G Protein-Coupled Oestrogen Receptor Actions Targeting the Hallmarks of Cancer in Human Prostate Cells: From Cell Fate to Metabolic Reprogramming.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) is commonly expressed in prostate cancer (PCa) cells and is associated with increased proliferation, metastases and androgen independence. Zearalenone (ZEA) is one of the most common mycotoxins contaminating food, which might mimic estrogens and bind to estrogen receptors (ERs). The ratio of androgens to estrogens in men decreases physiologically with age, and is believed to participate in prostate carcinogenesis. In this study, we evaluated the role of NFκB and ERβ in the induction of oxidative stress in human PCa cells by ZEA. As observed, ZEA at a dose of 30 µM induces oxidative stress in PCa cells associated with DNA damage and G2/M cell cycle arrest. We also observed that the inhibition of ERβ and NFΚB via specific inhibitors (PHTPP and BAY 117082) significantly increased ZEA-induced oxidative stress, although the mechanism seems to be different for androgen-dependent and androgen-independent cells. Based on our findings, it is possible that the activation of ERβ and NFΚB in PCa might protect cancer cells from ZEA-induced oxidative stress. We therefore shed new light on the mechanism of ZEA toxicity in human cells.

Regucalcin (RGN) is a calcium-binding protein and an oestrogen target gene, which has been shown to play essential roles beyond calcium homeostasis. Decreased RGN expression was identified in several cancers, including prostate cancer (PCa). However, it is unknown if the loss of RGN is a cause or a consequence of malignancy. Also, it needs confirmation if RGN oestrogenic regulation occurs through the G-protein-coupled oestrogen receptor (GPER). This study investigates how RGN knockdown affects prostate cell fate and metabolism and highlights the GPER/RGN interplay in PCa. Bioinformatic analysis assessed the relationship between RGN expression levels and patients' outcomes. RGN knockdown (siRNA) was performed in non-neoplastic prostate and castration-resistant PCa. Wild-type and RGN knockdown PCa cells were treated with the GPER agonist G1. Viability (MTT), proliferation (Ki-67 immunocytochemistry), apoptosis (caspase-3-like activity) and migration (Transwell assays) were evaluated. Spectrophotometric analysis was used to determine glucose consumption, lactate production and lactate dehydrogenase activity. Lipid content was assessed using the Oil Red assay. Bioinformatic analysis showed that the loss of RGN correlates with the development of metastatic PCa and poor survival outcomes. RGN knockdown induced a cancer-like phenotype in PNT1A cells, indicated by increased cell viability and proliferation and reduced apoptosis. In DU145 PCa cells, RGN knockdown augmented migration and enhanced the glycolytic profile, which indicates increased aggressiveness, in line with patients' data. GPER activation modulated RGN expression in PCa cells and RGN knockdown in DU145 cells influenced GPER actions, which highlighted an interplay between these molecular players with relevance for their potential use as biomarkers or therapeutic targets.

Castration-resistant prostate cancer (CRPC) often emerges within a few years following androgen deprivation therapy, and therapeutic options remain limited. Androgen deprivation induces oxidative stress in prostate cancer (PCa) cells, leading to aberrant activation of androgen receptor (AR) signaling. This study aims to clarify the molecular mechanism underlying oxidative stress-induced AR activation in CRPC. Transcriptional activity of the prostate-specific antigen (PSA) promoter was evaluated using a dual-luciferase reporter assay under various treatments. To identify AR-interacting proteins under oxidative stress, TurboID-mediated proximity biotin labeling coupled with mass spectrometry was employed following H2O2 exposure. Protein-protein interactions between AR and ubiquitin-specific peptidase 36 (USP36) were validated by co-immunoprecipitation (Co-IP). Subcellular AR expression was assessed via Immunofluorescence in PCa cells. Low doses H2O2 (10 and 20μM) enhanced viability and induced oxidative stress in PCa cells, and these concentrations were therefore selected for subsequent experiments. H2O2 treatment activated the AR-PSA signaling axis. The deubiquitinating enzyme USP36 was identified among the proteins that interact with AR upon H2O2 stimulation. Co-IP confirmed the specific binding between AR and USP36. Functional studies revealed that USP36 deubiquitinates and stabilizes AR. Notably, knockdown of USP36 abolished H2O2-induced activation of the AR-PSA pathway. H2O2 promotes the interaction between USP36 and AR, resulting in AR stabilization, transcriptional activation of PSA, and conferring androgen resistance. These findings provide mechanistic insights into how oxidative stress reactivates AR signaling in PCa and highlight potential therapeutic strategies for different stages of PCa.

