Cisplatin-activated ERß/DCAF8 positive feedback loop induces chemoresistance in non-small cell lung cancer via PTEN/Akt axis.

High levels of the estrogen receptor ß (ERß) predict poor prognosis following platinum-containing adjuvant chemotherapies in patients with non-small cell lung cancer (NSCLC). However, the precise role of ERß remains elusive. In this study, we demonstrated that targeting ERß could significantly increase the cytotoxicity of cisplatin both in vitro and in vivo. Mechanically, cisplatin directly binds to ERß, which facilitates its homodimerization and nuclear translocation. ERß activation transcriptionally represses the expression of DCAF8, an adaptor of CRL4 E3 ubiquitin ligase, which in turn attenuates the proteasomal degradation of ERß, leading to ERß accumulation; this positive feedback loop results in Akt activation and eventually cisplatin resistance in NSCLC through PTEN inhibition. Moreover, low expression of DCAF8 and high expression of ERß are associated with treatment resistance in patients receiving cisplatin-containing adjuvant chemotherapy. The present results provide insights into the underlying mechanism of ERß-induced cisplatin resistance and offer an alternative therapeutic strategy to improve the efficacy of platinum-based chemotherapy in patients with NSCLC.

Development of heterodimeric estrogen receptor alpha antagonists to target simultaneously the ligand and coactivator binding site.

One-third of breast cancer patients will develop recurrent cancer within 15 years of endocrine treatment. Notably, tumor growth in a hormone-refractory state still relies on the interaction between estrogen receptor alpha (ERα) and upregulated coactivators. Herein, we suggest that simultaneous targeting of the primary ligand binding site (LBS) and the coactivator binding site (CABS) at ERα represents a promising alternative therapeutic strategy to overcome mutation-driven resistance in breast cancer. We synthesized two series of compounds that connect the LBS-binder (E)-3-{4-[8-fluoro-4-(4-hydroxyphenyl)-2,3-dihydrobenzo[b]oxepin-5-yl]phenyl}acrylic acid 8 with the coactivator binding site inhibitors (CBIs) 4,6-bis(isobutyl(methyl)amino)pyrimidine or 3-(5-methoxy-1H-benzo[d]imidazol-2-yl)propanoic acid via covalent linkage. The most active benzoxepine-pyrimidine conjugate 31 showed strong inhibition of estradiol-induced transactivation (IC50 = 18.2 nM (ERα) and 61.7 nM (ERß)) in a luciferase reporter gene assay as well as high antiproliferative effects in MCF-7 (IC50 = 65.9 nM) and tamoxifen-resistant MCF-7/TamR (IC50 = 88.9 nM) breast cancer cells. All heterodimers exhibited two- to sevenfold higher antagonism at ERα (compared with ERß) and were superior to the acrylic acid precursor 8 in terms of ER antagonism and antiproliferative activity. It was demonstrated on the example of 31 that the compounds did not influence the ERα content in MCF-7 cells and therefore act as pure antiestrogens without downregulating potency. Possible interactions of the CBI at the receptor surface, which enhanced the biological activities, were evaluated using molecular docking studies.

Therapeutic Role of Tamoxifen for Triple-Negative Breast Cancer: Leveraging the Interaction Between ERß and Mutant p53.

The absence of effective therapeutic targets and aggressive nature of triple-negative breast cancer (TNBC) renders this disease subset difficult to treat. Although estrogen receptor beta (ERß) is expressed in TNBC, studies on its functional role have yielded inconsistent results. However, recently, our preclinical studies, along with other observations, have shown the potential therapeutic utility of ERß in the context of mutant p53 expression. The current case study examines the efficacy of the selective estrogen receptor modulator tamoxifen in p53-mutant TNBC with brain metastases. Significant increase in ERß protein expression and anti-proliferative interaction between mutant p53 and ERß were observed after cessation of tamoxifen therapy, with significant regression of brain metastases. This case study provides supporting evidence for the use of tamoxifen in p53-mutant, ERß+TNBC, especially in the setting of brain metastasis.

