Marcello Maggiolini

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 Receptor 30 and Estrogen Receptor-[Alpha] Are Involved in the Proliferative Effects Induced by Atrazine in Ovarian Cancer Cells

Background Atrazine, one of the most common pesticide contaminants, has been shown to up-regulate aromatase activity in certain estrogen-sensitive tumors without binding or activating the estrogen receptor (ER). Recent investigations have demonstrated that the orphan G-protein–coupled receptor 30 (GPR30), which is structurally unrelated to the ER, mediates rapid actions of 17β-estradiol and environmental estrogens. Objectives Given the ability of atrazine to exert estrogen-like activity in cancer cells, we evaluated the potential of atrazine to signal through GPR30 in stimulating biological responses in cancer cells. Methods and results Atrazine did not transactivate the endogenous ERα in different cancer cell contexts or chimeric proteins encoding the ERα and ERβ hormone-binding domain in gene reporter assays. Moreover, atrazine neither regulated the expression of ERα nor stimulated aromatase activity. Interestingly, atrazine induced extracellular signal-regulated kinase (ERK) phosphorylation and the expression of estrogen target genes. Using specific signaling inhibitors and gene silencing, we demonstrated that atrazine stimulated the proliferation of ovarian cancer cells through the GPR30–epidermal growth factor receptor transduction pathway and the involvement of ERα. Conclusions Our results indicate a novel mechanism through which atrazine may exert relevant biological effects in cancer cells. On the basis of the present data, atrazine should be included among the environmental contaminants potentially able to signal via GPR30 in eliciting estrogenic action.

G protein-coupled receptors: novel targets for drug discovery in cancer

G protein-coupled receptors (GPCRs) belong to a superfamily of cell surface signalling proteins that have a pivotal role in many physiological functions and in multiple diseases, including the development of cancer and cancer metastasis. Current drugs that target GPCRs — many of which have excellent therapeutic benefits — are directed towards only a few GPCR members. Therefore, huge efforts are currently underway to develop new GPCR-based drugs, particularly for cancer. We review recent findings that present unexpected opportunities to interfere with major tumorigenic signals by manipulating GPCR-mediated pathways. We also discuss current data regarding novel GPCR targets that may provide promising opportunities for drug discovery in cancer prevention and treatment.

G Protein–Coupled Receptor 30 (GPR30) Mediates Gene Expression Changes and Growth Response to 17β-Estradiol and Selective GPR30 Ligand G-1 in Ovarian Cancer Cells

Estrogens play a crucial role in the development of ovarian tumors; however, the signal transduction pathways involved in hormone action are still poorly defined. The orphan G protein-coupled receptor 30 (GPR30) mediates the nongenomic signaling of 17beta-estradiol (E2) in a variety of estrogen-sensitive cancer cells through activation of the epidermal growth factor receptor (EGFR) pathway. Whether estrogen receptor alpha (ERalpha) also contributes to GPR30/EGFR signaling is less understood. Here, we show that, in ERalpha-positive BG-1 ovarian cancer cells, both E2 and the GPR30-selective ligand G-1 induced c-fos expression and estrogen-responsive element (ERE)-independent activity of a c-fos reporter gene, whereas only E2 stimulated an ERE-responsive reporter gene, indicating that GPR30 signaling does not activate ERalpha-mediated transcription. Similarly, both ligands up-regulated cyclin D1, cyclin E, and cyclin A, whereas only E2 enhanced progesterone receptor expression. Moreover, both GPR30 and ERalpha expression are required for c-fos stimulation and extracellular signal-regulated kinase (ERK) activation in response to either E2 or G-1. Inhibition of the EGFR transduction pathway inhibited c-fos stimulation and ERK activation by either ligand, suggesting that in ovarian cancer cells GPR30/EGFR signaling relays on ERalpha expression. Interestingly, we show that both GPR30 and ERalpha expression along with active EGFR signaling are required for E2-stimulated and G-1-stimulated proliferation of ovarian cancer cells. Because G-1 was able to induce both c-fos expression and proliferation in the ERalpha-negative/GPR30-positive SKBR3 breast cancer cells, the requirement for ERalpha expression in GPR30/EGFR signaling may depend on the specific cellular context of different tumor types.

