Estrogen Binding Research Articles

Abstract PD6-7: In-silico discovery of novel estrogen receptor-α inhibitors as potential therapeutics for tamoxifen resistant breast cancer

Abstract Estrogen receptor-α (ER) positive breast cancer (BCa) represents 75% of all invasive BCas. Conventional ER-directed drug Tamoxifen targets the estrogen binding pocket (EBP) of the receptor. However, over prolonged periods of treatment, the therapeutic efficacy of Tamoxifen declines due to development of resistance. Numerous factors are causative for this phenomenon, including recently reported mutations in the receptor (T537S). Therefore, there is an urgency to develop novel anti-ER therapeutics that exhibit entirely different mode of ER inhibition. A promising alternative strategy is to prevent receptor-coactivator interaction and block further crucial steps in ER activity. ER-coactivator interface should be less prone to adaptive mutations as any mutations at this site would also likely block the coactivator recruitment, we therefore targeted the Activation Function-2 (AF2) site, a coactivator binding pocket on ER, called to overcome the limitations of Tamoxifen. Although AF2 is a shallow surface pocket, the pharmacophore-rich features of this site make it a druggable target. To identify potential ER AF2 inhibitors, virtual screening was performed. Initial hits were subjected to lead optimization and more potent analogues were rationally designed by exploiting critical features of this site. Potential compounds were tested for their ability to inhibit ER transcriptional activity using the T47D-KBluc cell line stably transfected with an ER-specific luciferase reporter. Consequently, the lead compound VPC-16339 (IC50=8.24µM) was identified. The direct binding between VPC-16339 and the receptor was confirmed by Biolayer Interferometry assay. More importantly, VPC-16339 prevents coactivator recruitment at the AF2 pocket in a dose dependent manner as measured by TR-FRET coactivator recruitment assay. Increasing concentrations of estradiol did not affect the IC50 of the lead compound, thereby ruling out the possibility of VPC-16339 binding to EBP. VPC-16339 demonstrated a strong anti-proliferative effect on MCF7 and Tamoxifen resistant cells, with no effect on ER- HeLa cells, suggesting its selective ER-mediated action, as further validated by ER luciferase assay in Tamoxifen resistant cells. VPC-16339 effectively inhibits mRNA and protein expression levels of the estrogen dependent genes such as pS2,CathD and CDC2. Due to AF2-guided mechanism, VPC-16339 successfully overcomes Tamoxifen resistance and inhibits the constitutively active Tamoxifen resistant form of ER (T537S). In summary, we report VPC-16339 as an ER AF2 specific inhibitor with promising anti-proliferative effect in BCa cell lines including Tamoxifen resistant cell lines. VPC-16339 effectively inhibits the mutant form of the receptor (T537S) which is responsible for acquired endocrine resistance. It can be anticipated that ER AF2 inhibitors will provide an alternative therapeutic strategy that can be applied concurrently or simultaneously with current anti-ER treatments for BCa patients with advanced disease. Citation Format: Kriti Singh, Ravi SN Munuganti, Miriam Butler, Artem Cherkasov, Paul S Rennie. In-silico discovery of novel estrogen receptor-α inhibitors as potential therapeutics for tamoxifen resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-7.

Ubiquitin-fold modifier 1 acts as a positive regulator of breast cancer.

Estrogen receptor-α (ERα) is a steroid hormone-sensitive transcription factor that plays a critical role in development of breast cancer. The binding of estrogen to ERα triggers the recruitment of transcriptional co-activators as well as chromatin remodeling factors to estrogen-responsive elements (ERE) of ERα target genes. This process is tightly associated with post-translational modifications (PTMs) of ERα and its co-activators for promotion of transcriptional activation, which leads to proliferation of a large subset of breast tumor cells. These PTMs include phosphorylation, acetylation, methylation, and conjugation by ubiquitin and ubiquitin-like proteins. Ubiquitin-fold modifier 1 (UFM1), one of ubiquitin-like proteins, has recently been shown to be ligated to activating signal co-integrator 1 (ASC1), which acts as a transcriptional co-activator of nuclear receptors. Here, we discuss the mechanistic connection between ASC1 modification by UFM1 and ERα transactivation, and highlight how the interplay of these processes is involved in development of breast cancer. We also discuss potential use of UFM1-conjugating system as therapeutic targets against not only breast cancer but also other nuclear receptor-mediated cancers.

