MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells

Abstract

IntroductionThe multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells.MethodsMolecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells.ResultsMIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments.ConclusionsOur findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor progression. Hence, the simultaneous inhibition of both ERα and GPER may guarantee major therapeutic benefits in respect to the use of a selective estrogen receptor antagonist.

International Union of Basic and Clinical Pharmacology. XCVII. G Protein–Coupled Estrogen Receptor and Its Pharmacologic Modulators

“…3 H]E2 in ER-negative GPER-expressing whole cells (Teng et al, 2008;Lappano et al, 2010Lappano et al, , 2012bChimento et al, 2014a). In one example, GPERexpressing SKBR3 cells were incubated with [ 3 H]E2 in the presence or absence of competing ligands for 2 hours at 37°C.…”

“…In one example, GPERexpressing SKBR3 cells were incubated with [ 3 H]E2 in the presence or absence of competing ligands for 2 hours at 37°C. The cells were washed with ice-cold phosphate-buffered saline, extracted with 100% ethanol and the radioactivity of the extracts measured by liquid scintillation counting, with competitor binding expressed as a percentage of maximal specific binding (Lappano et al, 2012b). Alternatively, trichloroacetic acid-based protein precipitation followed by solubilization in 0.1 N NaOH was employed (Chimento et al, 2014a).…”

Macromolecular Modelling and Docking Simulations for the Discovery of Selective GPER Ligands

“…To date, only a molecule exhibited inhibitory properties toward both GPER and ER. This agent named MIBE (30), which was tested in silico using as target ERα, displayed a binding mode similar to that adopted by the ER antagonist OHT in the crystal structure (47) (PDB code 3ERT). When GPER was used as the protein target, MIBE adopted a binding mode which differs from that adopted by G-1 (Fig.…”

“…Using the aforementioned approaches, several different natural and synthetic ligands of GPER (Fig. 1) have been identified along with their binding modes as resulting from docking simulations (13,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).…”

GPER Mediates Activation of HIF1α/VEGF Signaling by Estrogens

“…In addition, the G protein-coupled receptor (GPR)30/GPER has been shown to mediate estrogenic signaling in different normal and malignant cell contexts, including breast cancer (3)(4)(5)(6)(7). In this regard, the identification of selective GPER agonists or antagonists (8)(9)(10)(11)(12) has allowed the evaluation of certain biological responses elicited through GPER. Actually, GPER activates a network of transduction pathways involving the epidermal growth factor receptor (EGFR), the intracellular cyclic AMP (cAMP), the mitogen-activated protein kinases (MAPK) cascade, and calcium mobilization (13)(14)(15).…”