prediction as well as we perceived reasonably low MSE and positive coefficient of determination (R 2 ) scores in case of survival prediction. Additionally, we created unimodal and multimodal features from each input type and trained decision tree (DT), Naive Bayes (NB), K-nearest neighbors (KNN), logistic regression (LR), support vector machine (SVM), random forest (RF), and gradient boosting trees (GBT) as ML baseline models. Finally, we use the model averaging ensemble of top-3 models to report the final prediction.

Huaier polysaccharide inhibits the stem-like characteristics of ERα-36high triple negative breast cancer cells via inactivation of the ERα-36 signaling pathway.

Triple negative breast cancer (TNBC) is a highly aggressive cancer and lack of targeting therapies. It is believed that the breast cancer stem cells (BCSCs) are responsible for the aggressive characteristics of TNBC. Hence, developing BCSC-targeting agents may provide new therapeutic strategies for the patients. Huaier polysaccharide (HP), an active ingredient extracted from the mushroom Trametes robiniophila Murr, has been widely used in clinical anti-cancer treatments in China. Here we demonstrated that HP could target BCSCs in TNBC cells, resulting in decreased mammosphere formation, downregulated expression of stem-related genes and reduced proportion of aldehyde dehydrogenase positive cells in vitro, and inhibited xenograft tumor formation in vivo. Mechanically, HP markedly reduced the expression of estrogen receptor α-36 (ERα-36), a recently identified subtype of estrogen receptor α, and attenuated ERα-36-mediated activation of AKT/β-catenin signaling in ERα-36high TNBC cells. This study provides a new insight into the mechanism of HP on BCSC-targeting therapy and new ideas for comprehensive treatment strategies for TNBC.

Evaluation of endocrine activities of ellagic acid and urolithins using reporter gene assays

Urolithins are metabolites produced in the gut following consumption of ellagitannins and ellagic-acid-rich food, such as pomegranates, berries, and nuts. Compelling biological activities of urolithins together with variabilities between individuals in the metabolic capacity of the resident gut microbiota to produce urolithins, have suggested potential benefits of direct consumption of urolithins. Based on the structures of ellagic acid and urolithins, they might be expected to show endocrine effects. We report on their impact on the estrogen, androgen, glucocorticoid and thyroid-hormone receptors, as determined in vitro using reporter gene assays in the Hela9903 (estrogen receptor), MDA-kb2 (androgen and glucocorticoid receptors) and GH3.TRE-Luc (thyroid hormone receptor) cell lines. Urolithins A and B, but not ellagic acid and urolithin D, showed estrogenic activities on estrogen receptor subtype α under our assay conditions, with EC50 values of 5.59 μM and 32.60 μM, respectively. Moreover, ellagic acid and urolithins A and D showed anti-thyroid hormonal activities (IC50 values of 37.45 μM, 30.32 μM and 8.80 μM, respectively). Glucocorticoid and androgen agonist and antagonist activities were assessed using a luciferase reporter gene assay in the MDA-kb2 cell line. None of these tested compounds showed glucocorticoid agonist or antagonist activities, and ellagic acid showed weak androgen agonist activity, although only at the highest concentration tested. Detected estrogen and antithyroid activities warrant further risk assessment in relation to the exposure of urolithins in humans.

Non-Small Cell Lung Cancer with Estrogen Receptors and ALK Positivity.

Estrogens are steroid hormones that affect a number of physiological functions in cells; these compounds play an irreplaceable role in the reproductive system. Their effects are mediated by the estrogen receptor (ER), of which there are two subtypes - ERα and ERβ. ERs are expressed in regions outside the reproductive system, including bone, brain, intestine, endothelium, kidney, and lung. Expression of ER by lung cancer (LC) cells was first described in the early 1980s. The experimental literature also describes co-expression of ERβ and an epidermal growth factor activating mutation, and the additive antiproliferative effects of anti-estrogens plus tyrosine kinase inhibitors during treatment of lung adenocarcinoma. However, coincident expression of ER and ALK translocation were not decribed. The 4-year survival for generalized non-small cell lung cancer indicates the possibility that both molecular features (ER and ALK positivity) may be a favorable prognostic biomarker for this tumor. A 69-year-old patient presented with generalized lung adenocarcinoma that was positive for ERb and the ALK-EML4 fusion gene. The tumor was stage IV. Additional examinations excluded malignancy of the gynecological area. The patient was treated with chemotherapy and atyrosine kinase ALK inhibitor. The role of individual ER subtypes in LC carcinogenesis, and the possible therapeutic effects, is unclear. This is the first documented case of co-occurrence of ALK-EML4 and ERβ in LC. Key words non-small cell lung cancer - estrogen receptors - ALK translocation - prognosis - treatment This work was supported by Ministry of Health of the Czech Republic, grant AZV 16-32318A, and by Ministry of Education, Youth and Sports of the Czech Republic, grant NPU I LO1304. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 23. 9. 2018 Accepted: 25. 10. 2018.