Synthesis and in vitro evaluation of ferutinol aryl esters for estrogenic activity and affinity toward cannabinoid receptors

Ferutinin (1), the major constituent of Ferula hermonis and other Ferula species, is a sesquiterpene ester with remarkable estrogenic activity, beside other valuable medicinal properties. To investigate the influence of chemical modification of the ferutinin structure on its estrogenic effect and binding affinity toward the cannabinoid CB1 and CB2 receptors, twelve derivatives of 1 were prepared and evaluated in vitro, together with the parent compound, for the respective bioactivities, based on the recent evidence for estrogen–endocannabinoid interaction. Nine of the prepared derivatives (3–11) are new semisynthetic esters of 1. The parent compound ferutinin (1) exhibited the highest level of estrogenic activity (EC50 0.3 μM and a percent maximal 17β-estradiol response of 90 % at 1 µM). Compound 6 was found to be a selective agonist for CB2 receptor (EC50 0.051 μM, Ki 0.025 μM), with much less affinity for CB1 receptor (EC50 97 μM, Ki 48.5 μM). Compound 8 was a selective agonist for CB1 (EC50 62, Ki 0.031 μM) with no affinity toward CB2.

Use of recombinant oestrogen binding protein for the electrochemical detection of oestrogen

We have previously reported the development of an electrochemical method to quantitatively detect vertebrate oestrogens using an oestrogen binding protein (EBP1) present in wild type Saccharomyces cerevisiae and Candida albicans cells. However, the assays were complex and slow with both whole cells and cell lysate. In this work we report the transfer of the EBP1 gene to an industrial yeast, the addition of a his tag sequence to simplify purification of the protein, and the oestrogen binding characteristics of the protein. The recombinant protein (Ebp1p-6h) can now be produced by a non-pathogenic cell, and has shown good stability both when in use and when lyophilised. The detection range covers likely environmental concentrations of free oestrogens and the limit of detection is below the environmental concentration that has significant biological effect. In addition the assay period has been reduced to approximately 2min. This work reports progress toward the construction of a rapid, portable oestrogen sensor that is not restricted to use to the laboratory.

Combined action of estrogen receptor agonists and antagonists in two-hybrid recombinant yeast in vitro

Estrogen receptor (ER) antagonistic chemicals in aquatic environments are believed to influence the binding of both endogenous and exogenous estrogens to ERs in aquatic organisms. Although the combined effects of estrogenic compounds have attracted much scientific concern, little work has been done on the influence of such antiestrogens on the biological effects of estrogens. This study focused on how the presence of different amounts of antagonists affects the results of ER agonist activity tests. To achieve this, three questions were stated and answered in sequence. A two-hybrid recombinant yeast assay mediated by ER was adopted, providing a single mode of action and single target of action for this study. Mixtures created by an ER agonist and three antagonists following the fixed-ratio principle were assessed. The concentration of 17β-estradiol causing maximum induction was set as the fixed dose of estrogen in the antagonist activity test (question 1). When the two classes of chemicals coexisted, antiestrogens, which as a whole behaved according to the concentration addition model (question 2), decreased the response of estrogen and compressed the concentration–response curves along the y-axis in the agonist activity test (question 3). This may cause the estradiol equivalent to be underestimated and potentially mask the action of estrogenic effects in toxicity evaluation of environmental samples.

Cadmium and proliferation in human uterine leiomyoma cells: evidence of a role for EGFR/MAPK pathways but not classical estrogen receptor pathways.

Background:It has been proposed that cadmium (Cd) is an environmental “metalloestrogen” and that its action is mediated via the estrogen receptor (ER). Cd mimics the effects of estrogen in the rat uterus, and blood Cd concentrations positively correlate with ER levels in uteri of women with fibroids.Objectives:In the present study we explored whether Cd could stimulate proliferation of estrogen-responsive human uterine leiomyoma (ht-UtLM) cells and uterine smooth muscle cells (ht-UtSMCs) through classical interactions with ERα and ERβ, or by nongenomic mechanisms.Methods:We used estrogen response element (ERE) reporters, phosphorylated receptor tyrosine kinase arrays, Western blot analysis, estrogen binding, and cell proliferation assays to evaluate the effects of Cd on ht-UtLM cells and ht-UtSMCs.Results:Cd stimulated growth of both cell types at lower concentrations and inhibited growth at higher concentrations (≥ 50 μM). Cd did not significantly bind to ERα or ERβ, nor did it show transactivation in both cell types transiently transfected with ERE reporter genes. However, in both cells types, Cd (0.1 μM and 10 μM) activated p44/42 MAPK (ERK1/2), and a MAPK inhibitor (PD98059) abrogated Cd-induced cell proliferation. Cd in ht-UtLM cells, but not in ht-UtSMCs, activated the growth factor receptors EGFR, HGFR, and VEGF-R1 upstream of MAPK. Additional studies in ht-UtLM cells showed that AG1478, an EGFR inhibitor, abolished Cd-induced phosphorylation of EGFR and MAPK.Conclusions:Our results show that low concentrations of Cd stimulated cell proliferation in estrogen-responsive uterine cells by nongenomic activation of MAPK, but not through classical ER-mediated pathways.Citation:Gao X, Yu L, Moore AB, Kissling GE, Waalkes MP, Dixon D. 2015. Cadmium and proliferation in human uterine leiomyoma cells: evidence of a role for EGFR/MAPK pathways but not classical estrogen receptor pathways. Environ Health Perspect 123:331–336; http://dx.doi.org/10.1289/ehp.1408234

