Francesca De Amicis

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.

Androgen Receptor Overexpression Induces Tamoxifen Resistance in Human Breast Cancer Cells

Although the androgen receptor (AR) is a known clinical target in prostate cancer, little is known about its possible role in breast cancer. We have investigated the role of AR expression in human breast cancer in response to treatment with the antiestrogen tamoxifen. Resistance to tamoxifen is a major problem in treating women with breast cancer. By gene expression profiling, we found elevated AR and reduced estrogen receptor (ER) α mRNA in tamoxifen-resistant tumors. Exogenous overexpression of AR rendered ERα-positive MCF-7 breast cancer cells resistant to the growth-inhibitory effects of tamoxifen in anchorage-independent growth assays and in xenograft studies in athymic nude mice. AR-overexpressing cells remained sensitive to growth stimulation with dihydrotestosterone. Treatment with the AR antagonist Casodex™ (bicalutamide) reversed this resistance, demonstrating the involvement of AR signaling in tamoxifen resistance. In AR-overexpressing cells, tamoxifen induced transcriptional activation by ERα that could be blocked by Casodex, suggesting that AR overexpression enhances tamoxifen’s agonistic properties. Our data suggest a role for AR overexpression as a novel mechanism of hormone resistance, so that AR may offer a new clinical therapeutic target in human breast cancers.

Evidence that leptin through STAT and CREB signaling enhances cyclin D1 expression and promotes human endometrial cancer proliferation.

Obesity is a risk factor for endometrial cancer in pre‐ and post‐menopausal women. Leptin, an adipocyte‐derived hormone, in addition to the control weight homeostasis, is implicated in multiple biological actions. A recent study demonstrated that leptin promotes endometrial cancer growth and invasiveness through STAT/MAPK and Akt pathways, but the molecular mechanism involved in such processes still needs to be elucidated. In an attempt to understand the role of leptin in regulating endometrial cancer cells proliferation, we have demonstrated that leptin treatment reduced the numbers of cells in G0/G1‐phase while increased cell population in S‐phase. This effect is associated with an up‐regulation of cyclin D1 together with a down‐regulation of cyclin‐dependent kinase inhibitor p21WAF1/Cip1. Mutagenesis studies, eletrophoretic mobility shift, and chromatin immunoprecipitation analysis revealed that signal transducers and activators of transcription 3 (STAT3) and cyclic AMP‐responsive element (CRE) binding protein motifs, within cyclin D1 promoter, were required for leptin‐induced cyclin D1 expression in Ishikawa endometrial cancer cells. Silencing of STAT3 and CREB gene expression by RNA interference reversed the up‐regulatory effect of leptin on cyclin D1 expression and cells proliferation. These results support the hypothesis that STAT3 and CREB play an important role in leptin signaling pathway that leads to the proliferation of Ishikawa cells, thus establishing a direct association between obesity and endometrial tumorogenesis. J. Cell. Physiol. 218: 490–500, 2009.

Omega-3 PUFA ethanolamides DHEA and EPEA induce autophagy through PPARγ activation in MCF-7 breast cancer cells†‡§

The omega‐3 long chain polyunsaturated fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), elicit anti‐proliferative effects in cancer cell lines and in animal models. Dietary DHA and EPA can be converted to their ethanolamide derivatives, docosahexaenoyl ethanolamine (DHEA), and eicosapentaenoyl ethanolamine (EPEA), respectively; however, few studies are reported on their anti‐cancer activities. Here, we demonstrated that DHEA and EPEA were able to reduce cell viability in MCF‐7 breast cancer cells whereas they did not elicit any effects in MCF‐10A non‐tumorigenic breast epithelial cells. Since DHA and EPA are ligands of Peroxisome Proliferator‐Activated Receptor gamma (PPARγ), we sought to determine whether PPARγ may also mediate DHEA and EPEA actions. In MCF‐7 cells, both compounds enhanced PPARγ expression, stimulated a PPAR response element‐dependent transcription as confirmed by the increased expression of its target gene PTEN, resulting in the inhibition of AKT‐mTOR pathways. Besides, DHEA and EPEA treatment induced phosphorylation of Bcl‐2 promoting its dissociation from beclin‐1 which resulted in autophagy induction. We also observed an increase of beclin‐1 and microtubule‐associated protein 1 light chain 3 expression along with an enhanced autophagosomes formation as revealed by mono‐dansyl‐cadaverine staining. Finally, we demonstrated the involvement of PPARγ in DHEA‐ and EPEA‐induced autophagy by using siRNA technology and a selective inhibitor. In summary, our data show that the two omega‐3 ethanolamides exert anti‐proliferative effects by inducing autophagy in breast cancer cells highlighting their potential use as breast cancer preventive and/or therapeutic agents. J. Cell. Physiol. 228: 1314–1322, 2013.

