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

Introduction The 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. Methods Molecular 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. Results MIBE 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. Conclusions Our 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.


Introduction
Estrogens regulate many aspects of human physiology and influence diverse pathological processes, including the development of hormone-dependent tumors [1]. The biological actions of estrogens are mainly mediated by the estrogen receptor (ER)α and ERβ, which belong to the nuclear receptor superfamily [1]. Acting as ligand-activated transcription factors, ERs regulate gene expression by binding to responsive elements (ERE) located within the promoter region of estrogen target genes [1]. In addition, gene regulation can occur in response to estrogens through plasma membrane receptors, such as growth factor receptors or G proteincoupled receptors, and by protein kinase signaling cascades [2].
Prolonged exposure to estrogens represents a major risk factor for the progression of breast cancer [3], which expresses elevated levels of ERα in approximately 70% of cases [4]. Consequently, ERα antagonists like tamoxifen and raloxifene are currently used as frontline pharmacological interventions in ERα-positive breast cancer in order to inhibit the mitogenic stimulation of estrogens [5]. Although there is general concordance between ERα expression and responsiveness to ER-targeted agents, as indicated by a greater five-year disease-free survival for ERα-positive patients receiving tamoxifen, one in four patients does not respond to treatment from the onset and in most patients tamoxifen produces agonist effects after a few years [6].
In order to further characterize the molecular mechanisms involved in the action of estrogens, recent studies have demonstrated that the G protein-coupled receptor, named GPR30/GPER, mediates rapid biological responses to estrogens in diverse normal, as well as transformed, cell types [7]. The potential role of GPER in cancer was supported by numerous investigations performed in different tumor cells, including breast [8][9][10], endometrial [11], ovarian [12], thyroid [13], prostate [14] and testicular germ cells [15]. In accordance with these findings, GPER has been associated with aggressive features of breast cancer [16], high-grade endometrial tumors [17] and poor prognosis in ovarian cancer [18]. Since its identification to date, the transduction signaling and gene expression profile triggered by GPER have been extensively characterized. The early discovery [8] of a transmembrane receptor able to mediate estrogen responsiveness in ER-negative breast cancer cells was then confirmed by several reports by which GPER was considered as a genuine ER [10,19]. Indeed, a whole series of intracellular events, such as the rapid phosphorylation of mitogen-activated protein kinases (MAPK) ERK1/2, the activation of PI3-kinase (PI3K) and phospholipase C (PLC), the increase in cAMP concentrations and the intracellular calcium mobilization, was shown to follow GPER activation by both estrogens and anti-estrogens [20]. In particular, it was demonstrated that GPER-dependent ERK activation occurs via the transactivation of the epidermal growth factor receptor (EGFR) through matrix metalloproteinase activity and integrin α5β1, which trigger the extracellular release of heparan-bound epidermal growth factor (HB-EGF) [8,21]. Interestingly, a physical and functional cross-talk between GPER and EGFR contributes to the intricate signaling network involved in the stimulation of hormone-sensitive tumors [22,23].
The rapid responses to estrogenic signals mediated by GPER regulate a typical gene signature, as revealed in previous studies, including a microarray analysis [7,24]. Of note, GPER target genes were shown to contribute to the proliferation and migration in diverse cancer cell types [9,[11][12][13]22,24,25] as well as in cancer associated fibroblasts (CAFs) [26].
GPER exhibits many of the expected characteristics of an estrogen receptor, including the capability to bind to estrogens, phyto-and xenoestrogens and even the ER antagonists 4-hydroxytamoxifen (OHT) and fulvestrant (ICI 182 780) [10,19,27,28]. Surprisingly, unlike the antagonistic properties displayed by these anti-estrogens with respect to the classical ERs, both compounds act as GPER agonists [8,11,19,24]. Conversely, the well known ER agonist estriol exerts inhibitory effects on GPERmediated signaling [28], confirming the potential opposite functions elicited by estrogenic/anti-estrogenic agents through each type of estrogen receptor. In addition to the selective GPER agonist G-1 [29], GPER ligands showing antagonistic properties have been identified [30,31]. Recently, a GPER antagonist showed at high concentrations limited binding properties and stimulatory activity on ER-mediated transcription [30]. The use of these compounds has greatly advanced our understanding of the role of GPER in numerous biological systems as well as in cancer.
On the basis of the aforementioned findings, GPER may be considered as an additional therapeutic target in estrogen-sensitive tumors, such as breast cancer. In this regard, the opposite functional activity elicited by antiestrogens through the classical ERs and GPER as stated above, could represent a therapeutic concern toward the pharmacological inhibition of all types of estrogen receptor.
We discovered a novel compound, ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl] but-2-enoate (referred to as MIBE) (Figure 1), which displays the unique property to bind to and inhibit GPER-and ERα-mediated signaling in breast cancer cells. The antagonistic action exerted by MIBE on both estrogen receptor types could represent a novel, promising tool for a more comprehensive pharmacological approach to estrogen-dependent tumors such as breast cancer.

