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Eric R Prossnitz - One of the best experts on this subject based on the ideXlab platform.
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co expression of GPR30 and erβ and their association with disease progression in uterine carcinosarcoma
American Journal of Obstetrics and Gynecology, 2010Co-Authors: Gloria S Huang, Hugo Ariaspulido, Eric R Prossnitz, Marc J Gunter, Rebecca C Arend, Maomi Li, Gary L Goldberg, Harriet O SmithAbstract:Objective We sought to evaluate the expression of G protein-coupled receptor 30 (GPR30) and estrogen receptor (ER)β in uterine carcinosarcoma (CS). Study Design Immunohistochemistry was performed using antibodies to GPR30, ERβ, ERα, and progesterone receptor (PR). The staining intensity and percentage of positive cells were scored for each tissue section. Expression levels were compared using the Wilcoxon rank sum test. Correlation was evaluated by Spearman rho and logistic regression. Results Compared with normal endometrium, CS had lower ERα and PR expression (both P P = .03). Advanced-stage CS had higher GPR30 ( P P = .02) epithelial expression compared with early-stage CS. Expression of GPR30 and ERβ correlated with each other ( P Conclusion In uterine CS, GPR30 and ERβ are coordinately overexpressed and expression levels increase in advanced-stage disease, supporting the involvement of alternative ERs in disease progression.
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GPR30 and estrogen receptor expression new insights into hormone dependence of inflammatory breast cancer
Breast Cancer Research and Treatment, 2010Co-Authors: Hugo Ariaspulido, Melanie Royce, Nancy E Joste, Lesley Lomo, Nabila Chaher, J Lara, Yun Gong, Claire F. Verschraegen, Eric R Prossnitz, Massimo CristofanilliAbstract:GPR30 is a novel G protein-coupled estrogen receptor (ER) associated with metastases in breast cancer (BC) and poor survival in endometrial and ovarian tumors. The association of GPR30 expression with inflammatory breast cancer (IBC), an aggressive and commonly hormone-independent form of BC, has not been studied. GPR30, ER, progesterone receptor (PR), epidermal growth factor receptor (EGFR), and HER-2 expression were assessed by immunohistochemistry (and FISH for HER-2) in 88 primary IBCs. GPR30 expression was correlated with patient overall survival (OS), disease-free survival (DFS), pathologic variables, and other biomarkers. GPR30 expression was found in 69% of IBC cases. ER, PR, HER-2, and EGFR were found in 43, 35, 39, and 34% of IBC cases, respectively. GPR30 expression correlated inversely with ER expression (P = 0.02). Co-expression of ER and GPR30 was found in 24% of IBC samples; 19% expressed only ER and 46% expressed only GPR30. Univariate analysis showed no association between GPR30 expression and OS or DFS. However, co-expression of ER and GPR30 was associated with improved OS (P < 0.03) and marginally with DFS (P < 0.06); the absence of both ER and GPR30 was associated with worse OS and DFS (P = 0.03 for both). Multivariate analysis identified ER as an independent prognostic factor of OS (P = 0.008) and DFS (P = 0.02). The majority of IBC tumors are GPR30-positive, suggesting that estrogen signaling may be active in ER-negative IBC patients. These findings suggest potential new therapeutic targets for IBC such as novel endocrine agents or direct modulation of GPR30.
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synthesis and characterization of iodinated tetrahydroquinolines targeting the g protein coupled estrogen receptor GPR30
Journal of Medicinal Chemistry, 2010Co-Authors: Chinnasamy Ramesh, Larry Sklar, Ritwik Burai, Helen J. Hathaway, Eric R Prossnitz, Megan K Dennis, Tapan K Nayak, Jeffrey B. ArterburnAbstract:A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and specificity of binding to GPR30 versus the classical estrogen receptors ERα/β and functional responses associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines exhibited IC50 values lower than 20 nM in competitive binding studies with GPR30-expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of selected compounds were radiolabeled with 125I using the iodogen method. In vivo biodistribution studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal, and reproductive organs. Biodistribution and quantitati...
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GPR30 and HER-2 Expression in Invasive and Metastatic Breast Cancer.
