Antonio Madeo

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 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.

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.

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.

Nuclear Alternate Estrogen Receptor GPR30 Mediates 17β-Estradiol-Induced Gene Expression and Migration in Breast Cancer-Associated Fibroblasts

Fibroblasts are the principal cellular component of connective tissue and are associated with cancer cells at all stages of tumor progression. Structural and functional contributions of fibroblasts to the growth, survival, and invasive capacity of cancer cells are beginning to emerge. In breast carcinoma, approximately 80% of stromal fibroblasts termed cancer-associated fibroblasts (CAF) are thought to manifest an activated phenotype that promotes cancer cell proliferation tumor growth at metastatic sites similar to the primary tumor. In this report, we show that CAFs respond to physiologic concentrations of 17beta-estradiol (E2) by rapidly inducing extracellular signal-regulated kinase phosphorylation and immediate early gene expression, including c-fos and connective tissue growth factor, and cyclin D1. Notably, the E2 response is mediated by the alternate estrogen receptor GPR30, which interfaces with the epidermal growth factor receptor (EGFR) signaling pathway. In particular, E2 stimulates a physical interaction between GPR30 and phosphorylated EGFR, recruiting them to the cyclin D1 gene promoter. Nuclear localization induced by E2 was confirmed by cellular immunofluorescence methods. GPR30 was required for CAF proliferation and migration induced by E2. Our results provide important new mechanistic insights into how CAFs are stimulated by estrogen through a GPR30-mediated nuclear signaling pathway. More generally, they define estrogenic GPR30 signaling as a functionally important component of the tumor microenvironment.

A sexually dimorphic distribution pattern of the novel estrogen receptor G-protein-coupled receptor 30 in some brain areas of the hamster

The isolation of the G-protein-coupled receptor 30 (GPR30), an orphan membrane receptor unrelated to nuclear estrogen receptors (ERs), has become a key factor towards the unraveling of rapid estrogen action. This membrane receptor together with cellular signaling intermediaries, i.e., extracellular signal-dependent kinases 1 and 2, may promote neuronal proliferation and differentiation activities. In the present study, an evident gene expression pattern of GPR30 characterized postnatal 7 (young) and 60 (adult) days of age hamsters as shown by its heterogeneous mRNA distribution in hypothalamic, amygdalar and cerebellar areas of both sexes. In particular, most of the brain areas considered in the adult hamster plus only the amygdala and cerebellum of young animals behaved in a sexually dimorphic fashion. This similar pattern was also detected for the ERalpha and beta, as shown by the latter receptor prevailing in young and adult females, while the former predominated in young females. Even for the two kinases, a sexually dimorphic distribution was featured above all for young hamsters. Overall, the findings of the present study established a distinct expression pattern of the novel ER (GPR30) that may operate differently in some brain areas of the hamster and this may provide interesting insights regarding its probable neuroprotective role during the execution of some hibernating states, which are typical of our rodent model.

Effects of atrazine on estrogen receptor α- and G protein-coupled receptor 30-mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts.

Background: The pesticide atrazine does not bind to or activate the classical estrogen receptor (ER), but it up-regulates the aromatase activity in estrogen-sensitive tumor cells. The G protein estrogen receptor (GPR30/GPER) has been reported to be involved in certain biological responses to endogenous estrogens and environmental compounds exerting estrogen-like activity. Objectives: We aimed to evaluate the potential of atrazine to trigger GPER-mediated signaling in cancer cells and cancer-associated fibroblasts (CAFs). Methods and Results: Using gene reporter assays in diverse types of cancer cells, we found that atrazine did 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, appeared to rely on both GPER and ERα expression. As a biological counterpart, atrazine stimulated the proliferation of ovarian cancer cells that depend on GPER and ERα, as evidenced by gene silencing experiments and the use of specific signaling inhibitors. Of note, through GPER, atrazine elicited ERK phosphorylation, gene expression, and migration in CAFs, thus extending its stimulatory role to these main players of the tumor microenvironment. Conclusions: Our 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 GPER-mediated signaling. Citation: Albanito L, Lappano R, Madeo A, Chimento A, Prossnitz ER, Capello AR, Dolce V, Abonante S, Pezzi V, Maggiolini M. 2015. Effects of atrazine on estrogen receptor α– and G protein–coupled receptor 30–mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts. Environ Health Perspect 123:493–499; http://dx.doi.org/10.1289/ehp.1408586

c-Jun activation is required for 4-hydroxytamoxifen-induced cell death in breast cancer cells.

The c-Jun N-terminal kinase (JNK) has been shown to mediate tamoxifen-induced apoptosis in breast cancer cells. However, the downstream mediators of the JNK pathway linking tamoxifen to effectors of apoptosis have yet to be identified. In this study, we analysed whether c-Jun, the major nuclear target of JNK, has a role in tamoxifen-induced apoptosis of SkBr3 breast cancer cells. We show that before DNA fragmentation and caspase 3/7 activation, cytotoxic concentrations of 4-hydroxytamoxifen (OHT) induced JNK-dependent phosphorylation of c-Jun at JNK sites earlier shown to regulate c-Jun-mediated apoptosis. In addition, OHT induced ERK-dependent expression of c-Fos and transactivation of an AP-1-responsive promoter. In particular, the ectopic expression of dominant-negative constructs blocking either AP-1 activity or c-Jun N-terminal phosphorylation prevented DNA fragmentation after OHT treatment. Furthermore, both c-Fos expression and c-Jun N-terminal phosphorylation preceded OHT-dependent activation of caspase 3–7 in different types of tamoxifen-sensitive cancer cells, but not in OHT-resistant LNCaP prostate cancer cells. Taken together, our results indicate that the c-Jun/c-Fos AP-1 complex has a pro-apoptotic role in OHT-treated cancer cells and suggest that pharmacological boosts of c-Jun activation may be useful in a combination therapy setting to sensitize cancer cells to tamoxifen-mediated cell death.

Direct Evaluation of the Post-Buckling Behavior of Slender Structures Through a Numerical Asymptotic Formulation

The analysis of slender structures, characterized by complex buckling and postbuckling phenomena and by a strong imperfection sensitivity, is heavily penalized by the lack of adequate computational tools. Standard incremental iterative approaches are computationally expensive and unaffordable, while FEM implementation of the Koiter method is a convenient alternative. The analysis is very fast, its computational burden is of the same order as a linearized buckling load evaluation and the simulation of different imperfections costs only a fraction of that needed to characterize the perfect structure. In this respect it can be considered as a direct method for the evaluation of the critical and post-critical behaviour of geometrically nonlinear elastic structures. The main objective of the present work is to show that finite element implementations of the Koiter method can be both accurate and reliable and to highlight the aspects that require further investigation.

A Mixed 4-Node 3D Plate Element Based on Self-Equilibrated Isostatic Stresses

A new mixed stress 4-node flat shell finite element, designed for the linear and nonlinear analysis of folded plate structures, is presented. The kinematics of the element is defined by 24 dofs with in- and out-of-plane displacements assumed to be quadratic, controlled by displacement and rotation parameters through an Allman like interpolation, and flexural rotations assumed to be bilinear. The assumed stress approximation, described within a local Cartesian frame aligned with the element orientation, is self-equilibrated and ruled by the minimum number of parameters. The element does not suffer from kinematical locking and rank defectiveness. Many numerical tests show the very good performance of the element.