Hormonal therapies up-regulate MANF and overcome female susceptibility to immune checkpoint inhibitor myocarditis.

Immune checkpoint inhibitors (ICIs) have been increasingly used in combination for cancer treatment but are associated with myocarditis. Here, we report that tumor-bearing mice exhibited response to treatment with combinatorial anti-programmed cell death 1 and anti-cytotoxic T lymphocyte antigen-4 antibodies but also presented with cardiovascular toxicities observed clinically with ICI therapy, including myocarditis and arrhythmia. Female mice were preferentially affected with myocarditis compared to male mice, consistent with a previously described genetic model of ICI myocarditis and emerging clinical data. Mechanistically, myocardial tissue from ICI-treated mice, the genetic mouse model, and human heart tissue from affected patients with ICI myocarditis all exhibited down-regulation of MANF (mesencephalic astrocyte-derived neurotrophic factor) and HSPA5 (heat shock 70-kDa protein 5) in the heart; this down-regulation was particularly notable in female mice. ICI myocarditis was amplified by heart-specific genetic deletion of mouse Manf and was attenuated by administration of recombinant MANF protein, suggesting a causal role. Ironically, both MANF and HSPA5 were transcriptionally induced by liganded estrogen receptor ß and inhibited by androgen receptor. However, ICI treatment reduced serum estradiol concentration to a greater extent in female compared to male mice. Treatment with an estrogen receptor ß-specific agonist and androgen depletion therapy attenuated ICI-associated cardiac effects. Together, our data suggest that ICI treatment inhibits estradiol-dependent expression of MANF/HSPA5 in the heart, curtailing the cardiomyocyte response to immune injury. This endocrine-cardiac-immune pathway offers new insights into the mechanisms of sex differences in cardiac disease and may offer treatment strategies for ICI myocarditis.

Oleuropein suppresses endometriosis progression and improves the fertility of mice with endometriosis.

BACKGROUND: Endometriosis is an estrogen-dependent inflammatory reproductive disease. Therefore, systematic estrogen depletion and anti-inflammatory drugs are the current treatment for endometriosis. However, current endometriosis treatments have low efficacy and cause adverse effects in endometriosis patients. Consequently, alternative endometriosis treatments targeting endometriosis-specific factors are in demand. In this context, ERß was selected as a druggable target for endometriosis due to its critical role in progression. Therefore, selective targeting of ERß without inhibiting ERα activity would be a new paradigm for endometriosis treatment to overcome the low efficacy and adverse effects of hormonal endometriosis therapy. METHODS: Cell-based ERß and ERα activity assay systems were employed to define a selective ERß-inhibiting chemical product from a library of natural products. A surgically induced endometriosis mouse model was used to determine whether an ERß inhibitory drug suppressed endometriosis progression. Mice with endometriosis were randomly separated and then orally treated with vehicle or 25 mg/kg oleuropein (once a day for 21 days), an ERß inhibitory drug. The volume of endometriotic lesions or luciferase activity of endometriotic lesions was examined to define the growth of ectopic lesions in mice with endometriosis. The metabolite and levels of metabolic enzymes of the liver and kidney were determined in the serum of female mice treated with vehicle and oleuropein (25 mg/kg, once a day for 21 days) to define the toxicity of oleuropein. The in vitro decidualization assay was conducted with normal human endometrial stromal cells and endometriotic stromal cells to determine whether oleuropein overcomes decidualization in endometriosis patients. The pregnancy rate and pup numbers of C57BL/6 J female mice with endometriosis treated with vehicle or oleuropein (n = 10/group) were determined after mating with male mice. The cytokine profile in endometriotic lesions treated with vehicle and oleuropein (25 mg/kg) was determined with a Mouse Cytokine Array Kit. RESULTS: Among natural products, oleuropein selectively inhibited ERß but not ERα activity in vitro. Oleuropein treatment inhibited the nuclear localization of ERß in human endometrial cells upon estradiol treatment. Oleuropein (25 mg/kg) treatment suppressed the growth of mouse (6.6-fold) and human (sixfold) ectopic lesions in mice with endometriosis compared to the vehicle by inhibiting proliferation and activating apoptosis in endometriotic lesions. Oleuropein treatment did not cause reproductive toxicity in female mice. Additionally, mice with endometriosis subjected to oleuropein treatment had a higher pregnancy rate (100%) than vehicle-treated mice (70%). Furthermore, oleuropein treatment partially recovered the decidualization impact of human endometriotic stromal cells from endometriotic lesions compared to the vehicle. Oleuropein-treated mice with endometriosis exhibited significantly lower levels of cytokines directly regulated by ERß in ectopic lesions than vehicle-treated mice, illustrating the improvement in the hyperinflammatory state of mice with endometriosis. CONCLUSIONS: Oleuropein is a promising and novel nutraceutical product for nonhormonal therapy of endometriosis because it selectively inhibits ERß, but not ERα, to suppress endometriosis progression and improve the fertility of mice with endometriosis.

