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Estrogens have been implicated in the pathogenesis of various cancers, with increasing concern regarding the overall rising incidence of disease and exposure to environmental estrogens. Estrogens, both endogenous and environmental, manifest their actions through intracellular and plasma membrane receptors, named ERα, ERβ, and GPER. Collectively, they act to promote a broad transcriptional response that is mediated through multiple regulatory enhancers, including estrogen response elements (EREs), serum response elements (SREs), and cyclic AMP response elements (CREs). Yet, the design and rational assignment of antiestrogen therapy for breast cancer has strictly relied upon an endogenous estrogen-ER binary rubric that does not account for environmental estrogens or GPER. New endocrine therapies have focused on the development of drugs that degrade ER via ER complex destabilization or direct enzymatic ubiquitination. However, these new approaches do not broadly treat all cancer-involved receptors, including GPER. The latter is concerning since GPER is directly associated with tumor size, distant metastases, cancer stem cell activity, and endocrine resistance, indicating the importance of targeting this receptor to achieve a more complete therapeutic response. This review focuses on the critical importance and value of GPER-targeted therapeutics as part of a more holistic approach to the treatment of estrogen-driven malignancies.

Decisions regarding the assignment of hormonal therapy for breast cancer are based solely upon the presence of nuclear estrogen receptors (ERs) in biopsied tumor tissue. This is despite the fact that the G-protein-coupled estrogen receptor (GPER) is linked to advanced breast cancer and is required for breast cancer stem cell survival, an observation that suggests that effective endocrine therapy should also target this receptor. Here, two ER/GPER-targeting proteolytic chimeras (UI-EP001 and UI-EP002) are described that effectively degrade ERα, ERβ, and GPER. These chimeras form high-affinity interactions with GPER and ER with binding dissociation constants of ∼30 nM and 10-20 nM, respectively. Plasma membrane and intracellular GPER and nuclear ER were degraded by UI-EP001 and UI-EP002, but not by a partial proteolytic targeting chimera (PROTAC) lacking its estrogen-targeting domain. Pretreatment of cells with the proteasomal inhibitor, MG132, blocked UI-EP001 and UI-EP002 proteolysis, while the lysosomotrophic inhibitor, chloroquine, had no effect. The off-target activity was not observed against recombinant β1-adrenergic receptor or CXCR4. Target specificity was further demonstrated in human MCF-7 cells where both drugs effectively degraded ERα, ERβ, and GPER, sparing the progesterone receptor (PR). UI-EP001 and UI-EP002 induced cytotoxicity and G2/M cell cycle arrest in MCF-7 breast cancer and human SKBR3 (ERα-ERβ-GPER + ) breast cancer cells but not human MDA-MB-231 breast cancer cells that do not express functional GPER/ER. These results suggest that it is possible to develop a receptor-based strategy of antiestrogen treatment for breast cancer that targets both plasma membrane and intracellular estrogen receptors.

Estrogens exert their physiological and pathophysiological effects via cellular receptors, named ERα, ERβ, and G-protein coupled estrogen receptor (GPER). Estrogen-regulated physiology is tightly controlled by factors that regulate estrogen bioavailability and receptor sensitivity, while disruption of these control mechanisms can result in loss of reproductive function, cancer, cardiovascular and neurodegenerative disease, obesity, insulin resistance, endometriosis, and systemic lupus erythematosus. Restoration of estrogen physiology by modulating estrogen bioavailability or receptor activity is an effective approach for treating these pathological conditions. Therapeutic interventions that block estrogen action are employed effectively for the treatment of breast and prostate cancer as well as for precocious puberty and anovulatory infertility. Theoretically, treatments that block estrogen biosynthesis should prevent estrogen action at ERs and GPER, although drug resistance and ligand-independent receptor activation may still occur. In addition, blockade of estrogen biosynthesis does not prevent activation of estrogen receptors by naturally occurring or man-made exogenous estrogens. A more complicated scenario is provided by anti-estrogen drugs that antagonize ERs since these drugs function as GPER agonists. Based upon its association with metabolic dysregulation and advanced cancer, GPER represents a therapeutic target with promise for the treatment of several critical health concerns facing Western society. Selective ligands that specifically target GPER have been developed and may soon serve as pharmacological agents for treating human disease. Here, we review current forms of estrogen therapy and the implications that GPER holds for these therapies. We also discuss existing GPER targeted drugs, additional approaches towards developing GPER-targeted therapies and how these therapies may complement existing modalities of estrogen-targeted therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Rouhimoghadam, Lu, Salem and Filardo.)