Molecular modelling and docking simulations
For docking simulations we used as targets the crystallographic coordinates of ERα in complex with E2 (closed-conformation) as well as with OHT (open conformation) and a GPER molecular model built by homology as described elsewhere (PDB code 1G50; PDB code 3ERT) [28,32,33]. Docking studies were performed by GOLD 5.0.1 (the Cambridge Crystallographic Data Center, UK), a program using a genetic algorithm useful to investigate the full range of ligand conformational flexibility and a partial protein side chain flexibility. As active sites of ERα, we identified those atoms that are within 20 Å distance from each atom of the ligand experimental position. Regarding GPER, we identified the O atom of Phe 208 as the protein active site centre on the basis of our previous docking simulations [28]. In this case, the active site atoms were considered those located within 20 Å from the centre. For each structure, 10 docking solutions were generated allowing an early termination of the process, if the respective RMSDs of the three highest ranked docking solutions were within 1.5 Å of each other. The default GOLD settings were used for running the simulations. ERα protein side chains Met342, Glu353, Trp383, Met388, Arg394, Phe404, His524 and Leu525 were considered as flexible, while in the GPER model the residues Tyr123, Gln138, Phe206, Phe208, Glu275, Phe278 and His282 were defined flexible side chains allowing their free rotation. The molecular structures of the ligands screened in silico were built and energy minimized with the programs Insight II and Discover3 (Biosym/MSI, San Diego, CA, USA). All the figures were drawn with the program Chimera (UCSF, San Francisco, CA, USA) [34].
Procedure for the preparation of MIBE was as follows. Indium (III) chloride (10 mol%) was added under nitrogen to a mixture of 5-hydroxy-1-methyl-1H-indole and ethyl acetoacetate. The reaction mixture was heated under reflux for two hours, and then it was left to cool to room temperature. Ice water was added and then the reaction mixture was extracted by ethyl acetate. The organic layers were collected and washed with brine, dried over MgSO 4   Cell culture MCF7 breast cancer cells and human embryonal kidney Hek293 cells were maintained in DMEM with phenol red supplemented with 10% FBS. SkBr3 breast cancer cells were maintained in RPMI 1640 without phenol red supplemented with 10% FBS. All cell lines to be processed for immunoblot and RT-PCR assays were switched to medium without serum and phenol red the day before treatments. The experiments performed in this study do not require Institute Ethics Board approval, because only commercially available cell lines were used.

Transfection, Luciferase assays and gene silencing experiments
Cells were plated into 24-well plates with 500 μl of regular growth medium/well the day before transfection. Cell medium was replaced with medium supplemented with 1% charcoal-stripped (CS) FBS lacking phenol red and serum on the day of transfection, which was performed using the Fugene 6 Reagent as recommended by the manufacturer (Roche Diagnostics, Milan, Italy) with a mixture containing 0.5 μg of reporter plasmid, 2 ng of pRL-TK, 0.1 μg of effector plasmid and 0.1 μg of full length AR expression plasmid where applicable. After 6 h, the medium was replaced again with serum-free medium lacking phenol red and supplemented with 1% CS-FBS, treatments were added at this point and cells were incubated for an additional 18 h. Luciferase activity was then measured using the Dual Luciferase Kit (Promega, Milan, Italy) according to the manufacturer's recommendations. Firefly luciferase activity was normalized to the internal transfection control provided by the Renilla luciferase activity. The normalized relative light unit values obtained from cells treated with vehicle were set as one-fold induction upon which the activity induced by treatments was calculated.
For the gene silencing experiments, cells were plated into 10-cm dishes, maintained in serum-free medium for 24 h and then transfected for an additional 48 h before treatments using Fugene 6 (according to the manufacturer's recommendations) and control vector (shRNA) or shGPER.
In ligand binding assay for GPER, SkBr3 cells were grown in 10-cm cell culture dishes, washed two times and incubated with 1 nM [2,4,6,7-3H]E2 (89 Ci/mmol; Ge Healthcare, Milan, Italy) in the presence or absence of an increasing concentration of nonlabeled competitors (E2, G-1, OHT and MIBE). Then, cells were incubated for two hours at 37°C and washed three times with ice-cold PBS; the radioactivity collected by 100% ethanol extraction was measured by liquid scintillation counting. Competitor binding was expressed as a percentage of maximal specific binding. Each point is the mean of three observations.

