Expression Of G Protein-coupled Estrogen Receptor Research Articles

Differential expression of estrogen receptor subtypes in ovarian high-grade serous carcinoma and clear cell carcinoma.

PurposeTo investigate the role of estrogen receptors (ERs) in high‐grade serous carcinoma (HGSC) and clear cell carcinoma (CCC) of the ovary and evaluate ERs as prognostic biomarkers for ovarian cancer.MethodsThis study included 79 patients with HGSC (n = 38) or CCC (n = 41) treated at our institution between 2005 and 2014. Immunohistochemistry examined protein expression of ERα, ERβ, and G protein‐coupled estrogen receptor‐1 (GPER‐1); relationships between ERα, ERβ, and GPER‐1 with patient survival were evaluated. Additionally, cell proliferation assay and phosphokinase proteome profiling were performed.ResultsIn HGSC patients, expression of ERα, cytoplasmic GPER‐1, or nuclear GPER‐1 was associated with poor progression‐free survival (PFS) (P = .041, P = .010, or P = .013, respectively). Cytoplasmic GPER‐1 was an independent prognostic factor for PFS in HGSC patients (HR = 2.83, 95% CI = 1.03‐9.16, P = .007). ER expressions were not associated with prognosis in CCC patients. GPER‐1 knockdown by siRNA reduced the cells number to 60% of siRNA‐control‐treated cells (P < .05), and GPER‐1 antagonist, G‐15 inhibited two HGSC cell lines proliferation (KF and UWB1.289) in a dose‐dependent manner. Phosphoprotein array revealed that GPER‐1 silencing decreased relative phosphorylation of glycogen synthase kinase‐3.ConclusionsHigh GPER‐1 expression is an independent prognostic factor for PFS in HGSC patients, and GPER‐1 may play a role in HGSC cell proliferation.

Abstract 1225: LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of cancer

Abstract Women have a lower incidence of and improved clinical outcomes for most cancer types, but the mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that many cancer types lack classic nuclear estrogen receptors. The GPER agonist G-1, a racemate, has been demonstrated to have anticancer activities by many independent laboratories, though there are some contradictory findings in the literature. We resolved the enantiomers that compose G-1 and determined that LNS8801 is the sole enantiomer responsible for G-1's anticancer effects. Using CRISPR to deplete GPER, we determined that the anticancer activity of LNS8801 is dependent upon GPER expression within the tumor cells. Previously, we determined that the anticancer mechanism of GPER activation is through a rapid proteasomal degradation of c-Myc. The PRISM screen was performed on LNS8801 at the Broad Institute, and it was determined that this compound has universal anticancer activity across multiple cancer types, consistent with our inhouse data. Sensitivity to LNS8801 was highly correlated with expression of c-Myc target genes, which is consistent with the known mechanism of action. Lineage analysis identified two specific cancer types that were uniformly more sensitive to LNS8801 than other cancer types: diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL). DLBCL has historically been considered to be highly dependent on c-Myc, which is consistent with the known mechanism of action of LNS8801; however, the identification of ALCL was unexpected. Deeper analysis determined that high expression of anaplastic lymphoma kinase (ALK) fusion genes tightly correlated with sensitivity to LNS8801 in ALCL and other ALK+ cancer types. Using non-small cell lung cancer cell lines engineered to express EML4-ALK, we validated that ALK fusion expression results in increased sensitivity to LNS8801 relative to isogenic controls. This work has identified an enantiomerically pure GPER agonist that has potent activity across many cancer types, with c-Myc-dependent and ALK+ cancer types being enriched for heightened sensitivity. Citation Format: Chris Natale, Tina Garyantes, Todd Ridky. LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1225.

G Protein-Coupled Estrogen Receptor 1 (GPER1) Mediates Aldosterone-Induced Endothelial Inflammation in a Mineralocorticoid Receptor-Independent Manner.