GPER possesses structural and functional characteristics shared by members of the G-protein-coupled receptor (GPCR) superfamily, the largest class of plasma membrane receptors. This newly appreciated estrogen receptor is localized predominately within intracellular membranes in most, but not all, cell types and its surface expression is modulated by steroid hormones and during tissue injury. An intracellular staining pattern is not unique among GPCRs, which employ a diverse array of molecular mechanisms that restrict cell surface expression and effectively regulating receptor binding and activation. The finding that GPER displays an intracellular predisposition has created some confusion as the estrogen-inducible transcription factors, ERα and ERβ, also reside intracellularly, and has led to complex suggestions of receptor interaction. GPER undergoes constitutive retrograde trafficking from the plasma membrane to the endoplasmic reticulum and recent studies indicate its interaction with PDZ binding proteins that sort transmembrane receptors to synaptosomes and endosomes. Genetic targeting and selective ligand approaches as well as cell models that express GPER in the absence of ERs clearly supports GPER as a bonafide "stand alone" receptor. Here, the molecular details that regulate GPER action, its cell biological activities and its implicated roles in physiological and pathological processes are reviewed., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The G protein-coupled estrogen receptor-1, GPER-1, coordinates fibronectin (FN) matrix assembly and release of heparan-bound epidermal growth factor (HB-EGF). This mechanism of action results in the recruitment of FN-engaged integrin α5β1 to fibrillar adhesions and the formation of integrin α5β1-Shc adaptor protein complexes. Here, we show that GPER-1 stimulation of murine 4 T1 or human SKBR3 breast cancer cells with 17β-estradiol (E2β) promotes the formation of focal adhesions and actin stress fibers and results in increased cellular adhesion and haptotaxis on FN, but not collagen. These actions are also induced by the xenoestrogen, bisphenol A, and the estrogen receptor (ER) antagonist, ICI 182, 780, but not the inactive stereoisomer, 17α-estradiol (E2α). In addition, we show that GPER-1 stimulation of breast cancer cells allows for FN-dependent, anchorage-independent growth and FN fibril formation in "hanging drop" assays, indicating that these GPER-1-mediated actions occur independently of adhesion to solid substrata. Stable expression of Shc mutant Y317F lacking its primary tyrosyl phosphorylation site disrupts E2β-induced focal adhesion and actin stress fiber formation and abolishes E2β-enhanced haptotaxis on FN and anchorage-dependent growth. Collectively, these data demonstrate that E2β action via GPER-1 enhances cellular adhesivity and FN matrix assembly and allows for anchorage-independent growth, cellular events that may allow for cellular survival, and tumor progression.

Estrogens rapidly affect dopamine (DA) neurotransmission in the dorsal striatum (dSTR) and DA-related diseases, such as Parkinson's disease and schizophrenia. How estrogens influence DA function remains unclear, in part, because the ultrastructural localization of estrogen receptors (ER) in the dSTR is not known. Light microscopic studies of the dSTR have suggested the presence of ER. This experiment used electron microscopy to determine whether these ER are at extranuclear sites in the dSTR, providing evidence for a mechanism through which estrogen could rapidly affect DA transmission. The dSTR was labeled with antibodies for ERα, ERβ, and G protein-coupled ER 1 (GPER-1) to confirm whether these ER were present in this brain area. After this, the dSTR was dual labeled with antibodies for ERα or GPER-1 and tyrosine hydroxylase or vesicular acetylcholine transporter to determine whether ER are localized to dopaminergic and/or cholinergic processes, respectively. Ultrastructural analysis revealed immunoreactivity (IR) for ERα, ERβ, and GPER-1 exclusively at extranuclear sites throughout the dSTR. ERα-, ERβ-, and GPER-1-IR are mostly frequently observed in axons and glial profiles but are also localized to other neuronal profiles. Dual labeling revealed that ERα- and GPER-1-IR is not associated with DA axons and terminals but is sometimes associated with cholinergic neurons. Because these receptors are exclusively extranuclear in the dSTR, binding at these receptors likely affects neurotransmission via nongenomic mechanisms.