Western blotting
Cells were grown in 10-cm dishes, exposed to ligands, and then lysed in 500 μL of 50 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10% glycerol, 1% Triton X-100, 1% sodium dodecyl sulfate (SDS), and a mixture of protease inhibitors containing 1 mmol/L aprotinin, 20 mmol/L phenylmethylsulfonyl fluoride and 200 mmol/L sodium orthovanadate. Protein concentration was determined using Bradford reagent according to the manufacturer's recommendations (Sigma-Aldrich, Milan, Italy). Equal amounts of whole protein extract were resolved on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (GE Healthcare, Milan, Italy), probed overnight at 4°C with antibodies against Cyclin

Proliferation assay
For quantitative proliferation assay, cells (1 × 10 5 ) were seeded in 24-well plates in regular growth medium. Cells were washed once they had attached and then were incubated in medium containing 2.5% charcoalstripped FBS with the indicated treatments; medium was renewed every two days (with treatments) before counting, using the Countess Automated Cell Counter, as recommended by the manufacturer's protocol (Invitrogen S.r.l., Milan, Italy).

Statistical analysis
Statistical analysis was done using ANOVA followed by Newman-Keuls' testing to determine differences in means. P < 0.05 was considered as statistically significant.

Molecular modeling and binding assays demonstrate that MIBE is a ligand of both ERa and GPER
On the basis of the results obtained in docking simulations as described in the Materials and methods section, we evaluated the affinity of MIBE for the ligand binding pockets of both ERα and GPER with respect to E2 and G-1, respectively ( Figure 2). Docking E2 to the hormone binding pocket of a closed conformation of ERα ( Figure  2a), we observed a binding mode similar to that reported in the experimental crystallographic complex (superposition of the solution provided by GOLD to the crystallographic structure led to a RMSD of 0.092Å) [32]. Docking MIBE to the same pocket using ERα in both the closed and open conformation, we evidenced a better affinity for the last conformation ( Figure 2b) and a binding mode similar to that adopted by the ER antagonist OHT in the crystallographic structure (PDB code 3ERT) [33].
As it concerns the GPER ligand binding pocket, visual inspection showed that it lies within a deep cleft in where 10 hydrophobic residues (V116, Met133, Leu137, Phe206, Phe208, Phe 278, Ile279, Ile308, Val309 and Phe314) and 5 polar amino acids (Tyr123, Gln138, Asp210, Glu275 and His282) contribute to stabilize the ligands through Van der Waals interactions and hydrogen bonds, respectively. Using GPER as a target, docking simulations confirmed a good affinity of the protein for the agonist G-1 (Figure 2c) as previously demonstrated both in silico and in vitro [29]. Next, we docked MIBE to GPER using the same settings and parameters as for G-1. MIBE, which was positioned within the GPER binding site (Figure 2d), displayed a high affinity for GPER, even better than that exhibited by G-1. In particular, MIBE binds to GPER forming hydrogen bonds with the hydroxyl groups located on its branched arms, on one side with Y123 OH, on the other with Q215 NE2 and H282 ND1 atoms. MIBE is also stabilized in the protein binding pocket by Van der Waals interactions of its methyl groups with residues F208, I279, T305 and I308, while a π-π stacking interaction is formed by the aromatic rings of F208 and the indole ring of MIBE. Starting from the aforementioned observations, we performed diverse assays to fully evaluate the ligand binding properties and the potential agonist/antagonist activity of MIBE exerted through ERα and GPER.
In order to confirm whether MIBE is a ligand of ERα, we performed competitive binding experiments by using the recombinant ERα protein. MIBE displaced the radiolabeled E2 in a dose-dependent manner (Figure 3a) indubitably demonstrating its capability to bind to ERα in a direct fashion, although with a lower binding affinity in respect to E2 and OHT as 10 μM MIBE induced approximately 40% displacement of [3H]E2. On the basis of the ability of MIBE to interact with GPER in docking simulations, we also performed ligand binding studies using radiolabeled E2 as a tracer in ER-negative but GPER-positive SkBr3 breast cancer cells, as previously reported [28]. Hence, we performed binding experiments using cold E2, MIBE, the selective GPER ligand G-1 and OHT, which has been largely reported to act as a GPER agonist [7]. Interestingly, MIBE showed the capability to displace [3H]E2 (Figure 3b) in accordance with the results obtained in docking simulations. E2, G-1 and OHT confirmed the ability to compete with [3H]E2 as previously shown [28]. Collectively, our findings demonstrate that MIBE is a ligand of both ERα and GPER.