Cancer Research, 2009Co-Authors: Hugo Arias-pulido, Nancy E Joste, Lesley Lomo, Nabila Chaher, Claire F. Verschraegen, Eric R Prossnitz, A. Meisner, C. Martinez, Melanie RoyceAbstract:Background: GPR30 expression, a new estrogen receptor, has been previously associated with HER-2, tumor size and metastasis in invasive breast cancer (BC) but its role in paired normal (N), invasive (I), and metastatic (M), samples is unknown. We described GPR30 and HER-2 expression in a collection of paired N/I/M samples, derived from the same individual. Materials and Methods: GPR30 and HER-2 expression was assessed by immunohistochemistry (IHC) in tissue microarrays, containing paraffin-embedded cores from 100 patients diagnosed with invasive BC. N and M samples were also available from the same patient. GPR30 expression was evaluated by an H-score (Intensity (0, negative; 1+, weak; 2+, moderate; 3+, strong) x Percentage of stained epithelial cells). HER-2 expression was evaluated per standard criteria. Log rank tests and Wald tests were employed to assess the clinical impact of these molecular targets on patient outcome based on Kaplan-Meier Product estimator and Cox Proportional Hazard Regression. Results: GPR30 was expressed in 50%, 76%, and 72% of N, I and M, respectively, samples. HER-2 (3+) was found in 14% and 18% of I and M samples, respectively. GPR30 expression in I cases correlated with expression in M cases, and HER-2 expression in I but not M cases. GPR30 expression in M cases correlated with expression in HER-2 expression in M cases. HER-2 expression in I cases correlated with expression in M samples (P 0.05). GPR30 expression in I or M samples was not associated with either overall survival (OS) or BC-specific survival (BCSS)(P>0.5). HER-2 expression was marginally associated with OS in I (P=0.06; Hazard Ratios (HR): 1.91; 95%CI: 0.956, 3.83) but not in M (P=0.23) cases. HER-2 was significantly associated with BCSS in I cases (P=0.03; HR: 2.39; 95%CI: 1.07, 5.32) and marginally in M (P=0.08) cases. Discussion: A previous study suggested that GPR30 expression predicted the development of metastasis. We found high GPR30 expression in both the primary and metastatic sample but the difference was not significant. While GPR30 expression was not associated with OS or BCSS, HER-2 expression was marginally associated with OS and significantly associated with BCSS in invasive cases. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4158.
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GPR30 predicts poor survival for ovarian cancer
Gynecologic Oncology, 2009Co-Authors: Helen J. Hathaway, Hugo Ariaspulido, Nancy E Joste, Claire F. Verschraegen, Harriet O Smith, Clifford Qualls, Tamara Howard, Eric R ProssnitzAbstract:Objectives GPR30 is a 7-transmembrane G protein-coupled estrogen receptor that functions alongside traditional estrogen receptors to regulate cellular responses to estrogen. Recent studies suggest that GPR30 expression is linked to lower survival rates in endometrial and breast cancer. This study was conducted to evaluate GPR30 expression in ovarian tumors.
Edward J Filardo - One of the best experts on this subject based on the ideXlab platform.
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involvement of g protein coupled receptor 30 GPR30 in rapid action of estrogen in primate lhrh neurons
Molecular Endocrinology, 2009Co-Authors: Sekoni D Noel, Edward J Filardo, Kim L Keen, David I Baumann, Ei TerasawaAbstract:Previously, we have reported that 17β-estradiol (E2) induces an increase in firing activity of primate LH-releasing hormone (LHRH) neurons. The present study investigates whether E2 alters LHRH release as well as the pattern of intracellular calcium ([Ca2+]i) oscillations and whether G protein-coupled receptor 30 (GPR30) plays a role in mediating the rapid E2 action in primate LHRH neurons. Results are summarized: 1) E2, the nuclear membrane-impermeable estrogen, estrogen-dendrimer conjugate, and the plasma membrane-impermeable estrogen, E2-BSA conjugate, all stimulated LHRH release within 10 min of exposure; 2) whereas the estrogen receptor antagonist, ICI 182,780, did not block the E2-induced LHRH release, E2 application to cells treated with pertussis toxin failed to induce LHRH release; 3) GPR30 mRNA was expressed in olfactory placode cultures, and GPR30 protein was expressed in a subset of LHRH neurons; 4) pertussis toxin treatment blocked the E2-induced increase in [Ca2+]i oscillations; 5) knockdown of GPR30 in primate LHRH neurons by transfection with small interfering RNA (siRNA) for GPR30 completely abrogated the E2-induced changes in [Ca2+]i oscillations, whereas transfection with control siRNA did not; 6) the estrogen-dendrimer conjugate-induced increase in [Ca2+]i oscillations also did not occur in LHRH neurons transfected with GPR30 siRNA; and 7) G1, a GPR30 agonist, resulted in changes in [Ca2+]i oscillations, similar to those observed with E2. Collectively, E2 induces a rapid excitatory effect on primate LHRH neurons, and this rapid action of E2 appears to be mediated, in part, through GPR30.