Abstract 2521: Small molecule inhibitors targeting the activation function-2 site of estrogen receptor-α

Abstract Approximately 75% of Breast Cancers (BCas) are classified as Estrogen Receptor alpha (ERα) positive. Treatment with anti-estrogens such as Tamoxifen has been the main therapeutic approach for more than 30 years. However, one third of women treated with Tamoxifen for 5 years develop recurrent disease. Experimental and clinical observations have suggested that ERα signalling continues to play an important role even after the development of resistance. Moreover, biopsies from BCa patients who relapsed on Tamoxifen indicated that ERα expression was retained in more than 50% of the cases. Because of the emergence of hormone resistance, there is a clear need to develop entirely novel anti-ERα therapeutics, such as drugs that would directly disrupt the interaction between ERα and its coactivator proteins at the corresponding regulatory interfaces, exemplified by a well-characterized Activation Function-2 (AF-2) site.In the current study we have used state of artificial intelligence systems to rationally select new anti-ERα drug candidates. Using the power of modern computers, we performed large-scale docking using millions of existing chemicals from the ZINC database and identified several promising small molecules as candidate AF-2 binders. We then conducted biological screens to identify compounds that can bind to the AF-2 pocket and inhibit ERα transactivation. A reporter assay was developed using T47D-Kbluc breast cancer cells, a line which had been stably transfected with an estrogen responsive luciferase reporter gene construct consisting of three estrogen response elements (EREs) upstream of a TATA promoter, to evaluate the potential of these compounds to inhibit ERα transcriptional activity. Compounds that inhibited ERα-mediated transcription of the reporter gene in a concentration dependent manner were further analysed. These compounds do not displace estrogen, but block ERα-coactivator interaction, as measured by TR-FRET assay, thereby confirming that inhibition of coactivator recruitment is not by the allosteric mechanism of conventional antagonists. One of our best compounds, VPC-16046, shows direct reversible binding to the ERα ligand binding domain as detected by Biolayer Interferometry assay. This compound demonstrated a strong anti-proliferative effect on MCF7, T47D and Tamoxifen resistant cells without affecting the growth of ERα-negative HeLa cells, used as a control in MTS assay.In summary, our study has identified a novel class of ERα AF2 inhibitors that have the potential to effectively inhibit ERα transcriptional activity by a mechanism which does not target the estrogen binding site and thereby circumvents treatment resistance seen with conventional, clinically used anti-estrogens. Treatment with these inhibitors should lead to a substantial improvement in the survival rate of women with advanced Tamoxifen-resistant BCa. Citation Format: Kriti Singh, Ravi Shashi Nayana Munuganti, Eric Leblanc, Artem Cherkasov, Paul S. Rennie. Small molecule inhibitors targeting the activation function-2 site of estrogen receptor-α. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2521. doi:10.1158/1538-7445.AM2014-2521

Estradiol Binds to Insulin and Insulin Receptor Decreasing Insulin Binding in vitro.

