Activation of ERß hijacks the splicing machinery to trigger R-loop formation in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with aggressive behavior and poor prognosis. Current therapeutic options available for TNBC patients are primarily chemotherapy. With our evolving understanding of this disease, novel targeted therapies, including poly ADP-ribose polymerase (PARP) inhibitors, antibody-drug conjugates, and immune-checkpoint inhibitors, have been developed for clinical use. Previous reports have demonstrated the essential role of estrogen receptor ß (ERß) in TNBC, but the detailed molecular mechanisms downstream ERß activation in TNBC are still far from elucidated. In this study, we demonstrated that a specific ERß agonist, LY500307, potently induces R-loop formation and DNA damage in TNBC cells. Subsequent interactome experiments indicated that the residues 151 to 165 of U2 small nuclear RNA auxiliary factor 1 (U2AF1) and the Trp439 and Lys443 of ERß were critical for the binding between U2AF1 and ERß. Combined RNA sequencing and ribosome sequencing analysis demonstrated that U2AF1-regulated downstream RNA splicing of 5-oxoprolinase (OPLAH) could affect its enzymatic activity and is essential for ERß-induced R-loop formation and DNA damage. In clinical samples including 115 patients from The Cancer Genome Atlas (TCGA) and 32 patients from an in-house cohort, we found a close correlation in the expression of ESR2 and U2AF1 in TNBC patients. Collectively, our study has unraveled the molecular mechanisms that explain the therapeutic effects of ERß activation in TNBC, which provides rationale for ERß activation-based single or combined therapy for patients with TNBC.

Estrogen signaling in the dorsal raphe regulates binge-like drinking in mice.

Estrogens promote binge alcohol drinking and contribute to sex differences in alcohol use disorder. However, the mechanisms are largely unknown. This study aims to test if estrogens act on 5-hydroxytryptamine neurons in the dorsal raphe nucleus (5-HTDRN) to promote binge drinking. We found that female mice drank more alcohol than male mice in chronic drinking in the dark (DID) tests. This sex difference was associated with distinct alterations in mRNA expression of estrogen receptor α (ERα) and 5-HT-related genes in the DRN, suggesting a potential role of estrogen/ERs/5-HT signaling. In supporting this view, 5-HTDRN neurons from naïve male mice had lower baseline firing activity but higher sensitivity to alcohol-induced excitation compared to 5-HTDRN neurons from naïve female mice. Notably, this higher sensitivity was blunted by 17ß-estradiol treatment in males, indicating an estrogen-dependent mechanism. We further showed that both ERα and ERß are expressed in 5-HTDRN neurons, whereas ERα agonist depolarizes and ERß agonist hyperpolarizes 5-HTDRN neurons. Notably, both treatments blocked the stimulatory effects of alcohol on 5-HTDRN neurons in males, even though they have antagonistic effects on the activity dynamics. These results suggest that ERs' inhibitory effects on ethanol-induced burst firing of 5-HTDRN neurons may contribute to higher levels of binge drinking in females. Consistently, chemogenetic activation of ERα- or ERß-expressing neurons in the DRN reduced binge alcohol drinking. These results support a model in which estrogens act on ERα/ß to prevent alcohol-induced activation of 5-HTDRN neurons, which in return leads to higher binge alcohol drinking.

No detectable changes in anxiety-related and locomotor behaviors in adult ovariectomized female rats exposed to estradiol, the ERß agonist DPN or the ERα agonist PPT.

The sex steroid hormone 17ß-estradiol (estradiol) and its Estrogen Receptors (ERs) have been linked to modulation of anxiety-related and locomotor behaviors in female rodents. Research suggests that estradiol mitigates anxiety-related behaviors through activating Estrogen Receptor (ER)ß and increases locomotor behaviors through ERα. The influence of ERs on these behaviors cannot always be detected. Here we discuss two experiments in which we tested the hypothesis that anxiety-related behaviors would decrease after ERß activation and locomotor behaviors would increase after ERα activation, and also assessed the persistence of these behavioral effects by varying the timing of behavioral testing. Two cohorts of adult female ovariectomized rats were exposed to estradiol, the ERß agonist DPN, the ERα agonist PPT, or oil for four consecutive days. Body mass was assessed throughout as a positive control. In both cohorts, open field behaviors were assessed on the first day of exposure. In one cohort (Experiment 1), open field, light/dark box, and elevated plus maze behaviors were assessed on the final day of injections. In the second cohort (Experiment 2), these behaviors were assessed 24 h after the final exposure. As expected, significant differences in body mass were detected in response to estradiol and PPT exposure, validating the estradiol and ER manipulation. No significant differences were observed in anxiety-related or locomotor behaviors across treatment groups, indicating that the efficacy of these agonists as therapeutic agents may be limited. We review these results in the context of previous literature, emphasizing relevant variables that may obscure ER-related actions on behavior.

