G Protein-coupled Estrogen Receptor Signaling Research Articles

GPER deletion triggers inhibitory effects in triple negative breast cancer (TNBC) cells through the JNK/c-Jun/p53/Noxa transduction pathway

The G protein-coupled estrogen receptor (GPER) mediates estrogen action in different pathophysiological conditions, including cancer. GPER expression and signaling have been found to join in the progression of triple-negative breast cancer (TNBC), even though controversial data have been reported. In present study, we aimed at providing new mechanistic and biological discoveries knocking out (KO) GPER expression by CRISPR/Cas9 technology in MDA-MB-231 TNBC cells. GPER KO whole transcriptome respect to wild type (WT) MDA-MB-231 cells was determined through total RNA sequencing (RNA-Seq) and gene ontology (GO) enrichment analysis. We ascertained that anti-proliferative and pro-apoptotic gene signatures characterize GPER KO MDA-MB-231 cells. Thereafter, we determined that these cells exhibit a reduced proliferative, clonogenic and self-renewal potential along with an increased mitochondria-dependent apoptosis phenotype. In addition, we recognized that decreased cAMP levels trigger the JNK/c-Jun/p53/Noxa axis, which in turn orchestrates the pro-apoptotic effects observed in GPER KO cells. In accordance with these data, survival analyses in TNBC patients of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset indicated that high Noxa expression correlates with improved outcomes in TNBC patients. Furthermore, we demonstrated that GPER KO in TNBC cells impairs the expression and secretion of the well-acknowledged GPER target gene named CTGF, thus resulting in the inhibition of migratory effects in cancer-associated fibroblasts (CAFs). Overall, the present study provides novel mechanistic and biological insights on GPER KO in TNBC cells suggesting that GPER may be considered as a valuable target in comprehensive therapeutic approaches halting TNBC progression.

232-OR: STIM1 Interacts with G Protein–Coupled Estrogen Receptor Signaling to Maintain ß-Cell Identity in Female Mice

Calcium (Ca2+) plays a vital role in normal β cell function, and the β cell endoplasmic reticulum (ER) serves as the dominant intracellular store of Ca2+. ER Ca2+ depletion triggers a rescue mechanism known as store-operated Ca2+ entry (SOCE), which replenishes ER Ca2+ stores through the ER-localized Ca2+ sensor stromal interaction molecule 1 (STIM1). We have demonstrated pathogenic reductions in β cell SOCE and loss of STIM1 expression in rodent and human models of type 2 diabetes (T2D) and shown that β cell-specific loss of STIM1 (STIM1Δβ) leads to obesity-induced glucose intolerance, decreased β cell and increased cell mass in female but not male STIM1Δβ mice. To test mechanisms underlying these findings, lineage tracing experiments were performed and revealed increased colocalization of tdTomato and glucagon in STIM1Δβ mice compared to control mice, suggesting dedifferentiation of β cells to ⍺ cells. RNA sequencing of islets isolated from female STIM1Δβ mice revealed reductions in G-protein coupled estrogen receptor (GPER) expression and a transcriptional signature indicative of reduced estradiol mediated signaling. To test whether GPER is required for the maintenance of β cell identity, INS-1 cells were transfected with siRNA targeted to Gper1 or treated with the GPER1 antagonist, G-15. RT-qPCR showed that markers of β cell identity were significantly reduced and Gcg expression was significantly increased in Gper1 knockdown and G-15-treated cells. Lastly, to explore the functional connection between SOCE and GPER, STIM1KO and GPER knockdown INS-1 cells were stimulated with glucose, and Ca2+ responses were evaluated. First and second phase Ca2+ responses after glucose treatment were impaired in both STIM1KO and GPER knockdown INS-1 cells compared to WT INS-1 cells, suggesting that STIM1 and GPER may be functionally linked through Ca2+ signaling. Together, these data identify a novel connection between β cell SOCE, GPER signaling, and β cell differentiation status. Disclosure M.Mclaughlin: None. P.Sohn: None. P.Krishnan: None. C.Lee: None. T.Kono: None. C.Evans-molina: Advisory Panel; Provention Bio, Inc., DiogenX, Avotres Inc., Neurodon, MaiCell Therapeutics, Other Relationship; Isla Technology, Bristol-Myers Squibb Company, Nimbus Therapeutics, Research Support; Lilly, Astellas Pharma Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK093954, R01DK127236, U01DK127786, R01DK127308, UC4DK104166); U.S. Department of Veterans Affairs (I01BX001733)

Nuclear receptor co-repressor NCOR2 and its relation to GPER with prognostic impact in ovarian cancer.