Repeated Exposure to 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), an Active Metabolite of Bisphenol A, Aggressively Stimulates Breast Cancer Cell Growth in an Estrogen Receptor β (ERβ)-Dependent Manner.

Bisphenol A (BPA), recognized as an endocrine disruptor, is thought to exert its activity through a mechanism involving the activation of estrogen receptors (ERs) α/β However, a major problem is that very high concentrations of BPA are required (i.e., those in excess of environmental levels) for effective activation of ERα/β-mediated transcriptional activities in vitro, despite the BPA-induced estrogenic effects observed in vivo. To elucidate the causal reasons, we successfully identified a BPA metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which exhibits highly potent estrogenic activity both in vivo and in vitro. We have focused on the biologic relationship between breast tumor promotion and MBP/BPA, because BPA is considered to be a human carcinogen owing to its breast tumor-promoting properties. In general, humans are exposed to many endocrine disruptors, including BPA. In the present study, we used the ERα/β-positive human breast cancer cell line MCF-7 as an experimental model to investigate the effects of repeated exposure to BPA/MBP at concentrations found in the environment on the expression of ERα/β and to determine the particular ER subtype involved. We demonstrated that repeated exposure to MBP, but not to BPA, significantly downregulated ERα protein expression and stimulated the proliferation of MCF-7 cells through the activation of ERβ-mediated signaling.

Oestrogen differentially modulates lymphoid and myeloid cells of the European sea bass in vitro by specifically regulating their redox biology

Besides their obvious role in sex determination and reproduction, oestrogens display a prominent and complex immunomodulatory role across all vertebrates. To date, our knowledge on the oestrogenic immunomodulation in non-mammalian species is, however, scarce. In both teleosts and mammals, the direct immunomodulatory function of oestrogen is underscored by the presence of multiple oestrogen receptor subtypes in the various immune cells. For a better understanding of the regulatory processes, we investigated the oestrogen receptor expression in two major lymphoid organs of European sea bass: the head-kidney and the spleen. All oestrogen receptor subtypes, including nuclear and membrane oestrogen receptors, were present in both immune organs as well as in the isolated leucocytes. The same findings have been previously made for the thymus. To determine the oestrogen responsiveness of the different immune cell populations and to evaluate the importance of non-genomic and genomic pathways, we assessed the kinetics and the concentration dependent effects of 17β-oestradiol on isolated leucocytes from the head-kidney, the spleen and the thymus in vitro. Given the importance of reactive oxygen species as signalling and defence components in mammalian immune cells, the oxidative burst capacity, the redox status and the viability of both lymphoid and myeloid cells were measured by flow cytometry. The treatment with 17β-oestradiol specifically modulated these parameters depending on (1) the time kinetic, (2) the concentration of 17β-oestradiol, (3) the immune cell population (lymphoid and myeloid cells) as well as (4) the lymphoid organs from which they originated. The observed in vitro oestrogenic effects as well the presence of various oestrogen receptor subtypes in the immune cells of sea bass suggest a complex and direct oestrogenic action via multiple interconnected oestrogen-signalling pathways. Additionally, our study suggests that the oestrogenic regulation of the sea bass immune function involves a direct and tissue specific modulation of the immune cell redox biology comprising redox signalling, NADPH-oxidase activity and H2O2-permeability, thus changing oxidative burst capacity and immature T cell fate because oestrogen impacted thymocyte viability. Importantly, immune cells from both primary and secondary lymphoid organs have shown specific in vitro oestrogen-responsiveness. As established in mammals, oestrogen is likely to be specifically and directly involved in immature T cell differentiation and mature immunocompetent cell function in sea bass too.