MIBE inhibits both ER transactivation and gene expression induced by E2
On the basis of these results, we aimed to ascertain whether MIBE could function as an agonist or antagonist for ERα and GPER. Initially, we evaluated the potential of MIBE in activating or inhibiting the ERα-mediated signaling. Hence, we transiently transfected an ER-reported gene in MCF7 breast cancer cells, which express ERα but not ERβ as judged by RT-PCR (data not shown). The reporter plasmid used carries firefly luciferase sequences under the control of an ERE upstream of the thymidine kinase promoter. As an internal transfection control, we co-transfected a plasmid expressing renilla luciferase which is enzymatically distinguishable from firefly luciferase by the strong cytomegalovirus enhancer/promoter. MIBE did not show any capability to transactivate ERα; however, it abrogated the luciferase activity induced by E2 like the ER antagonist OHT (Figure 4a, b). To confirm these data and to examine the response of ERβ, we transiently transfected the ER-negative Hek293 cells with chimeric proteins consisting of the DNA binding domain (DBD) of the yeast transcription factor Gal4 and the ligand binding domain (LBD) of ERα (GalERα) or ERβ (GalERβ), respectively. MIBE did not activate GalERα and GalERβ (Figure 4c, d), but prevented the transactivation of these chimeric proteins by E2 mimicking the inhibitory activity of OHT (Figure 4e, f). In order to evaluate whether MIBE acts through a further member of the steroid receptor superfamily as the AR, we transiently transfected the ER-negative Hek293 cells with an AR reporter gene along with the expression vector encoding AR. DHT transactivated the AR reporter gene, whereas MIBE neither activated AR nor prevented the DHT-induced activation of AR (Additional file 1). Together, these results provide evidence regarding the specific action of MIBE on ERmediated signaling.
In order to further demonstrate that MIBE acts as an ERα antagonist, we evaluated its ability to repress in MCF7 cells the mRNA expression of well known E2 target genes like pS2, Cyclin D1, PR and IRS-1. As determined by real-time PCR, the E2-dependent increase of all genes examined was prevented by MIBE as obtained using OHT (Figure 5a). Similarly, the protein expression of cyclin D1 and IRS-1 induced by E2 in MCF7 cells was inhibited by MIBE (and OHT) (Figure 5b, c).

MIBE prevents the proliferative effects triggered by E2
Considering that the regulation of estrogen target genes connects the signaling of E2 with the proliferation of breast cancer cells [40,41], we wanted to determine the biological significance of the antagonist action elicited by MIBE through ERα. MIBE as OHT did not stimulate growth effects used alone ( Figure  5d); however, both compounds abolished the proliferation of MCF7 cells induced by E2 (Figure 5e). Hence, MIBE can be considered as an ER antagonist on the basis of its full inhibitory activity elicited on ERmediated signaling.

MIBE prevents the GPER-mediated EGFR and ERK activation
Having established that MIBE is an inhibitor of ERα, we aimed to determine its functional activity on the GPERmediated transduction pathway. Previous studies have indicated that GPER activation triggers the EGFRdependent signaling in cancer cells, even involving a functional cross-talk between these receptors [8,9,23]. Then, we sought to evaluate the role played by GPER in EGFR phosphorylation upon exposure to its cognate ligand. Notably, in SkBr3 cells the EGFR activation induced by EGF was prevented by knocking down GPER expression (Figure 6a-d) as observed in the presence of MIBE (Figure 6e, f), which further demonstrated that it acts as an inhibitor of GPER-mediated function. Accordingly, the activation of EGFR triggered by G-1 was abolished in the presence of MIBE, hence confirming its inhibitory activity on GPER-mediated signaling (Additional file 2). Corroborating the aforementioned findings, MIBE showed the capability to inhibit the ERK activation upon EGF exposure (Figure 6g, h) as well as by the GPER activators E2, G-1 and OHT (Figure 6i-l). Overall, these results suggest that MIBE acting as an inhibitor of GPER blocks the EGFR activation and the ERK phosphorylation induced by EGF and the ligands of GPER, thus preventing the functional crosstalk between GPER and EGFR.