The significance of the non-classical G-protein-coupled estrogen receptor (GPER) as positive or negative prognostic factor for ovarian cancer patients remains still controversial. Recent results indicate that an imbalance of both co-factors and co-repressors of nuclear receptors regulates ovarian carcinogenesis by altering the transcriptional activity through chromatin remodeling. The present study aims to investigate whether the expression of the nuclear co-repressor NCOR2 plays a role in GPER signaling which thereby could positively impact overall survival rates of ovarian cancer patients. NCOR2 expression was evaluated by immunohistochemistry in a cohort of 156 epithelial ovarian cancer (EOC) tumor samples and correlated with GPER expression. The correlation and differences in clinical and histopathological variables as well as their effect on prognosis were analyzed by Spearman's correlation, Kruskal-Wallis test and Kaplan-Meier estimates. Histologic subtypes were associated with different NCOR2 expression patterns. More specifically, serous and mucinous EOC demonstrated a higher NCOR2 expression (P = 0.008). In addition, high nuclear NCOR2 expression correlated significantly with high GPER expression (cc = 0.245, P = 0.008). A combined evaluation of both high NCOR2 (IRS > 6) and high GPER (IRS > 8) expression revealed an association of a significantly improved overall survival (median OS 50.9 versus 105.1months, P = 0.048). Our results support the hypothesis that nuclear co-repressors such as NCOR2 may influence the transcription of target genes in EOC such as GPER. Understanding the role of nuclear co-repressors on signaling pathways will allow a better understanding of the factors involved in prognosis and clinical outcome of EOC patients.

Comparative G-Protein-Coupled Estrogen Receptor (GPER) Systems in Diabetic and Cancer Conditions: A Review.

For many patients, diabetes Mellitus and Malignancy are frequently encountered comorbidities. Diabetes affects approximately 10.5% of the global population, while malignancy accounts for 29.4 million cases each year. These troubling statistics indicate that current treatment approaches for these diseases are insufficient. Alternative therapeutic strategies that consider unique signaling pathways in diabetic and malignancy patients could provide improved therapeutic outcomes. The G-protein-coupled estrogen receptor (GPER) is receiving attention for its role in disease pathogenesis and treatment outcomes. This review aims to critically examine GPER' s comparative role in diabetes mellitus and malignancy, identify research gaps that need to be filled, and highlight GPER's potential as a therapeutic target for diabetes and malignancy management. There is a scarcity of data on GPER expression patterns in diabetic models; however, for diabetes mellitus, altered expression of transport and signaling proteins has been linked to GPER signaling. In contrast, GPER expression in various malignancy types appears to be complex and debatable at the moment. Current data show inconclusive patterns of GPER expression in various malignancies, with some indicating upregulation and others demonstrating downregulation. Further research should be conducted to investigate GPER expression patterns and their relationship with signaling pathways in diabetes mellitus and various malignancies. We conclude that GPER has therapeutic potential for chronic diseases such as diabetes mellitus and malignancy.

A pilot study of the impact of estrogen signaling on tumor immunity in a syngeneic model of ovarian cancer (213)

A pilot study of the impact of estrogen signaling on tumor immunity in a syngeneic model of ovarian cancer (213)

Modulatory Effects of Estradiol and Its Mixtures with Ligands of GPER and PPAR on MAPK and PI3K/Akt Signaling Pathways and Tumorigenic Factors in Mouse Testis Explants and Mouse Tumor Leydig Cells.