Objective It has been increasingly appreciated that G protein-coupled estrogen receptor 1 (GPER1) mediates both proinflammatory and anti-inflammatory response of estrogen. It is also involved in some rapid vascular effects of aldosterone in a mineralocorticoid receptor (MR) independent manner. However, whether GPER1 mediates aldosterone-induced inflammation response in endothelial cells and its relationship with MR are yet undetermined and therefore require further explanation. Method Based on the hypothesis that GPER1 plays a role in the aldosterone-related vascular inflammation, the present study utilized a model of human umbilical vein endothelial cells transfected with MR siRNA and induced for inflammatory response with increasing concentration of aldosterone. Results It was discovered that induction of aldosterone had no effect on the expression of GPER1 but promoted the expression of MR. Suppression of MR did not influence GPER1 expression, and GPER1 was capable of mediating part of aldosterone-induced endothelial inflammatory response. This effect may involve phosphoinositide 3-kinases (PI3K) pathway signaling. Conclusion These findings not only demonstrated the role of GPER1 in aldosterone-induced vascular inflammation but also suggested an alternative for pharmaceutical treatment of hyperaldosteronism considering the unsatisfying effect on cardiovascular risks with MR antagonists.

G Protein‐Coupled Estrogen Receptor 1 is Required for Greater Endothelin‐1 Excretion in Female Mice

Estrogen signaling via G protein-coupled estrogen receptor 1 (GPER1) can elicit cardiovascular and renal protective actions. Endothelin-1 (ET-1) is a pro-natriuretic peptide that contributes to sex differences in blood pressure regulation and Na+ homeostasis. We recently showed that activation of GPER1 in the renal medulla promotes ET-1-dependent natriuresis in female but not male rats. Also, renal GPER1 expression is significantly higher in female rats than in male rats. In the current study, we hypothesized that genetic deletion of GPER1 would elevate blood pressure and impair renal ET-1 production/release in a sex-specific manner. Twelve-week-old male and female GPER1-knockout (KO) and wild-type (WT) littermate mice were implanted with telemeters to monitor blood pressure. Parallel groups of mice were placed into metabolic cages, and 24-h urine samples were collected while food and water intake were monitored. Then, animals were euthanized to obtain plasma. Urinary levels of electrolytes and ET-1 were measured. GPER1 deletion did not significantly affect mean arterial blood pressure in female (KO: 102±1 mmHg; WT: 102±2 mmHg; n=7-8/group, p=0.9) or male mice (KO: 106±3 mmHg; WT: 106±2 mmHg; n=6-10/group, p=1). No genotypic differences were observed in food intake, water intake, or urine flow rate. GPER1 deletion did not significantly affect urinary Na+excretion in female (KO: 0.25±0.05 μg/24 h; WT: 0.17±0.05 μg/24 h; n=9-12/group, p=0.52) or male mice (KO: 0.34±0.06 μg/24 h; WT: 0.35±0.04 μg/24 h; n=10-12/group, p=1.00). Similarly, GPER1 deletion did not affect urinary K+ excretion in either sex. However, GPER1 deletion decreased urinary ET-1 excretion in females (KO: 75.16±16.35 ng/24 h; WT: 172.83±24.67 ng/24 h; n=6-10/group, p<0.005) but not males (KO: 95.18±17.52 ng/24 h; WT: 76.03± 9.90 ng/24 h; n=10-12/group, p=0.69). Of note, WT females had significantly greater urinary ET-1 excretion than WT males (p=0.001), whereas KO mice had no sex differences in ET-1 excretion. Importantly, urinary excretion of ET-1 reflects intrarenal ET-1 production/release. GPER1 deletion did not significantly change plasma ET-1 concentration in either sex. Finally, GPER1 deletion did not affect urinary aldosterone excretion in females (KO: 315.98±73.13 pg/24 h; WT: 306.79±81.45 pg/24 h; n=7-8, p=1.00) or males (KO: 544.09±130.20 pg/24 h; WT: 481.84±68.30 pg/24 h; n=8/group, p=0.87). However, male mice had significantly greater urinary aldosterone excretion than female mice (p=0.04), regardless of genotype. Overall, we demonstrate that WT female mice have higher urinary ET-1 excretion than WT males, and that GPER1 deletion eliminates this sex-related difference in intrarenal ET-1 production/release. Our data suggest that GPER1 may contribute to the enhanced capacity of the female kidney to maintain Na+ homeostasis and have led us to hypothesize this as a mechanism for protecting females against salt-sensitive hypertension.