In vivo potentiation of muscle torque is enhanced in female mice through estradiol-estrogen receptor signaling.

Estradiol affects several properties of skeletal muscle in females including strength. Here, we developed an approach to measure in vivo posttetanic twitch potentiation (PTP) of the anterior crural muscles of anesthetized mice and tested the hypothesis that 17ß-estradiol (E2) enhances PTP through estrogen receptor (ER) signaling. Peak torques of potentiated twitches were ∼40%-60% greater than those of unpotentiated twitches and such PTP was greater in ovary-intact mice, or ovariectomized (Ovx) mice treated with E2, compared with Ovx mice (P ≤ 0.047). PTP did not differ between mice with and without ERα ablated in skeletal muscle fibers (P = 0.347). Treatment of ovary-intact and Ovx mice with ERß antagonist and agonist (PHTPP and DPN, respectively) did not affect PTP (P ≥ 0.258). Treatment with G1, an agonist of the G protein-coupled estrogen receptor (GPER), significantly increased PTP in Ovx mice from 41 ± 10% to 66 ± 21% (means ± SD; P = 0.034). Collectively, these data indicate that E2 signals through GPER, and not ERα or ERß, in skeletal muscles of female mice to augment an in vivo parameter of strength, namely, PTP.NEW & NOTEWORTHY A novel in vivo approach was developed to measure potentiation of skeletal muscle torque in female mice and highlight another parameter of strength that is impacted by estradiol. The enhancement of PTP by estradiol is mediated distinctively through the G-protein estrogen receptor, GPER.

Comprehensive analysis of cellular estrogen signaling in representative estrogen receptor ligands.

The estrogen receptor (ER)-mediated signaling pathway in physiological and biochemical aspects is very important in the environment, including food. The physiological action of estrogen is mediated by ER alpha (ERα) and beta (ERß), whose physiological action on estrogenic substances is complex because of the relatively low ligand-binding domain (LBD) similarity of the two ERs. In this study, the comprehensive activity of representative ER ligands was evaluated by using BRET-based ERα and ERß dimerization and ER transactivation assays to differentiate the specific binding and function of ERα and ERß from 12 representative natural and synthetic estrogenic substances. Results revealed that 11 chemicals mediated receptor ERα and ERß dimerization, 7 out of 12 chemicals were confirmed to be estrogen agonists, and 5 chemicals were antagonistic. Overall, this study demonstrated consistency between BRET dimerization and transactivation responses, supporting potential supplementary application of mechanism-based BRET assays as high-throughput screening methods for evaluation of potential endocrine-disrupting activity of environmental agents. This study also provided information about receptor specificity of ligand-mediated estrogenic activity via dimerization assays and elucidated cellular estrogen signaling pathways.

Effects of estrogen receptor ß or G protein-coupled receptor 30 activation on anxiety-like behaviors in relation to GABAergic transmission in stress-ovariectomized rats.

For mental disorders such as anxiety and depression, stress and stressful events are considered as precipitating causes that may be enhanced by estrogen variability. This condition is proven by the higher vulnerability of women than men. Despite the complexity of underlying mechanisms, the gamma-aminobutyric acid (GABA) system piques interest as its receptor contains multiple psychoactive modulatory sites including neurosteroids. Moreover, according to clinical and experimental reports, GABA-associated genes can be altered by stress and hormonal status. Therefore, this study investigated the effects of estrogen receptor ß (ERß) or G protein-coupled receptor 30 (GPR30) activation on anxiety/depression-like behaviors and the alterations in the GABA-associated gene of ovariectomized rats under chronic mild stress (CMS). Mild stressors were focused on because they represent a realistic simulation of daily life stress. In this study, ovariectomized rats were treated with vehicle, estradiol (E2), diarylpropionitrile (DPN; ERß agonist) or G1 (GPR30 agonist) and exposed to 4-week CMS. The results showed that E2, DPN, and G1 treatments reduced anxiety-like behaviors without affecting depression-like behaviors. Concurrently, the GABA level and most GABA- and neurosteroid-associated mRNAs were altered by E2. Similar mRNA profiles were observed in DPN- and E2-administrations but not in G1 treatment. Collectively, these data suggest that estrogen exerts an anxiolytic-like action through either ERß and/or GPR30 activation, and the modulatory effects of estrogen on GABAergic system are likely to be modulated through ERß. The findings of this study therefore further provide insights into the roles of estrogen and daily mild stressors in GABA-related activity and behavioral responses, especially anxiety.