The present study was designed to evaluate how estradiol alone or in combination with G protein-coupled estrogen receptor (GPER) agonists and GPER and peroxisome proliferator-activated receptor (PPAR) antagonists alter the expression of tumor growth factor β (TGF-β), cyclooxygenase-2 (COX-2), hypoxia inducible factor 1-alpha (HIF-1α), and vascular endothelial growth factor (VEGF) in mouse testis explants and MA-10 mouse tumor Leydig cells. In order to define the hormone-associated signaling pathway, the expression of MAPK and PI3K/Akt was also examined. Tissue explants and cells were treated with estradiol as well as GPER agonist (ICI 182,780), GPER antagonist (G-15), PPARα antagonist (GW6471), and PPARγ antagonist (T00709072) in various combinations. First, we showed that in testis explants GPER and PPARα expressions were activated by the GPER agonist and estradiol (either alone or in mixtures), whereas PPARγ expression was activated only by GPER agonist. Second, increased TGF-β expression and decreased COX-2 expression were found in all experimental groups of testicular explants and MA-10 cells, except for up-regulated COX-2 expression in estradiol-treated cells, compared to respective controls. Third, estradiol treatment led to elevated expression of HIF-1α and VEGF, while their lower levels versus control were noted in the remaining groups of explants. Finally, we demonstrated the up-regulation of MAPK and PI3Kp85/Akt expressions in estradiol-treated groups of both ex vivo and in vitro models, whereas estradiol in mixtures with compounds of agonistic or antagonistic properties either up-regulated or down-regulated signaling kinase expression levels. Our results suggest that a balanced estrogen level and its action together with proper GPER and PPAR signaling play a key role in the maintenance of testis homeostasis. Moreover, changes in TGF-β and COX-2 expressions (that disrupted estrogen pathway) as well as disturbed GPER-PPAR signaling observed after estradiol treatment may be involved in testicular tumorigenesis.

PD16-09 THE ROLE OF GPER SIGNALING IN DEVELOPMENT OF PROSTATIC ENLARGEMENT IN HIGH FAT DIET INDUCED OBESITY RAT MODEL

You have accessJournal of UrologyCME1 May 2022PD16-09 THE ROLE OF GPER SIGNALING IN DEVELOPMENT OF PROSTATIC ENLARGEMENT IN HIGH FAT DIET INDUCED OBESITY RAT MODEL Shinsuke Mizoguchi, Kenichi Mori, Toshitaka Shin, and Hiromitsu Mimata Shinsuke MizoguchiShinsuke Mizoguchi More articles by this author , Kenichi MoriKenichi Mori More articles by this author , Toshitaka ShinToshitaka Shin More articles by this author , and Hiromitsu MimataHiromitsu Mimata More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002548.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although there has been increasing evidence that indicates positive relationship between metabolic syndrome and emergence in benign prostatic hyperplasia (BPH), the detailed pathophysiological mechanism is still unknown. Estrogen, which is increased in obese patients, is considered to be one of the key factors in development of BPH and G protein-coupled estrogen receptor (GPER) has been reported to be implicated in symptomatic BPH. To elucidate a role of GPER signaling in development of BPH, we evaluated changes in prostatic weight and inflammatory profiles in the prostate by GPER inhibition using high fat diet induced obesity rat model. METHODS: Male Wistar rats (8 weeks old) were divided into three groups; rats with normal diet group (ND, n=6), rats with high fat diet induced obesity group (HFD, n=6), and HFD treated by G15 (G15, n=6) which is GPER antagonist. The high fat diet contains 32% fat. These rats were maintained for twelve weeks followed by two weeks treatment by oral administration of placebo or G15 (0.1mg/kg) daily. Lateral lobes of prostate were harvested to assess prostatic weight, histological change and mRNA expression of GPER, IL1β, TGFβ, ERK1/2 by qPCR. RESULTS: HFD showed significantly increased body weight, visceral fat and prostatic weight as well as significant upregulation of gene expressions in GPER, IL1β, TGFβ, and ERK1/2 compared to ND. Hematoxylin and eosin (HE) staining showed inflammatory cell infiltration in prostatic tissue section from HFD. In contrast, HFD treated by G15 demonstrated significantly decreased prostatic weight in association with downregulation of IL1β, TGFβ and ERK1/2 compared to HFD with placebo. mRNA expression of GPER was not significantly changed between HFD and G15 group. CONCLUSIONS: We demonstrated improvement of prostatic enlargement and inflammation by GPER inhibition, which suggest GPER signaling has an important role in development of BPH in obese patients. Therefore, GPER inhibition could be an therapeutic target of BPH with metabolic syndrome. Source of Funding: KAKENHI 20K18094 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e272 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shinsuke Mizoguchi More articles by this author Kenichi Mori More articles by this author Toshitaka Shin More articles by this author Hiromitsu Mimata More articles by this author Expand All Advertisement PDF DownloadLoading ...

