Marco Pupo

Bisphenol A Induces Gene Expression Changes and Proliferative Effects through GPER in Breast Cancer Cells and Cancer-Associated Fibroblasts

Background: Bisphenol A (BPA) is the principal constituent of baby bottles, reusable water bottles, metal cans, and plastic food containers. BPA exerts estrogen-like activity by interacting with the classical estrogen receptors (ERα and ERβ) and through the G protein-coupled receptor (GPR30/GPER). In this regard, recent studies have shown that GPER was involved in the proliferative effects induced by BPA in both normal and tumor cells. Objectives: We studied the transduction signaling pathways through which BPA influences cell proliferation and migration in human breast cancer cells and cancer-associated fibroblasts (CAFs). Methods and results: We used as a model system SKBR3 breast cancer cells and CAFs that lack the classical ERs. Specific pharmacological inhibitors and gene-silencing procedures were used to show that BPA induces the expression of the GPER target genes c-FOS, EGR-1, and CTGF through the GPER/EGFR/ERK transduction pathway in SKBR3 breast cancer cells and CAFs. Moreover, we observed that GPER is required for growth effects and migration stimulated by BPA in both cell types. Conclusions: Results indicate that GPER is involved in the biological action elicited by BPA in breast cancer cells and CAFs. Hence, GPER-mediated signaling should be included among the transduction mechanisms through which BPA may stimulate cancer progression.

G protein-coupled estrogen receptor mediates the up-regulation of fatty acid synthase induced by 17β-estradiol in cancer cells and cancer-associated fibroblasts.

Background: Fatty acid synthase (FASN) is a key lipogenic enzyme regulated by various factors, including estrogens. Results: GPER mediates FASN expression and activity induced by estrogens in cancer cells. Conclusion: Fatty acid biogenesis is regulated by estrogens through GPER. Significance: GPER may be included among the transduction mediators involved by estrogens in regulating FASN expression and activity. Activation of lipid metabolism is an early event in carcinogenesis and a central hallmark of many tumors. Fatty acid synthase (FASN) is a key lipogenic enzyme catalyzing the terminal steps in the de novo biogenesis of fatty acids. In cancer cells, FASN may act as a metabolic oncogene, given that it confers growth and survival advantages to these cells, whereas its inhibition effectively and selectively kills tumor cells. Hormones such as estrogens and growth factors contribute to the transcriptional regulation of FASN expression also through the activation of downstream signaling and a cross-talk among diverse transduction pathways. In this study, we demonstrate for the first time that 17β-estradiol (E2) and the selective GPER ligand G-1 regulate FASN expression and activity through the GPER-mediated signaling, which involved the EGF receptor/ERK/c-Fos/AP1 transduction pathway, as ascertained by using specific pharmacological inhibitors, performing gene-silencing experiments and ChIP assays in breast SkBr3, colorectal LoVo, hepatocarcinoma HepG2 cancer cells, and breast cancer-associated fibroblasts. In addition, the proliferative effects induced by E2 and G-1 in these cells involved FASN as the inhibitor of its activity, named cerulenin, abolished the growth response to both ligands. Our data suggest that GPER may be included among the transduction mediators involved by estrogens in regulating FASN expression and activity in cancer cells and cancer-associated fibroblasts that strongly contribute to cancer progression.

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

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.

The nuclear localization signal is required for nuclear GPER translocation and function in breast Cancer-Associated Fibroblasts (CAFs).

Cancer associated fibroblasts (CAFs) actively contribute to the growth and invasion of cancer cells. In recent years, the G protein estrogen receptor (GPER) has been largely involved in the estrogenic signals in diverse types of normal and tumor cells. In CAFs, GPER was localized into the nucleus, however the molecular mechanisms which regulate its nuclear shuttle remain to be clarified. In the present study, we demonstrate that in breast CAFs GPER translocates into the nucleus through an importin-dependent mechanism. Moreover, we show that a nuclear localization signal is involved in the nuclear import of GPER, in the up-regulation of its target genes c-fos and CTGF and in the migration of CAFs induced by estrogens. Our data provide novel insights into the nuclear localization and function of GPER in CAFs toward a better understanding of the estrogen action elicited through these key players of the tumor microenvironment.

Two Novel GPER Agonists Induce Gene Expression Changes and Growth Effects in Cancer Cells.