α1-adrenoceptors (α1-ARs) and “cannabinoid-like” G Protein Coupled Receptor 55 (GPR55) belong to the G-protein coupled receptor (GPCR) family and play a crucial role in regulating prostate function. Although physical and functional interactions between the cannabinoid and adrenergic systems have been reported, analysis of functional interactions between α1-AR and GPR55 in normal and neoplastic prostate has not been reported. Since GPR55 levels are high in rodent adrenal gland, we propose a function link between the adrenergic system and GPR55 receptor. Confocal Laser Scanning Microscopy (CLSM) was employed to examine the endogenous α1-AR and GPR55 expression and their co-localization, expressed as fluorescence, in vitro in human andro-gen-insensitive PC-3 and androgen-sensitive LNCaP prostatic carcinoma cell lines, using the fluo-rescent ligands—Syto 62 (nuclear stain), BODIPY FL-Prazosin (QAPB; fluorescent quinazoline α1-AR ligand) and Tocriflour (T1117; a novel fluorescent diarylpyrazole cannabinoid/GPR55 ligand). Fluorescent ligand binding in untreated PC-3 cells and LNCaP cells and spheroids showed hetero-geneous expression of both α1-ARs and GPR55. A small proportion of cells had both α1-ARs and GPR55 in relatively equal numbers indicating a degree of co-localization. Co-localization of fluo-rescent ligand binding exhibited a stronger correlation in LNCaP (0.87) as compared to PC-3 (0.63) cells. Upregulation of α1-AR was observed in PC-3 cells following chronic doxazosin incubation. Robust T1117 binding, suggestive of GPR55 upregulation, was also observed in these cells. The presence of subtype-rich cells with a degree of co-localization between α1-ARs and GPR55 indicates a possibility for dimerisation or functional interaction and a new paradigm for functional synergism in which interactions may be either between cells or involve converging intracellular signaling processes.

Diosgenin is a phytosteroid sapogenin with reported antitumoral activity. Despite the evidence indicating a lower incidence of prostate cancer (PCa) associated with a higher consumption of phytosteroids and the beneficial role of these compounds, only a few studies have investigated the effects of diosgenin in PCa, and its mechanisms of action remain to be disclosed. The present study investigated the effect of diosgenin in modulating PCa cell fate and glycolytic metabolism and explored its potential interplay with G protein-coupled oestrogen receptor (GPER). Non-neoplastic (PNT1A) and neoplastic (LNCaP, DU145, and PC3) human prostate cell lines were stimulated with diosgenin in the presence or absence of the GPER agonist G1 and upon GPER knockdown. Diosgenin decreased the cell viability, as indicated by the MTT assay results, which also demonstrated that castrate-resistant PCa cells were the most sensitive to treatment (PC3 > DU145 > LNCaP > PNT1A; IC50 values of 14.02, 23.21, 56.12, and 66.10 µM, respectively). Apoptosis was enhanced in diosgenin-treated cells, based on the increased caspase-3-like activity, underpinned by the altered expression of apoptosis regulators evaluated by Western blot analysis, which indicated the activation of the extrinsic pathway. Exposure to diosgenin also altered glucose metabolism. Overall, the effects of diosgenin were potentiated in the presence of G1. Moreover, diosgenin treatment augmented GPER expression, and the knockdown of the GPER gene suppressed the proapoptotic effects of diosgenin in PC3 cells. Our results support the antitumorigenic role of diosgenin and its interest in PCa therapy, alone or in combination with G1, mainly targeting the more aggressive stages of the disease.

Estrogens are involved in growth, differentiation and pathogenesis of human prostate through the mediation of the classical estrogen receptors ERα and ERβ. The G protein-coupled estrogen receptor (GPER) is a 'novel' mediator of estrogen signaling which has been recently recognized in some human reproductive tissues, but its expression in the prostate gland is still unknown. Here, we investigated GPER in benign (from 5 patients) and neoplastic prostatic tissues (from 50 patients) by immunohistochemical analysis and Western blotting. Normal areas of benign prostates revealed a strong GPER immunoreactivity in the basal epithelial cells while luminal epithelial cells were unreactive and stromal cells were weakly immunostained. GPER was also immunolocalized in adenocarcinoma samples but the immunoreactivity of tumoral areas decreased from Gleason pattern 2 to Gleason pattern 4. Furthermore, a strong GPER immunostaining was also revealed in cells of pre-neoplastic lesions (high-grade prostatic intra-epithelial neoplasia). Western blot analysis of benign and tumor protein extracts showed the presence of a ~42 kDa band, consistent with the GPER molecular weight. An increase in both pAkt and p cAMP-response-binding protein (pCREB) levels was also observed in poorly differentiated PCa samples. Finally, this work identified GPER in the epithelial basal cells of benign human prostate, with a different localization with respect to the classical estrogen receptors. Furthermore, the expression of GPER in prostatic adenocarcinoma cells was also observed but with a modulation of the immunoreactivity according to tumor cell arrangements.

