Paola De Marco

G Protein-Coupled Receptor 30 Expression Is Up-Regulated by EGF and TGFα in Estrogen Receptor α-Positive Cancer Cells

In the present study, we evaluated the regulation of G protein-coupled receptor (GPR)30 expression in estrogen receptor (ER)-positive endometrial, ovarian, and estrogen-sensitive, as well as tamoxifen-resistant breast cancer cells. We demonstrate that epidermal growth factor (EGF) and TGF alpha transactivate the GPR30 promoter and accordingly up-regulate GPR30 mRNA and protein levels only in endometrial and tamoxifen-resistant breast cancer cells. These effects exerted by EGF and TGF alpha were dependent on EGF receptor (EGFR) expression and activation and involved phosphorylation of the Tyr(1045) and Tyr(1173) EGFR sites. Using gene-silencing experiments and specific pharmacological inhibitors, we have ascertained that EGF and TGF alpha induce GPR30 expression through the EGFR/ERK transduction pathway, and the recruitment of c-fos to the activator protein-1 site located within GPR30 promoter sequence. Interestingly, we show that functional cross talk of GPR30 with both activated EGFR and ER alpha relies on a physical interaction among these receptors, further extending the potential of estrogen to trigger a complex stimulatory signaling network in hormone-sensitive tumors. Given that EGFR/HER2 overexpression is associated with tamoxifen resistance, our data may suggest that ligand-activated EGFR could contribute to the failure of tamoxifen therapy also by up-regulating GPR30, which in turn could facilitates the action of estrogen. In addition, important for resistance is the ability of tamoxifen to bind to and activate GPR30, the expression of which is up-regulated by EGFR activation. Our results emphasize the need for new endocrine agents able to block widespread actions of estrogen without exerting any stimulatory activity on transduction pathways shared by the steroid and growth factor-signaling networks.

Estriol acts as a GPR30 antagonist in estrogen receptor-negative breast cancer cells.

Estrogens are structurally related steroids that regulate important physiological processes. 17beta-estradiol (E2) is reversibly oxidized to estrone (E1) and both E2 and E1 can be irreversibly converted to estriol (E3), which also originates directly from androstenedione. The action of E2 has been traditionally explained by the binding to the estrogen receptor (ER) alpha and ER beta, however the G protein-coupled receptor (GPR) 30 has been recently involved in the rapid signaling triggered by estrogens. Although the role of E2 in the development of breast cancer has been largely documented, the contribution of E3 still remains to be completely evaluated. Here, we demonstrate for the first time that E3 acts as a GPR30 antagonist since it was able to inhibit the GPR30-mediated responses such as the rapid ERK activation, the up-regulation of target genes like c-fos and connective tissue growth factor, the proliferative effects observed in ER-negative SkBr3 cells.

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.

GPER mediates the Egr-1 expression induced by 17β-estradiol and 4-hydroxitamoxifen in breast and endometrial cancer cells.

Early growth response-1 (Egr-1) is an immediate early gene involved in relevant biological events including the proliferation of diverse types of cell tumors. In a microarray analysis performed in breast cancer cells, 17β-estradiol (E2) and the estrogen receptor antagonist 4-hydroxitamoxifen (OHT) up-regulated Egr-1 through the G protein-coupled receptor named GPR30/GPER. Hence, in this study, we aimed to provide evidence regarding the ability of E2, OHT and the selective GPER ligand G-1 to regulate Egr-1 expression and function through the GPER/EGFR/ERK transduction pathway in both Ishikawa (endometrial) and SkBr3 (breast) cancer cells. Interestingly, we demonstrate that Egr-1 is involved in the transcription of genes regulating cell proliferation like CTGF and cyclin D1 and required for the proliferative effects induced by E2, OHT, and G-1 in both Ishikawa and SkBr3 cells. In addition, we show that GPER mediates the expression of Egr-1 also in carcinoma-associated fibroblasts (CAFs). Our data suggest that Egr-1 may represent an important mediator of the biological effects induced by E2 and OHT through GPER/EGFR/ERK signaling in breast and endometrial cancer cells. The results obtained in CAFs provide further evidence regarding the potential role exerted by the GPER-dependent Egr-1 up-regulation in tumor development and progression. Therefore, Egr-1 may be included among the bio-markers of estrogen and antiestrogen actions and may be considered as a further therapeutic target in both breast and endometrial tumors.