The adverse outcome pathway (AOP) of estrogen interference effect induced by triphenyl phosphate (TPP): Integrated multi-omics and molecular dynamics approaches

Triphenyl phosphate (TPP) has been detected with increasing frequency in various biota and environmental media, and it has been confirmed that G protein-coupled estrogen receptor (GPER) was involved in the estrogenic activity of TPP. Therefore, it is necessary to link the estrogen-interfering effects and possible mechanisms of action of TPP with the molecular initiation event (MIE) to improve its adverse outcome pathway framework. In this study, transcriptomic and proteomic methods were used to analyze the estrogen interference effect of TPP mediated by GPER, and the causal relationship was supplemented by molecular dynamics simulation and fluorescence analysis. The omics results showed that TPP could regulate the response of key GPER signaling factors and the activation of downstream pathways including PI3K-Akt signaling pathway, MAPK signaling pathway, and estrogen signaling pathway. The similar activation effect of TPP and agonist G1 change of GPER was proved by molecular dynamics simulation. After TPP binding, the conformation of GPER will change from the inactive to active state. Therefore, TPP may affect cell proliferation, metastasis, and apoptosis and regulate gene transcription and kinase activity, leading to abnormal immune function and other estrogen-dependent cell processes and cancer through GPER, ultimately causing the estrogen interference effect.

Chlorobisphenol A activated kisspeptin/GPR54-GnRH neuroendocrine signals through ERα and GPER pathway in neuronal GT1-7 cells

Chlorobisphenol A (ClxBPA) is a kind of novel estrogenic compounds. The present study aims to investigate the effects of three ClxBPA compounds on the kisspeptin/G protein-coupled receptor 54 (GPR54, also named KissR1)-gonadotropin-releasing hormone (GnRH) (KGG) system in neuronal GT1–7 cells with mechanistic insights by estrogen receptor signaling pathways. The study demonstrated that low-concentration ClxBPA induced the cell proliferation, promoted GnRH secretion, upregulated the expression of KGG neuroendocrine signal-related proteins (KissR1, GnRH1 and kisspeptin) and genes including Kiss1, GnRH1, KissR1, luteinizing hormone receptor (Lhr) and follicle-stimulating hormone receptor (Fshr) in GT1–7 cells. Additionally, ClxBPA activated nuclear estrogen receptor alpha (ERα) and member estrogen receptor G protein-coupled estrogen receptor (GPER)-regulated phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinase (Erk1/2) signaling pathways. Pretreatment of GT1–7 cells with GPER inhibitor G15 and ERα inhibitor ICI reduced the expression of KissR1, GnRH1 and kisspeptin proteins, attenuated mRNA levels of Kiss1, GnRH1, KissR1, Fshr and Lhr genes, and decreased ClxBPA-induced GT1–7 cell proliferation. The results suggested that ClxBPA activated the KGG neuroendocrine signals and induced the proliferation of GT1–7 cells via ERα and GPER signaling pathways. This study provides a new perspective to explore the neuroendocrine toxicity mechanism of ClxBPA. CapsuleClxBPA activated KGG neuroendocrine signaling pathway via ERα and GPER and induced the proliferation of GT1–7 cells.

Natural and Synthetic Estrogens in Chronic Inflammation and Breast Cancer.

Simple SummaryEstrogens regulate key physiological functions in the human body, including the development of female reproductive organs. However, dysregulated estrogen signaling—mainly mediated by estrogen receptors (ER)—is associated with many developmental, mental, and other diseases, including cancer. An increasing number of studies have demonstrated that estrogen modulates inflammatory processes within many tissues. In addition to estrogens made within the body, humans are also constantly exposed to many outside estrogens, naturally occurring in plants and those artificially synthesized in industries. Here, we will also discuss the link between chronic exposure to environmental estrogens with tissue inflammation and breast cancer development.The oncogenic role of estrogen receptor (ER) signaling in breast cancer has long been established. Interaction of estrogen with estrogen receptor (ER) in the nucleus activates genomic pathways of estrogen signaling. In contrast, estrogen interaction with the cell membrane-bound G-protein-coupled estrogen receptor (GPER) activates the rapid receptor-mediated signaling transduction cascades. Aberrant estrogen signaling enhances mammary epithelial cell proliferation, survival, and angiogenesis, hence is an important step towards breast cancer initiation and progression. Meanwhile, a growing number of studies also provide evidence for estrogen’s pro- or anti-inflammatory roles. As other articles in this issue cover classic ER and GPER signaling mediated by estrogen, this review will discuss the crucial mechanisms by which estrogen signaling influences chronic inflammation and how that is involved in breast cancer. Xenoestrogens acquired from plant diet or exposure to industrial products constantly interact with and alter innate estrogen signaling at various levels. As such, they can modulate chronic inflammation and breast cancer development. Natural xenoestrogens generally have anti-inflammatory properties, which is consistent with their chemoprotective role in breast cancer. In contrast, synthetic xenoestrogens are proinflammatory and carcinogenic compounds that can increase the risk of breast cancer. This article also highlights important xenoestrogens with a particular focus on their role in inflammation and breast cancer. Improved understanding of the complex relationship between estrogens, inflammation, and breast cancer will guide clinical research on agents that could advance breast cancer prevention and therapy.