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extra nuclear estrogen receptor GPR30 regulates serotonin function in rat hypothalamus
Neuroscience, 2009Co-Authors: H Xu, Edward J Filardo, Eric R Prossnitz, Gonzalo A Carrasco, George Battaglia, Lydia L Doncarlos, Nancy A MumaAbstract:Abstract Selective serotonin reuptake inhibitors (SSRIs), such as Prozac®, are used to treat mood disorders. SSRIs attenuate (i.e. desensitize) serotonin 1A (5-HT1A) receptor signaling, as demonstrated in rats through decreased release of oxytocin and adrenocorticotropin hormone (ACTH) following 5-HT1A receptor stimulation. Maximal therapeutic effects of SSRIs for treatment of mood disorders, as well as effects on hypothalamic 5-HT1A receptor signaling in animals, take 1 to 2 weeks to develop. Estradiol also attenuates 5-HT1A receptor signaling, but, in rats, these effects occur within 2 days; thus, estrogens or selective estrogen receptor modulators may serve as useful short-term tools to accelerate desensitization of 5-HT1A receptors in response to SSRIs if candidate estrogen receptor targets in the hypothalamus are identified. We found high levels of GPR30, which has been identified recently as a pertussis-toxin (PTX) sensitive G-protein-coupled estrogen receptor, in the hypothalamic paraventricular nucleus (PVN) of rats. Double-label immunohistochemistry revealed that GPR30 co-localizes with 5-HT1A receptors, corticotrophin releasing factor (CRF) and oxytocin in neurons in the PVN. Pretreatment with PTX to the PVN before peripheral injections of 17-β-estradiol 3-benzoate completely prevented the reduction of the oxytocin response to the 5-HT1A receptor agonist, (+)-8-hydroxy-2-dipropylaminotetralin (DPAT). Treatment with the selective GRP30 agonist, G-1, attenuated 5-HT1A receptor signaling in the PVN as measured by an attenuated oxytocin (by 29%) and ACTH (by 31%) response to DPAT. This study indicates that a putative extra-nuclear estrogen receptor, GPR30, may play a role in estradiol-mediated attenuation of 5-HT1A receptor signaling, and potentially in accelerating the effects of SSRIs in treatment of mood disorders.
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Assessment of GPR30, a Seven Transmembrane-Spanning Estrogen Receptor, as an Oncogene
2007Co-Authors: Edward J FilardoAbstract:Abstract : Our prior work has linked the seven transmembrane estrogen receptor GPR30 to specific estrogen binding (Thomas et al, 2005), rapid estrogen action (Filardo and Thomas, 2005) and the development of metastatic breast cancer in man (Filardo et al, 2006). To further address the role of GPR30 in experimental breast cancer biology, transgenic mice were created for the purpose of overexpressing wild type or active GPR30 in the mammary gland using the mouse mammary tumor virus tissue specific promoter. Two suitable candidate founder mice with stably integrated wild-type GPR30 were generated (T6- 1A and T6-2E). We were unsuccessful in our attempts to generate an active GPR30 allele as assessed by in vitro assays outline in this proposal during this time frame. A no cost extension was requested to further propagate the wild-type mice and to analyze their capacity to develop mammary adenocarcinoma.
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activation of the novel estrogen receptor g protein coupled receptor 30 GPR30 at the plasma membrane
Endocrinology, 2007Co-Authors: Edward J Filardo, Curt Graeber, S Shaw, Yefei Pang, Jeffrey A Quinn, Jing Dong, Peter ThomasAbstract:G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17β-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments...