Rationale: Insulin (INS) resistance associated with hyperestrogenemias occurs in gestational diabetes mellitus, polycystic ovary syndrome, ovarian hyperstimulation syndrome, estrogen therapies, metabolic syndrome, and obesity. The mechanism by which INS and estrogen interact is unknown. We hypothesize that estrogen binds directly to INS and the insulin receptor (IR) producing INS resistance.Objectives: To determine the binding constants of steroid hormones to INS, the IR, and INS-like peptides derived from the IR; and to investigate the effect of estrogens on the binding of INS to its receptor.Methods: Ultraviolet spectroscopy, capillary electrophoresis, and NMR demonstrated estrogen binding to INS and its receptor. Horse-radish peroxidase-linked INS was used in an ELISA-like procedure to measure the effect of estradiol on binding of INS to its receptor.Measurements: Binding constants for estrogens to INS and the IR were determined by concentration-dependent spectral shifts. The effect of estradiol on INS binding to its receptor was determined by shifts in the INS binding curve.Main Results: Estradiol bound to INS with a Kd of 12 × 10−9 M and to the IR with a Kd of 24 × 10−9 M, while other hormones had significantly less affinity. Twenty-two nanomolars of estradiol shifted the binding curve of INS to its receptor 0.8 log units to the right.Conclusion: Estradiol concentrations in hyperestrogenemic syndromes may interfere with INS binding to its receptor producing significant INS resistance.

A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells

Since over 60% of breast cancers are estrogen receptor positive (ER+), many therapies have targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth factor promoting phosphorylation of the ER. Other proliferation pathways such as the phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting multiple cellular proliferation and survival pathways at the onset of treatment is critical for the development of more effective therapies. The grapefruit flavanone naringenin (Nar) is an inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use serum containing medium in order to retain all the potential inducers of cell proliferation so as not to exclude any targets of Nar. Here we show that a Nar–Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar–Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar–Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar–Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells.

Environmental Estrogen Exposure During Fetal Life: A Time Bomb for Prostate Cancer

In this issue, Prins et al (1) elegantly describe how exposure to bisphenol A (BPA) during development could modify prostate cell fate in adulthood. This group, involved in research to understand prostate cancer (PCa) development for many years, first determined the effect of BPA on human prostate epithelial stem-like cells obtained from young men. This allowed them to determine that these cells are highly and equally sensitive to estrogens and BPA and are able to amplify. Estradiol and BPA maintain the stem-like state within the normal prostate epithelial population as demonstrated by a higher accumulation of genes such as TBX3 and NANOG. Conversely, epithelial differentiation genes such as NKX3.1 and CK18 are at a low level. In addition to this work on cell culture, Prins et al (1) demonstrated estradiol and BPA effects during prostate development using an ingenious mouse model by xenografting chimeric human-rat prostate tissues in Nude mice (2) and found that low-dose BPA exposure increases estrogen-driven carcinogenesis of human prostate epithelium. As a result of this experimental tour de force, this team has described the missing link between estrogen-like endocrine disrupters and PCa. The Holy Grail has been found: environmental estrogen exposure will modify the fate of prostate stem cells, making them more sensitive to estrogen during adulthood and more prone to develop PCa. Development is a tightly controlled process that involves thousands of genes and hundreds of signaling molecules (hormones and growth factors) and cell interactions (junctions and synapses) that will allow correct cell migration. Like a Lego model, all of the interlocking “bricks” must be assembled correctly to produce a normal individual. Such precise mechanisms are involved that it is easy to understand that any defect in a signaling molecule or, on the contrary, an abnormal activation of a usually silent pathway can produce cell defects. Estrogen-like endocrine disruptors have been considered for many years as putatively responsible for various health defects by interfering with the synthesis, metabolism, binding, or cellular responses of natural estrogens (3). These molecules can be found in various plastic products, flame retardants, pesticides, and many other products that are needed for daily use. Among them, BPA is considered as the paradigm of these environmental estrogen-like molecules. Even though BPA was banned from being used in baby bottles by the European Union in 2011 (4) and by the United States in 2012, over 3 million tons per year of this estrogenic monomer have been used to manufacture polycarbonate plastic products, in resins lining metal cans, in dental sealants, and in blends with other types of plastic products, thus suggesting that adults can be overexposed to it. The ester bond linking BPA molecules in polycarbonate and resins undergoes hydrolysis, resulting in the release of free BPA into food, beverages, and the environment, and numerous monitoring studies now show almost ubiquitous human exposure to biologically active levels of this chemical. BPA exerts estrogenic effects through the classical nuclear estrogen receptors, and BPA acts as a selective estrogen receptor modulator (5). However, BPA also initiates rapid responses via estrogen receptors presumably associated with the plasma membrane. Similar to estradiol, BPA causes changes in some cell functions at the tiniest con-

Preliminary study of quercetin affecting the hypothalamic-pituitary-gonadal axis on rat endometriosis model.

