[Show abstract][Hide abstract] ABSTRACT: Estrogens influence multiple physiological processes and are implicated in many diseases as well. Cellular responses to estrogens are mainly mediated by the estrogen receptors (ER)α and ERβ, which act as ligand-activated transcription factors. Recently, a member of the G protein-coupled receptor (GPCR) superfamily, namely GPER/GPR30, has been identified as a further mediator of estrogen signalling in different pathophysiological conditions, including cancer. Today, computational methods are commonly used in all areas of health science research. Among these methods, virtual ligand screening has become an established technique for hit discovery and optimization. The absence of an established three-dimensional structure of GPER promoted studies of structure-based drug design in order to build reliable molecular models of this receptor. Here, we discuss the results obtained through the structure-based virtual ligand screening for GPER, which allowed the identification and synthesis of different selective agonist and antagonist moieties. These compounds led significant advances in our understanding of the GPER function at the cellular, tissue, and organismal levels. In particular, selective GPER ligands were critical toward the evaluation of the role elicited by this receptor in several pathophysiological conditions, including cancer. Considering that structure-based approaches are fundamental in drug discovery, future research breakthroughs with the aid of computer-aided molecular design and chemo-bioinformatics could generate a new class of drugs that, acting through GPER, would be useful in a variety of diseases as well as in innovative anticancer strategies.
The AAPS Journal 11/2015; DOI:10.1208/s12248-015-9844-3 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Copper promotes tumor angiogenesis, nevertheless the mechanisms involved remain to be fully understood. We have recently demonstrated that the G-protein estrogen receptor (GPER) cooperates with hypoxia inducible factor-1α (HIF-1α) toward the regulation of the pro-angiogenic factor VEGF. Here, we show that copper sulfate (CuSO4) induces the expression of HIF-1α as well as GPER and VEGF in breast and hepatic cancer cells through the activation of the EGFR/ERK/c-fos transduction pathway. Worthy, the copper chelating agent TEPA and the ROS scavenger NAC prevented the aforementioned stimulatory effects. We also ascertained that HIF-1α and GPER are required for the transcriptional activation of VEGF induced by CuSO4. In addition, in human endothelial cells, the conditioned medium from breast cancer cells treated with CuSO4 promoted cell migration and tube formation through HIF-1α and GPER. The present results provide novel insights into the molecular mechanisms involved by copper in triggering angiogenesis and tumor progression. Our data broaden the therapeutic potential of copper chelating agents against tumor angiogenesis and progression.
[Show abstract][Hide abstract] ABSTRACT: We report the rational design, based on docking simulations, and synthesis of the first fluorescent and selective probe of GPER for bioimaging purposes and functional dissecting studies. It has been conceived as a Bodipy derivative and obtained by accessible and direct synthesis. Its optical properties have been measured in different solvents, showing insensitivity to their polarity. Its binding to GPER was achieved by competition assays with [3H]E2 and [5,6-3H] nicotinic acid in ER-negative and GPER-positive SkBr3 breast cancer cells. SkBr3 cells, transfected with a GPER expression vector containing a FLAG tag, were used to confirm that the fluorophore binds to GPER in a specific manner.
[Show abstract][Hide abstract] ABSTRACT: A number of tumors exhibit an altered expression of sirtuins, including NAD +-dependent histone deacetylase silent information regulator 1 (SIRT1) that may act as a tumor suppressor or tumor promoter mainly depending on the tumor types. For instance, in breast cancer cells SIRT1 was shown to exert an essential role toward the oncogenic signaling mediated by the estrogen receptor-α (ERα). In accordance with these findings, the suppression of SIRT1 led to the inhibition of the transduction pathway triggered by ERα. As the regulation of SIRT1 has not been investigated in cancer cells lacking ER, in the present study we ascertained the expression and function of SIRT1 by estrogens in ER-negative breast cancer cells and cancer-associated fibroblasts obtained from breast cancer patients. Our results show that 17β-estradiol (E2) and the selective ligand of GPER, namely G-1, induce the expression of SIRT1 through GPER and the subsequent activation of the EGFR/ERK/c-fos/AP-1 transduction pathway. Moreover, we demonstrate that SIRT1 is involved in the pro-survival effects elicited by E2 through GPER, like the prevention of cell cycle arrest and cell death induced by the DNA damaging agent etoposide. Interestingly, the aforementioned actions of estrogens were abolished silencing GPER or SIRT1, as well as using the SIRT1 inhibitor Sirtinol. In addition, we provide evidence regarding the involvement of SIRT1 in tumor growth stimulated by GPER ligands in breast cancer cells and xenograft models. Altogether, our data suggest that SIRT1 may be included in the transduction network activated by estrogens through GPER toward the breast cancer progression.