Associations of adult-attained height and early life energy restriction with postmenopausal breast cancer risk according to estrogen and progesterone receptor status.

Adult-attained height is a marker for underlying mechanisms, such as cell growth, that may also influence postmenopausal breast cancer (BC) risk, perhaps specifically hormone-sensitive BC subtypes. Early life energy restriction may inhibit these mechanisms, resulting in shorter height and a reduced postmenopausal BC risk. Women (62,573) from the Netherlands Cohort Study completed a self-administered questionnaire in 1986 when 55-69 years old, and were followed-up for 20.3 years (case-cohort: Nsubcohort = 2,438; Ncases = 3,354). Cox multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI) were estimated for BC risk overall and by estrogen and progesterone receptor subtypes in relation to height and early life energy restriction during the Hunger Winter, War Years, and Economic Depression. Although energy restriction can only influence longitudinal growth in women exposed before and/or during the growth spurt, it may also influence BC risk when occurring after the growth spurt, possibly through different growth processes. Therefore, Cox analyses were additionally conducted according to timing of energy restriction in relation to the growth spurt. Height was associated with an increased BC risk (HRper 5cm = 1.07, 95%CI:1.01-1.13), particularly hormone receptor-positive BC. Energy restriction before and/or during the growth spurt was associated with a decreased hormone receptor-positive BC risk. Energy restriction during the Hunger Winter increased the estrogen receptor-negative BC risk regardless of the timing of energy restriction. In conclusion, height and energy restriction before and/or during the growth spurt were both associated with hormone receptor-positive BC risk, in the direction as expected, indicating critical exposure windows for hormonal growth-related mechanisms.

Differential involvement of the three nuclear estrogen receptors during oogenesis in European sea bass (Dicentrarchus labrax)†.

Estrogens are involved in a wide range of processes in vertebrate reproduction through ligand activation of their specific cognate receptors. In most teleosts, three nuclear estrogen receptor subtypes have been identified (Esr1, Esr2a, and Esr2b). Differences in ligand binding affinity and seasonal expression patterns in reproductive tissues among these Esr subtypes suggest distinct roles during oogenesis, vitellogenesis, and spermatogenesis. This study focuses on the role of the Esr subtypes in European sea bass (Dicentrarchus labrax) oogenesis and their endocrine regulation. The coding genes of the three Esr subtypes are highly expressed in reproduction-related tissues such as pituitary, gonad, and liver. Quantification of esr1, esr2a, and esr2b expression in the ovary and liver during a whole reproductive cycle showed different patterns depending on stage and subtype, suggesting differential roles of the three receptors in the regulation of oogenesis and vitellogenesis. Esr2a and Esr2b also showed differences in transcriptional activity and ligand affinity when functionally characterized in HEK293 cells. Finally, for the first time in teleosts, the localization of the three Esr subtypes in ovarian follicles and their regulation by gonadotropins is described. Immunodetection of the receptors revealed different distribution patterns in follicular cells and various subcellular locations of the oocyte. Gonadotropin stimulation of ovarian follicles in different stages of vitellogenesis showed a consistent induction of esrb2b expression by Fsh. All together, these data reinforce the hypothesis that each estrogen receptor plays a specific role in oogenesis.

Combinatorial anti-proliferative effects of tamoxifen and naringenin: The role of four estrogen receptor subtypes

Breast cancer is the most diagnosed diseases and the second-leading cause of death in females, among which the estrogen receptor positive (ER+) patients are more common of all cases. In present study, we selected MCF-7 as an in vitro model and investigated the combinatorial anti-proliferative effects of tamoxifen and naringenin on ER+ breast cancer, and then explored the potential mechanisms involved in mitochondrial dysfunction and oxidative stress mediated by several estrogen receptor subtypes. Six assessment endpoints including cell viability, cell migration, cell cycle, cell apoptosis, mRNA, and protein expression were estimated. Tamoxifen and naringenin were shown to inhibit the cell growth of MCF-7 cells at higher concentrations, and co-exposure with them significantly inhibited cell proliferation in an additive manner. Results from a wound healing assay indicated that the combined treatment of tamoxifen and naringenin markedly suppressed cell migration compared with the single exposure by downregulating the expression of MMP-9 and MMP-2. Flow cytometry analysis revealed that the combined treatment of tamoxifen and naringenin blocked cell cycle in G2/M phase through suppressing the transcription of cell cycle regulation proteins. Simultaneously, co-treatment with them also induced cell apoptosis by regulating the expression of mitochondrial apoptotic proteins as well as by simulating the production of reactive oxygen species (ROS). Real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB) analysis results further demonstrated that the two nuclear estrogen receptors—ERα66 and ERβ, as well as the two membrane estrogen receptors—ERα36 and GPR30 were downregulated when cells were exposed to single tamoxifen, whereas naringenin treatment group not only downregulated the expression of ERα66 and GPR30 but also upregulated ERβ and ERα36. Interestingly, co-treatment group resulted in significant downregulation of ERα66, ERα36 and GPR30 but upregulation of ERβ. Taken together, co-treatment of tamoxifen and naringenin could inhibit cell proliferation more effectively in ER+ breast cancer cells, which was associated with a modulation of the expression levels of several estrogen receptors.