Differential contribution of estrogen receptors to the intestinal therapeutic effects of 17ß-estradiol in a murine model of Parkinson's disease.

Beneficial effects of estrogens have been reported in Parkinson's disease (PD) for many years. We previously reported their neuroprotective and anti-inflammatory potentials in the enteric nervous system of the intestine, a region possibly affected during the early stages of the disease according to Braak's hypothesis. Three different estrogen receptors have been characterized to date: the estrogen receptor alpha (ERα), the estrogen receptor beta (ERß) and the G protein coupled estrogen receptor 1 (GPER1). The aim of the present study was to decipher the individual contribution of each estrogen receptor to the therapeutic properties of 17ß-estradiol (E2) in the myenteric plexus of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Different agonists, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT; ERα), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ERß), G1 (GPER1), and antagonists, ICI 182,780 (ERα and ERß), G15 (GPER1), were used to analyze the involvement of each receptor. We confirmed that G1 protects dopamine (DA) neurons to a similar extent as E2. An anti-inflammatory effect on proinflammatory macrophages and cultured human monocytes was also demonstrated with E2 and G1. The effects of PPT and DPN were less potent than G1 with only a partial neuroprotection of DA neurons by PPT and a partial reduction of interleukin (IL)- 1ß production in monocytes by PPT and DPN. Overall, the present results indicate that the positive outcomes of estrogens are mainly through activation of GPER1. Therefore, this suggests that targeting GPER1 could be a promising approach for future estrogen-based hormone therapies during early PD.

Epigenetic restoration and activation of ERß: an inspiring approach for treatment of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. TNBC lacks targeted therapy receptors, rendering endocrine and HER2-targeted therapies ineffective. TNBC is typically treated with cytotoxic chemotherapy followed by surgery. Targeting epigenetic modifications could potentially be a new effective TNBC target therapy. The aim of this study is to examine the effects of epigenetic drugs, decitabine as DNA methyltransferase inhibitor (DNMTI) and vorinostat as histone deacetylase inhibitor (HDACI), and the ERß agonist DPN on ERα and ERß re-expressions in the MDA-MB-231 cells as a model of TNBC. METHODS: Using MTT assay, the IC50 of decitabine, vorinostat, and DPN on MDA-MB-231 cells were determined. The effects of all drugs alone or in combinations on MDA-MB-231 cells were evaluated. qRT-PCR was used to determine ERα & ERß gene expression. Caspase-3 activity and the protein expression levels of VEGF, Cyclin D1, and IGF-1 were assessed. RESULTS: Both ERα and ERß mRNA were re-expressed in different high levels in all treated groups, especially in the triple therapy group compared with control. Significantly, the triple drugs therapy showed the lowest levels of VEGF, Cyclin D1, and IGF-1 and the highest level of Caspase-3 activity, indicating a possible antitumor effect of ERß activation through decreasing proliferation and angiogenesis and increasing apoptosis in MDA-MB-231 cells. CONCLUSIONS: The antiproliferative effect of ERß could be retained when co-expressed with Erα using a powerful epigenetic combination of Decitabine and vorinostat with DPN.

Estrogen receptor α activation modulates the gut microbiome and type 2 diabetes risk factors.