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distribution of GPR30 a seven membrane spanning estrogen receptor in primary breast cancer and its association with clinicopathologic determinants of tumor progression
Clinical Cancer Research, 2006Co-Authors: Edward J Filardo, Curt Graeber, Jeffrey A Quinn, Murray B Resnick, Dilip Giri, Ronald A Delellis, Margaret M Steinhoff, Edmond SaboAbstract:Purpose: The seven transmembrane receptor, GPR30, is linked to estrogen binding and heparan-bound epidermal growth factor release. Here, the significance of GPR30 in human breast cancer was evaluated by comparing its relationship to steroid hormone receptor expression and tumor progression variables. Experimental Design: Immunohistochemical analysis of a National Cancer Institute–sponsored tumor collection comprised of 361 breast carcinomas obtained at first diagnosis (321 invasive and 40 intraductal tumors). Biopsies from 12 reduction mammoplasties served as controls. The distribution pattern of GPR30, estrogen receptor (ER), and progesterone receptor (PR) was correlated with clinicopathologic variables obtained at diagnosis. Results: GPR30, ER, and PR were positive in all 12 normal controls. In contrast, GPR30 expression varied in breast tumors, in which 62% (199 of 321) of invasive tumors and 42% (17 of 40) of intraductal tumors were positive. Codistribution of ER and GPR30 was measured in 43% (139 of 321) of invasive breast tumors, whereas both receptors were lacking (ER − GPR30 − ) in 19% (61 of 321) of the tumors analyzed, indicating a significant association between ER and GPR30 ( P P = 0.014; odds ratio, 1.9). Conclusions: GPR30 and ER exhibited distinct patterns of association with breast tumor progression variables, including HER-2/neu, tumor size, and metastatic disease. Thus, these results support the hypothesis that GPR30 and ER have an independent influence on estrogen responsiveness in breast carcinoma.
Tudor I Oprea - One of the best experts on this subject based on the ideXlab platform.
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the g protein coupled receptor GPR30 inhibits proliferation of estrogen receptor positive breast cancer cells
Cancer Research, 2010Co-Authors: Eric A Ariazi, Jeffrey B. Arterburn, Eugen Brailoiu, Smitha Yerrum, Heather A Shupp, Michael Slifker, Heather E Cunliffe, Michael A Black, Anne L Donato, Tudor I OpreaAbstract:The G protein–coupled receptor GPR30 binds 17β-estradiol (E 2 ) yet differs from classic estrogen receptors (ERα and ERβ). GPR30 can mediate E 2 -induced nongenomic signaling, but its role in ERα-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERα-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E 2 and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca 2+ mobilization studies, GPR30, but not ERα, mediated E 2 -induced Ca 2+ responses because E 2 , 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca 2+ increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E 2 -induced and G-1–induced Ca 2+ mobilization, but ERα depletion did not. Interestingly, GPR30-coupled Ca 2+ responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G 1 phase. Thus, GPR30 antagonizes growth of ERα-positive breast cancer and may represent a new target to combat this disease. Cancer Res; 70(3); 1184–94
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The G Protein–Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Receptor–Positive Breast Cancer Cells
Cancer Research, 2010Co-Authors: Eric A Ariazi, Jeffrey B. Arterburn, Eugen Brailoiu, Smitha Yerrum, Heather A Shupp, Michael Slifker, Heather E Cunliffe, Michael A Black, Anne L Donato, Tudor I OpreaAbstract:The G protein–coupled receptor GPR30 binds 17β-estradiol (E 2 ) yet differs from classic estrogen receptors (ERα and ERβ). GPR30 can mediate E 2 -induced nongenomic signaling, but its role in ERα-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERα-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E 2 and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca 2+ mobilization studies, GPR30, but not ERα, mediated E 2 -induced Ca 2+ responses because E 2 , 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca 2+ increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E 2 -induced and G-1–induced Ca 2+ mobilization, but ERα depletion did not. Interestingly, GPR30-coupled Ca 2+ responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G 1 phase. Thus, GPR30 antagonizes growth of ERα-positive breast cancer and may represent a new target to combat this disease. Cancer Res; 70(3); 1184–94
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expression of estrogen receptor GPR30 in the rat spinal cord and in autonomic and sensory ganglia