[Show abstract][Hide abstract] ABSTRACT: Estrogens control a wide number of aspects of human physiology and play a key role in multiple diseases, including cancer. Estrogens act by binding to and activating the cognate receptor (ER), however numerous studies have revealed that the G protein-coupled receptor named GPR30/GPER mediates also estrogen signals. As ER and GPER share the ability to bind to same compounds, the use of GPER-selective ligands has allowed a better understanding of the biological responses mediated by GPER. In the present study, we designed and synthesized two novel carbazole derivatives and then investigated their ability to interact with and activate the GPER-mediated transduction pathway in breast cancer cells. Both compounds did not activate the classical ER in MCF7 cells, whereas one of the two compounds synthesized triggered through GPER the rapid ERK activation in ER-negative SkBr3 cells, demonstrating a good affinity for GPER in docking studies. The characterization of this novel selective GPER agonist could represent a potential useful tool to provide further insights into the physiopathological role exerted by GPER.
Current topics in medicinal chemistry 03/2015; 15(11). DOI:10.2174/1568026615666150317221549 · 3.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pesticide atrazine does not bind or activate the classical estrogen receptor (ER), but up-regulates the aromatase activity in estrogen-sensitive tumor cells. It has recently been reported that the G protein estrogen receptor (GPR30/GPER) is involved in certain biological responses to endogenous estrogens and environmental compounds exerting estrogen-like activity.
We aimed to evaluate the potential of atrazine to trigger the GPER-mediated signaling in cancer cells and cancer-associated fibroblasts (CAFs).
Using gene reporter assays in diverse types of cancer cell, we found that atrazine does not transactivate endogenous ERα or chimeric proteins that encode the ERα and ERβ hormone binding domains. Conversely, atrazine was able to bind to GPER to induce ERK activation and the expression of estrogen target genes, which interestingly relied on both GPER and ERα expression. As a biological counterpart, atrazine stimulated the proliferation of ovarian cancer cells depending on GPER and ERα, as evidenced by gene silencing experiments and using specific signaling inhibitors. Of note, atrazine through GPER elicited ERK phosphorylation, gene expression and migration in CAFs, thus extending its stimulatory role to these main players of the tumor microenvironment.
The current results suggest a novel mechanism through which atrazine may exert relevant biological effects in cancer cells and CAFs. On the basis of our data, atrazine should be included among the environmental contaminants that may elicit estrogenic activity through the GPER-mediated signaling.
Environmental Health Perspectives 01/2015; 123(5). DOI:10.1289/ehp.1408586 · 7.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fulvenes represent a class of molecules very interesting under a chemical point of view because are easily accessible starting materials and are still poorly characterized for their biological activities, with the exception of acylfulvene and irofulvenes which have been reported to exert cytotoxic properties. Here, we describe the synthesis and characterization of several aryl-fulvenes together with their effects on cancer cell growth by MTT method. The cytotoxic potential was investigated on a panel of tumor cell lines such as breast MCF7 and SkBr3, endometrial Ishikawa, prostate LnCaP and lung A549, in comparison with the cis-diamminedichloroplatinum(II) (cisplatin) which is largely used for the treatment of different types of cancer. The evaluation of the cytotoxic activity of these compounds indicated that they are able to inhibit the proliferation of the aforementioned cancer cell types. In particular, the compound 4 exhibited the most powerful antiproliferative activity on all tumor cells evaluated with higher inhibitory effects respect to cisplatin and without altering the proliferation of human mammary MCF-10A epithelial cells.
Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents) 10/2014; 15(4). DOI:10.2174/1871520614666141019190855 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biological responses to estrogens in normal and malignant tissues are mainly mediated by the estrogen receptors ERα and ERβ, which function as ligand-activated transcription factors. Additionally, the G protein-coupled receptor GPR30 (GPER) mediates estrogenic signaling in breast cancer cells and cancer-associated fibroblasts (CAF) that contribute to cancer progression. In the present study, we evaluated the role elicited by GPER in the estrogen-regulated expression and function of VEGF in ER-negative breast cancer cells and CAF. We demonstrated that 17β-estradiol (E2) and the GPER selective ligand G-1 triggered a GPER/EGFR/ERK/c-fos signaling pathway that leads to increased VEGF via upregulation of HIF-1α. In further extending the mechanisms involved in E2-supported angiogenesis, we also showed that conditioned medium from CAF treated with E2 and G-1 promoted human endothelial tube formation in a GPER-dependent manner. In vivo, ligand-activated GPER was sufficient to enhance tumor growth and the expression of HIF-1α, VEGF and the endothelial marker CD34 in a mouse xenograft model of breast cancer. Our findings offer important new insights into the ability of estrogenic GPER signaling to trigger HIF-1α-dependent VEGF expression that supports angiogenesis and progression in breast cancer.