MIBE inhibits gene transcription and cell proliferation mediated by GPER
The characterization of the transcriptional response to GPER signaling has recently identified a set of target genes that mediate the stimulatory effects triggered by GPER activation in cancer cells [24]. Hence, we performed real-time PCR experiments to evaluate the potential of MIBE in regulating the expression of GPERdependent genes. Of note, the up-regulation of c-fos, CTGF, Cyr61 and EGR1 induced by the GPER agonists E2, G-1 and OHT in SKBr3 cells was abolished in the presence of MIBE (Figure 7a). In accordance with these results, MIBE also prevented the increase of both c-fos and CTGF at the protein level (Figure 7b, c). Next, we wondered what might be the biological significance of the inhibitory action of MIBE through GPER signaling. As shown in panel d of Figure 7, the proliferative effects elicited by E2, G-1 and OHT in SKBr3 cells were inhibited by MIBE. Altogether, these findings demonstrate that MIBE acts as an antagonist of both ERα and GPER in breast cancer cells.

Discussion
In the present study, we identified the first ligand of ERα and GPER, referred to as MIBE, which acts as an  antagonist of both receptors in breast cancer cells. By molecular modeling and binding experiments we demonstrated that MIBE binds to both receptors, while through functional assays we showed that MIBE inhibits the ERα-and GPER-mediated signaling. In particular, using the ER-positive MCF7 and ER-negative SkBr3 breast cancer cells as a model system, we characterized the biological properties of MIBE. We found that in MCF7 cells MIBE blocks the ER  transactivation induced by E2 as well as the ERmediated gene regulation and cell proliferation. In addition, in SkBr3 cells MIBE prevented the GPERdependent responses, such as rapid ERK phosphorylation, gene transcription and growth effects induced by the GPER agonists E2, OHT and G-1. The exclusive antagonistic action exerted by MIBE on both ERα and GPER could represent a novel promising tool for a more comprehensive pharmacological approach in estrogen-dependent tumors like breast cancer, which express one or both receptors from the onset or following tumor progression.
Breast cancer is the most commonly diagnosed invasive malignancy and the second leading cause of cancer death in women [42]. Endocrine treatment along with surgery, chemotherapy, radiotherapy and targeted therapy are fundamental modalities for the therapeutic management of breast cancer. The expression of ERα in breast carcinomas correlates with the beneficial response to anti-estrogens [43], whereas the lacking of ERα is coupled to a worse prognosis and to short disease-free survival rates [44]. On the basis of the main role exerted by ERα in the development and progression of breast cancer and considering that this receptor is expressed in approximately 70% of breast tumors, the ER antagonist tamoxifen has been widely used, although its effectiveness is limited by de novo and acquired resistance [45]. In accordance with these data, comparative clinical studies have indicated that aromatase inhibitors blocking estrogen biosynthesis may provide major benefits in respect to ERα antagonists in breast cancer patients [46]. Among the various mechanisms involved in the resistance to endocrine treatment, the activation of transduction pathways different from those mediated by ERα has been proposed. For instance, an increased expression and/or activation of growth factor receptors, such as EGFR/HER2, have been associated with the failure of endocrine therapy in breast tumors [47]. Moreover, the existence of alternative ERs able to mediate estrogen signaling without exhibiting any sensitivity to the repressive action of the ER antagonists could be also involved in the resistance to endocrine agents. In this scenario, it has been recently demonstrated that GPER acts as an additional receptor mediating the effects of estrogens in a wide number of cell types, such as breast, endometrial and ovarian cancer cells [7]. Of note, diverse studies have shown that E2 as well as the antiestrogens tamoxifen and ICI bind to and activate GPER signaling, including ERK phosphorylation and gene transcription, which in turn lead to cancer cell proliferation and migration [7].
The activation of the GPER transduction pathway requires the EGFR transactivation [8], in accordance with evidence showing that the agonist stimulation of diverse G-protein coupled receptors (GPCRs) triggers the transactivation of EGFR through the release of EGFlike ligands tethered at the cell surface and the subsequent generation of intracellular signaling [48]. In addition, the functional crosstalk which occurs between members of GPCR and growth factor receptor families contributes to the progression of different tumors [8,48]. In this regard, we have previously reported that GPER and EGFR physically and functionally interact in both ER-negative and ER-positive cancer cells [22,23].