Overexpression of estrogen receptor ß inhibits cellular functions of human hepatic stellate cells and promotes the anti-fibrosis effect of calycosin via inhibiting STAT3 phosphorylation.

BACKGROUND: Estrogen receptor ß (ERß) is the major ER subtype in hepatic stellate cells (HSCs). Previously we reported phytoestrogen calycosin suppressed liver fibrosis progression and inhibited HSC-T6 cell functions, suggesting the effects may be related to ERß. Here, we explore the effect of overexpressed ERß on human HSCs and the role of ERß in pharmacological action of calycosin. METHODS: LX-2 cells were transfected with lentivirus to overexpress ERß. In the presence or absence of overexpressed ERß, the effects of ERß and calycosin on proliferation, migration, activation, collagen production and degradation of TGF-ß1-induced LX-2 cells and the role of ERß in the inhibition effect of calycosin were investigated. LX-2 cells overexpressed with ERß or treated with ER non-selective antagonist ICI182,780 were used to investigate the regulation of ERß on JAK2/STAT3 signaling pathway. CCK-8 method was used to screen effective doses of calycosin and investigate cell proliferation. The cell migration was detected by transwell chamber assay. The expression of α-SMA was detected by immunofluorescence and western blot. The protein expressions of Col-I, MMP1, TIMP1, JAK2, p-JAK2, STAT3 and p-STAT3 were detected by western blot. RESULTS: ERß overexpressed lentivirus was successfully transfected into LX-2 cells with high efficiency. Overexpressed ERß or calycosin alone inhibited the TGF-ß1-induced LX-2 cell proliferation and migration, downregulated the protein expressions of α-SMA, Col-I, TIMP-1, p-STAT3 and upregulated MMP-1. Both overexpressed ERß and calycosin had no significant effect on JAK2, p-JAK2 and STAT3 expressions. ERß overexpression further enhanced the above effects of calycosin. However, after the cells were treated with ICI182,780, downregulation of STAT3 phosphorylation induced by calycosin was reversed. CONCLUSIONS: ERß mediated the inhibition of major functions of LX-2 cell possibly by inhibiting the phosphorylation of STAT3, and was an important pathway through which calycosin exerted anti-liver fibrosis effect.

Estrogen Receptor-ß Agonists Modulate T-Lymphocyte Activation and Ameliorate Left Ventricular Remodeling During Chronic Heart Failure.