In this study, the endometriosis rats model was randomly divided into 6 groups: model control group, ovariectomized group, Gestrinone group, and quercetin high/medium/low dose group. Rats were killed after 3 weeks of administration. The expression levels of serum FSH and LH were detected by ELISA. The localizations and quantities of ERα, ERβ, and PR were detected by immunohistochemistry and western blot. The results showed that the mechanism of quercetin inhibiting the growth of ectopic endometrium on rat endometriosis model may be through the decreasing of serum FSH and LH levels and then reducing local estrogen content to make the ectopic endometrium atrophy. Quercetin can decrease the expression of ERα, ERβ, and PR in hypothalamus, pituitary, and endometrium, thereby inhibiting estrogen and progesterone binding to their receptors to play the role of antiestrogen and progesterone.

Abstract P5-09-14: Plant-derived estrogen receptorb agonists alone or in combination with aromatase inhibitors restore sensitivity in endocrine therapy resistant breast tumors

Abstract Estrogen receptors (ER) play an important role in breast cancer. Over two-thirds of all breast cancers express ERa, and current endocrine therapies target its signaling. Antiestrogens (AE) block the binding of estrogen to ERα, while aromatase inhibitors (AI) inhibit local and systemic estrogen production. Both treatments improve outcomes for about 50% of patients with early or advanced ERα positive breast cancer. Unfortunately, therapeutic resistance frequently arises. There is a critical need to develop effective alternate strategies to prevent or delay the development of resistance to endocrine therapy and the resulting tumor progression. Both normal and tumor tissue express ERβ which has anti-proliferative activity and recent studies have identified several natural compounds that have the potential to function as ERβ agonists. Plant-derived ERβ-agonists, Liquiritigenin (Liq), from the plant Glycyrrhizae uralensis and S-equol, from the soy isoflavone daidzein, are currently in clinical trials for the treatment of vasomotor hot flashes associated with menopause. In this study we sought to determine if ERβ agonists alone or in combination with AI could resensitize Letrozole-resistant breast cancer cells in vivo. Xenografts were established using cells which model post-menopausal breast cancer. Endocrine therapy sensitive (MCF-7aro) and Letrozole resistant (LTLT-Ca) tumors were treated with either ERβ agonists or in combination with AI and progression was measured. In MCF-7aro tumors, ERβ agonist treatment reduced tumor volume and prolonged sensitivity to AI. In the therapy resistant tumors (LTLT-Ca), ERβ agonist treatment blocked tumor growth and restored sensitivity to AI therapy. To determine the molecular mechanism by which ERβ agonists inhibit tumor growth and prolong and restore sensitivity, qRT-PCR and western blot analysis was performed. Our results show changes in the levels of p53, NFkB, cyclin D1, and KLF5 in addition to other genes. To determine the role of ERβ agonists on the activation of these genes specific gene promoter-luciferase reporter constructs were used and the specificity of ERβ-mediated actions was confirmed using a siRNA approach. This study suggests that ERβ acts as a tumor suppressor by suppressing cell growth through the inhibition of cell cycle genes and by inducing apoptosis through the regulation of p53. In conclusion, this study demonstrates the potential role for ERβ agonists to improve adjuvant endocrine therapy to treat both hormone-responsive and hormone-resistant breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-14.