Estradiol and exercise can decrease risk factors associated with type 2 diabetes (T2D) including total body weight gain and abdominal fat gain. Estradiol functions through estrogen receptor (ER) α and ERß. Some studies suggest that activation of ERα may provide protection against T2D. Female Wistar rats were ovariectomized and fed a high-fat diet for 10 weeks and divided into the following 5 experimental groups: (1) no treatment (control), (2) exercise, (3) estradiol, (4) propylpyrazoletriyl (a selective ERα agonist), and (5) diarylpropionitrile (a selective ERß agonist). ERα activation decreased the abundance of Firmicutes, and ERα and ERß activation increased the abundance of Bacteroidetes. ERα activation decreased food consumption, and ERα and ERß activation increased voluntary activity. Exercise was the only treatment to decrease the blood glucose and serum insulin levels. ERα activation, but not ERß, increased hepatic protein expression of ACC and FAS and decreased hepatic protein expression of LPL. ERα activation also decreased hepatic mRNA expression of PPARα and PPARγ. This study elucidates the functions of estradiol by assessing specific activation of ERα and ERß. As obesity increases the abundance of Firmicutes and decreases the abundance of Bacteroidetes, our study shows that ERα activation can restore the gut microbiome to non-obese abundances. This study further provides novel insights into ERα's role in hepatic fat metabolism via regulation of ACC, FAS, LPL, PPARα, and PPARγ.

ERα, but not ERß and GPER, Mediates Estradiol-Induced Secretion of TSH in Mouse Pituitary.

Although estradiol (E2) plays a critical role in the promotion of pituitary development and in the regulation of various pituitary hormones, its effects on the thyroid-stimulating hormone (TSH) remain unaddressed. The actions of E2 are mediated by two classical nuclear estrogen receptors α (ERα) and ß (ERß) and the G protein-coupled estrogen receptor (GPER). However, the types of estrogen receptor involvement in the regulation of thyrotropes are still limited. In this study, we demonstrate that ERα, but not ERß and GPER, is localized to thyrotropes in the pituitary of female mouse. In agreement with the presence of ERα in thyrotropes, E2 was shown to stimulate TSH release in vitro from primary culture of female mouse pituitary cells. PPT, a ERα-selective agonist, but not DPN (a ERß-selective agonist) and G-1 (a GPER-selective agonist), was shown to stimulate TSH release in mouse pituitary cells. This effect could be prevented by the specific ER antagonist fulvestrant and the selective ERα antagonist MPP. The findings of this study suggest that E2 may bind to ERα to trigger TSH release and provide novel information on the differential regulation of multiple estrogen receptors in the pituitary.

The Natural Estrogen Receptor Beta Agonist Silibinin as a Promising Therapeutic Tool in Diffuse Large B-cell Lymphoma.

BACKGROUND/AIM: About 40% of patients with diffuse large cell lymphoma (DLBCL) still have a poor prognosis. Additionally, DLBCL patients treated with doxorubicin are at risk of cardiac failure. Growing evidence suggests an antitumor and cardioprotective activity exerted by estrogen via its binding to estrogen receptor (ER) ß. The aim of this study was to evaluate the anticancer activity of the phytoestrogen silibinin, an ERß selective agonist, on DLBCL growth, and its potential cardioprotective effect. MATERIALS AND METHODS: DLBCL cell lines SUDHL-8, SUDHL-6, and RIVA were used. The anti-tumor activity of silibinin was also evaluated in vivo in NOD/SCID/IL2Rg-/- (NSG) xenografted mice. AC16 human ventricular cardiomyocytes were used to investigate the cardioprotective effects of silibinin. RESULTS: In vitro silibinin induced apoptosis and autophagy, and blocked tumor cell proliferation, also protecting AC16 cardiomyocytes from doxorubicin-induced toxicity. In vivo silibinin induced cell death and autophagy, and reduced tumor volume. CONCLUSION: Silibinin represents a promising therapeutic tool.

The estrogen receptor beta agonist liquiritigenin enhances the inhibitory effects of the cholesterol biosynthesis inhibitor RO 48-8071 on hormone-dependent breast-cancer growth.