New advances on the functional cross-talk between insulin-like growth factor-I and estrogen signaling in cancer.

There is increasing awareness that estrogens may affect cell functions through the integration with a network of signaling pathways. The IGF system is a phylogenetically highly conserved axis that includes the insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) pathways, which are of crucial importance in the regulation of metabolism and cell growth in relationship to nutrient availability. Numerous studies nowadays document that estrogens cooperate with IGF system at multiple levels both in physiology and in disease. Several studies have focused on this bidirectional cross-talk in central nervous system, in mammary gland development and in cancer. Notably, cancer cells show frequent deregulation of the IGF system with overexpression of IR and/or IGF-IR and their ligands as well as frequent upregulation of the classical estrogen receptor (ER)α and the novel ER named GPER. Recent studies have, therefore, unraveled further mechanisms of cross-talk involving membrane initiated estrogen actions and the IGF system in cancer, that converge in the stimulation of pro-tumoral effects. These studies offer hope for new strategies aimed at the treatment of estrogen related cancers in order to prevent an estrogen-independent and more aggressive tumor progression.

Copper activates HIF-1α/GPER/VEGF signalling in cancer cells

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.

GPER1 is regulated by insulin in cancer cells and cancer-associated fibroblasts

Elevated insulin levels have been associated with an increased cancer risk as well as with aggressive and metastatic cancer phenotypes characterized by a poor prognosis. Insulin stimulates the proliferation, migration, and invasiveness of cancer cells through diverse transduction pathways, including estrogen signaling. As G protein estrogen receptor 1 (GPER1) mediates rapid cell responses to estrogens, we evaluated the potential of insulin to regulate GPER1 expression and function in leiomyosarcoma cancer cells (SKUT-1) and breast cancer-associated fibroblasts (CAFs), which were used as a model system. We found that insulin transactivates the GPER1 promoter sequence and increases the mRNA and protein expression of GPER1 through the activation of the PRKCD/MAPK1/c-Fos/AP1 transduction pathway, as ascertained by means of specific pharmacological inhibitors and gene-silencing experiments. Moreover, cell migration triggered by insulin occurred through GPER1 and its main target gene CTGF, whereas the insulin-induced expression of GPER1 boosted cell-cycle progression and the glucose uptake stimulated by estrogens. Notably, a positive correlation between insulin serum levels and GPER1 expression was found in cancer fibroblasts obtained from breast cancer patients. Altogether, our data indicate that GPER1 may be included among the complex network of transduction signaling triggered by insulin that drives cells toward cancer progression.

GPER is involved in the stimulatory effects of aldosterone in breast cancer cells and breast tumor-derived endothelial cells

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.

Novel aspects concerning the functional cross-talk between the insulin/IGF-I system and estrogen signaling in cancer cells

The insulin/IGF system plays an important role in cancer progression. Accordingly, elevated levels of circulating insulin have been associated with an increased cancer risk as well as with aggressive and metastatic cancer phenotypes. Numerous studies have documented that estrogens cooperate with the insulin/IGF system in multiple pathophysiological conditions. The biological responses to estrogens are mainly mediated by the estrogen receptors (ER)α and ERβ, which act as transcription factors; however, several studies have recently demonstrated that a member of the G protein-coupled receptors, named GPR30/G-protein estrogen receptor (GPER), is also involved in the estrogen signaling in normal and malignant cells as well as in cancer-associated fibroblasts (CAFs). In this regard, novel mechanisms linking the action of estrogens through GPER with the insulin/IGF system have been recently demonstrated. This review recapitulates the relevant aspects of this functional cross-talk between the insulin/IGF and the estrogenic GPER transduction pathways, which occurs in various cell types and may account for cancer progression.

GPER, IGF-IR, and EGFR transduction signaling are involved in stimulatory effects of zinc in breast cancer cells and cancer-associated fibroblasts.

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.