Neuroprotective effect of Asparagus racemosus root extract via the enhancement of brain-derived neurotrophic factor and estrogen receptor in ovariectomized rats

Ethnopharmacological relevanceAsparagus racemosus (AR) is well known as an Ayurvedic rasayana which used traditionally by Ayurvedic practitioners for nervous disorders and prevent aging. In our previous study it was found that ethanol AR root extract can improve learning and memory impairment, induced by an ovariectomy, but the extract's mechanisms as a neuroprotective property are still unknown. Aim of the studyThis study aimed to examine the effects and mechanisms of ethanol AR root extract on the alteration of brain-derived neurotrophic factor (BDNF) and estrogen receptor (ER) subtypes in ovariectomized (OVX) rats. Materials and methodsAdult female Wistar rats were divided into five groups, 4 groups underwent ovariectomy, and one group was designed to be the sham control group. Two groups were gavaged with propylene glycol for sham, and a second group similarly prepared for OVX. Two further groups of OVX rats were gavaged once daily, one group with 100 mg/kg b.w. of ethanol AR root extract and the second group with 1000 mg/kg b.w. of ethanol AR root extract. The fifth group was gavaged once daily with 0.1 mg/kg b.w. of 17α-ethynylestradiol (EE). BDNF, ERα and ERβ expression were evaluated by western blot analysis. ResultsThe western blot analysis revealed that the OVX rats showed a significant decrease in BDNF and a down-regulation of ERα and ERβ in the frontal cortex and hippocampus. It was also demonstrated that EE and AR root extract increased BDNF, ERα and ERβ in the frontal cortex and hippocampus of ovariectomized rats. ConclusionsBased on these results, the enhancement of BDNF and ERs up-regulation may be involved in the neuroprotective effects of ethanol AR root extract in ovariectomized rat.

17β‐Estradiol inhibits testosterone‐induced cell proliferation in HepG2 by modulating the relative ratios of 3 estrogen receptor isoforms to the androgen receptor

Sex hormones, especially 17β-estradiol (E2) and testosterone (TEST), play crucial roles in the oncogenesis and progression of liver cancer via hormone-related receptors. As women have a lower rate of hepatocellular carcinoma (HCC) than men, estrogens might attenuate the occurrence and development of HCC. This study aimed to investigate the inhibitory effects and mechanisms of E2 on TEST-induced HCC development; the HepG2 cell line was used as an in vitro model. Five endpoints, including cell viability, cell apoptosis, cell cycle, receptor protein expression, and messenger RNA transcription, were investigated. Different roles and the ratios of androgen receptor (AR) and 3 estrogen receptor (ER) subtypes were also estimated. Cell viability assay showed that co-treatment of E2 and TEST resulted in a significant inhibition of E2-induced or TEST-induced cell proliferation. Flow cytometry analysis revealed that combined treatment of E2 and TEST blocked the cell cycle in the G0/G1 phase as well as induced cell early apoptosis, characterized by decreased cyclin-dependent kinase transcription and the ratio of Bcl-2/Bax. Real-time quantitative polymerase chain reaction and Western blot analysis results further demonstrated that estrogen receptor estrogen receptor α66 (ERα66) and estrogen receptor β (ERβ) were upregulated, whereas AR and estrogen receptorα36 (ERα36) were downregulated, irrespective of whether E2 and TEST were considered separately or together, whereas the combined treatment of E2 and TEST resulted in a decrease in the ERα66/ERβ ratio, the ERα66/ERα36 ratio, and the ERβ/ERα36 ratio, but with an increase in the ERα66/AR ratio, the ERα36/AR ratio, and the ERβ/AR ratio. To sum up, E2 could inhibit TEST-induced cell proliferation by modulating the ratio of different hormone-related receptors.