PURPOSE: Most hormone-dependent human breast cancers develop resistance to anti-hormone therapy over time. Our goal was to identify novel treatment strategies to avoid this drug resistance and thereby control hormone-dependent breast cancer. METHODS: Sulforhodamine B assays were used to measure viability of cultured human breast-cancer cells. BT-474 cell tumor xenografts in nude mice were used to evaluate tumor growth. Immunohistochemistry was used to assess estrogen-receptor and angiogenesis-marker expression, as well as apoptosis, in tumor-xenograft tissues. RESULTS: MCF-7 and BT-474 breast-cancer cells treated with either RO 48-8071 <[4'-[6-(Allylmethylamino)hexyloxy]-4-bromo-2'-fluorobenzophenone fumarate] [RO]; a small-molecule inhibitor of oxidosqualene cyclase, a key enzyme in cholesterol biosynthesis> or liquiritigenin [LQ; an estrogen receptor (ER) ß agonist] exhibited significantly reduced viability in vitro. RO + LQ treatment further significantly reduced cell viability. Administration of RO, LQ, or RO + LQ significantly inhibited growth of BT-474 tumor xenografts in vivo. RO, LQ, or RO + LQ reduced ERα but induced ER ß expression in tumor xenografts. Both compounds significantly reduced angiogenesis-marker expression and increased apoptosis in tumor xenografts; use of RO + LQ significantly enhanced the effects observed with a single agent. CONCLUSION: The ERß ligand LQ significantly enhanced the inhibition of breast-cancer cell viability and tumor-xenograft growth by RO. The anti-tumor properties of RO may in part be due to an off-target effect that reduces ERα and increases ERß, the latter of which can then interact with LQ to promote anti-proliferative effects. The RO + LQ combination may have value when considering novel treatment strategies for hormone-dependent breast cancer.

SERMs (selective estrogen receptor modulator), acting as estrogen receptor ß agonists in hepatocellular carcinoma cells, inhibit the transforming growth factor-α-induced migration via specific inhibition of AKT signaling pathway.

Selective estrogen receptor modulator (SERM) interacts with estrogen receptors and acts as both an agonist or an antagonist, depending on the target tissue. SERM is widely used as a safer hormone replacement therapeutic medicine for postmenopausal osteoporosis. Regarding hepatocellular carcinoma (HCC), accumulating evidence indicates gender differences in the development, and that men are at higher morbidity risk than premenopausal women, suggesting that estrogen protects against HCC. However, it remains unclear whether SERM affects the HCC progression. Previously, we have shown that transforming growth factor (TGF)-α promotes the migration of HCC cells via p38 mitogen-activated protein kinases (MAPK), c-Jun N-terminal kinase and AKT. In the present study, we investigated whether SERM such as tamoxifen, raloxifene and bazedoxifene, affects the HCC cell migration using human HCC-derived HuH7 cells. Raloxifene and bazedoxifene but not tamoxifen, significantly suppressed the TGF-α-induced HuH7 cell migration. ERB041 and DPN, estrogen receptor (ER) ß agonists, inhibited the TGF-α-induced cell migration whereas PPT, an ERα agonist, did not show the suppressive effect on the cell migration. ERB041 attenuated the TGF-α-induced phosphorylation of AKT without affecting the phosphorylation of p38 MAPK and c-Jun N-terminal kinase. Raloxifene and bazedoxifene also inhibited the phosphorylation of AKT by TGF-α. Furthermore, PHTPP, an ERß antagonist, significantly reversed the suppression by both raloxifene and bazedoxifene of the TGF-α-induced cell migration. Taken together, our results strongly indicate that raloxifene and bazedoxifene, SERMs, suppress the TGF-α-induced migration of HCC cells through ERß-mediated inhibition of the AKT signaling pathway.

Estradiol promotes cell survival and induces Greb1 expression in granulosa cell tumors of the ovary through an ERα-dependent mechanism.

Granulosa cell tumor (GCT) is a form of ovarian tumor characterized by its tendency to recur years after surgical ablation. Little is known about the mechanisms involved in GCT development and progression. GCTs can produce estradiol (E2), but whether this hormone could play a role in this cancer through its nuclear receptors, i.e. ERα and ERß, remains unknown. Here, we addressed this issue by cell-based and molecular studies on human GCTs and GCT cell lines. Importantly, we observed that E2 significantly increased the growth of GCT cells by promoting cell survival. The use of selective agonists of each type of receptor, together with Esr1 (ERα) or Esr2 (ERß)-deleted GCT cells, revealed that E2 mediated its effects through ERα-dependent genomic mechanisms and ERß/ERα-dependent extra-nuclear mechanisms. Notably, the expression of Greb1, a prototypical ER target gene, was dose-dependently upregulated by E2 specifically through ERα in GCT cells. Accordingly, using GCTs from patients, we found that GREB1 mRNA abundance was positively correlated to intra-tumoral E2 concentrations. Tissue microarray analyses showed that there were various combinations of ER expression in primary and recurrent GCTs, and that ERα expression persisted only in combination with ERß in ~40% of recurrent tumors. Altogether, this study demonstrates that E2 can promote the progression of GCTs, with a clear dependence on ERα. In addition to demonstrating that GCTs can be classified as a hormone-related cancer, our results also highlight that the nature of ER forms present in recurrent GCTs could underlie the variable efficiency of endocrine therapies. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel targeted approach to delineate a role for estrogen receptor-ß in ameliorating murine mammary tumor-associated neuroinflammation.