Eplerenone inhibits oxidized low-density lipoprotein-induced proliferation and migration of vascular smooth muscle cells by downregulating GPER expression.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are associated with the pathogenesis of atherosclerosis (AS). Eplerenone (EPL), a novel selective aldosterone receptor blocker, plays a substantial role in the treatment of cardiovascular disease. The G protein-coupled estrogen receptor (GPER) is a target of EPL as the STITCH website predicated. We aimed to investigate the roles of EPL in AS and identify its potential mechanisms of action. Oxidized low-density lipoprotein (ox-LDL) was employed to stimulate VSMCs to establish a cellular model of AS. The ability of cell proliferation was examined using a Cell Counting Kit-8, and the expression of proliferation-related proteins was tested using immunofluorescence staining and western blot analysis. Subsequently, cell migration and the expression of migration-associated proteins were evaluated with a wound healing assay, transwell assay and western blot analysis. Then, GPER expression was determined using western blot analysis in the absence or presence of EPL. To explore the regulatory mechanisms of EPL in ox-LDL-stimulated VSMCs, GPER was overexpressed, followed by measurement of cell proliferation and migration. The Ox-LDL stimulation notably upregulated GPER expression, whereas EPL treatment downregulated GPER expression in a dose-dependent manner. Additionally, EPL markedly inhibited proliferation and migration of VSMCs, and the highest dose of EPL resulted in the most marked effect. By contrast, GPER overexpression reversed the inhibitory effects of EPL on proliferation and migration of VSMCs. Eplerenone suppressed ox-LDL-induced proliferation and migration of VSMCs partly through downregulation of GPER, providing a new mechanism of support for EPL use in the clinical treatment of AS.

Activation of GPER by E2 promotes proliferation, invasion and migration of breast cancer cells by regulating the miR-124/CD151 pathway.

Breast cancer is one of the most common malignancies worldwide and is responsible for a high mortality rate. However, the underlying pathological mechanism of breast cancer remains unclear. MicroRNAs (miRNAs/miRs) play critical roles in the progression of breast cancer. Recent studies have reported that miR-124/CD151 participates in the development of breast cancer. However, the exact molecular mechanism of miR-124/CD151 action in 17β-estradiol (E2)-treated breast cancer cells remains unknown. Thus, the present study aimed to investigate miR-124 and CD151 expression levels in MCF-7 cells treated with E2 via reverse transcription-quantitative PCR and western blot analyses. Bioinformatic analysis was performed to predict and identify whether CD151 is a potential target of miR-124. The Cell Counting Kit-8 and colony formation assays were performed to detect proliferation of MCF-7 cells. In addition, the invasive and migratory abilities of MCF-7 cells were assessed via the Transwell and wound healing assays, respectively. The results demonstrated that E2 downregulated miR-124 expression, while upregulating G protein -coupled estrogen receptor (GPER) expression in MCF-7 cells. Following treatment with the GPER antagonist, G15, miR-124 expression was significantly enhanced and E2-induced proliferation, invasion and migration of MCF-7 cells were notably inhibited. In addition, CD151 was confirmed as a direct target of miR-124. CD151 silencing remarkably suppressed the proliferation, invasion and migration of E2-induced MCF-7 cells. Taken together, these results suggest that upregulation of GPER expression induced by E2 promotes proliferation, invasion and migration of breast cancer cells by regulating the miR-124/CD151 pathway. Thus, the results of the present study provide a potential novel method for the treatment and prognosis of breast cancer.

The G-Protein-Coupled Estrogen Receptor (GPER) Regulates Trimethylation of Histone H3 at Lysine 4 and Represses Migration and Proliferation of Ovarian Cancer Cells In Vitro.

Histone H3 lysine 4 trimethylation (H3K4me3) is one of the most recognized epigenetic regulators of transcriptional activity representing, an epigenetic modification of Histone H3. Previous reports have suggested that the broad H3K4me3 domain can be considered as an epigenetic signature for tumor-suppressor genes in human cells. G-protein-coupled estrogen receptor (GPER), a new membrane-bound estrogen receptor, acts as an inhibitor on cell growth via epigenetic regulation in breast and ovarian cancer cells. This study was conducted to evaluate the relationship of GPER and H3K4me3 in ovarian cancer tissue samples as well as in two different cell lines (Caov3 and Caov4). Silencing of GPER by a specific siRNA and two selective regulators with agonistic (G1) and antagonistic (G15) activity were applied for consecutive in vitro studies to investigate their impacts on tumor cell growth and the changes in phosphorylated ERK1/2 (p-ERK1/2) and H3K4me3. We found a positive correlation between GPER and H3K4me3 expression in ovarian cancer patients. Patients overexpressing GPER as well as H3K4me3 had significantly improved overall survival. Increased H3K4me3 and p-ERK1/2 levels and attenuated cell proliferation and migration were observed in Caov3 and Caov4 cells via activation of GPER by G1. Conversely, antagonizing GPER activity by G15 resulted in opposite effects in the Caov4 cell line. In conclusion, interaction of GPER and H3K4me3 appears to be of prognostic significance for ovarian cancer patients. The results of the in vitro analyses confirm the biological rationale for their interplay and identify GPER agonists, such as G1, as a potential therapeutic approach for future investigations.