Using cDNA cloning strategies commonly employed for G protein-coupled receptors (GPCR), GPCR-30 (GPR30), was isolated from mammalian cells before knowledge of its cognate ligand. GPR30 is evolutionarily conserved throughout the vertebrates. A broad literature suggests that GPR30 is a Gs-coupled heptahelical transmembrane receptor that promotes specific binding of naturally occurring and man-made estrogens but not cortisol, progesterone, or testosterone. Its "pregenomic" signaling actions are manifested by plasma membrane-associated actions familiar to GPCR, namely, stimulation of adenylyl cyclase and Gβγ-subunit protein-dependent release of membrane-tethered heparan bound epidermal growth factor. These facts regarding its mechanism of action have led to the formal renaming of this receptor to its current functional designate, G protein-coupled estrogen receptor (ER) (GPER)-1. Further insight regarding its biochemical action and physiological functions in vertebrates is derived from receptor knockdown studies and the use of selective agonists/antagonists that discriminate GPER-1 from the nuclear steroid hormone receptors, ERα and ERβ. GPER-1-selective agents have linked GPER-1 to physiological and pathological events regulated by estrogen action, including, but not limited to, the central nervous, immune, renal, reproductive, and cardiovascular systems. Moreover, immunohistochemical studies have shown a positive association between GPER-1 expression and progression of female reproductive cancer, a relationship that is diametrically opposed from ER. Unlike ER knockout mice, GPER-1 knockout mice are fertile and show no overt reproductive anomalies. However, they do exhibit thymic atrophy, impaired glucose tolerance, and altered bone growth. Here, we discuss the role of GPER-1 in female reproductive cancers as well as renal and vascular physiology.

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases function as a common signaling conduit for membrane receptors that lack intrinsic enzymatic activity, such as G-protein coupled receptors and integrins. GPR30, an orphan member of the seven transmembrane receptor (7TMR) superfamily has been linked to specific estrogen binding, rapid estrogen-mediated activation of adenylyl cyclase and the release of membrane-tethered proHB-EGF. More recently, GPR30 expression in primary breast adenocarcinoma has been associated with pathological parameters commonly used to assess breast cancer progression, including the development of extramammary metastases. This newly appreciated mechanism of cross communication between estrogen and EGF is consistent with the observation that 7TMR-mediated transactivation of the EGFR is a recurrent signaling paradigm and may explain prior data reporting the EGF-like effects of estrogen. The molecular details surrounding GPR30-mediated release of proHB-EGF, the involvement of integrin beta1 as a signaling intermediary in estrogen-dependent EGFR action, and the possible implications of these data for breast cancer progression are discussed herein.

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<0.05). The coexpression of PR and ER did not influence GPR30 expression, yet coexpression of GPR30 and ER was linked to PR positivity. Unlike ER, which varied inversely with HER-2/neu and tumor size, GPR30 positively associated with HER-2/neu and tumor size. In addition, GPR30 showed a positive association with metastasis (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.

Heterotrimeric G proteins and seven-transmembrane-spanning (7TM) receptors are implicated in rapid estrogen signaling. The orphan 7TM receptor GPR30 is linked to estrogen-mediated activation of adenylyl cyclase, release of epidermal growth factor (EGF)-related ligands, and specific estrogen binding. GPR30 acts independently of estrogen receptors, ERalpha and ERbeta, and probably functions as a heptahelical ER. 7TM receptors elicit signals that stimulate second messengers, and convey intracellular signals via EGF receptors. Identification of GPR30 as a Gs-coupled 7TM receptor that triggers release of heparin-binding EGF establishes its role in cell signaling cascades initiated by estrogens, and explains their capacity to activate second messengers and promote EGF-like effects. Thus, estrogen can signal by the same mechanism as various other hormones, through a specific 7TM receptor.

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-17beta (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 treatment of transfected cell membranes caused activation of a stimulatory G protein that is directly coupled to the receptor, indicating GPR30 is a G protein-coupled receptor (GPCR), and also increased adenylyl cyclase activity. The finding that the antiestrogens tamoxifen and ICI 182,780, and an environmental estrogen, ortho,para-dichlorodiphenyldichloroethylene (o,p'-DDE), have high binding affinities to the receptor and mimic the actions of E2 has important implications for both the development and treatment of estrogen-dependent breast cancer. GPR30 is structurally unrelated to the recently discovered family of GPCR-like membrane progestin receptors. The identification of a second distinct class of GPCR-like steroid membrane receptors suggests a widespread role for GPCRs in nonclassical steroid hormone actions.