Background: Endocrine therapy is an important therapeutic choice for patients with estrogen receptor (ER)a-positive breast carcinoma and functions by blocking proliferative estrogen signalling through the classical nuclear ERa. G protein-coupled estrogen receptor 1 (GPER1) is a novel membrane estrogen receptor responsible for unique estrogen responses in vitro and in vivo and that is activated by tamoxifen. The aim of this study was to determine the correlation of GPER1 with clinicopathological variables and distant disease-free survival (DDFS) in breast cancer patients treated with and without adjuvant tamoxifen, and whether the prognostic impact is dependent on ERa-status. Material and Methods: GPER1 was investigated by immunohistochemistry in tissue microarrays of breast tumors from 208 premenopausal node-negative patients (median age 47 years; range 30–57), mainly (87%) not subjected to any adjuvant systemic treatment, and from 273 stage II patients (median age 63 years; range 26–81), all treated with adjuvant tamoxifen for 2 years. Because almost 90% of the samples had a high percentage (>50%) GPER1-stained cells, we decided to evaluate only the staining intensity (negative, very weak, weak, moderate, and strong) for this variable. Pearson's χ2 test for trend was used for analyzing association between GPER1 and categorical clinicopathological variables, a test for trend based on ranks for association with age and tumor size, and a log-rank test for trend for evaluating the impact of GPER1 on DDFS after 5 years of follow-up. Results: GPER1 positively correlated with ERa (P=0.0005 and P=0.01, respectively) and progesterone receptor expression (P=0.004 and P=0.01, respectively) in both the premenopausal and tamoxifentreated groups, but not with HER2 expression (P=0.45 and P=0.42, respectively). In the premenopausal group, GPER1 negatively correlated with tumor size (P=0.02) and positively with age (P=0.003), whereas in the tamoxifen group GPER1 did not correlate with either tumor size or age. During 5 years of follow up, 64 patients were diagnosed with distant recurrences in the tamoxifen group and 34 patients in the premenopausal group. In univariate analysis, GPER1 positively correlated with DDFS in the tamoxifen group (P=0.04), but non-significantly in the premenopausal group (P=0.08). When stratifying for ERa-status, GPER1 was a prognostic factor in the ERa-positive subgroup (P=0.02 in tamoxifen group and P=0.08 in premenopausal group), but not in the ERa-negative subgroup (P=0.57 and P=0.95, respectively). Conclusion: We propose that GPER1 is a prognostic marker for increased DDFS in ERa-positive breast cancer. While our results suggest that GPER1 is also a tamoxifen-predictive factor, this needs to be further studied, ideally in a randomized trial comparing clinical outcome for patients treated with and without adjuvant tamoxifen. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-02.

G-protein coupled estrogen receptor 1 (GPER1): A potential target for chemoprevention of prostate cancer.

BackgroundEstrogenic signals are suggested to have protection roles in the development of colorectal cancer (CRC). The G protein-coupled estrogen receptor (GPER) has been reported to mediate non-genomic effects of estrogen in hormone related cancers except CRC. Its expression and functions in CRC were investigated.MethodsThe expression of GPER and its associations with clinicopathological features were examined. The mechanisms were further investigated using cells, mouse xenograft models, and clinical human samples.ResultsGPER was significantly (p < 0.01) down regulated in CRC tissues compared with their matched adjacent normal tissues in our two cohorts and three independent investigations from Oncomine database. Patients whose tumors expressing less (n = 36) GPER showed significant (p < 0.01) poorer survival rate as compared with those with greater levels of GPER (n = 54). Promoter methylation and histone H3 deacetylation were involved in the down regulation of GPER in CRC cell lines and clinical tissues. Activation of GPER by its specific agonist G-1 inhibited proliferation, induced cell cycle arrest, mitochondrial-related apoptosis and endoplasmic reticulum (ER) stress of CRC cells. The upregulation of reactive oxygen species (ROS) induced sustained ERK1/2 activation participated in G-1 induced cell growth arrest. Further, G-1 can inhibit the phosphorylation, nuclear localization, and transcriptional activities of NF-κB via both canonical IKKα/ IκBα pathways and phosphorylation of GSK-3β. Xenograft model based on HCT-116 cells confirmed that G-1 can suppress the in vivo progression of CRC.ConclusionsEpigenetic down regulation of GPER acts as a tumor suppressor in colorectal cancer and its specific activation might be a potential approach for CRC treatment.