GPER-Induced ERK Signaling Decreases Cell Viability of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with a poor prognosis. Effective biomarkers and specific therapeutic targets for HCC are therefore urgently needed. G protein-coupled estrogen receptor (GPER) plays a crucial role in numerous cancer types; however, its functions in HCC require further exploration. In the present study, we found a remarkable difference in GPER staining between tumor tissue (100/141, 70.9%) and matched non-tumor tissue (27/30, 90.0%). Compared with the GPER-negative patients, the GPER-positive patients with HCC were closely associated with female sex, negative hepatitis B surface antigen, small tumor size, low serum alpha fetoprotein level, and longer overall survival. Treatment with GPER-specific agonist G1 led to the sustained and transient activation of the EGFR/ERK and EGFR/AKT signaling pathways, respectively, in the HCC cell lines HCCLM3 and SMMC-7721, which express high levels of GPER. Interestingly, G1-induced EGFR/ERK signaling, rather than EGFR/AKT signaling mediated by GPER, was involved in decreasing cell viability by blocking cell cycle progression, thereby promoting apoptosis and inhibiting cell growth. Clinical analysis indicated that simultaneous high expression of GPER and phosphorylated-ERK (p-ERK) predicted improved prognosis for HCC. Finally, the activation of GPER/ERK signaling remarkably suppressed tumor growth in an HCC xenograft model, and this result was consistent with the in vitro data. Our findings suggest that specific activation of the GPER/ERK axis may serve as a novel tumor-suppressive mechanism and that this axis could be a therapeutic target for HCC.

A novel transcript variant of human G-protein coupled estrogen receptor.

The G-protein coupled estrogen receptor (GPER) mediates short-term non-genomic effects of estrogen in diverse cell types and tissues. According to the NCBI nucleotide database, three variants of GPER are known. They are NM_001505.2 (GPER-v2), NM_001039966.1 (GPER-v3), and NM_001098201.1 (GPER-v4). Investigations on GPER expression are key to understand its physiological and pathological roles. However, most studies on GPER mRNA expression have considered total GPER mRNA expression regardless of the individual variants. The present study is motivated by a novel transcript observed in the UCSC Genome Browser (uc010ksd.1), which is annotated as GPER. The novel variant is similar to the known transcriptvariants of GPER in terms of the protein-coding sequence and the 3'UTR. However, it has a unique 5'UTR, which distinguishes it from other GPER variants. Using primers specific for uc010ksd.1, we have performed RT-PCR to show that the novel GPER transcript (hereafter referred to as GPER-v5) is expressed in human cancer cell lines, such as MCF-7, SW-620, COLO-205, and HT-29. Preliminary evidences indicate that GPER-v5 is a novel GPER mRNA variant. The expression of GPER-v5 in primary cells and tissues should be investigated before probing into its role and relevance in physiological and pathological conditions.

G Protein-Coupled Estrogen Receptor Correlates With Dkk2 Expression and Has Prognostic Impact in Ovarian Cancer Patients.