Estrogen relaxes gastric muscle cells via a nitric oxide- and cyclic guanosine monophosphate-dependent mechanism: A sex-associated differential effect.

Various gastrointestinal (GI) disorders have a higher prevalence in women than in men. In addition, estrogen has been demonstrated to have an inhibitory effect on the contractility of GI smooth muscle. Although increased plasma estrogen levels have been implicated in GI disorders, the role of gastric estrogen receptor (ER) in these sex-specific differences remains to be fully elucidated. The present study was designed to investigate the sex-associated differences in the expression of the two ER isoforms, ERα and ERβ, and the effect of estrogen on gastric muscle contraction via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway. Experiments were performed on single gastric smooth muscle cells (GSMCs) isolated from male and female Sprague Dawley rats. The effect of acetylcholine (ACh), a muscarinic agonist, on the contraction of GSMCs was measured via scanning micrometry in the presence or absence of 1 µM 17β-estradiol (E2), an agonist to the majority of ERs, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), an ERα agonist, or diarylpropionitrile (DPN), an ERβ agonist. The protein expression levels of ER subtypes in GSMCs were measured using a specifically designed ELISA. GSMCs from female rats had a higher expression of ERα and ERβ protein compared with GSMCs from males. ACh induced less contraction in female that in male GSMCs. Pre-treatment of GSMCs with E2 reduced the contraction of GSMCs from both sexes, but to a greater extent in those from females. PPT and DPN inhibited ACh-induced contraction in GSMCs from females. Furthermore, E2 increased NO and cGMP levels in GSMCs from males and females; however, higher levels were measured in females. Of note, pre-incubation of female GSMCs with Nω-nitro-L-arginine, a NO synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylyl cyclase inhibitor, reduced the inhibitory effect of estrogen on GSMC contraction. In conclusion, estrogen relaxes GSMCs via an NO/cGMP-dependent mechanism, and the reduced contraction in GSMCs from females by estrogen may be associated with the sex-associated increased expression of ERα and ERβ, and greater production of NO and cGMP, compared with that in GSMCs from males.

Abstract 5421: Roles of sex hormone pathways in the gender disparity of esophageal squamous-cell carcinoma

Abstract Objectives: Esophageal squamous-cell carcinoma (ESCC) incidence has been increasing rapidly, with an imbalanced ratio of occurrence in men more often than in women. The expression of sex hormone receptors is associated with the prognosis of ESCC. However, the exact roles of sex hormones and hormone receptors in ESCC remain unclear. We systematically evaluated the effects and the underlying mechanism of sex hormones and their receptors on esophageal carcinogenesis and malignant proliferation to clarify the extent to which sex hormones pathways contribute to the gender disparity of ESCC. Methods: To determine the roles of hormones in vivo, we used a 4NQO-induced esophageal carcinogenesis model, a xenograft model and the castrated BALB/c Nude mouse model. To observe the function of hormones on the proliferation of ESCC cells in vitro, we performed colony formation, Cell Counting Kit-8, and EdU assays. To elucidate the precise molecular mechanism by which hormone receptors exert their functions, we performed ChIP-seq and RNA-seq to comprehensively identify the ESCC-specific hormone responsive genes and regulatory networks. Results: There is a large male predominance in the 4NQO-induced esophageal carcinogenesis C57BL/6J mouse model. AR-positive esophageal cancer cells grow more rapidly in the male BALB/c Nude mice than the female mice. ERα-positive esophageal cancer cells possess faster proliferative capability in the female BALB/c Nude mice compared to the male mice, whereas ERβ-positive cells exhibit the opposite gender disparity in phenotypes. Moreover, the above proliferative effect is attenuated in castrated or ovariectomized mice. Furthermore, exogenous supplementation of estrogen pellet in ovariectomized female mice enhances ERα-positive cell growth but suppresses ERβ-positive cell growth. In vitro experimental data show that dihydrotestosterone treatment promotes the growth of AR positive ESCC cells, while 17-β estradiol (E2) exerts opposite functions depending on the estrogen receptor subtypes. Integrative analysis of ChIP-seq and RNA-seq data reveals that AR/ER is transactivated upon hormone treatment and binds to the promoter or enhancer of hormone responsive genes to induce differential gene expression. These genes are responsible for the malignant development of ESCC and may provide a new explanation of the gender disparity of ESCC. Conclusions: Androgen promotes the proliferation of ESCC cells via AR while estrogen exerts dual functions dependent on the specific estrogen receptor subtypes, which may provide new insight into the etiology, prevention, and hormone-based treatment of ESCC. Citation Format: Furong Huang, Hongyan Chen, Qianben Wang, Zhihua Liu. Roles of sex hormone pathways in the gender disparity of esophageal squamous-cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5421.