GPER and IGF-1R mediate the anti-inflammatory effect of genistein against lipopolysaccharide (LPS)-induced nigrostriatal injury in rats

Neuroinflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Genistein is an estrogen-like phytoestrogen that can exert biological effects via the crosstalk of estrogen receptor and insulin-like growth factor 1 receptor (IGF-1R). The present study aimed to evaluate the involvement of G protein-coupled estrogen receptor (GPER) and IGF-1R in the anti-inflammatory effects of genistein against lipopolysaccharide (LPS)-induced nigrostriatal injury in ovariectomized rats. Our results showed that genistein treatment could ameliorate the apomorphine-induced rotational behavior in LPS-induced inflammatory PD rat model. Genistein attenuated LPS-induced decrease of the contents of dopamine (DA) and its metabolites in striatum as well as the loss of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the substantia nigra (SN) of the lesioned side, which could be blocked by GPER antagonist G15 or IGF-1R antagonist JB1. Meanwhile, G15 or JB1 could attenuate the anti-inflammatory effects of genistein in LPS-induced microglial activation and production of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, genistein could inhibit the LPS-induced phosphorylation of p38, JNK, ERK and IκB in the lesioned side of SN and these effects could also be blocked by G15 or JB1. Taken together, our data provide the first evidence that genistein can inhibit the increase of microglia and protect dopaminergic neurons at least in part via GPER and IGF-1R signaling pathways in ovariectomized PD rat model.

Phase 1 trial of a novel, first-in-class G protein-coupled estrogen receptor (GPER) agonist, LNS8801, in patients with advanced or recurrent treatment-refractory solid malignancies.

3084 Background: The G protein-coupled estrogen receptor (GPER) is a broadly expressed G protein-coupled receptor that is tumor suppressive. LNS8801 is an oral, highly selective small molecule agonist of GPER. GPER activation results in c-Myc depletion, inhibition of tumor proliferation, and enhancement of tumor immune recognition. Preclinically, LNS8801 demonstrates potent single-agent and combinatorial anti-cancer activity and can overcome established resistance to standard-of-care anti-cancer therapies including immune checkpoint inhibitors. Methods: The primary objective of this phase 1/1B first-in-human, open-label, multicenter study (NCT04130516) was to determine the safety and tolerability and recommended phase 2 dose (RP2D) of LNS8801 in patients (pts) with locally advanced or metastatic solid tumor malignancies, both as monotherapy and in combination with the anti-PD-1 antibody, pembrolizumab. Dose levels were escalated in a 3+3 fashion and included 10, 40 and 125 mg dosed 3/7, 125 mg daily, and 125 and 250 mg twice daily. Dose limiting toxicity (DLT) was defined via NCI CTCAE v5.0 during the first 21 days of treatment. An increase in prolactin over the initial 12 hrs of dosing was measured to assess systemic GPER signaling. Tumor c-Myc expression was measured as a surrogate of treatment-related biologic response. Radiographic response (RECIST v1.1) was evaluated every 8 weeks until progression. Results: 33 pts (19 M/14 F) with median age 58.8 y and 4 (1-9) prior therapies enrolled. Median duration of treatment was 66 d (1–367+). With monotherapy (n = 28), no DLTs, treatment-related SAEs, or treatment-related study discontinuations were observed up through the maximum administered dose (250 mg bid). Possibly related AEs were grade 1 or 2 and did not correlate with dose level. Exposure was above that predicted for efficacy and t1/2 was ̃10 hr at all doses. Of 26 evaluable monotherapy pts, 8 (27%) experienced stable disease (SD) for up to a year. All SD pts had a prolactin response. Among tumors expressing GPER, c-Myc depletion was observed in 100% (5/5) of paired pre and on-treatment biopsies. In the combination cohort (n = 5), 2/2 evaluable pts demonstrated net tumor reductions on initial f/up scans, including one RECIST partial response. Based on PK/PD data, 125 mg daily has provisionally been identified as the monotherapy and combination RP2D. Conclusions: LNS8801 is well tolerated and demonstrates signals of anti-tumor activity when administered both as monotherapy and in combination with pembrolizumab. Confirmation of RP2Ds and updated efficacy data will be presented in June. A phase 2A expansion study to evaluate these RP2Ds in clinical settings of high unmet need is now in development. Clinical trial information: NCT04130516.