PURPOSE: Circulating estrogens in breast cancer patients and survivors are often extremely low due to menopause and estrogen-reducing cancer treatments. Simultaneously, circulating inflammatory markers, and inflammatory proteins in brains of rodent tumor models, can be elevated and correlate with debilitating neurological and psychological comorbidities. Because estrogen has anti-inflammatory properties in the brain, we hypothesized that mammary tumor-induced neuroinflammation is driven, in part, by reduced brain estrogen signaling. METHODS: An ovariectomized mouse model of postmenopausal breast cancer utilizing the ERα-positive 67NR mammary tumor cell line was used for these experiments. A novel, orally bioavailable, and brain penetrant ERß agonist was administered daily via oral gavage. Following treatment, estrogen-responsive genes were measured in brain regions. Central and circulating inflammatory markers were measured via RT-qPCR and a multiplex cytokine array, respectively. RESULTS: We present novel findings that peripheral mammary tumors alter estrogen signaling genes including receptors and aromatase in the hypothalamus, hippocampus, and frontal cortex. Mammary tumors induced peripheral and central inflammation, however, pharmacological ERß activation was not sufficient to reduce this inflammation. CONCLUSIONS: Data presented here suggest that compensating for low circulating estrogen with ERß brain activation is not sufficient to attenuate mammary tumor-induced neuroinflammation, and is therefore not a likely candidate for the treatment of behavioral symptoms in patients. The novel finding that mammary tumors alter estrogen signaling-related genes is a clinically relevant advancement to the understanding of how peripheral tumor biology modulates neurobiology. This is necessary to predict and prevent behavioral comorbidities (e.g., cognitive impairment) prevalent in cancer patients and survivors.

Clinicopathological Significance of Estrogen Receptor ß and Estrogen Synthesizing/Metabolizing Enzymes in Urothelial Carcinoma of Urinary Bladder.

Sex-specific differences in the incidence of urinary bladder carcinomas are well known, and the possible involvement of sex steroids has been proposed. We previously reported the association of the loss of androgen receptors and androgen-producing enzymes with tumor progression of urinary bladder cancer patients. Clinically, the selective estrogen receptor modulators (SERMs) were reported to suppress the progression of these tumors but the status of estrogen receptors (ERs) has not been well studied in patients with bladder urinary cancer. Moreover, not only ERs but also estrogen-related enzymes, such as aromatase, steroid sulfatase (STS), and estrogen sulfotransferase (EST), have been reported in the biological/clinical behavior of various hormone-dependent carcinomas but not studied in urinary bladder carcinoma. Therefore, in this study, we immunolocalized ERs as well as estrogen metabolizing enzymes in urinary bladder carcinoma and performed immunoblotting and cell proliferation assays using the bladder urothelial carcinoma cell line, T24. The results revealed that the loss of STS and aromatase was significantly correlated with advanced stages of the carcinoma. In vitro studies also revealed that T24 cell proliferation rates were significantly ameliorated after treatment with estradiol or diarylpropionitrile (DPN). EST and aromatase were also significantly correlated with the nuclear grade of the carcinoma. The results of our present study, for the first time, demonstrated that biologically active estrogens that bind to ERs could suppress tumor progression and the inactive ones could promote its progression and the potential clinical utility of SERM treatment in selective patients with urinary bladder carcinoma.

Structure-Activity Relationship of para-Carborane Selective Estrogen Receptor ß Agonists.

Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERß, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17ß-estradiol geometry in the design of ERß selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERß selective structure-activity relationship. We report ERß agonists with low nanomolar potency, greater than 200-fold selectivity for ERß over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERß selective agonists measure favorably against clinically developed ERß agonists and support further evaluation of carborane-based selective estrogen receptor modulators.

Perinatal activation of ERα and ERß but not GPER-1 masculinizes female rat caudate-putamen medium spiny neuron electrophysiological properties.