Background: G protein-coupled estrogen receptor (GPER) is a putative estrogen receptor (ER) with potential clinical implications in breast cancer. Depending on subcellular location, cut-off and treatment, GPER has been proposed as both a marker of aggressive disease and of good prognosis. The correlation with known prognostic factors appears to be biphasic. Furthermore, plasma membrane GPER-overexpression (GPERPM) is associated to a worse prognosis, suggesting potential receptor mutation or amplification. In vitro studies also show that tamoxifen upregulates GPER in breast cancer cells, suggesting GPER as a mediator of endocrine resistance. To further clarify the role of GPER in breast cancer and endocrine treatment we studied GPER during evolution from primary tumor to lymph node metastasis (lgl-met), and from first tumor (BC1) to a second contralateral tumor (CBC), with/without endocrine treatment (mainly tamoxifen) in between. As a CBC developed despite adjuvant treatment given for BC1 is presumably resistant to this treatment, it may be used to study endocrine treatment escape mechanisms in vivo. Patients and methods: From a well-defined population-based cohort of CBC-patients we constructed a unique tissue-microarray including 688 patients with metachronous CBC. Overall GPER staining intensity (0-5) (GPERoverall) and GPERPM (present/absent) was evaluated by two investigators in BC1 (n=559), CBC (n=595) and corresponding lgl-met (nBC1=151 and nCBC=158). Impact on survival was analyzed using the Kaplan-Meier method and Cox regression, and p-values calculated with the log rank test. Association was tested with a chi-square test (for trend when appropriate), and differences between paired samples with Wilcoxon signed rank test. Results: GPR30overall showed the expected biphasic association with ER-α and the progesterone receptor (PgR) in BC1 and CBC, where high and low GPER expression associated with ER/PgR negativity (p&amp;lt;0.001). High GPER expression was also associated with increased Ki67 expression (p=0.04 and p=0.08). Similar results were seen for GPERPM (ER/PgR-negativity p&amp;lt;0.001, high Ki67 p&amp;lt;0.001). There was no association between GPER in BC1 and CBC, and prior endocrine treatment did not seem to affect GPER expression in CBC. However, a significant association between primary tumor and corresponding lgl-met was seen in both BC1 and CBC. Further, a comparison between BC1/CBC and corresponding lgl-met showed a decrease in GPERoverall (p&amp;lt;0.001), but an increase in GPERPM (p=0.01). A high CBC GPER expression was associated with a worse OS, both when considering GPERoverall (p=0.02, HR=1.18, 95%CI: 1.05-1.32) and GPERPM (p=0.007, HR=1.61 95%CI: 1.14-2.29). Similar results were seen when analyzing survival in relation to CBC lgl-met GPER expression. Conclusion: A high GPER expression in CBC and matched lgl-met is associated with worse OS. The overall expression of GPER decreases from primary tumor to lgl-met, but plasma membrane staining increases, supporting a mutation or amplification of the receptor. In addition, GPER expression correlates with other markers of aggressive disease, highlighting the potential as a novel treatment target. However, prior tamoxifen treatment did not alter tumor GPER expression. Citation Format: Sjöström M, Fernö M, Rydén L, Leeb-Lundberg FLM, Alkner S. G protein-coupled estrogen receptor expression in lymph node metastasis and contralateral breast cancer during endocrine treatment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-05-01.