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.

Abstract PR16: Specific activation of the G protein-coupled estrogen receptor inhibits melanoma and other cancers and potentiates immune and targeted therapies

Abstract Modern immune and targeted therapies appear more effective against advanced melanoma in women than in men. Melanoma incidence is also lower in women. Similar sex differences are observed in other tumors that are not classically considered sex hormone responsive. Here, we show that much of the protective effect likely results from estrogen signaling through the G protein-coupled estrogen receptor (GPER) on tumor cells. Selective GPER activation in primary male and female melanocytes, and in melanoma cells, induced long-term changes that maintained a more differentiated cell state as defined by increased expression of well-established melanocyte differentiation antigens, increased pigment production, decreased proliferative capacity, and decreased expression of the oncodriver and stem cell marker c-Myc. GPER signaling also rendered melanoma cells more vulnerable to immunotherapy. Systemic delivery of a small-molecule, selective GPER agonist was well tolerated, and cooperated with PD-1 directed immune checkpoint blockade in melanoma-bearing mice to dramatically extend survival, with up to half of mice clearing their tumor. Complete responses were associated with immune memory that protected against rechallenge with naive tumor cells. Specific GPER activation in the BRaf-driven murine melanoma model (YUMM1.7) also delayed resistance to targeted BRaf inhibition in vivo. Similar antitumor responses were observed in genetically defined KRas-driven syngeneic pancreatic ductal carcinoma (PDAC) models. The GPER agonist used has favorable pharmacokinetics and oral bioavailability in mice and higher vertebrates and appears to have a large therapeutic window. Together, these data suggest that GPER may be a useful, pharmacologically accessible target for melanoma and other common cancers. This abstract is also being presented as Poster B15. Citation Format: Chris Natale, Jinyang Li, Jun Zhang, Cristina Aquirre, Ankit Dahal, Tzvete Dentchev, John Seykora, Ben Stanger, Todd Ridky. Specific activation of the G protein-coupled estrogen receptor inhibits melanoma and other cancers and potentiates immune and targeted therapies [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR16.

Non-genomic mechanisms in the estrogen regulation of glycolytic protein levels in endothelial cells.

Few studies have explored the mechanisms coupling estrogen signals to metabolic demand in endothelial cells. We recently showed that 17β-estradiol (E2) triggers angiogenesis via the membrane G-protein coupled estrogen receptor (GPER) and the key glycolytic protein PFKFB3 as a downstream effector. We herein investigated whether estrogenic agents regulate the stability and/or degradation of glycolytic proteins in human umbilical vein endothelial cells (HUVECs). Similarly to E2, the GPER selective agonist G1 rapidly increased PFKFB3 protein amounts, without affecting mRNA levels. In the presence of cycloheximide, E2 and G1 treatment counteracted PFKFB3 degradation over time, whereas E2-induced PFKFB3 stabilization was abolished by the GPER antagonist G15. Inhibitors of selective SCF E3 ubiquitin ligase (SMER-3) and proteasome (MG132) rapidly increased PFKFB3 protein levels. Accordingly, ubiquitin-bound PFKFB3 was lower in E2- or G1-treated HUVECs. Both agents increased deubiquitinase USP19 levels through GPER signaling. Notably, USP 19 siRNA decreased PFKFB3 levels and abolished E2- and G1-mediated HUVEC tubularization. Finally, E2 and G1 treatments rapidly enhanced glucose transporter GLUT1 levels via GPER independent of transcriptional activation. These findings provide new evidence on mechanisms coupling estrogen signals with the glycolytic program in endothelium and unravel the role of USP19 as a target of the pro-angiogenic effect of estrogenic agents.