PurposeWnt pathway modulator Dickkopf 2 (Dkk2) and signaling of the G protein-coupled estrogen receptor (GPER) seem to have essential functions in numerous cancer types. For epithelial ovarian cancer (EOC), it has not been proven if either Dkk2 or the GPER on its own have an independent impact on overall survival (OS). So far, the correlation of both factors and their clinical significance has not systematically been investigated before.MethodsExpression levels of Dkk2 were immunohistochemically analyzed in 156 patient samples from different histologic subtypes of EOC applying the immune-reactivity score (IRS). Expression analyses were correlated with clinical and pathological parameters to assess for prognostic relevance. Data analysis was performed using Spearman’s correlations, Kruskal-Wallis-test and Kaplan-Meier estimates.ResultsHighest Dkk2 expression of all subtypes was observed in clear cell carcinoma. In addition, Dkk2 expression differed significantly (p<0.001) between low and high grade serous ovarian cancer. A significant correlation of Dkk2 with the cytoplasmic GPER expression was noted (p=0.001) but not for the nuclear estrogen receptor alpha (ERα) or beta (ERβ). Patients exhibiting both, high expression Dkk2 (IRS>4) and GPER (IRS>8), had a significantly better overall survival compared to patients with low expression (61 months vs. 33 months; p=0.024).ConclusionDkk2 and GPER expression correlates in EOC and combined expression of both is associated with improved OS. These findings underline the clinical significance of both pathways and indicate a possible prognostic impact as well as a potential for treatment strategies addressing interactions between estrogen and Wnt signaling in ovarian cancer.

Insight into the mechanism of tetrachlorobisphenol A (TCBPA)-induced proliferation of breast cancer cells by GPER-mediated signaling pathways.

Tetrachlorobisphenol A (TCBPA), a chlorinated derivative of bisphenol A, is an endocrine disruptor based on interaction with nuclear estrogen receptor alpha (ERα). However, there is only limited data on the mechanisms through which TCBPA-associated estrogenic activity is related to the membrane G protein-coupled estrogen receptor (GPER) pathway. In this study, three human breast cancer cell lines-MCF-7, SKBR3, and MDA-MB-231cells were used to evaluate whether, as well as how, TCBPA at concentration range of 0.001-50μM affect cell proliferation. The role of GPER signaling in TCBPA-induced cell proliferation was studied by analyzing the protein expression and mRNA levels of relevant signal targets. The results showed that low concentrations of TCBPA significantly induced the proliferation of MCF-7, SKBR3, and MDA-MB-231cells, with MCF-7cells being the most sensitive to TCBPA exposure. Low-concentration TCBPA also upregulated the expression of GPER, CyclinD1, c-Myc, and c-Fos proteins, as well as increased the phosphorylation of extracellular signal-regulated-kinase 1/2 (Erk1/2) and protein kinase B (Akt). Additionally, the mRNA levels of genes associated with estrogen signaling pathways also increased upon exposure to TCBPA. However, the phosphorylation of Erk1/2 and Akt decreased when the cells were treated with GPER inhibitor G15 and phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin (WM) prior to TCBPA exposure. Besides, the increased proliferation of breast cancer cells induced by TCBPA were also inhibited. In ERα-positive MCF-7cells, TCBPA also upregulated ERα expression, and ERα was found to interact with GPER-mediated signaling. The results indicate that GPER activates the PI3K/Akt and Erk1/2 signal cascades to drive the cell proliferation observed for low concentrations of TCBPA. The presented results suggest a new mechanism by which TCBPA exerts estrogenic action in breast cancer cells, namely, GPER signaling in an ERα-independent manner, and also highlights the potential risks to human health of the usage of TCBPA.

Alterations in the estrogen receptor profile of cardiovascular tissues during aging.

Estrogen exerts protective effects on the cardiovascular system via three known estrogen receptors: alpha (ERα), beta (ERß), and the G protein-coupled estrogen receptor (GPER). Our laboratory has previously showed the importance of GPER in the beneficial cardiovascular effects of estrogen. Since clinical studies indicate that the protective effects of exogenous estrogen on cardiovascular function are attenuated or reversed 10 years post-menopause, the hypothesis was that GPER expression may be reduced during aging. Vascular reactivity and GPER protein expression were assessed in female mice of varying ages. Physiological parameters, blood pressure, and estrogen receptor transcripts via droplet digital PCR (ddPCR) were assessed in the heart, kidney, and aorta of adult, middle-aged, and aged male and female C57BL/6 mice. Vasodilation to estrogen (E2) and the GPER agonist G-1 were reduced in aging female mice and were accompanied by downregulation of GPER protein. However, ERα and GPER were the predominant receptors in all tissues, whereas ERß was detectable only in the kidney. Female sex was associated with higher mRNA for both ERα and GPER in both the aorta and the heart. Aging impacted receptor transcript in a tissue-dependent manner. ERα transcript decreased in the heart with aging, while GPER expression increased in the heart. These data indicate that aging impacts estrogen receptor expression in the cardiovascular system in a tissue- and sex-specific manner. Understanding the impact of aging on estrogen receptor expression is critical for developing selective hormone therapies that protect from cardiovascular damage.