Chemoprevention of LA7-Induced Mammary Tumor Growth by SM6Met, a Well-Characterized Cyclopia Extract.

Breast cancer (BC) is the leading cause of cancer-related deaths in women. Chemoprevention of BC by using plant extracts is gaining attention. SM6Met, a well-characterized extract of Cyclopia subternata with reported selective estrogen receptor subtype activity, has shown tumor suppressive effects in a chemically induced BC model in rats, which is known to be estrogen responsive. However, there is no information on the estrogen sensitivity of the relatively new orthotopic model of LA7 cell-induced mammary tumors. In the present study, the potential chemopreventative and side-effect profile of SM6Met on LA7 cell-induced tumor growth was evaluated, as was the effects of 17β-estradiol and standard-of-care (SOC) endocrine therapies, such as tamoxifen (TAM), letrozole (LET), and fulvestrant (FUL). Tumor growth was observed in the tumor-vehicle control group until day 10 post tumor induction, which declined afterward on days 12–14. SM6Met suppressed tumor growth to the same extent as TAM, while LET, but not FUL, also showed substantial anti-tumor effects. Short-term 17β-estradiol treatment reduced tumor volume on days prior to day 10, whereas tumor promoting effects were observed during long-term treatment, which was especially evident at later time points. Marked elevation in serum markers of liver injury, which was further supported by histological evaluation, was observed in the vehicle-treated tumor control, TAM, LET, and long-term 17β-estradiol treatment groups. Alterations in the lipid profiles were also observed in the 17β-estradiol treatment groups. In contrast, SM6Met did not augment the increase in serum levels of liver injury biomarkers caused by tumor induction and no effect was observed on lipid profiles. In summary, the results from the current study demonstrate the chemopreventative effect of SM6Met on mammary tumor growth, which was comparable to that of TAM, without eliciting the negative side-effects observed with this SOC endocrine therapy. Furthermore, the results of this study also showed some responsiveness of LA7-induced tumors to estrogen and SOC endocrine therapies. Thus, this model may be useful in evaluating potential endocrine therapies for hormone responsive BC.

The correlation between mammographic densities and molecular pathology in breast cancer

This study aimed to analyze the correlation between mammographic density obtained by density analysis software (DAS)/radiologists visual (RV) classification with molecular subtype, and the expression levels of estrogen receptor (ER), progesterone receptor (PR), Ki67 antigen (Ki-67), p53 gene (p53), and human epidermal growth factor receptor-2 (HER2). A total of 688 breast cancer patients with digital mammography and complete molecular pathological results in Tianjin Medical University Cancer Institute and Hospital between February 2015 and February 2016 were collected. The DAS-density grade (DASD) and the radiologists visually classified density grade (RVD) were evaluated by 3 radiologists. The correlation between density grade and the expression levels of ER, PR, Ki-67, p53, HER2 and breast cancer molecular subtype (PMS) were analyzed. The agreement between DASD and RVD was explored. ER, PR and HER-2 positive rate were significantly different among patients with different RVD grades (P< 0.05). HER2 positive rates showed an increasing trend following RVD upgrading (P𝑡𝑟𝑒𝑛𝑑< 0.05). HER-2 positive rate in RVD D1 + D2 was 7.69%, which was higher than that in D3 + D4 (P< 0.05). The ER and Ki-67 expressions in patients were markedly different among DASD (P= 0.009 and 0.002) and RVD (P= 0.012 and 0.036) with different grades. The kappa value of each DASD to RVD was 0.31 (P< 0.01). The RVD 3 proportion was 14.58% (63/432) in HER2 Over-expressing subtype, which was apparently higher than RVD1 (2.43%, 1/41) (P< 0.05). Breast density may be partial correlated with molecular pathology in breast cancer.