<b>Abstract ID 14042</b> <b>Poster Board 508</b> Hypertension, which is a leading cardiovascular risk factor impacting 40% of American women, is more frequent in aged compared to younger women. Postmenopausal women undergo a decline in the circulating level of estrogen, along with the natural aging process. It has been shown that G protein-coupled estrogen receptor 1 (GPER1) contributes to the cardiovascular protective actions of estrogen in different experimental models. Pregnancy augments GPER1-induced vasodilation in rats. However, the potential interaction between GPER1 and pregnancy in impacting the cardiovascular health later in life is not clear. We hypothesize that GPER1 signaling and pregnancy protects against hypertension in aged female mice. In our study, 16-20 months-old GPER1 knock-out (KO) and wild-type (WT) female mice with and without a history of former pregnancies were implanted with radio-telemeters for blood pressure recording. Animals were placed into metabolic cages for collection of 24h urine samples. Then, animals were euthanized and plasma was collected. To examine whether GPER1 and/or former pregnancies affects circadian blood pressure rhythm in aged female mice, blood pressure was analyzed by cosinor analysis. No significant differences in body weight were observed between groups. GPER1 deletion in virgin mice did not impact food intake or urinary Na⁺excretion. Assessment of plasma sex hormone levels revealed that genetic deletion of GPER1 did not alter plasma estradiol or progesterone levels in virgin mice. However, previous pregnancies increased plasma levels of estradiol (<i>p</i>=0.0063), but not progesterone (<i>p</i>=0.6748) in GPER1 KO mice. Of note, GPER1 deletion increased mean arterial pressure in virgin mice (KO: 126±3; WT: 107±2 mmHg; <i>p</i>&lt;0.0001). Interestingly, prior pregnancies eliminated this genotypic difference in blood pressure. Cosinor analysis revealed augmented mean arterial blood pressure amplitude in virgin GPER1 KO mice, that was attenuated in previously-pregnant GPER1 KO mice. We also measured urinary excretion of endothelin and aldosterone which are major contributors to blood pressure regulation. Previous pregnancies lowered urinary levels of endothelin-1 (<i>p</i>=0.0049) and aldosterone (<i>p</i>=0.0099) in GPER1 KO mice. Overall, our data demonstrate an elevated blood pressure in GPER1 KO virgin mice compared to WT virgin and previously-pregnant GPER1 KO aged mice. The current data suggest that GPER1 and former pregnancies provide protection against hypertension in aged females. Funded by R00DK119413 to EG.

Hypertension prevalence is lower in women than in men. Enhanced renal sodium (Na+) handling in females has been implicated in sex differences in hypertension. Epithelial Na+ channel (ENaC) is a key contributor to Na+ homeostasis and is regulated by estrogen. Recent evidence suggests G protein-coupled estrogen receptor 1 (GPER1) evokes a female-specific natriuresis that involves endothelin-1 (ET-1). ET-1 has been shown to downregulate ENaC activity, but whether GPER1 regulates ENaC to modulate natriuresis is unknown. We tested the hypothesis that renal GPER1 functionally interacts with ENaC to promote natriuresis in a sex-specific manner. RNAscope confirmed coexpression of GPER1 and ENaC in rat renal tubules in a sex- and region-specific manner. Within the renal medulla, the number of ENaC/GPER1-positive tubules was greater in females than males. Renal medullary inhibition of ENaC or activation of GPER1 evoked comparable natriuresis in female rats. Electrophysiology revealed that pharmacological GPER1 activation downregulated ENaC activity, whereas genetic deletion of GPER1 from the principal cells of the collecting duct caused ENaC hyperactivity. The hyperactivity of ENaC caused by deletion of GPER1 in the principal cells was greater in female than male mice. RNAscope coexpression of aquaporin 2 (AQP2) and GPER1 confirmed the knockout (KO) of GPER1 from the principal cell (PC) in the kidney. Thus, renal GPER1 functionally interacts with ENaC in a sex-specific manner to promote natriuresis.NEW & NOTEWORTHY This study identified GPER1 as a sex-specific upstream regulator of ENaC. We found that GPER1 and ENaC were coexpressed in the rat renal tubules in a sex and region-specific manner. Activation of GPER1 inhibited ENaC activity in isolated mouse collecting ducts, whereas deletion of GPER1 from the principal cells caused ENaC hyperactivity to a greater extent in female mice. Our data suggest GPER1 functionally interacts with ENaC in a sex-specific manner to promote natriuresis.