Interstitial Leydig Cell Tumorigenesis—Leptin and Adiponectin Signaling in Relation to Aromatase Expression in the Human Testis

Although epidemiological studies from the last years report an increase in the incidences of Leydig cell tumors (previously thought to be a rare disease), the biochemical characteristics of that tumor important for understanding its etiology, diagnosis, and therapy still remains not completely characterized. Our prior studies reported G-protein coupled estrogen receptor signaling and estrogen level disturbances in Leydig cell tumors. In addition, we found that expressions of multi-level-acting lipid balance- and steroidogenesis–controlling proteins including peroxisome proliferator-activated receptor are altered in this tumor. In order to get deeper into the other molecular mechanisms that regulate lipid homeostasis in the Leydig cell tumor, here we investigate the presence and expression of newly-described hormones responsible for lipid homeostasis balancing (leptin and adiponectin), together with expression of estrogen synthase (aromatase). Samples of Leydig cell tumors (n = 20) were obtained from patients (31–45 years old) and used for light and transmission electron microscopic, western blotting, and immunohistochemical analyses. In addition, body mass index (BMI) was calculated. In tumor mass, abundant lipid accumulation in Leydig cells and various alterations of Leydig cell shape, as well as the presence of adipocyte-like cells, were observed. Marked lipid content and various lipid droplet size, especially in obese patients, may indicate alterations in lipid homeostasis, lipid processing, and steroidogenic organelle function in response to interstitial tissue pathological changes. We revealed significantly increased expression of leptin, adiponectin and their receptors, as well as aromatase in Leydig cell tumors in comparison to control. The majority of patients (n = 13) were overweight as indicated by their BMI. Moreover, a significant increase in expression of phospholipase C (PLC), and kinases Raf, ERK which are part of adipokine transductional pathways, was demonstrated. These data expand our previous findings suggesting that in human Leydig cell tumors, estrogen level and signaling, together with lipid status, are related to each other. Increased BMI may contribute to certain biochemical characteristics and function of the Leydig cell in infertile patients with a tumor. In addition, altered adipokine-estrogen microenvironment can have an effect on proliferation, growth, and metastasis of tumor cells. We report here various targets (receptors, enzymes, hormones) controlling lipid balance and estrogen action in Leydig cell tumors indicating their possible usefulness for diagnostics and therapy.

A novel GPER antagonist protects against the formation of estrogen-induced cholesterol gallstones in female mice

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (-/-) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.

Antagonistic Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Reveal GRK5-Mediated Cardioprotection via Mineralocorticoid Receptor Inhibition

Aldosterone (Aldo), when overproduced, is a cardiotoxic hormone underlying heart failure and hypertension. Aldo exerts damaging effects via the mineralocorticoid receptor (MR) but also activates the antiapoptotic G protein-coupled estrogen receptor (GPER) in the heart. G protein-coupled receptor (GPCR)-kinase (GRK)-2 and -5 are the most abundant cardiac GRKs and phosphorylate GPCRs as well as non-GPCR substrates. Herein, we investigated whether they phosphorylate and regulate cardiac MR and GPER. To this end, we used the cardiomyocyte cell line H9c2 and adult rat ventricular myocytes (ARVMs), in which we manipulated GRK5 protein levels via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and GRK2 activity via pharmacological inhibition. We report that GRK5 phosphorylates and inhibits the cardiac MR whereas GRK2 phosphorylates and desensitizes GPER. In H9c2 cardiomyocytes, GRK5 interacts with and phosphorylates the MR upon β2-adrenergic receptor (AR) activation. In contrast, GRK2 opposes agonist-activated GPER signaling. Importantly, GRK5-dependent MR phosphorylation of the MR inhibits transcriptional activity, since aldosterone-induced gene transcription is markedly suppressed in GRK5-overexpressing cardiomyocytes. Conversely, GRK5 gene deletion augments cardiac MR transcriptional activity. β2AR-stimulated GRK5 phosphorylates and inhibits the MR also in ARVMs. Additionally, GRK5 is necessary for the protective effects of the MR antagonist drug eplerenone against Aldo-induced apoptosis and oxidative stress in ARVMs. In conclusion, GRK5 blocks the cardiotoxic MR-dependent effects of Aldo in the heart, whereas GRK2 may hinder beneficial effects of Aldo through GPER. Thus, cardiac GRK5 stimulation (e.g., via β2AR activation) might be of therapeutic value for heart disease treatment via boosting the efficacy of MR antagonists against Aldo-mediated cardiac injury.