A Clinicopathologic Study on the Role of Estrogen, Progesterone, and Their Classical and Nonclassical Receptors in Cutaneous Neurofibromas of Individuals With Neurofibromatosis 1.

To evaluate the expression of progesterone receptor (PR), estrogen receptor (ER), and G protein-coupled estrogen receptor 1 (GPER-1) in cutaneous neurofibromas (cNFs) and their correlation with demographic, clinical, and laboratory data of individuals with neurofibromatosis 1 (NF1). The association of PROGINS polymorphism and PR expression in cNFs, as well as the serum steroidal hormones and the number of cNFs, was investigated. The sample comprised 80 large and 80 small cNFs from 80 individuals with NF1. PR, ER, GPER-1, and Ki-67 expression were investigated by immunohistochemistry in tissue micro- and macroarrays and quantified using a digital computer-assisted method. The number of cNFs, the levels of serum 17β estradiol and progesterone, and the PROGINS polymorphism were identified. Twelve (8.5%) small cNFs were weakly positive for ER, 131 (92.3%) cNFs expressed PR, and all (100%) cNFs expressed GPER-1. Large cNFs showed a higher expression of PR (P < .0001) and GPER-1 (P = .019) and had a higher intensity of staining for these receptors (P < .0001). The cell proliferation index was positively correlated with PR (P = .001). Persons with more cNFs had higher serum levels of progesterone (P = .001). These findings emphasize the role of estrogen and progesterone in cNF development and suggest that these hormones may act on cNF cells via a noncanonical pathway through GPER-1.

G-Protein-Coupled Estrogen Receptor 1 Promotes Gender Disparities in Hepatocellular Carcinoma via Modulation of SIN1 and mTOR Complex 2 Activity.

Due to its intricate heterogeneity and limited treatment, hepatocellular carcinoma (HCC) has been considered a major cause of cancer-related mortality worldwide. Increasing evidence indicates that G-protein-coupled estrogen receptor 1 (GPER1) can promote estrogen-dependent hepatocellular proliferation by activating AKT signaling. The mTOR complex 2 (mTORC2), whose integrity and activity are modulated by its subunit Sin1, controls the activation of AKT by phosphorylation at position S473. In this study, we investigate the modulation of Sin1 and how estrogen signaling may influence the mTORC2-AKT cascade in HCC cells and a DEN-induced mouse model. We have found that estradiol-dependent Sin1 expression is transcriptionally modulated by GPER1 as well as ERα. GPER1 is able to regulate Sin1 stability via nuclear translocation, therefore increasing Sin1-mTORC2-AKT activation. Moreover, Sin1 interacts with ERα and further enhances its transcriptional activity. Sin1 is highly expressed in acute liver injury and in cases of HCC harboring high expression of GPER1 and constitutive activation of mTORC2-AKT signaling. GPER1 inhibition using the antagonist G-15 reverses DEN-induced acute liver injury by suppressing Sin1 expression and mTORC2-AKT activation. Notably, SIN1 expression varies between male and female mice in the context of both liver injury and liver cancer. In addition, high SIN1 expression is predictive of good prognosis in both male and female patients with HCC who are free from hepatitis virus infection and who report low alcohol consumption. Hence, here we demonstrate that Sin1 can be regulated by GPER1 both through nongenomic and indirect genomic signaling. IMPLICATIONS: This study suggests that Sin1 may be a novel HCC biomarker which is gender-dependent and sensitive to particular risk factor.

G protein-coupled oestrogen receptor promotes cell growth of non-small cell lung cancer cells via YAP1/QKI/circNOTCH1/m6A methylated NOTCH1 signalling.