G protein-coupled estrogen receptor 1 mediates estrogen effect in red common carp (Cyprinus carpio)

Uterine leiomyomas are ovarian hormone-responsive tumors originating in the smooth muscle layer of the uterus called the myometrium. These benign tumors require surgery or other clinical intervention in ∼25% of all premenopausal women in the United States. Despite the high frequency and clinical impact of these tumors, the pathogenesis of uterine leiomyomas is not understood. The estrogen-responsive growth of uterine leiomyomas has been well established, however the role of G protein-coupled estrogen receptor (GPER) in leiomyoma cell regulation has not been studied. The membrane bound estrogen receptor GPER, formerly known as GPR30, is a 7-transmembrane G protein-coupled receptor that is activated by 17β-estradiol (E2). A role for GPER has been previously established in cancers that originate in E2-dependent tissues such as the breast and uterus. Other reports show that GPER is expressed in myometrial cells and activation of GPER modulates myometrial contraction suggesting an important regulatory role for this receptor in the myometrium. Based on these reports, we hypothesized that GPER regulates uterine leiomyoma cells and may represent a putative therapeutic target for this tumor type. The Eker rat model-derived uterine leiomyoma (ELT-3) cell line was used as a model system to study GPER in uterine leiomyoma cells. First, the expression of GPER in uterine leiomyoma cells was determined using RNA and protein samples from ELT-3 cells and real-time PCR and immunoblot analysis. Our findings indicate that GPER transcript and protein were expressed in ELT-3 cells. To determine if activation of GPER regulated uterine leiomyoma cell proliferation, ELT-3 cells were treated with G1, a GPER-specific ligand, or vehicle control and counted 5 days after treatment. Data from these experiments indicated that G1 treatment, and potentially GPER activation, reduced the number of cells over time compared to vehicle control. GPER activation is known to result in the accumulation of pERK and intracellular free Ca2+ and such accumulation is used as a biomarker for GPER activation. Upon G1 treatment, pERK and intracellular free Ca2+ were elevated compared to vehicle treatment in ELT-3 cells suggesting that GPER is functional in uterine leiomyoma cells and is activated by G1. In an effort to determine the mechanism of inhibition of uterine leiomyoma cell proliferation by G1 treatment, we hypothesized that treatment of estrogen-stimulated ELT-3 cells with G1 would inhibit the characteristic E2-induced proliferation of this cell type. Growth curve experiments were performed and data obtained in these experiments suggested that G1 treatment resulted in the inhibition of E2-induced ELT-3 cell proliferation. Taken together, these data suggest that GPER is an important regulator of uterine leiomyoma cell proliferation and is a putative target for novel molecular therapeutics for this tumor type. Citation Format: Maryann Castillo, Angelique M. Wimbley, Jacob J. Mayfield, Jenifer C. Lascano, Kevin D. Houston. Activation of G-protein coupled estrogen receptor (GPER) inhibits ELT-3 uterine leiomyoma cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1305. doi:10.1158/1538-7445.AM2013-1305

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; G protein–coupled estrogen receptor 1 (GPER1), previously named GPR30, is a membrane receptor reported to mediate nongenomic estrogen responses. We investigated if GPER1 expression correlates with any clinicopathologic variables and distant disease-free survival (DDFS) in patients with breast cancer, if any prognostic impact of the receptor is dependent on estrogen receptor-α (ER-α) status, and if the receptor impacts apoptotic signaling in ER-positive breast cancer cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; GPER1 expression was analyzed by immunohistochemistry in breast tumors from 273 pre- and postmenopausal stage II patients, all treated with adjuvant tamoxifen for 2 years (cohort I) and from 208 premenopausal lymph node-negative patients, of which 87% were not subjected to any adjuvant systemic treatment (cohort II). GPER1-dependent proapoptotic signaling was analyzed in MCF7 cells with and without GPER1 knockdown, T47D cells, HEK293 cells (HEK), and HEK stably expressing GPER1 (HEK-R).&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; GPER1 positively correlates with ER and progesterone receptor expression. Multivariate analysis showed that GPER1 is an independent prognostic marker of increased 10-year DDFS in the ER-positive subgroup. HEK-R has higher basal proapoptotic signaling compared with HEK including increased cytochrome C release, caspase-3 cleavage, PARP cleavage, and decreased cell viability. Treating HEK-R with the proteasome inhibitor epoxomicin, to decrease GPER1 degradation, further increases receptor-dependent proapoptotic signaling. Also, GPER1 knockdown decreases basal and agonist-stimulated proapoptotic receptor signaling in MCF7 cells.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; GPER1 is a prognostic indicator for increased DDFS in ER-positive breast cancer, which may be associated with constitutive GPER1-dependent proapoptotic signaling in ER-positive breast cancer cells. &lt;i&gt;Clin Cancer Res; 19(7); 1681–92. ©2013 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;