BACKGROUND: CD4+ T cells temporally transition from protective to pathological during ischemic heart failure (HF; 8 weeks postmyocardial infarction). Cellular mechanisms mediating this shift are unknown. METHODS: RNA-sequencing of cardiac CD4+ T cells and flow cytometric analysis of immune cells was conducted. RESULTS: RNA-sequencing of CD4+ T cells from the failing hearts of male mice indicated activation of ER (estrogen receptor)-α signaling. Flow cytometric analysis showed that ERα in CD4+ T cells decreases significantly at 3-day postmyocardial infarction but increases during HF. To antagonize ERα, we tested a novel ERß agonist (OSU-ERb-012) to inhibit T cells and blunt left ventricular remodeling. Proliferation assays showed that OSU-ERb-012 dose-dependently inhibited proliferation and proinflammatory cytokine expression in anti-CD3/CD28 stimulated splenic T cells isolated from both the sexes. For in vivo efficacy, 10- to 12-week-old male and ovariectomized female mice were randomized at 4 weeks postmyocardial infarction and treated with either vehicle or drug (60 mg/kg per day; oral). While vehicle-treated HF mice displayed progressive left ventricular dilatation with significantly increased end-systolic and end-diastolic volumes from 4 to 8 weeks postmyocardial infarction, treatment with OSU-ERb-012 significantly blunted these changes and stopped left ventricular remodeling in both the sexes. Reduction in tibia-normalized heart and left ventricular weights, cardiomyocyte hypertrophy and interstitial fibrosis further supported these results. Additionally, OSU-ERb-012 treatment selectively inhibited cardiac, splenic, and circulating CD4+ T cells without affecting other myeloid and lymphoid cells in the HF mice. CONCLUSIONS: Our studies indicate that ERß agonists and OSU-ERb-012, in particular, could be used as selective immunomodulatory drugs to inhibit CD4+ T cells during chronic HF.

Identification of the estrogen receptor beta as a possible new tamoxifen-sensitive target in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype. Despite the proven efficacy of combined immunochemotherapy (R-CHOP) in the majority of patients, ~40% of DLBCL patients do not respond or will relapse and consequently have a very poor prognosis. The development of targeted therapies has not improved patient survival, underscoring the need for new treatment approaches. Using an unbiased genome-wide CD20 guilt-by-association approach in more than 1800 DLBCL patients, we previously identified the estrogen receptor beta (ERß) as a new target in DLBCL. Here, we demonstrate that ERß is expressed at significantly higher levels in DLBCL compared to normal B cells, and ERß plays a role in the protection against apoptosis in DLBCL. Targeting of the ERß with the selective estrogen receptor modulator tamoxifen reduces cell viability in all tested DLBCL cell lines. Tamoxifen-induced cell death was significantly decreased in an ERß knock-out cell line. The activity of tamoxifen was confirmed in a xenograft human lymphoma model, as tumor growth decreased, and survival significantly improved. Finally, tamoxifen-treated breast cancer (BC) patients showed a significantly reduced risk of 38% for DLBCL compared to BC patients who did not receive tamoxifen. Our findings provide a rationale to investigate tamoxifen, a hormonal drug with a good safety profile, in DLBCL patients.

Chrysin reduces inflammation and oxidative stress and improves ovarian function in D-gal-induced premature ovarian failure.

Premature ovarian failure (POF), a frequently occurring pathology. Chrysin has antioxidant, anti-inflammatory, anti-apoptotic and other pharmacological activities. This study was designed to detect the effect of Chrysin on POF. The establishment of POF was depended on the subcutaneous injection of D-gal (200 mg/kg/d). With the adoption of ELISA, the levels of hormones and release of inflammatory cytokines were assayed. The expression of MDA, GSH-px, SOD and ROS was evaluated with corresponding kits. In addition, the pathological changes of ovary and apoptosis of ovarian granulosa cells in D-gal-induced mice were detected using H&E staining and TUNEL, respectively. Moreover, the levels of FSH receptor and apoptosis-related proteins were measured with western blot. Finally, ERß expression was measured with RT-qPCR and western blot. In this study, we found that chrysin regulated the expression of hormones and weight of D-gal-induced mice. It was also found that chrysin inhibited the inflammation and oxidative stress in mice with D-gal induction. In addition, the number and advancement of follicle in D-gal-induced mice treated with chrysin revealed that chrysin could improve the ovarian function of mice with POF. Furthermore, chrysin exhibited inhibitory effects on the apoptosis of ovarian granulosa cells in D-gal-induced mice. More importantly, chrysin molecule targeted ERß and activated ERß expression in POF. Overall, Chrysin reduces inflammation and oxidative stress and improves ovarian function in D-gal-induced premature ovarian failure, suggesting that chrysin is valuable for the treatment of POF.