Recently, it has also been found that a crosstalk among EGFR, the nerve growth factor (NGF) receptor TrkA and the GPCR Formyl Peptide Receptor (FPR) occurs in monocytes [49]. In particular, the inhibition of EGFR prevented the ligand-dependent responses mediated by the other two receptors, while the inhibition of FPR abolished the EGFR and TrkA phosphorylation induced by EGF and NGF, respectively. Accordingly, the silencing of each receptor suppressed the capability of the other receptors to mediate the ligand-induced actions like ERK phosphorylation [49]. In line with these findings, our current results provide novel insight into the functional crosstalk between GPER and EGFR in cancer cells. Notably, we show for the first time that the activation of EGFR induced by its cognate ligand EGF is abolished by knocking down GPER expression or in the presence of MIBE, which is an inhibitor of GPER as ascertained in the present study. Nevertheless, further studies are needed to better understand the role played by GPER in the activation of EGFR by its cognate ligand EGF and to appreciate the potential of MIBE in preventing the crosstalk between GPER and EGFR which was previously well described [23].
On the basis of these remarks, it remains to be evaluated that the potential of MIBE to interfere with the functional crosstalk between EGFR and ERα, toward a better characterization of its inhibitory activity elicited in cell contexts expressing both receptors. In particular, considering that a physical and functional interaction between EGFR and ER leads to the activation of multiple intracellular cascades, including MAPK, phosphoinositide 3-kinase (PI3K) and other protein kinases [50][51][52][53], it would be interesting to ascertain whether MIBE could alter these transduction signals that have been involved in the proliferation of cancer cells [50,[54][55][56][57][58].
In 2005, two reports provided evidence on the capability of estrogens and anti-estrogens to bind to GPER [10,19]. In particular, the ER antagonists tamoxifen and ICI displayed a high binding affinity for GPER, as assessed in competition assays. Surprisingly, unlike the antagonistic properties exhibited by these agents on the classical ER-mediated pathways, both tamoxifen and ICI act as GPER agonists [8,9,19]. In the following years, further ER ligands and activators showed the ability to bind to GPER eliciting promiscuous actions through the two receptors. For instance, the phytoestrogen genistein and the xenoestrogen bisphenol A, which exert estrogen-like activities binding to and activating ERα [9,59], displayed the ability to bind to and activate GPER signaling [9,27,60]. As it concerns the pesticide atrazine, it exerted estrogenic effects without binding to ERs [61] and exhibiting the capability to activate the GPERmediated pathway despite a low binding affinity for this receptor [25,27]. Unlike E2 which exhibited ERα and GPER agonism in several investigations [7], the well known ERα ligand and activator estriol showed antagonistic properties for GPER-mediated signaling [28]. Besides, G-1 [29] and G-15, along with its derivatives [30,31] as ligands activated or inhibited, respectively, the GPER-mediated signaling, while some GPER antagonists triggered at high concentrations ER-dependent transcriptional responses [30].
GPER expression was indicated as a potential predictor of biological aggressive features in breast carcinomas [16]. Although a significant association between ERα and GPER was observed, approximately 50% of ERαnegative breast tumors retained GPER suggesting that the expression of these receptors may not be interdependent [16]. On the basis of these and the aforementioned findings, tumor cells that express GPER but lack ERα may be stimulated by estrogens and even by antiestrogens, such as tamoxifen. In this regard, it should be noted that the stimulatory effects on cancer progression elicited by estrogens via both ERα and GPER and by ERα antagonists through GPER address the need to discover novel drugs targeting simultaneously both receptors, in order to obtain major therapeutic benefits in respect to the use of the current selective antagonists.

Conclusions
The exclusive antagonistic activity exerted by MIBE on ERα-and GPER-mediated signaling as shown in the present study (Figure 8), could represent a promising pharmacological approach either at the beginning or during the progression of breast tumors which express one or both receptors. In this respect, further studies are needed to examine whether MIBE could be considered a useful tool towards a more comprehensive treatment in breast cancer.

Additional material
Additional file 1: MIBE does not activate AR. Hek293 cells were transfected with AR luciferase reporter gene (ARE-luc) and AR expression plasmid along with the internal transfection control Renilla Luciferase, and treated with 10 nM DHT alone and in combination with 10 μM MIBE, as indicated. The normalized luciferase activities of cells treated with vehicle (-) were set as one-fold induction, upon which the activities induced by treatments were calculated. Each data point represents the mean ± SD of three experiments performed in triplicate.