Cancer Research 06/2014; 74(15). DOI:10.1158/0008-5472.CAN-13-3590 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The G-protein-coupled estrogen receptor-1 (GPER, formerly known as GPR30) has attracted increasing interest, considering its ability to mediate estrogenic signaling in different cell types, including the hormone-sensitive tumors like breast cancer. As observed for other GPCR-mediated responses, the activation of the epidermal growth factor receptor is a fundamental integration point in the biological action triggered by GPER. A wide number of natural and synthetic compounds, including estrogens and anti-estrogens, elicit stimulatory effects in breast cancer through GPER up-regulation and activation, suggesting that GPER function is associated with breast tumor progression and tamoxifen resistance. GPER has also been proposed as a candidate biomarker in triple-negative breast cancer, opening a novel scenario for a more comprehensive assessment of breast tumor patients.
Frontiers in Endocrinology 05/2014; 5:66. DOI:10.3389/fendo.2014.00066
[Show abstract][Hide abstract] ABSTRACT: Nicotinic acid, also known as niacin, is the water soluble vitamin B3 used for decades for the treatment of dyslipidemic diseases. Its action is mainly mediated by the G protein-coupled receptor (GPR) 109A, however certain regulatory effects on lipid levels occur in a GPR109A- independent manner. The amide form of nicotinic acid, named nicotinamide, acts as a vitamin although neither activates the GPR109A nor exhibits the pharmacological properties of nicotinic acid. In the present study, we demonstrate for the first time that nicotinic acid and nicotinamide bind to and activate the GPER-mediated signalling in breast cancer cells and cancer-associated fibroblasts (CAFs). In particular, we show that both molecules are able to promote the up-regulation of well established GPER target genes through the EGFR/ERK transduction pathway. As a biological counterpart, nicotinic acid and nicotinamide induce proliferative and migratory effects in breast cancer cells and CAFs in a GPER-dependent fashion. Moreover, nicotinic acid prevents the up-regulation of ICAM-1 triggered by the pro-inflammatory cytokine TNF-α and stimulates the formation of endothelial tubes through GPER in HUVECs. Together, our findings concerning the agonist activity for GPER displayed by both nicotinic acid and nicotinamide broaden the mechanisms involved in the biological action of these molecules and further supports the potential of a ligand to induce different responses mediated in a promiscuous manner by distinct GPCRs.
[Show abstract][Hide abstract] ABSTRACT: The synthesis and characterization of some new titanocene-complexes, having a ethenyl-phenoxide or a benzyl group as substituents of the cyclopentadienyl rings, are reported. The synthesized compounds have been evaluated for their cytotoxic potential against two human breast cancer cell lines, that is: MCF7 and SkBr3. Most of these compounds have shown significant cytotoxic effects, compared to cisplatin, in MTT-based cell tests.
[Show abstract][Hide abstract] ABSTRACT: Carcinoma-associated fibroblasts (CAFs) play a pivotal role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors possess a unique microenvironment characterized by local hypoxia, which induces gene expression changes and biological features leading to poor outcomes. Hypoxia inducible factor 1 (HIF-1) is the main transcription factor that mediates the cell response to hypoxia through different mechanisms, that include the regulation of genes strongly associated with cancer aggressiveness. Among the HIF-1 target genes, the G-protein estrogen receptor (GPER) exerts a stimulatory role in diverse types of cancer cells and in CAFs.
We evaluated the regulation and function of the key angiogenic mediator VEGF in CAFs exposed to hypoxia. Gene expression studies, western blotting analysis and immunofluorescence experiments were performed in CAFs and breast cancer cells in presence of CoCl2 or cultured under low oxygen tension (2% O2), in order to analyze the involvement of the HIF-1alpha/GPER signaling in the biological responses to hypoxia. We also explored the role of the HIF-1alpha/GPER transduction pathway in functional assays like tube formation in HUVECs and cell migration in CAFs.
We first determined that hypoxia induces the expression of HIF-1alpha and GPER in CAFs, then we ascertained that the HIF-1alpha/GPER signaling is involved in the regulation of VEGF expression in breast cancer cells and in CAFs exposed to hypoxia. We also assessed by ChIP assay that HIF-1alpha and GPER are both recruited to the VEGF promoter sequence and required for the VEGF promoter stimulation upon hypoxic condition. As a biological counterpart of these findings, conditioned medium from hypoxic CAFs promoted tube formation in HUVECs in a HIF-1alpha/GPER dependent manner. The functional cooperation between HIF-1alpha and GPER in CAFs was also evidenced in the hypoxia-induced cell migration, which involved a further target of the HIF-1alpha/GPER signaling like the connective tissue growth factor (CTGF).