Exposure to steroid sex hormones such as 17ß-estradiol (estradiol) during early life potentially permanently masculinize neuron electrophysiological phenotype. In rodents, one crucial component of this developmental process occurs in males, with estradiol aromatized in the brain from testes-sourced testosterone. However, it is unknown whether most neuron electrophysiological phenotypes are altered by this early masculinization process, including medium spiny neurons (MSNs) of the rat caudate-putamen. MSNs are the predominant and primary output neurons of the caudate-putamen and exhibit increased intrinsic excitability in females compared to males. Here, we hypothesize that since perinatal estradiol exposure occurs in males, then a comparable exposure in females to estradiol or its receptor agonists would be sufficient to induce masculinization. To test this hypothesis, we injected perinatal female rats with estradiol or its receptor agonists and then later assessed MSN electrophysiology. Female and male rats on postnatal day 0 and 1 were systemically injected with either vehicle, estradiol, the estrogen receptor (ER)α agonist PPT, the ERß agonist DPN, or the G-protein-coupled receptor 1 (GPER-1) agonist G1. On postnatal days 19 ± 2, MSN electrophysiological properties were assessed using whole cell patch clamp recordings. Estradiol exposure abolished increased intrinsic excitability in female compared to male MSNs. Exposure to either an ERα or ERß agonist masculinized female MSN evoked action potential firing rate properties, whereas exposure to an ERß agonist masculinized female MSN inward rectification properties. Exposure to ER agonists minimally impacted male MSN electrophysiological properties. These findings indicate that perinatal estradiol exposure masculinizes MSN electrophysiological phenotype via activation of ERα and ERß.NEW & NOTEWORTHY This study is the first to demonstrate that estradiol and estrogen receptor α and ß stimulation during early development sexually differentiates the electrophysiological properties of caudate-putamen medium spiny neurons, the primary output neuron of the striatal regions. Overall, this evidence provides new insight into the neuroendocrine mechanism by which caudate-putamen neuron electrophysiology is sexually differentiated and demonstrates the powerful action of early hormone exposure upon individual neuron electrophysiology.

Estrogen receptor ß and treatment with a phytoestrogen are associated with inhibition of nuclear translocation of EGFR in the prostate.

Knockout of ERß in the mouse leads to nuclear expression of epidermal growth factor receptor (EGFR) in the prostate. To examine whether ERß plays a similar role in the human prostate, we used four cohorts of men: 1) a Swedish cohort of normal prostates and PCa (prostate cancer) of different Gleason grades; 2) men with benign prostatic hyperplasia (BPH) treated with the 5α-reductase inhibitor, finasteride, and finasteride together with the ERß agonists, soy isoflavones; 3) men with PCa above Gleason grade 4 (GG4), treated with ADT (androgen deprivation therapy) and abiraterone (AA), the blocker of androgen synthesis for different durations; and 4) men with GG4 PCa on ADT or ADT with the AR (androgen receptor) blocker, enzalutamide, for 4 mo to 6 mo. In men with BPH, finasteride treatment induced EGFR nuclear expression, but, when finasteride was combined with isoflavones, EGFR remained on the cell membrane. In GG4 patients, blocking of AR for 4 mo to 6 mo resulted in loss of ERß and PTEN expression and increase in patients with nuclear EGFR from 10 to 40%. In the men with GG4 PCa, blocking of adrenal synthesis of testosterone for 2 mo to 7 mo had the beneficial effect of increasing ERß expression, but, on treatment longer than 8 mo, ERß was lost and EGFR moved to the nucleus. Since nuclear EGFR is a predictor of poor outcome in PCa, addition of ERß agonists together with abiraterone should be considered as a treatment that might sustain expression of ERß and offer some benefit to patients.

Structure-Activity Relationship of Phytoestrogen Analogs as ERα/ß Agonists with Neuroprotective Activities.

A set of isoflavononid and flavonoid analogs was prepared and evaluated for estrogen receptor α (ERα) and ERß transactivation and anti-neuroinflammatory activities. Structure-activity relationship (SAR) study of naturally occurring phytoestrogens, their metabolites, and related isoflavone analogs revealed the importance of the C-ring of isoflavonoids for ER activity and selectivity. Docking study suggested putative binding modes of daidzein 2 and dehydroequol 8 in the active site of ERα and ERß, and provided an understanding of the promising activity and selectivity of dehydroequol 8. Among the tested compounds, equol 7 and dehydroequol 8 were the most potent ERα/ß agonists with ERß selectivity and neuroprotective activity. This study provides knowledge on the SAR of isoflavonoids for further development of potent and selective ER agonists with neuroprotective potential.