Journal of Neuroscience Research, 2009Co-Authors: Cristina G Brailoiu, Jeffrey B. Arterburn, Eric R Prossnitz, Eugen Brailoiu, Tudor I OpreaAbstract:The G protein-coupled receptor GPR30 has recently been identified as a nonnuclear estrogen receptor. Reverse transcriptase-polymerase chain reaction revealed expression of GPR30 mRNA in varying quantities in the rat spinal cord, dorsal root ganglia, nodose ganglia, trigeminal ganglia, hippocampus, brain stem, and hypothalamus. Immunohistochemical studies that used a rabbit polyclonal antiserum against the human GPR30 C-terminus revealed a fine network of GPR30-immunoreactive (irGPR30) cell processes in the superficial layers of the spinal cord; some of which extended into deeper laminae. A population of neurons in the dorsal horn and ventral horn were irGPR30. Dorsal root, nodose, and trigeminal ganglionic neurons displayed varying intensities of irGPR30. Positively labeled neurons were detected in the major pelvic ganglion, but not in the superior cervical ganglion. A population of chromaffin cells in the adrenal medulla was irGPR30, so were cells of the zona glomerulosa. Double-labeling the adrenal medulla with GPR30 antiserum and tyrosine hydroxylase antibody or phenylethanolamine-N-methyltransferase antiserum revealed that irGPR30 is expressed in the majority of tyrosine hydroxylase-positive chromaffin cells. Last, some of the myenteric ganglion cells were irGPR30. Tissues processed with preimmune serum resulted in no staining. Voltage-sensitive dye imaging studies showed that the selective GPR30 agonist G-1 (1, 10, and 100 nM) depolarized cultured spinal neurons in a concentration-dependent manner. Collectively, our result provides the first evidence that GPR30 is expressed in neurons of the dorsal and ventral horn as well as in sensory and autonomic neurons, and activation of GPR30 by the selective agonist G-1 depolarizes cultured spinal neurons.
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the ins and outs of GPR30 a transmembrane estrogen receptor
The Journal of Steroid Biochemistry and Molecular Biology, 2008Co-Authors: Eric R Prossnitz, Larry Sklar, Tudor I Oprea, Jeffrey B. ArterburnAbstract:Abstract Estrogen is an important hormone in human physiology. It acts both via transcriptional regulation as well as via modulation of intracellular signaling through second messengers. Although estrogen's transcriptional effects occur through classical nuclear steroid receptors (ERs), recent studies reveal the existence of a novel 7-transmembrane G protein-coupled receptor, GPR30, which responds to estrogen and tamoxifen stimulation with rapid cellular signaling including ERK activation, PI3K activation, calcium mobilization and cAMP production. To distinguish between ER- and GPR30-mediated signaling, we have identified a novel GPR30 agonist that exhibits high specificity for GPR30. In this review, we will describe recent work to further our understanding of the role of GPR30 in estrogen biology.
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g protein coupled receptor 30 GPR30 mediates gene expression changes and growth response to 17beta estradiol and selective GPR30 ligand g 1 in ovarian cancer cells
Cancer Research, 2007Co-Authors: Lidia Albanito, Eric R Prossnitz, Tudor I Oprea, Antonio Madeo, Rosamaria Lappano, Adele Vivacqua, Vittoria Rago, Amalia Carpino, Anna Maria Musti, Sebastiano AndoAbstract:Estrogens play a crucial role in the development of ovarian tumors; however, the signal transduction pathways involved in hormone action are still poorly defined. The orphan G protein-coupled receptor 30 (GPR30) mediates the nongenomic signaling of 17beta-estradiol (E2) in a variety of estrogen-sensitive cancer cells through activation of the epidermal growth factor receptor (EGFR) pathway. Whether estrogen receptor alpha (ERalpha) also contributes to GPR30/EGFR signaling is less understood. Here, we show that, in ERalpha-positive BG-1 ovarian cancer cells, both E2 and the GPR30-selective ligand G-1 induced c-fos expression and estrogen-responsive element (ERE)-independent activity of a c-fos reporter gene, whereas only E2 stimulated an ERE-responsive reporter gene, indicating that GPR30 signaling does not activate ERalpha-mediated transcription. Similarly, both ligands up-regulated cyclin D1, cyclin E, and cyclin A, whereas only E2 enhanced progesterone receptor expression. Moreover, both GPR30 and ERalpha expression are required for c-fos stimulation and extracellular signal-regulated kinase (ERK) activation in response to either E2 or G-1. Inhibition of the EGFR transduction pathway inhibited c-fos stimulation and ERK activation by either ligand, suggesting that in ovarian cancer cells GPR30/EGFR signaling relays on ERalpha expression. Interestingly, we show that both GPR30 and ERalpha expression along with active EGFR signaling are required for E2-stimulated and G-1-stimulated proliferation of ovarian cancer cells. Because G-1 was able to induce both c-fos expression and proliferation in the ERalpha-negative/GPR30-positive SKBR3 breast cancer cells, the requirement for ERalpha expression in GPR30/EGFR signaling may depend on the specific cellular context of different tumor types.