Results from various studies reveal that the role of G protein‐coupled oestrogen receptor (GPER) is cancer‐context dependent, and the function of GPER in non–small‐cell lung cancer (NSCLC) is still unclear. The present study demonstrated that neoplasm lung tissues expressed higher level of GPER compared with the normal lung tissues. The clinical data also showed that GPER expression level was positively correlated with the tumour stage of NSCLC. Our experimental data confirmed that GPER played an oncogenic role to promote cell growth of NSCLC cells. Mechanistic dissection revealed that GPER could modulate the NOTCH1 pathway to regulate cell growth in NSCLC cells. Further exploration of the mechanism demonstrated that GPER could up‐regulate circNOTCH1, which could compete with NOTCH1 mRNA for METTL14 binding. Because of the lack of m6A modification by METTL14 on the NOTCH1 mRNA, NOTCH1 mRNA was more stable and much easier to undergo protein translation. Subsequently, we found that GPER could prevent YAP1 phosphorylation and promote YAP1‐TEAD's transcriptional regulation on QKI, a transacting RNA‐binding factor involved in circRNA biogenesis, to facilitate circNOTCH1 generation. Supportively, data from preclinical mice model with implantation of H1299 cells also demonstrated that knock‐down of circNOTCH1 could block GPER‐induced NOTCH1 to suppress NSCLC tumour growth. Together, our data showed that GPER could promote NSCLC cell growth via regulating the YAP1/QKI/circNOTCH1/m6A methylated NOTCH1 pathway, and targeting our identified molecules may be a potentially therapeutic approach to suppress NSCLC development.

Soy Isoflavones Accelerate Glial Cell Migration via GPER-Mediated Signal Transduction Pathway.

Soybean isoflavones, such as genistein, daidzein, and its metabolite, S-equol, are widely known as phytoestrogens. Their biological actions are thought to be exerted via the estrogen signal transduction pathway. Estrogens, such as 17β-estradiol (E2), play a crucial role in the development and functional maintenance of the central nervous system. E2 bind to the nuclear estrogen receptor (ER) and regulates morphogenesis, migration, functional maturation, and intracellular metabolism of neurons and glial cells. In addition to binding to nuclear ER, E2 also binds to the G-protein-coupled estrogen receptor (GPER) and activates the nongenomic estrogen signaling pathway. Soybean isoflavones also bind to the ER and GPER. However, the effect of soybean isoflavone on brain development, particularly glial cell function, remains unclear. We examined the effects of soybean isoflavones using an astrocyte-enriched culture and astrocyte-derived C6 clonal cells. Isoflavones increased glial cell migration. This augmentation was suppressed by co-exposure with G15, a selective GPER antagonist, or knockdown of GPER expression using RNA interference. Isoflavones also activated actin cytoskeleton arrangement via increased actin polymerization and cortical actin, resulting in an increased number and length of filopodia. Isoflavones exposure increased the phosphorylation levels of FAK (Tyr397 and Tyr576/577), ERK1/2 (Thr202/Tyr204), Akt (Ser473), and Rac1/cdc42 (Ser71), and the expression levels of cortactin, paxillin and ERα. These effects were suppressed by knockdown of the GPER. Co-exposure of isoflavones to the selective RhoA inhibitor, rhosin, selective Cdc42 inhibitor, casin, or Rac1/Cdc42 inhibitor, ML-141, decreased the effects of isoflavones on cell migration. These findings indicate that soybean isoflavones exert their action via the GPER to activate the PI3K/FAK/Akt/RhoA/Rac1/Cdc42 signaling pathway, resulting in increased glial cell migration. Furthermore, in silico molecular docking studies to examine the binding mode of isoflavones to the GPER revealed the possibility that isoflavones bind directly to the GPER at the same position as E2, further confirming that the effects of the isoflavones are at least in part exerted via the GPER signal transduction pathway. The findings of the present study indicate that isoflavones may be an effective supplement to promote astrocyte migration in developing and/or injured adult brains.

Estrogen Actions in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks estrogen receptor (ER) α, but the expression of estrogen receptors ERβ and G protein-coupled estrogen receptor 1 (GPER-1) is able to trigger estrogen-responsivity in TNBC. Estrogen signaling in TNBC can also be activated and modulated by the constitutively active estrogen-related receptors (ERRs). In this review article, we discuss the role of ERβ and GPER-1 as mediators of E2 action in TNBC as well as the function of ERRs as activators and modulators of estrogen signaling in this cancer entity. For this purpose, original research articles on estrogen actions in TNBC were considered, which are listed in the PubMed database. Additionally, we performed meta-analyses of publicly accessible integrated gene expression and survival data to elucidate the association of ERβ, GPER-1, and ERR expression levels in TNBC with survival. Finally, options for endocrine therapy strategies for TNBC were discussed.