Although the action of estrogens has been traditionally explained by the binding to and transactivation of the nuclear estrogen receptor (ER)α and ERβ, recently the G protein-coupled receptor GPR30/GPER has been involved in the rapid estrogen signaling. We investigated the ability of two original molecules, which were named GPER-L1 and GPERL2, to bind to and activate the GPER transduction pathway in cancer cells. Competition assays, docking simulations, transfection experiments, real-time PCR, immunoblotting, gene silencing technology and growth assays were performed to ascertain the selective action of GPER-L1 and GPER-L2 in activating the GPER-mediated signaling. Both compounds, which did not show any ability to bind to and activate the classical ERs, were able to bind to GPER and to trigger the rapid activation of the GPER/EGFR/ERK transduction pathway which led to the up-regulation of GPER-target genes. Notably, GPER-L1 and GPER-L2 induced the proliferation of SkBr3 breast and Ishikawa endometrial cancer cells at nM concentrations through GPER, hence providing further evidence on their capability to elicit relevant biological responses mediated by GPER. The identification and characterization of these novel compounds as selective GPER agonists represent a valuable tool to further dissect the pharmacology of this novel estrogen receptor and to better differentiate the specific functions elicited by each estrogen receptor subtype in cancer cells.

GPER Mediates Cardiotropic Effects in Spontaneously Hypertensive Rat Hearts

Estrogens promote beneficial effects in the cardiovascular system mainly through the estrogen receptor (ER)α and ERβ, which act as ligand-gated transcription factors. Recently, the G protein-coupled estrogen receptor (GPER) has been implicated in the estrogenic signaling in diverse tissues, including the cardiovascular system. In this study, we demonstrate that left ventricles of male Spontaneously Hypertensive Rats (SHR) express higher levels of GPER compared to normotensive Wistar Kyoto (WKY) rats. In addition, we show that the selective GPER agonist G-1 induces negative inotropic and lusitropic effects to a higher extent in isolated and Langendorff perfused hearts of male SHR compared to WKY rats. These cardiotropic effects elicited by G-1 involved the GPER/eNOS transduction signaling, as determined by using the GPER antagonist G15 and the eNOS inhibitor L-NIO. Similarly, the G-1 induced activation of ERK1/2, AKT, GSK3β, c-Jun and eNOS was abrogated by G15, while L-NIO prevented only the eNOS phosphorylation. In hypoxic Langendorff perfused WKY rat heart preparations, we also found an increased expression of GPER along with that of the hypoxic mediator HIF-1α and the fibrotic marker CTGF. Interestingly, G15 and L-NIO prevented the ability of G-1 to down-regulate the expression of both HIF-1α and CTGF, which were found expressed to a higher extent in SHR compared to WKY rat hearts. Collectively, the present study provides novel data into the potential role played by GPER in hypertensive disease on the basis of its involvement in myocardial inotropism and lusitropism as well as the expression of the apoptotic HIF-1α and fibrotic CTGF factors. Hence, GPER may be considered as a useful target in the treatment of some cardiac dysfunctions associated with stressful conditions like the essential hypertension.

GPER activates Notch signaling in breast cancer cells and cancer-associated fibroblasts (CAFs)

The G protein-coupled receptor GPR30/GPER has been shown to mediate rapid effects of 17β-estradiol (E2) in diverse types of cancer cells. Here, we provide evidence for a novel crosstalk between GPER and the Notch signaling pathway in breast cancer cells and cancer-associated fibroblasts (CAFs). We show that E2 and the GPER selective ligand G-1 induce both the γ-secretase-dependent activation of Notch-1 and the expression of the Notch target gene Hes-1. These inductions are prevented by knocking down GPER or by using a dominant-negative mutant of the Notch transcriptional co-activator Master-mind like-1 (DN-MAML-1), hence suggesting the involvement of GPER in the Notch-dependent transcription. By performing chromatin-immunoprecipitation experiments and luciferase assays, we also demonstrate that E2 and G-1 induce the recruitment of the intracellular domain of Notch-1 (N1ICD) to the Hes-1 promoter and the transactivation of a Hes-1-reporter gene, respectively. Functionally, the E2 and G-1-induced migration of breast cancer cells and CAFs is abolished in presence of the γ-secretase inhibitor GSI or DN-MAML-1, which both inhibit the Notch signaling pathway. In addition, we demonstrate that E2 and G-1 prevent the expression of VE-Cadherin, while both compounds induce the expression of Snail, a Notch target gene acting as a repressor of cadherins expression. Notably, both GSI and DN-MAML-1 abolish the up-regulation of Snail-1 by E2 and G-1, whereas the use of GSI rescues VE-Cadherin expression. Taken together, our results prove the involvement of the Notch signaling pathway in mediating the effects of estrogenic GPER signaling in breast cancer cells and CAFs.