The expression and clinical significance of ERß/ERα in ovarian cancer: can we predict the effectiveness of platinum plus taxane therapy?

Estrogens play an extremely important role in regulating the proliferation of ovarian cancer. The estrogen receptor alpha (ERα) stimulates cell growth, whereas ERß can be attributed to tumor suppressors. The study aims to assess the relationship between the expression of estrogen receptors in tumors and the efficacy of front-line platinum plus taxane chemotherapy in ovarian cancer patients. MATERIALS AND METHODS: ERα and ERß tumor expression was evaluated quantitatively by flow cytometry in a narrowly defined group (31 patients): stage III high-grade serous ovarian carcinoma (HGSOC), suboptimal surgical cytoreduction, front-line platinum plus taxane chemotherapy (front-line, six cycles). RESULTS: The median of progression-free survival (PFS) was 2 times greater (18 vs 8 months, p = 0.04) and the recurrence risk (HR) was 2.2 times (95 % CI: 1.1-6.2, p = 0.04) lower in the group with high (in more than 40% of the cells) vs low level of ERß tumor expression. The statistically significant difference between PFS in the groups with high vs low tumor ERα expression was not revealed. CONCLUSION: A high level of ERß and not ERα expression can predict the efficacy of front-line platinum plus taxane chemotherapy in stage III HGSOC patients. The status of estrogen receptor beta can be considered as one of the possible predictors for evaluating the effectiveness of ovarian cancer therapy.

Targeting estrogen receptors in colorectal cancer.

Hormones have become a useful therapeutic aspect of clinical endocrinology but how to use them to optimize the health benefits and avoid adverse effects is a major challenge. Estrogen is an indispensable hormone for proper biological functioning but is also implicated with the pathology of both the reproductive and non-reproductive tissues. Abnormal estrogen receptor signaling may increase the risk of development of a variety of diseases including colorectal cancer (CRC). Estrogen receptor beta (ERß) is the predominant subtype in the colonic epithelium and confers the anti-tumor effect through various mechanisms. Many investigators have embarked on the search for the biological mechanisms by which estrogen and estrogen-like compounds may influence the pathogenesis of CRC. This review explores the recent findings on the therapeutic role of ERß in the colonic epithelium as a prospective candidate for targeted endocrine therapy in CRC.

Estrogen receptor ß: Potential target for therapy in adult granulosa cell tumors?

OBJECTIVE: Adult granulosa cell tumor (AGCT) is a rare form of sex-cord stromal ovarian tumors. Due to their origin, AGCTs secrete estrogens, and thus, estrogen receptor (ER)-mediated signaling has been considered as a possible target for therapy. The aim of the present study was to get insights into estrogen receptor status and activity in AGCTs, as a strategy to provide molecular support for personalized hormonal treatments. METHODS: We evaluated by immunohistochemistry the expression of ERα, ERß isoforms (i.e. ERß1, ERß2 and ERß5), progesterone and androgen receptor (PR, AR) in 20 untreated AGCTs and 12 unmatched recurrent lesions. Thereafter, we visualized by immunofluorescence, the subcellular distribution of cytoplasmic receptors, and by the proximity ligation assays (PLA) we characterized in situ their ability to interact with other proteins involved in the apoptotic cascade. RESULTS: Primary AGCTs predominantly expressed ERß isoforms, along with PR and AR, while only 30% of patients showed ERα expression. Recurrent tumors were associated with a decrease in AR levels. From mechanistic studies it emerges that ERß2, and to a lesser extent ERß1 and AR, are mitochondrial components in cancer cells and that ERß2 can act as a binding partner of proteins involved in the apoptotic cascade, in turn potentially inhibiting apoptosis. CONCLUSIONS: As in other endocrine tumors, ERß may play a role in the pathogenesis of AGCT; it is crucial to understand estrogen receptor-mediated pathways before planning hormonal treatment strategies in AGCT.

Estrogen via estrogen receptor beta partially inhibits mandibular condylar cartilage growth.