The unfolding stories of GPR30, a new membrane-bound estrogen receptor.

Steroid hormones such as estrogens are known to signal through ligand-regulated transcription factors of the nuclear receptor superfamily. In addition, they elicit rapid nongenomic responses from membrane-associated receptors. One of these receptors belongs to an entirely different family of proteins. The G protein-coupled and seven-transmembrane receptor, GPR30, is now widely recognized as an estrogen receptor (ER), hence its official new acronym GPER. It appears to mediate a wide range of responses to estrogen in a large variety of cell types. Its functions are clearly distinct from those of the classical nuclear ERs, although these pathways may overlap and interact in some cases. Here, we review the history of the discovery of this new ER, the evidence for the claim that it is an ER, its signal transduction, and its potential functions in physiology and disease.

17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the g protein-coupled receptor GPR30.

The higher incidence of thyroid carcinoma (TC) in women during reproductive years compared with men and the increased risk associated with the therapeutic use of estrogens have suggested a pathogenetic role exerted by these steroids in the development of TC. In the present study, we evaluated the potential of 17β-estradiol (E2), genistein (G), and 4-hydroxyta-moxifen (OHT) to regulate the expression of diverse estrogen target genes and the proliferation of human WRO, FRO, and ARO thyroid carcinoma cells, which were used as a model system. We have ascertained that ARO cells are devoid of estrogen receptors (ERs), whereas both WRO and FRO cells express a single variant of ERα that was neither transactivated, modulated, nor translocated into the nucleus upon treatment with ligands. However, E2, G, and OHT were able either to induce the transcriptional activity of c-fos promoter constructs, including those lacking the estrogen-responsive elements, or to increase c-fos, cyclin A, and D1 expression. It is noteworthy that we have demonstrated that the G protein-coupled receptor 30 (GPR30) and the mitogen-activated protein kinase (MAPK) pathway mediate both the up-regulation of c-fos and the growth response to E2, G, and OHT in TC cells studied, because these stimulatory effects were prevented by silencing GPR30 and using the MEK inhibitor 2′-amino-3′-methoxyflavone (PD 98059). Our findings provide new insight into the molecular mechanisms through which estrogens may induce the progression of TC.

Mechanisms of estrogen signaling and gene expression via GPR30.

The effects of estrogen are widespread throughout the body. Although the classical nuclear estrogen receptors have been known for many years to decades and their primary modes of action as transcriptional regulators is well understood, certain aspects of estrogen biology remain inconsistent with the mechanisms of action of these receptor. More recently, the G protein-coupled receptor, GPR30/GPER, has been suggested to contribute to some of the cellular and physiological effects of estrogen. Not only does GPR30 mediate some of the rapid signal transduction events following cell stimulation, such as calcium mobilization and kinase activation, it also appears to regulate rapid transcriptional activation of genes such as c-fos. Since many cells and tissues co-express classical estrogen receptors and GPR30, there exists great diversity in the possible avenues of synergism and antagonism. In this review, we will provide an overview of GPR30 function, focusing on the rapid signaling events that culminate in the transcriptional activation of certain genes.

