ABSTRACT
Zinc (Zn) is an essential trace mineral that contributes to the regulation of several cellular functions; however, it may be also implicated in the progression of breast cancer through different mechanisms. It has been largely reported that the classical estrogen receptor (ER), as well as the G protein estrogen receptor (GPER, previously known as GPR30) can exert a main role in the development of breast tumors. In the present study, we demonstrate that zinc chloride (ZnCl2 ) involves GPER in the activation of insulin-like growth factor receptor I (IGF-IR)/epidermal growth factor receptor (EGFR)-mediated signaling, which in turn triggers downstream pathways like ERK and AKT in breast cancer cells, and main components of the tumor microenvironment namely cancer-associated fibroblasts (CAFs). Further corroborating these findings, ZnCl2 stimulates a functional crosstalk of GPER with IGF-IR and EGFR toward the transcription of diverse GPER target genes. Then, we show that GPER contributes to the stimulatory effects induced by ZnCl2 on cell-cycle progression, proliferation, and migration of breast cancer cells as well as migration of CAFs. Together, our data provide novel insights into the molecular mechanisms through which zinc may exert stimulatory effects in breast cancer cells and CAFs toward tumor progression. © 2016 Wiley Periodicals, Inc.
Subject(s)
Breast Neoplasms/metabolism , Cancer-Associated Fibroblasts/pathology , Chlorides/metabolism , ErbB Receptors/metabolism , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Somatomedin/metabolism , Signal Transduction , Zinc Compounds/metabolism , Breast/metabolism , Breast/pathology , Breast Neoplasms/pathology , Cancer-Associated Fibroblasts/metabolism , Cell Line, Tumor , Cell Proliferation , Disease Progression , Female , HumansABSTRACT
Aldosterone induces relevant effects binding to the mineralcorticoid receptor (MR), which acts as a ligand-gated transcription factor. Alternate mechanisms can mediate the action of aldosterone such as the activation of epidermal growth factor receptor (EGFR), MAPK/ERK, transcription factors and ion channels. The G-protein estrogen receptor (GPER) has been involved in the stimulatory effects of estrogenic signalling in breast cancer. GPER has been also shown to contribute to certain responses to aldosterone, however the role played by GPER and the molecular mechanisms implicated remain to be fully understood. Here, we evaluated the involvement of GPER in the stimulatory action exerted by aldosterone in breast cancer cells and breast tumor derived endothelial cells (B-TEC). Competition assays, gene expression and silencing studies, immunoblotting and immunofluorescence experiments, cell proliferation and migration were performed in order to provide novel insights into the role of GPER in the aldosterone-activated signalling. Our results demonstrate that aldosterone triggers the EGFR/ERK transduction pathway in a MR- and GPER-dependent manner. Aldosterone does not bind to GPER, it however induces the direct interaction between MR and GPER as well as between GPER and EGFR. Next, we ascertain that the up-regulation of the Na+/H+ exchanger-1 (NHE-1) induced by aldosterone involves MR and GPER. Biologically, both MR and GPER contribute to the proliferation and migration of breast and endothelial cancer cells mediated by NHE-1 upon aldosterone exposure. Our data further extend the current knowledge on the molecular mechanisms through which GPER may contribute to the stimulatory action elicited by aldosterone in breast cancer.
Subject(s)
Aldosterone/pharmacology , Endothelial Cells/drug effects , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Endothelial Cells/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , Gene Expression/drug effects , Humans , Immunoblotting , Microscopy, Fluorescence , Protein Binding/drug effects , RNA Interference , Receptors, Estrogen/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, Mineralocorticoid/genetics , Receptors, Mineralocorticoid/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sodium-Hydrogen Exchanger 1 , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/metabolism , Time-Lapse Imaging/methodsABSTRACT
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 ERß, 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.