OBJECTIVE: Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role for female hormones in the disease process. However, little is known about the role of estrogen receptor (ER) signaling in regulating mandibular condylar cartilage growth. Therefore, the goal of this study was to examine the effects of altered estrogen levels on the mandibular condylar cartilage in wild type (WT) and ER beta Knockout (KO) mice. MATERIALS AND METHODS: 21-day-old female WT (n = 37) and ER beta KO mice (n = 36) were either sham operated or ovariectomized, and treated with either placebo or estradiol. The mandibular condylar cartilage was evaluated by histomorphometry, proliferation was analyzed by double ethynyl-2'-deoxyuridine/bromodeoxyuridine (EdU/BrdU) labeling, and assays on gene and protein expression of chondrocyte maturation markers were performed. RESULTS: In WT mice, ovariectomy caused a significant increase in mandibular condylar cartilage cell numbers, a significant increase in Sox9 expression and a significant increase in proliferation compared with sham operated WT mice. In contrast, ovariectomy did not cause any of these effects in the ER beta KO mice. Estrogen replacement treatment in ovariectomized WT mice caused a significant decrease in ER alpha expression and a significant increase in Sost expression compared with ovariectomized mice treated with placebo. Estrogen replacement treatment in ovariectomized ER beta KO mice caused a significant increase in Col2 expression, no change in ER alpha expression, and a significant increase in Sost expression. CONCLUSION: Estrogen via ER beta inhibits proliferation and ER alpha expression while estrogen independent of ER beta induces Col2 and Sost expression.

Inhibitory effects of ERß on proliferation, invasion, and tumor formation of MCF-7 breast cancer cells--prognostication for the use of ERß-selective therapy.

CONTEXT: Estrogen is well-known as an important factor in the physiological functions and pathological processes of breast. Estrogen receptor ß (ERß) is expressed in the majority of breast cancers at lower levels compared with the normal breast tissue. OBJECTIVE: The effect of ERß on the characteristics of breast tumor cells and its prognostication for the use of ERß-selective therapy were investigated here for the first time. MATERIALS AND METHODS: ERß was overexpressed in ERα positive MCF-7 breast cancer cells by gene transfection. The proliferation, motility, and xenografts growth of MCF-7 cells were investigated by MTT assays, wound-healing assay and animal study. RESULTS: Results demonstrated that ERß-GFP localized in both the cytoplasm and the nucleus in the presence of 17ß-estradiol (E2), with stronger fluorescence-signal intensity in the nucleus, 2.8-times higher than that in the cytoplasm. The ERß overexpressed MCF-7 cells resulted in a 38.7% decreased growth rate and motility in vitro. Furthermore, ERß overexpression enhanced the antiproliferative effects of phytoestrogen, antiestrogen, and histone deacetylase inhibitor. Exogenous ERß expression reduced tumor volume by 99% at 27 days postadministration, indicated that overexpression of ERß led to retardation of tumor formation and growth in immunodeficient mice. DISCUSSION AND CONCLUSION: This study provided a relatively new evidence to support that ERß is an important modulator of proliferation and motility of breast cancer cells, and implied for the first time a possibility for the use of novel ERß-selective therapies in breast cancer treatment.

ERbeta impedes prostate cancer EMT by destabilizing HIF-1alpha and inhibiting VEGF-mediated snail nuclear localization: implications for Gleason grading.

High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit diminished estrogen receptor beta (ERbeta) expression. We report that a key function of ERbeta and its specific ligand 5alpha-androstane-3beta,17beta-diol (3beta-adiol) is to maintain an epithelial phenotype and repress mesenchymal characteristics in prostate carcinoma. Stimuli (TGF-beta and hypoxia) that induce an epithelial-mesenchymal transition (EMT) diminish ERbeta expression, and loss of ERbeta is sufficient to promote an EMT. The mechanism involves ERbeta-mediated destabilization of HIF-1alpha and transcriptional repression of VEGF-A. The VEGF-A receptor neuropilin-1 drives the EMT by promoting Snail1 nuclear localization. Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF-1alpha and VEGF expression, and Snail1 nuclear localization compared to low Gleason grade cancers.