New Clues To The Mechanism Of Cardioprotection By Estrogen

Platelets are circulating anucleated cell fragments that play a major role in thrombosis and hemostasis in response to a number of stimuli. Platelet activation is a multi-step processes that can be modulated via several known mechanisms. It has long been suspected that sex hormones, like estrogen, have an effect on platelet aggregation and thrombosis; the exact role which estrogen plays in these processes remains unclear. Pre-menopausal women have a lower risk for coronary thrombosis than men– presumably due to higher estrogen levels. However, in postmenopausal women, hormone replacement paradoxically increases the risk for thrombotic events such as coronary and venous thrombosis. Current literature on the subject of estrogen effect on platelets varies considerably. Previous and current research indicates potential estrogen mechanisms for thrombosis; yet the complete biochemical pathway has not been elucidated. Current literature describes the aggregatory effects of estrogen, but little is said about the fact that premenopausal women still have a lower risk for coronary thrombosis in relation to their male counterparts despite having comparatively higher estrogen levels. The goal of our study was to better delineate estrogen mediated biochemical pathways involved in platelet aggregation. Blood was collected from healthy male donors and fractionated into platelet rich plasma (PRP). PRP was centrifuged to prepare platelet poor plasma (PPP) to be used as a control during platelet aggregation assays. PRP samples were incubated with varying physiologic concentrations of 17-b-estradiol for 1-10 minutes and then activated with agonist, and compared to activated control samples in the absence of estrogen. Agonists tested include TRAP-1 (activates PAR-1), gamma thrombin (activates PAR-4), collagen, and ADP. Our results show that estrogen at physiologic levels (60-80 nM) has a synergistic effect on platelet aggregation when combined with sub-threshold doses of agonist. We also showed that this synergy is at least in part mediated by lipid rafts, a fact demonstrated in other studies. A role for lipid rafts in the estrogen effect was demonstrated by incubating PRP with the lipid raft disruptor, methyl β cyclodextrin (MβCD), which resulted in the inhibition of estrogen-induced synergy. Incubation with MβCD alone had no effect on platelet aggregation. We demonstrated that estrogen-induced synergy was absent when platelets, derived from male donors, were washed free of plasma components. Upon the addition of PPP (10-25 uL), synergy was restored. We postulated the necessity of some plasma protein in presenting estrogen to its receptor (ERα and ERβ) for there to be any synergistic effect. Using immunoflourescent microscopy we were able to identify that platelets contain both ERα and ERβ and that the expression of each is altered upon binding of estrogen and TRAP-1. Using filtration techniques we were able to determine that the molecular weight of the necessary protein to restore synergy is greater than 90-100,000 kDa. The most likely candidate was the dimerized plasma sex hormone binding globulin (SHBG), due to its high affinity for estrogens. Support for this comes from the ability of anti-SHBG antibodies to block the estrogen-induced synergy of agonist-induced platelet aggregations. Platelets derived from premenopausal females showed no estrogen-induced synergy of platelet aggregations under the identical conditions used with platelets derived from males. However, platelets derived from postmenopausal women did show synergistic effects of estrogen mirroring effects observed with male platelets samples. In all cases where estrogen-induced synergy of aggregation was observed, incubation of the same platelet samples with estrogen for 2-24hrs totally reversed the synergistic effect. This would be comparable to the premenopausal woman whose platelets in vivo are constantly exposed to high levels of estrogen. Our studies demonstrate that the SHBG must present estrogen to the platelet membrane estrogen receptors and that these receptors are down-regulated upon prolonged exposure to estrogen resulting in reduced levels of platelet aggregation in the presence of sub-optimal concentrations of agonists. Results of our studies would account for the varied effects of estrogen on platelet activation reported in the literature. Studies are in progress with testosterone and second messengers. Disclosures: No relevant conflicts of interest to declare.

Anatomical location and redistribution of G protein-coupled estrogen receptor-1 during the estrus cycle in mouse kidney and specific binding to estrogens but not aldosterone

Prior studies have linked renoprotective effects of estrogens to G-protein-coupled estrogen receptor-1 (GPER-1) and suggest that aldosterone may also activate GPER-1. Here, the role of GPER-1 in murine renal tissue was further evaluated by examining its anatomical distribution, subcellular distribution and steroid binding specificity. Dual immunofluorescent staining using position-specific markers showed that GPER-1 immunoreactivity primarily resides in distal convoluted tubules and the Loop of Henle (stained with Tamm-Horsfall Protein-1). Lower GPER-1 expression was observed in proximal convoluted tubules marked with megalin, and GPER-1 was not detected in collecting ducts. Plasma membrane fractions prepared from whole kidney tissue or HEK293 cells expressing recombinant human GPER-1 (HEK-GPER-1) displayed high-affinity, specific [3H]-17β-estradiol ([3H]-E2) binding, but no specific [3H]-aldosterone binding. In contrast, cytosolic preparations exhibited specific binding to [3H]-aldosterone but not to [3H]-E2, consistent with the subcellular distribution of GPER-1 and mineralocorticoid receptor (MR) in these preparations. Aldosterone and MR antagonists, spironolactone and eplerenone, failed to compete for specific [3H]-E2 binding to membranes of HEK-GPER-1 cells. Furthermore, aldosterone did not increase [35S]-GTP-γS binding to membranes of HEK-GPER-1 cells, indicating that it is not involved in G protein signaling mediated through GPER-1. During the secretory phases of the estrus cycle, GPER-1 is upregulated on cortical epithelia and localized to the basolateral surface during proestrus and redistributed intracellularly during estrus. GPER-1 is down-modulated during luteal phases of the estrus cycle with significantly less receptor on the surface of renal epithelia. Our results demonstrate that GPER-1 is associated with specific estrogen binding and not aldosterone binding and that GPER-1 expression is modulated during the estrus cycle which may suggest a physiological role for GPER-1 in the kidney during reproduction.

Are typical human serum BPA concentrations measurable and sufficient to be estrogenic in the general population?