Identifying an early treatment window for predicting breast cancer response to neoadjuvant chemotherapy using immunohistopathology and hemoglobin parameters

BackgroundBreast cancer pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) varies with tumor subtype. The purpose of this study was to identify an early treatment window for predicting pCR based on tumor subtype, pretreatment total hemoglobin (tHb) level, and early changes in tHb following NAC.MethodsTwenty-two patients (mean age 56 years, range 34–74 years) were assessed using a near-infrared imager coupled with an Ultrasound system prior to treatment, 7 days after the first treatment, at the end of each of the first three cycles, and before their definitive surgery. Pathologic responses were dichotomized by the Miller-Payne system. Tumor vascularity was assessed from tHb; vascularity changes during NAC were assessed from a percentage tHb normalized to the pretreatment level (%tHb). After training the logistic prediction models using the previous study data, we assessed the early treatment window for predicting pathological response according to their tumor subtype (human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), triple-negative (TN)) based on tHb, and %tHb measured at different cycles and evaluated by the area under the receiver operating characteristic (ROC) curve (AUC).ResultsIn the new study cohort, maximum pretreatment tHb and %tHb changes after cycles 1, 2, and 3 were significantly higher in responder Miller-Payne 4–5 tumors (n = 13) than non-or partial responder Miller-Payne 1–3 tumors (n = 9). However, no significance was found at day 7. The AUC of the predictive power of pretreatment tHb in the cohort was 0.75, which was similar to the performance of the HER2 subtype as a single predictor (AUC of 0.78). A greater predictive power of pretreatment tHb was found within each subtype, with AUCs of 0.88, 0.69, and 0.72, in the HER2, ER, and TN subtypes, respectively. Using pretreatment tHb and cycle 1 %tHb, AUC reached 0.96, 0.91, and 0.90 in HER2, ER, and TN subtypes, respectively, and 0.95 regardless of subtype. Additional cycle 2 %tHb measurements moderately improved prediction for the HER2 subtype but did not improve prediction for the ER and TN subtypes.ConclusionsBy combining tumor subtypes with tHb, we predicted the pCR of breast cancer to NAC before treatment. Prediction accuracy can be significantly improved by incorporating cycle 1 and 2 %tHb for the HER2 subtype and cycle 1 %tHb for the ER and TN subtypes.Trial registrationClinicalTrials.gov, NCT02092636. Registered in March 2014.

Estrogenic Mechanisms and Cardiac Responses Following Early Life Exposure to Bisphenol A (BPA) and Its Metabolite 4-Methyl-2,4-bis( p-hydroxyphenyl)pent-1-ene (MBP) in Zebrafish.

Environmental exposure to Bisphenol A (BPA) has been associated with a range of adverse health effects, including on the cardiovascular system in humans. Lack of agreement on its mechanism(s) of action likely stem from comparisons between in vivo and in vitro test systems and potential multiple effects pathways. In rodents, in vivo, metabolic activation of BPA produces 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which is reported to be up to 1000 times more potent as an estrogen than BPA. We investigated the estrogenic effects and estrogen receptor signaling pathway(s) of BPA and MBP following early life exposure using a transgenic, estrogen responsive (ERE-TG) zebrafish and a targeted morpholino approach to knockdown the three fish estrogen receptor (ER) subtypes. The functional consequences of BPA exposure on the cardiovascular system of zebrafish larvae were also examined. The heart atrioventricular valves and the bulbus arteriosus were primary target tissues for both BPA and MBP in the ERE-TG zebrafish, and MBP was approximately 1000-fold more potent than BPA as an estrogen in these tissues. Estrogen receptor knockdown with morpholinos indicated that the estrogenic responses in the heart for both BPA and MBP were mediated via an estrogen receptor 1 (esr1) dependent pathway. At the highest BPA concentration tested (2500 μg/L), alterations in the atrial:ventricular beat ratio indicated a functional impact on the heart of 5 days post fertilization (dpf) larvae, and there was also a significantly reduced heart rate in these larvae at 14 dpf. Our findings indicate that some of the reported adverse effects on heart function associated with BPA exposure (in mammals) may act through an estrogenic mechanism, but that fish are unlikely to be susceptible to adverse effects on heart development for environmentally relevant exposures.