Jeffrey B. Arterburn - One of the best experts on this subject based on the ideXlab platform.
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synthesis and characterization of iodinated tetrahydroquinolines targeting the g protein coupled estrogen receptor GPR30
Journal of Medicinal Chemistry, 2010Co-Authors: Chinnasamy Ramesh, Larry Sklar, Ritwik Burai, Helen J. Hathaway, Eric R Prossnitz, Megan K Dennis, Tapan K Nayak, Jeffrey B. ArterburnAbstract:A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and specificity of binding to GPR30 versus the classical estrogen receptors ERα/β and functional responses associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines exhibited IC50 values lower than 20 nM in competitive binding studies with GPR30-expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of selected compounds were radiolabeled with 125I using the iodogen method. In vivo biodistribution studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal, and reproductive organs. Biodistribution and quantitati...
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the g protein coupled receptor GPR30 inhibits proliferation of estrogen receptor positive breast cancer cells
Cancer Research, 2010Co-Authors: Eric A Ariazi, Jeffrey B. Arterburn, Eugen Brailoiu, Smitha Yerrum, Heather A Shupp, Michael Slifker, Heather E Cunliffe, Michael A Black, Anne L Donato, Tudor I OpreaAbstract:The G protein–coupled receptor GPR30 binds 17β-estradiol (E 2 ) yet differs from classic estrogen receptors (ERα and ERβ). GPR30 can mediate E 2 -induced nongenomic signaling, but its role in ERα-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERα-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E 2 and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca 2+ mobilization studies, GPR30, but not ERα, mediated E 2 -induced Ca 2+ responses because E 2 , 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca 2+ increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E 2 -induced and G-1–induced Ca 2+ mobilization, but ERα depletion did not. Interestingly, GPR30-coupled Ca 2+ responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G 1 phase. Thus, GPR30 antagonizes growth of ERα-positive breast cancer and may represent a new target to combat this disease. Cancer Res; 70(3); 1184–94
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The G Protein–Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Receptor–Positive Breast Cancer Cells
Cancer Research, 2010Co-Authors: Eric A Ariazi, Jeffrey B. Arterburn, Eugen Brailoiu, Smitha Yerrum, Heather A Shupp, Michael Slifker, Heather E Cunliffe, Michael A Black, Anne L Donato, Tudor I OpreaAbstract:The G protein–coupled receptor GPR30 binds 17β-estradiol (E 2 ) yet differs from classic estrogen receptors (ERα and ERβ). GPR30 can mediate E 2 -induced nongenomic signaling, but its role in ERα-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERα-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E 2 and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca 2+ mobilization studies, GPR30, but not ERα, mediated E 2 -induced Ca 2+ responses because E 2 , 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca 2+ increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E 2 -induced and G-1–induced Ca 2+ mobilization, but ERα depletion did not. Interestingly, GPR30-coupled Ca 2+ responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G 1 phase. Thus, GPR30 antagonizes growth of ERα-positive breast cancer and may represent a new target to combat this disease. Cancer Res; 70(3); 1184–94
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expression of estrogen receptor GPR30 in the rat spinal cord and in autonomic and sensory ganglia
Journal of Neuroscience Research, 2009Co-Authors: Cristina G Brailoiu, Jeffrey B. Arterburn, Eric R Prossnitz, Eugen Brailoiu, Tudor I OpreaAbstract:The G protein-coupled receptor GPR30 has recently been identified as a nonnuclear estrogen receptor. Reverse transcriptase-polymerase chain reaction revealed expression of GPR30 mRNA in varying quantities in the rat spinal cord, dorsal root ganglia, nodose ganglia, trigeminal ganglia, hippocampus, brain stem, and hypothalamus. Immunohistochemical studies that used a rabbit polyclonal antiserum against the human GPR30 C-terminus revealed a fine network of GPR30-immunoreactive (irGPR30) cell processes in the superficial layers of the spinal cord; some of which