Mammalian estrogen receptors modulate many physiological processes. Chemicals with structural features similar to estrogens can interact with estrogen receptors to produce biological effects similar to those caused by endogenous estrogens in the body. Bisphenol A (BPA) is a structural analogue of estrogen that binds to estrogen receptors. Exposure to BPA in humans is virtually ubiquitous in industrialized societies, but BPA is rapidly detoxified by metabolism and does not accumulate in the body. Whether or not serum concentrations of BPA in humans are sufficiently high to disrupt normal estrogen-related biology is the subject of intense political and scientific debate. Here we show a convergence of robust methods for measuring or calculating serum concentrations of BPA in humans from 93 published studies of more than 30,000 individuals in 19 countries across all life stages. Typical serum BPA concentrations are orders of magnitude lower than levels measurable by modern analytical methods and below concentrations required to occupy more than 0.0009% of Type II Estrogen Binding Sites, GPR30, ERα or ERβ receptors. Occupancies would be higher, but ≤0.04%, for the highest affinity receptor, ERRγ. Our results show limited or no potential for estrogenicity in humans, and question reports of measurable BPA in human serum.

Pharmacophore Based 3D-QSAR Study of Biphenyl Derivatives as Nonsteroidal Aromatase Inhibitors in JEG-3 Cell Lines

Breast cancer is one of the most high-profile malignant diseases in modern society. Among postmenopausal women affected by the disease, a substantial portion has breast tumors that are estrogen-receptor positive. A common therapeutic intervention for this type of cancer is through endocrine therapy. Endocrine agents can act by either diminishing the availability or inhibiting the binding of estrogens to ER. Aromatase catalyzes the conversion of androgens to estrogens in the final step of the biosynthesis of estrogens and is therefore an attractive therapeutic target for inhibition. 3DQSAR pharmacophore modeling studies were undertaken for biphenyl derivatives as aromatase inhibitors in JEG-3 cell lines. A four-point pharmacophore with two H-bond acceptors and two aromatic rings as pharmacophoric features was developed. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a correlation coefficient of R² = 0.977 for training set molecules. The generated model showed excellent predictive power, with a correlation coefficient of Q² = 0.946 for an external test set. The 3D-QSAR plots illustrated insights into the structure activity relationship of these compounds which may help in the design and development of potent biphenyl derivatives as new aromatase inhibitors.

Regulation of the gene encoding human thrombin-activatable fibrinolysis inhibitor by estrogen and progesterone

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-carboxypeptidase B-like pro-enzyme that upon activation attenuates fibrinolysis and inflammatory processes. There is a large inter-individual variability in plasma TAFI levels within the population which is primarily due to epigenetic factors. Novel associations between plasma TAFI levels and sex steroids have triggered interest in determining the role of TAFI as a mediator of the cardioprotective effects of estrogens and progestins, or as a mediator of the increased thrombotic risk that accompanies use of oral contraceptives or hormone replacement therapy (HRT). In this study, we measured the effect of sex steroids on hepatic expression of CPB2, the human gene encoding TAFI. Using human hepatocellular carcinoma cells cultured in the presence of progesterone and 17β-estradiol, we demonstrated that the level of TAFI protein is decreased by those sex steroids. These changes in protein expression were paralleled by decreases in CPB2 mRNA abundance and promoter activity. We did not find evidence of estrogen or progesterone receptor binding sites in the CPB2 promoter region, suggesting that the genomic effects of progesterone and 17β-estradiol are mediated indirectly, without receptor binding to the CPB2 promoter. Indeed, we found that the effect of estrogen was independent of the estrogen receptor and was mediated through a novel signaling pathway dependent on phosphatidylinositol 3-kinase and Akt that did not involve GPR30. Our findings provide the molecular explanation for the ability of female sex steroids to decrease plasma TAFI concentrations.

D538G acquired mutation of estrogen receptor (ER) alpha: A novel mechanism of endocrine resistance in metastatic breast cancer.