Subject(s)
Drug Discovery/methods , Macromolecular Substances/chemistry , Molecular Docking Simulation/methods , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/metabolism , Animals , Computational Biology , Humans , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Copper Sulfate/toxicity , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neovascularization, Pathologic/metabolism , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/metabolism , Blotting, Western , Cell Line, Tumor , Fluorescent Antibody Technique , Humans , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , TranscriptomeABSTRACT
Estrogens regulate numerous pathophysiological processes, mainly by binding to and activating estrogen receptor (ER)α and ERß. Increasing amounts of evidence have recently demonstrated that G-protein coupled receptor 30 (GPR30; also known as GPER) is also involved in diverse biological responses to estrogens both in normal and cancer cells. The classical ER and GPER share several features, including the ability to bind to identical compounds; nevertheless, some ligands exhibit opposed activity through these receptors. It is worth noting that, owing to the availability of selective agonists and antagonists of GPER for research, certain differential roles elicited by GPER compared with ER have been identified. Here, we provide evidence on the molecular mechanisms through which a calixpyrrole derivative acts as a GPER antagonist in different model systems, such as breast tumor cells and cancer-associated fibroblasts (CAFs) obtained from breast cancer patients. Our data might open new perspectives toward the development of a further class of selective GPER ligands in order to better dissect the role exerted by this receptor in different pathophysiological conditions. Moreover, calixpyrrole derivatives could be considered in future anticancer strategies targeting GPER in cancer cells.
Subject(s)
Models, Biological , Models, Molecular , Pyrroles/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Animals , Biological Assay , Cell Line, Tumor , Cell Movement/drug effects , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Fibroblasts/metabolism , Humans , Ligands , Neoplasms/pathology , Pyrroles/chemistry , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effects , XenopusABSTRACT
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.
Subject(s)
Carbazoles/pharmacology , Hydrazines/pharmacology , Receptors, G-Protein-Coupled/agonists , Binding Sites , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor/drug effects , Female , Humans , MCF-7 Cells/drug effects , MCF-7 Cells/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Molecular Docking Simulation , Phosphorylation/drug effects , Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Agents/chemistry , Cyclopentanes/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation , Cisplatin/pharmacology , Cyclopentanes/chemical synthesis , Cyclopentanes/pharmacology , Drug Screening Assays, Antitumor , Humans , Structure-Activity RelationshipABSTRACT
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.
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.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Organometallic Compounds/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , MCF-7 Cells , Molecular Structure , Organometallic Compounds/chemical synthesis , Organometallic Compounds/chemistry , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The G protein-coupled receptor GPR30/GPER has been shown to mediate rapid effects of 17ß-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.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Notch/metabolism , Breast Neoplasms/genetics , Cell Line, Tumor , Cyclopentanes/pharmacology , Estradiol/pharmacology , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Quinolines/pharmacology , Receptor Cross-Talk , Signal Transduction/drug effects , Up-Regulation/drug effectsABSTRACT
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.
Subject(s)
Breast Neoplasms/metabolism , Cell Nucleus/metabolism , Fibroblasts/metabolism , Gene Expression Regulation, Neoplastic , Nuclear Localization Signals/metabolism , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Movement , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Cell Proliferation , Connective Tissue Growth Factor/genetics , Connective Tissue Growth Factor/metabolism , Estrogens/pharmacology , Female , Fibroblasts/drug effects , Fibroblasts/pathology , Humans , Karyopherins/genetics , Karyopherins/metabolism , Nuclear Localization Signals/chemistry , Primary Cell Culture , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , Receptors, Estrogen/genetics , Receptors, G-Protein-Coupled/genetics , Signal Transduction , Tumor Microenvironment/geneticsABSTRACT
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.
Subject(s)
Antineoplastic Agents/chemical synthesis , Organometallic Compounds/chemistry , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/toxicity , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Female , Humans , MCF-7 Cells , Organometallic Compounds/therapeutic use , Organometallic Compounds/toxicityABSTRACT
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 ERß) 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.