Estrogenic GPR30 signalling induces proliferation and migration of breast cancer cells through CTGF

The steroid hormone oestrogen can signal through several receptors and pathways. Although the transcriptional responses mediated by the nuclear oestrogen receptors (ER) have been extensively characterized, the changes in gene expression elicited by signalling through the membrane‐associated ER GPR30 have not been studied. We show here for ER‐negative human breast cancer cells that the activation of GPR30 signalling by oestrogen or by hydroxytamoxifen (OHT), an ER antagonist but GPR30 agonist, induces a transcription factor network, which resembles that induced by serum in fibroblasts. The most strongly induced gene, CTGF, appears to be a target of these transcription factors. We found that the secreted factor connective tissue growth factor (CTGF) not only contributes to promote proliferation but also mediates the GPR30‐induced stimulation of cell migration. These results provide a framework for understanding the physiological and pathological functions of GPR30. As the activation of GPR30 by OHT also induces CTGF in fibroblasts from breast tumour biopsies, these pathways may be involved in promoting aggressive behaviour of breast tumours in response to endogenous oestrogens or to OHT being used for endocrine therapy.

Estrogenic and antiproliferative activities of isoliquiritigenin in MCF7 breast cancer cells

Licorice root contains chemically diverse compounds that exhibit estrogenic effects in vitro and in vivo. The chalcone isoliquiritigenin (ISL) is a component of licorice extract exhibiting either antitumorigenic activity or estrogen receptor (ER) alpha-dependent growth promoting effects on breast cancer cells. In order to contribute to a better understanding of this apparent paradox, we synthesized and ascertained the estrogenic properties of ISL using, as model systems, the hormone-sensitive MCF7 breast cancer cells and the steroid-independent HeLa cells. Transfection experiments reveal that ISL is able to transactivate the endogenous ER alpha in MCF7 cells and this is supported by the capability to induce down-regulation of ER alpha protein levels and up-regulation of pS2 mRNA. Moreover, by using chimeric proteins consisting of the hormone binding domains of ER alpha and ER beta fused to the Gal4 DNA binding domain, we have determined that ISL is an estrogenic agonist of both ER isoforms. As a biological counterpart, low and intermediate ISL concentrations that induce substantial transcriptional activity stimulate the proliferation of MCF7 cells. However, high levels of ISL become cytotoxic even in steroid-receptor negative HeLa cells. Thus, the activity of ISL and the balance between risk or chemopreventive factor for estrogen-dependent breast cancer may depend on dietary intake.

Breast cancer: from estrogen to androgen receptor

To investigate the link existing between androgens and human breast cancer, the hormonal milieu present in pre- and post-menopausal women has been translated in an in vitro model utilizing a hormone dependent breast cancer cell line MCF-7 exposed to DHEA, DHEAS, androstenediol, T, DHT with or w/o E(2). DHEAS and androstenediol stimulate the growth of MCF-7 cell line but reduce cell proliferation induced by E(2) (1 nM). T and DHT (1-100 nM) instead inhibit MCF-7 cell proliferation independently on E(2) presence. When we focused our study on the most powerful androgen, DHT alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal growth rate and counteracts E(2) proliferative action by 68%. These data correlate well with cell cycle analysis showing an enhanced number of cells in G(0)/G(1) phase after 6 days of DHT treatment. Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while immunohistochemistry indicates that it was mostly translocated into the nucleus. So we assumed that the enhanced activation of the AR might inhibit MCF-7 cells proliferation. This assumption is corroborated by the fact that the inhibitory effects induced by DHT on MCF-7 cell proliferation are abrogated in the presence of hydroxyflutamide. Therefore to better investigate the role of AR in inhibiting E(2) action at genomic level, MCF-7 cells were transiently cotransfected with the reporter plasmid XETL carrying firefly luciferase sequence under the control of an estrogen responsive element and the full length AR or with an AR carrying a mutation (Cis 574-->Arg 574) which abolishes its binding to DNA. The over-expression of the AR markedly decreases E(2) signalling which furthermore appears inhibited by simultaneous exposure to DHT but reversed by addition of hydroxyflutamide. The inhibitory effect was no longer noticeable when MCF-7 cells were cotransfected with XETL and the mutant AR. Taken together these data demonstrate that gonadal androgens antagonize MCF-7 proliferation induced by E(2). This seems to be related to the inhibitory effects of the over-expressed AR on E(2) genomic action.