The present results provide novel insight into the role elicited by the HIF-1alpha/GPER transduction pathway in CAFs towards the hypoxia-dependent tumor angiogenesis. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression.
Breast cancer research: BCR 08/2013; 15(4):R64. DOI:10.1186/bcr3458 · 5.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: G protein-coupled receptors (GPCRs) and growth factor receptors mediate multiple physio-pathological responses to a diverse array of extracellular stimuli. In this regard, it has been largely demonstrated that GPCRs and growth factor receptors generate a multifaceted signaling network, which triggers relevant biological effects in normal and cancer cells. For instance, some GPCRs transactivate the epidermal growth factor receptor (EGFR), which stimulates diverse transduction pathways leading to gene expression changes, cell migration, survival and proliferation. Moreover, it has been reported that a functional interaction between growth factor receptors and steroid hormones like estrogens is involved in the growth of many types of tumors as well as in the resistance to endocrine therapy. This review highlights recent findings on the cross-talk between a member of the GPCR family, the G protein-coupled estrogen receptor 1 (GPER, formerly known as GPR30) and two main growth factor receptors like EGFR and insulin-like growth factor-I receptor (IGF-IR). The biological implications of the functional interaction between these important mediators of cell responses particularly in cancer are discussed.
The Journal of steroid biochemistry and molecular biology 03/2013; 137. DOI:10.1016/j.jsbmb.2013.03.005 · 3.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A series of novel titanocene-complexes has been prepared and evaluated for their growth regulatory effects in MCF7 and SkBr3 breast cancer cells. The capability of some of these compound to elicit relevant repressive effects on cancer cell growth could be taken into account towards novel pharmacological approaches in cancer therapy.
[Show abstract][Hide abstract] ABSTRACT: 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 like estrogens and growth factors contribute to the transcriptional regulation of FASN expression also through the activation of downstream signaling and a crosstalk 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 EGFR/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 (CAFs). 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 CAFs that strongly contribute to cancer progression.
Journal of Biological Chemistry 11/2012; DOI:10.1074/jbc.M112.417303 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: G-Protein Coupled Receptor (GPCR) superfamily, which comprises approximately 900 members, is the largest family of protein targets with proven therapeutic value. Although at least 500 GPCRs have been identified as therapeutically relevant, only thirteen GPCRs have been structurally characterized in apo-form or in complex with ligands. GPCRs share relatively low sequence similarity making hard the process of homology modelling, nevertheless some successful hits have been determined. Recently, the G-protein-coupled estrogen receptor 1 (GPER, formerly known as GPR30) has attracted increasing interest due to its ability in mediating estrogen signaling in different normal and cancer tissues. In this regard, the identification of selective GPER ligands has provided valuable tools in order to differentiate the specific functions elicited by this novel estrogen receptor respect to those exerted by the classical estrogen receptors (ERs). In this review, we focus on GPER examining "in silico" docking simulations and evaluating the different binding modes of diverse natural and synthetic ligands.
Current Medicinal Chemistry 10/2012; 19(36). DOI:10.2174/092986712804485755 · 3.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Environmental Health Perspectives 05/2012; 120(8):1177-82. DOI:10.1289/ehp.1104526 · 7.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Functional cross talk between insulin-like growth factor-I (IGF-I) system and estrogen signaling has been largely reported, although the underlying molecular mechanisms remain to be fully elucidated. As GPR30/GPER mediates rapid cell responses to estrogens, we evaluated the potential of IGF-I to regulate GPER expression and function in estrogen receptor (ER)α-positive breast (MCF-7) and endometrial (Ishikawa) cancer cells. We found that IGF-I transactivates the GPER promoter sequence and upregulates GPER mRNA and protein levels in both cells types. Similar data were found, at least in part, in carcinoma-associated fibroblasts. The upregulation of GPER expression by IGF-I involved the IGF-IR/PKCδ/ERK/c-fos/AP1 transduction pathway and required ERα, as ascertained by specific pharmacological inhibitors and gene-silencing. In both MCF-7 and Ishikawa cancer cells, the IGF-I-dependent cell migration required GPER and its main target gene CTGF, whereas the IGF-I-induced proliferation required both GPER and cyclin D1. Our data demonstrate that the IGF-I system regulates GPER expression and function, triggering the activation of a signaling network that leads to the migration and proliferation of cancer cells.Oncogene advance online publication, 19 March 2012; doi:10.1038/onc.2012.97.