extended into deeper laminae. A population of neurons in the dorsal horn and ventral horn were irGPR30. Dorsal root, nodose, and trigeminal ganglionic neurons displayed varying intensities of irGPR30. Positively labeled neurons were detected in the major pelvic ganglion, but not in the superior cervical ganglion. A population of chromaffin cells in the adrenal medulla was irGPR30, so were cells of the zona glomerulosa. Double-labeling the adrenal medulla with GPR30 antiserum and tyrosine hydroxylase antibody or phenylethanolamine-N-methyltransferase antiserum revealed that irGPR30 is expressed in the majority of tyrosine hydroxylase-positive chromaffin cells. Last, some of the myenteric ganglion cells were irGPR30. Tissues processed with preimmune serum resulted in no staining. Voltage-sensitive dye imaging studies showed that the selective GPR30 agonist G-1 (1, 10, and 100 nM) depolarized cultured spinal neurons in a concentration-dependent manner. Collectively, our result provides the first evidence that GPR30 is expressed in neurons of the dorsal and ventral horn as well as in sensory and autonomic neurons, and activation of GPR30 by the selective agonist G-1 depolarizes cultured spinal neurons.
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Pre-clinical development of non-steroidal estrogen receptor GPR30 targeted Tc-99m labeled analogues for breast and endometrial cancer imaging
Cancer Research, 2008Co-Authors: Tapan Nayak, Larry Sklar, Ritwik Burai, Helen J. Hathaway, Jeffrey Norenberg, Chinnasamy Ramesh, Jeffrey B. Arterburn, Eric ProssnitzAbstract:3156 Introduction and rationale Currently, the estrogen-based radioimaging agents used in clinical applications are not specific for either ER alpha or ER beta and do not differentiate between classical estrogen receptors and novel classes of estrogen receptors such as GPR30. Classical ER antagonist such as tamoxifen is agonists for GPR30, inducing cell proliferation, which may lead to secondary cancers of reproductive organs. As GPR30 is overexpressed in breast, endometrial and ovarian cancers, it is critical to evaluate GPR30 expression in cancer. Recently, we had identified the first GPR30-selective agonist G1, a non-steroidal tetrahydro-3H-cyclopenta[c]quinoline. Towards this end we have developed non-steroidal G1-modified Tc-99m-labeled GPR30-targeted agents for cancer imaging. Methods The parent GPR30-targeted ligand G1 was modified by adding a chelating moiety through various linkers and radiolabeled with Tc-99m using the tricarbonyl approach. Radioligand receptor binding studies were performed on ER alpha/beta- and GPR30-expressing human breast cancer MCF-7 cells and GPR30-expressing human endometrial carcinoma Hec50 cells. Blocking studies were performed with the GPR30-specific ligand G1 and 17 beta-estradiol (E2) to determine GPR30 specificity. In vivo biodistribution studies were performed on ovariectomized female athymic mice bearing human endometrial cancer Hec50 tumors and human breast cancer MCF-7/18 tumors. Blocking studies were performed by co-injecting either G1 or E2. NanoSPECT/CT imaging studies were performed on live tumor-bearing animals to evaluate the potential of Tc-99m agents as cancer imaging agents. Results All Tc-99m labeled derivatives were highly stable in plasma for up to 24 hours. The LogP values of the Tc-99m labeled agents ranged from 4.6 - 5.5. In cell binding studies, the Tc-99m labeled agents demonstrated GPR30 specificity. Metabolism and animal studies revealed high liver and intestine uptake as well as radiometabolites in the urine. The Tc-99m-G-ethyne-pyridin-2-yl hydrazine displayed liver uptake of 13-16% ID/g as compared to 21-28% ID/g for Tc-99m-G-ethane- pyridin-2-yl hydrazine. In terms of tumor uptake, the Tc-99m-G-ethyne-pyridin-2-yl hydrazine yielded tumor uptake of 1.05% ID/g at 3 hr PI as compared to 0.40 % ID/g for the Tc-99m-G-ethane-pyridin-2-yl hydrazine and 0.69% ID/g for the Tc-99m-G-ethyne-picolylamine. In imaging studies, the Hec50 tumor was visualized in a live animal for up to 6 hours after injecting the Tc-99m labeled agent. Conclusion We have synthesized and evaluated a series of first generation GPR30 targeted radioimaging agents. These GPR30-targeted agents exhibit promising biodistribution characteristics and tumor uptake, and are important lead compounds for ongoing efforts to optimize the GPR30 targeting and imaging characteristics for diagnostic applications.