Estrogen receptor modulators genistein, daidzein and ERB-041 inhibit cell migration, invasion, proliferation and sphere formation via modulation of FAK and PI3K/AKT signaling in ovarian cancer

BackgroundOvarian cancer is the most lethal gynaecological malignancy. Chemotherapy is the main stay of treatment for metastatic disease, with modest response rates but significant side effects. Therefore, there is a need for alternative therapies that can control the disease while offering good quality of life. Ovarian cancer cells express both estrogen receptor subtypes (ERα and ERβ). There is growing evidence that ERβ is anti-oncogenic. Genistein and daidzein are phytoestrogens found in soybeans and they display higher affinity to bind ERβ. ERB-041 is a potent selective ERβ agonist. In this study, we aimed to investigate the effects of genistein, daidzein and ERB-041 on ovarian cancer.MethodsOvarian cancer cell lines were treated with genistein, daidzein and ERB-041 in pharmacological doses. Cell migration, invasion, proliferation, cell cycle arrest, apoptosis and sphere formation were assessed by Transwell migration and invasion assays, XTT assay, focus formation, flow cytometry and sphere formation assay, respectively. Immunoblotting analysis was performed to determine the downstream signaling pathways.ResultsWe found that genistein, daidzein and ERB-041 significantly inhibited ovarian cancer cell migration, invasion, proliferation, as well as induced cell cycle arrest and apoptosis. Significantly inhibitory effect on the size and number of sphere formed in genistein, daidzein and ERB-041 treated cells was also demonstrated. Moreover, genistein, daidzein and ERB-041 treatment reduced p-FAK, p-PI3K, p-AKT, p-GSK3β, p21 or cyclin D1 expression in ovarian cancer cells.ConclusionGenistein, daidzein and ERB-041 decreased ovarian cancer cell migration, invasion, proliferation and sphere formation, and induced cell cycle arrest and apoptosis with altered FAK and PI3K/AKT/GSK signaling and p21/cyclin D1 expression, suggesting their roles on ovarian cancer cell metastasis, tumorigenesis and stem-like properties and their potential as alternative therapies for ovarian cancer patients.

Mechanism Underlying the Effects of Estrogen Deficiency on Otoconia.

Otoconia-related vertigo and balance deficits, particularly benign paroxysmal positional vertigo (BPPV), are common. Our recent studies in humans show that, while BPPV prevalence greatly increases with age in both genders, peri-menopausal women are especially susceptible. In the present study, we show that bilateral ovariectomized (OVX) mice have significant balance behavioral deficits, and that estrogen deficiency compromises otoconia maintenance and anchoring by reducing the expression of otoconial component and anchoring proteins. There is ectopic debris formation in the ampulla under estrogen deficiency due to aberrant matrix protein expression. Furthermore, phytoestrogen is effective in rescuing the otoconia abnormalities. By comparing the expression levels of known estrogen receptor (Esr) subtypes, and by examining the otoconia phenotypes of null mice for selected receptors, we postulate that Esr2 may be critical in mediating the effects of estrogen in otoconia maintenance.

Synthesis and biological evaluation of novel cyclopropyl derivatives as subtype-selective ligands for estrogen receptor.

Tamoxifen is the most commonly used selective estrogen receptor modulators (SERMs); however, patients often develop the acquired drug resistance on tamoxifen therapy. The aim of this study was to develop new SERMs. Several novel cyclopropyl derivatives were designed and synthesized. The binding affinities of these compounds as well as the selectivity on subtype of estrogen receptor (ER) were assessed by fluorescence polarization. The antagonistic activity was also evaluated by dual-luciferase reporter assay. Our data identified five compounds (9a, 9b, 9d, 9e and 9f) with a higher selectivity on ERα than ERβ subtype, warranting further development as a subtype-selective ER modulator. The study of antiestrogen activity also demonstrated that compounds 9a, 9c-f acted as full functional antagonists for ERα. These compounds had no or very low cytotoxicity. Although these cyclopropyl derivatives showed lower binding affinities on ERs compared to 17β-estradiol, five of these compounds exhibited binding to ERα only and therefore might serve as a promising lead compound for further development of novel subtype-selective SERMs.