GPER signalling in both cancer-associated fibroblasts and breast cancer cells mediates a feedforward IL1β/IL1R1 response

Cancer-associated fibroblasts (CAFs) contribute to the malignant aggressiveness through secreted factors like IL1β, which may drive pro-tumorigenic inflammatory phenotypes mainly acting via the cognate receptor named IL1R1. Here, we demonstrate that signalling mediated by the G protein estrogen receptor (GPER) triggers IL1β and IL1R1 expression in CAFs and breast cancer cells, respectively. Thereby, ligand-activation of GPER generates a feedforward loop coupling IL1β induction by CAFs to IL1R1 expression by cancer cells, promoting the up-regulation of IL1β/IL1R1 target genes such as PTGES, COX2, RAGE and ABCG2. This regulatory interaction between the two cell types induces migration and invasive features in breast cancer cells including fibroblastoid cytoarchitecture and F-actin reorganization. A better understanding of the mechanisms involved in the regulation of pro-inflammatory cytokines by GPER-integrated estrogen signals may be useful to target these stroma-cancer interactions.

GPER Mediates Non-Genomic Effects of Estrogen

Estrogens are important modulators of a broad spectrum of physiological functions in humans. However, despite their beneficial actions, a number of lines of evidence correlate the sustained exposure to exogenous estrogen with increased risk of the onset of various cancers. Mainly these steroid hormones induce their effects by binding and activating estrogen receptors (ERα and ERβ). These receptors belong to the family of ligand-regulated transcription factors, and upon activation they regulate the expression of different target genes by binding directly to specific DNA sequences. On the other hand, in recent years it has become clear that the G protein-coupled estrogen receptor 30 (GPR30/GPER) is able to mediate non-genomic action of estrogens in different cell contexts. In particular, GPER has been shown to specifically bind estrogens, and in turn to functionally cross-react with diverse cell signaling systems such as the epidermal growth factor receptor (EGFR) pathway, the Notch signaling pathway and the mitogen-activated protein kinases (MAPK) pathway. In this chapter we will present some of the different experimental techniques currently used to demonstrate the functional role of GPER in mediating non-genomic actions of estrogens, such as the dual luciferase assay, assessment of the involvement of GPER in the stimulation of cell migration in breast cancer cell lines and in cancer-associated fibroblasts, and chromatin immunoprecipitation assay. Overall, the experimental procedures described herein represent key instruments for assessing the biological role of GPER in mediating non-genomic signals of estrogen.

A genetic polymorphism repurposes the G-protein coupled and membrane-associated estrogen receptor GPER to a transcription factor-like molecule promoting paracrine signaling between stroma and breast carcinoma cells

GPER is a membrane-associated estrogen receptor of the family of G-protein coupled receptors. For breast cancer, the contribution of GPER to promoting the proliferation and migration of both carcinoma cells and cancer-associated fibroblasts (CAFs) in response to estrogen and other agonists has extensively been investigated. Intriguingly, GPER was previously found to be localized to the nucleus in one isolate of breast CAFs. Moreover, this nuclear GPER was shown to bind regulatory sequences of cancer-relevant target genes and to induce their expression. We decided to find out what induces the nuclear localization of GPER, how general this phenomenon is, and what its functional significance is. We discovered that interfering with N-linked glycosylation of GPER, either by mutation of the predicted glycosylation sites or pharmacologically with tunicamycin, drives GPER into the nucleus. Surveying a small set of CAFs from breast cancer biopsies, we found that a relatively common single nucleotide polymorphism, which results in the expression of a GPER variant with the amino acid substitution P16L, is associated with the nuclear localization of GPER. GPER with P16L fails to be glycosylated, presumably because of a conformational effect on the nearby glycosylation sites. GPER P16L is defective for membrane-associated signaling, but instead acts like an estrogen-stimulated transcription factor. In CAFs, it induces the secretion of paracrine factors that promote the migration of carcinoma cells. This raises the possibility that the GPER P16L polymorphism could be a risk factor for breast cancer.