e11578 Background: Resistance to endocrine therapy occurs in virtually all patients with ER-positive metastatic breast cancer (MBC) and is attributed to various mechanisms including loss of ER expression, altered activity of coregulators and cross-talk between the ER and growth factor signaling pathways. To our knowledge, acquired mutations of the ER have not been described as mediating endocrine resistance to endocrine treatment. Methods: Deep sequencing of the ER was conducted, as part of a commercially available next-generation sequencing of 182 cancer-related genes, on samples obtained from 10 heavily pre-treated, endocrine resistant patients with ER positive MBC. In one patient, a sample obtained at diagnosis was also available. Mutated ER was cloned and overexpressed in breast cancer cells. Transcriptional activity was tested using estrogen response element (ERE)-Luciferase construct, effects on viability were tested using MTT assays and computerized modeling was used to assess structural effects. Results: A novel mutation resulting in substitution of aspartic acid at position 538 to glycine was identified in 4 of 10 samples. Moreover, in one case in which samples were available prior to and following development of resistance, the D538G mutation was noted only in the latter sample. Structural modeling indicated that the substitution leads to a conformational change in the ligand binding domain which prevents binding of either estrogen or tamoxifen and mimics the conformation of activated receptor. Experiments in cell lines indicated constitutive ligand-independent transcriptional activity of the mutated receptor. Overexpression of the receptor in MCF-7 cells enhanced proliferation and conferred resistance to treatment with tamoxifen and fulvestarnt. Conclusions: We report here a novel mutation D538G of the ER in human breast cancer and demonstrate its role in mediating resistance to endocrine treatment. Our study also indicates, for the first time, acquired mutation of the ER as a novel mechanism of endocrine resistance. Further studies on larger populations and on less selected patients are needed in order to assess the accurate frequency of this novel mutation.

An in vitro investigation of endocrine disrupting effects of the mycotoxin alternariol

Alternariol (AOH) is a mycotoxin commonly produced by Alternaria alternata on a wide range of foods. Few studies to date have been performed to evaluate the effects of AOH on endocrine activity. The present study makes use of in vitro mammalian cellular based assays and gene expression to investigate the ability of AOH to act as an endocrine disruptor by various modes of action. Reporter gene assays (RGAs), incorporating natural steroid hormone receptors for oestrogens, androgens, progestagens and glucocorticoids were used to identify endocrine disruption at the level of nuclear receptor transcriptional activity, and the H295R steroidogenesis assay was used to assess endocrine disruption at the level of gene expression and steroid hormone production. AOH exhibited a weak oestrogenic response when tested in the oestrogen responsive RGA and binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of AOH. H295R cells when exposed to 0.1–1000ng/ml AOH, did not cause a significant change in testosterone and cortisol hormones but exposure to 1000ng/ml (3.87μM) AOH resulted in a significant increase in estradiol and progesterone production. In the gene expression study following exposure to 1000ng/ml (3.87μM) AOH, only one gene NR0B1 was down-regulated, whereas expression of mRNA for CYP1A1, MC2R, HSD3B2, CYP17, CYP21, CYP11B2 and CYP19 was up-regulated. Expression of the other genes investigated did not change significantly. In conclusion AOH is a weak oestrogenic mycotoxin that also has the ability to interfere with the steroidogenesis pathway.

Model of tumor-associated epigenetic changes of HER2, ER, and PgR expression in invasive breast cancer phenotypes

This theoretic paper is an attempt to apply the epigenetic progenitor model of human cancer origin, proposed by Feinberg et al. (Nat Rev Genet 7:21-33, 2006), to the reported phenotype features of invasive breast cancer. The model is based on the idea that expression of estrogen receptors (ER), progesterone receptors (PgR), and HER2 molecules in breast tumors is either remnants of the tissue stem cell from which the tumor has developed or a newly acquired tumor-associated epigenetic feature. HER2 overexpression is considered as an example of the tumor-associated epigenetic changes. The model makes a simple distinction regarding the possible types of ER and PgR expression: the "functional" steroid hormone receptors are inherited from pretumoral tissue stem cells, while the "dysfunctional" steroid hormone receptors are acquired during tumorigenesis from initially ER-PgR-negative cells. In the former, estrogen binding increases the PgR expression while progesterone binding decreases the expression of ER and PgR. Since the estrogen-dependent PgR expression works only in cells with functional ERs, the expected share of tumors with functional ER and PgR receptors is in the model calculated as the squared probability of expressing the PgRs. Reported data from various trials are pooled together to find out phenotype shares (ER+PgR+ makes 62.03 %, ER+PgR- 16.43 %, ER-PgR+ 3.06, and ER-PgR- 18.48 %). By applying the model on these shares, the proposed share of tumors with the functional ER+PgR+ phenotype was 38.48 %, while the share of tumors with the dysfunctional ER+PgR+ was 23.55 %. The presented model suggests that both luminal A and luminal B tumor types are heterogeneous regarding the steroid receptor expression. Some tumors have functional and some have dysfunctional steroid receptors. If these predicted subgroups exist, their detection in the clinical practice might substantially improve treatment options, particularly in the premenopausal setting.