G-protein Coupled Estrogen Receptor Expression in Growth Hormone Secreting and Non-Functioning Adenomas.

To evaluate the expression of G-protein coupled estrogen receptor (GPER1), aromatase, estrogen receptor α (ERα), estrogen receptor β (ERβ), pituitary tumor transforming gene (PTTG), and fibroblast growth factor 2 (FGF2) in GH-secreting and non-functioning adenomas (NFA). Thirty patients with acromegaly and 27 patients with NFA were included. Gene expression was determined via quantitative reverse transcription polymerase chain reaction (QRT-PCR). Protein expression was determined via immunohistochemistry. There was no difference, in terms of gene expression of aromatase, ERα, PTTG, and FGF2 between the two groups (p>0.05 for all). ERβ gene expression was higher and GPER1 gene expression was lower in GH-secreting adenomas than NFAs (p<0.05 for all). Aromatase and ERβ protein expression was higher in GH-secreting adenomas than NFAs (p=0.01). None of the tumors expressed ERα. GPER1 expression was detected in 62.2% of the GH-secreting adenomas and 45% of NFAs. There was no difference in terms of GPER1, PTTG, FGF2 H scores between the two groups (p>0.05 for all). GPER1 gene expression was positively correlated to ERα, ERβ, PTTG, and FGF2 gene expression (p<0.05 for all). There was a positive correlation between aromatase and GPER1 protein expression (r=0.31; p=0.04). GPER1 is expressed at both gene and protein level in a substantial portion of GH-secreting adenomas and NFAs. The finding of a positive correlation between GPER1 and ERα, ERβ, PTTG, and FGF2 gene expression and aromatase and GPER1 protein expression suggests GPER1 along with aromatase and classical ERs might mediate the effects of estrogen through upregulation of PTTG and FGF2.

G Protein-Coupled Estrogen Receptor Regulates Actin Cytoskeleton Dynamics to Impair Cell Polarization.

Mechanical forces regulate cell functions through multiple pathways. G protein-coupled estrogen receptor (GPER) is a seven-transmembrane receptor that is ubiquitously expressed across tissues and mediates the acute cellular response to estrogens. Here, we demonstrate an unidentified role of GPER as a cellular mechanoregulator. G protein-coupled estrogen receptor signaling controls the assembly of stress fibers, the dynamics of the associated focal adhesions, and cell polarization via RhoA GTPase (RhoA). G protein-coupled estrogen receptor activation inhibits F-actin polymerization and subsequently triggers a negative feedback that transcriptionally suppresses the expression of monomeric G-actin. Given the broad expression of GPER and the range of cytoskeletal changes modulated by this receptor, our findings position GPER as a key player in mechanotransduction.

Mechanisms of Estradiol-induced EGF-like Factor Expression and Oocyte Maturation via G Protein-coupled Estrogen Receptor.

Estrogen is an important modulator of reproductive activity through nuclear receptors and G protein-coupled estrogen receptor (GPER). Here, we observed that both estradiol and the GPER-specific agonist G1 rapidly induced cyclic adenosine monophosphate (cAMP) production in cumulus cells, leading to transient stimulation of phosphorylated cAMP response element binding protein (CREB), which was conducive to the transcription of epidermal growth factor (EGF)-like factors, amphiregulin, epiregulin, and betacellulin. Inhibition of GPER by G15 significantly reduced estradiol-induced CREB phosphorylation and EGF-like factor gene expression. Consistently, the silencing of GPER expression in cultured cumulus cells abrogated the estradiol-induced CREB phosphorylation and EGF-like factor transcription. In addition, the increase in EGF-like factor expression in the cumulus cells is associated with EGF receptor (EFGR) tyrosine kinase phosphorylation and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Furthermore, we demonstrated that GPER-mediated phosphorylation of EGFR and ERK1/2 was involved in reduced gap junction communication, cumulus expansion, increased oocyte mitochondrial activity and first polar body extrusion. Overall, our study identified a novel function for estrogen in regulating EGFR activation via GPER in cumulus cells during oocyte maturation.