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estrogen signaling characteristics of atlantic croaker g protein coupled receptor 30 GPR30 and evidence it is involved in maintenance of oocyte meiotic arrest
Endocrinology, 2008Co-Authors: Yefei Pang, Jing Dong, Peter ThomasAbstract:Human G protein-coupled receptor 30 (GPR30) mediates estradiol-17β (E2) activation of adenylyl cyclase in breast cancer cells and displays E2 binding typical of membrane estrogen receptors (mERs). We identified a mER in Atlantic croaker ovaries with characteristics similar to those of human GPR30. To confirm the proposed role of GPR30 as a mER in this distantly related vertebrate group, we cloned GPR30 from croaker ovaries and examined its distribution, steroid binding, and signaling characteristics. Western blot analysis showed the GPR30 protein (∼40 kDa) is expressed on the plasma membranes of croaker oocytes and HEK293 cells stably transfected with GPR30 cDNA. Plasma membranes prepared from croaker GPR30-transfected cells displayed high-affinity, limited-capacity, and displaceable binding specific for estrogens, characteristic of mERs. Consistent with previous findings with human GPR30, estrogen treatment of plasma membranes from both croaker ovaries and GPR30-transfected cells caused activation of a stimulatory G protein (Gs) resulting in increased cAMP production. Treatment with E2 as well as G-1, a specific GPR30 ligand, significantly reduced both spontaneous and progestin-induced maturation of both croaker and zebrafish oocytes in vitro, suggesting a possible involvement of GPR30 in maintaining oocyte meiotic arrest in these species. Injection of antisense oligonucleotides to GPR30 into zebrafish oocytes blocked the inhibitory effects of estrogen on oocyte maturation, confirming a role for GPR30 in the control of meiotic arrest. These findings further support our previous suggestion that GPR30 is a vertebrate mER. In addition, the results suggest GRP30 may play a critical role in regulating reentry into the meiotic cell cycle in fish oocytes.
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activation of the novel estrogen receptor g protein coupled receptor 30 GPR30 at the plasma membrane
Endocrinology, 2007Co-Authors: Edward J Filardo, Curt Graeber, S Shaw, Yefei Pang, Jeffrey A Quinn, Jing Dong, Peter ThomasAbstract:G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17β-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments...
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identity of an estrogen membrane receptor coupled to a g protein in human breast cancer cells
Endocrinology, 2005Co-Authors: Peter Thomas, Yefei Pang, Edward J Filardo, Jing DongAbstract:Although nonclassical estrogen actions initiated at the cell surface have been described in many tissues, the identities of the membrane estrogen receptors (mERs) mediating these actions remain unclear. Here we show that GPR30, an orphan receptor unrelated to nuclear estrogen receptors, has all the binding and signaling characteristics of a mER. A high-affinity (dissociation constant 2.7 nm), limited capacity, displaceable, single binding site specific for estrogens was detected in plasma membranes of SKBR3 breast cancer cells that express GPR30 but lack nuclear estrogen receptors. Progesterone-induced increases and small interfering RNA-induced decreases in GPR30 expression in SKBR3 cells were accompanied by parallel changes in specific estradiol-17β (E2) binding. Plasma membranes of human embryonic kidney 293 cells transfected with GPR30, but not those of untransfected cells, and human placental tissues that express GPR30 also displayed high-affinity, specific estrogen binding typical of mERs. E2 treatm...