Therapeutic Nrf2 Activation improves LV function in the cardiomyocyte‐specific GPER knockdown mouse

BackgroundThe G protein‐coupled estrogen receptor (GPER) plays an important role in the maintenance of cardiac health partly via its link to anti‐oxidant and anti‐inflammatory pathways. The NLRP3 inflammasome is upregulated in hearts of mice with cardiomyocyte GPER gene deletion, and NLRP3 inhibition limits left ventricle (LV) dysfunction from cardiac GPER deficiency. Studies from others show that GPER mediates the inductive effect of estrogen on the transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2), the master regulator of detoxification and antioxidant systems. This in vivo animal study was designed to further confirm that lack of GPER signaling (from gene deletion or menopause) impedes Nrf2 activation, and in so doing contributes to ROS accumulation and inflammation in the cardiomyocyte. We hypothesize that chronic Nrf2 activation counteracts the adverse effects of cardiac GPER knockdown on LV function, irrespective of estrogen.MethodsMale GPER KO and WT littermates (7 months of age) were randomized to receive, via drinking water, either 8 weeks of oral treatment with the Nrf2 activator, dimethyl fumarate (KO‐DMF: n=5; WT‐DMF: n=5) at 30mg/kg/day, or its vehicle 0.08% methyl cellulose (KO‐VEH: n=5; WT‐VEH: n=5). At 9 months of age, echocardiographic indices of LV function (isoflurane anesthesia) were obtained prior to sacrifice. Data were analyzed by two‐way ANOVA for gene (GPER), treatment (DMF) and gene x treatment. P<0.05 was considered significant.ResultsSomatic characteristics (e.g. body weight, hair coat appearance) were not overtly affected by GPER knockdown nor DMF treatment. As reported previously, GPER gene deletion led to significant reductions in systolic function (% fractional shortening (FS)), myocardial relaxation (e′) and increases in filling pressure (E/e′) (gene effect: P<0.05). Chronic treatment with the Nrf2 activator significantly attenuated LV systolic dysfunction (treatment effect: P<0.05) and nearly normalized filling pressures (E/e′) in GPER KO without modifying the normal functioning heart of the WT (gene x treatment: P<0.05) (Figure ).ConclusionsThese data suggest that targeting the transcription factor Nrf2 may be a promising strategy to limit LV dysfunction as a result of cardiac oxidative stress and low‐grade inflammation from GPER deactivation (e.g., estrogen loss from ovarian extirpation or menopause).Support or Funding InformationHL051952 (CMF, LG) and AG033727 (LG)Figure 1

G-Protein-Coupled Estrogen Receptor Antagonist G15 Decreases Estrogen-Induced Development of Non-Small Cell Lung Cancer.

G-protein-coupled estrogen receptor (GPER) was found to promote non-small cell lung cancer (NSCLC) by estrogen, indicating the potential necessity of inhibiting GPER by a selective antagonist. This study was performed to elucidate the function of GPER-selective inhibitor G15 in NSCLC development. Cytoplasmic GPER (cGPER) and nuclear GPER (nGPER) were detected by immunohistochemical analysis in NSCLC samples. The relation of GPER and estrogen receptor β (ERβ) expression and correlation between GPER, ERβ, and clinical factors were analyzed. The effects of activating GPER and function of G15 were analyzed in the proliferation of A549 and H1793 cell lines and development of urethane-induced adenocarcinoma. Overexpression of cGPER and nGPER was detected in 80.49% (120/150) and 52.00% (78/150) of the NSCLC samples. High expression of GPER was related with higher stages, poorer differentiation, and high expression of ERβ. The protein level of GPER in the A549 and H1793 cell lines was increased by treatment with E2, G1 (GPER agonist), or fulvestrant (Ful; ERβ antagonist) and decreased by G15. Administration with G15 reversed the E2- or G1-induced cell growth by inhibiting GPER. In urethane-induced adenocarcinoma mice, the number of tumor nodules and tumor index increased in the E2 or G1 group and decreased by treatment with G15. These findings demonstrate that using G15 to block GPER signaling may be considered as a new therapeutic target in NSCLC.