Resveratrol, through NF-Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor α gene expression via p38MAPK/CK2 signaling in human breast cancer cells

Agents to counteract acquired resistance to hormonal therapy for breast cancer would substantially enhance the long‐term benefits of hormonal therapy. In the present study, we demonstrate how resveratrol (Res) inhibits human breast cancer cell proliferation, including MCF‐7 tamoxifen‐resistant cells (IC50 values for viability were in the 30–45 μM range). We show that Res, through p38MAPK phosphorylation, causes induction of p53, which recruits at the estrogen receptor α (ERα) proximal promoter, leading to an inhibition of ERα expression in terms of mRNA and protein content. These events appear specifically p53 dependent, since they are drastically abrogated with p53‐targeting siRNA. Coimmunoprecipitation assay showed specific interaction between p53, the Sin3A corepressor, and histone deacetylase 1 (HDAC1), which was phosphorylated. The enhancement of the tripartite complex p53/Sin3A/HDAC1, together with NF‐Y on Res treatment, was confirmed by chromatin immunoprecipitation analyses, with a concomitant release of Sp1 and RNA polymerase II, thereby inhibiting the cell transcriptional machinery. The persistence of such effects in MCF‐7 tamoxifen‐resistant cells at a higher extent than parental MCF‐7 cells addresses how Res may be considered a useful pharmacological tool to be exploited in the adjuvant settings for treatment of breast cancer developing hormonal resistance.—De Amicis, F., Giordano, F., Vivacqua, A., Pellegrino, M., Panno, M. L., Tramontano, D., Fuqua, S. A. W., Andò, S. Resveratrol, through NF‐Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor α gene expression via p38MAPK/CK2 signaling in human breast cancer cells. FASEB J. 25, 3695–3707 (2011). www.fasebj.org

Estrogen receptor beta as a novel target of androgen receptor action in breast cancer cell lines

IntroductionThe two isoforms of estrogen receptor (ER) alpha and beta play opposite roles in regulating proliferation and differentiation of breast cancers, with ER-alpha mediating mitogenic effects and ER-beta acting as a tumor suppressor. Emerging data have reported that androgen receptor (AR) activation inhibits ER-positive breast cancer progression mainly by antagonizing ER-alpha signaling. However, to date no studies have specifically evaluated a potential involvement of ER-beta in the inhibitory effects of androgens.MethodsER-beta expression was examined in human breast cancer cell lines using real-time PCR, Western blotting and small interfering RNA (siRNA) assays. Mutagenesis studies, electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were performed to assess the effects of mibolerone/AR on ER-beta promoter activity and binding.ResultsIn this study, we demonstrate that mibolerone, a synthetic androgen ligand, up-regulates ER-beta mRNA and protein levels in ER-positive breast cancer cells. Transient transfection experiments, using a vector containing the human ER-beta promoter region, show that mibolerone increases basal ER-beta promoter activity. Site-directed mutagenesis and deletion analysis reveal that an androgen response element (ARE), TGTTCT motif located at positions −383 and −377, is critical for mibolerone-induced ER-beta up-regulation in breast cancer cells. This occurs through an increased recruitment of AR to the ARE site within the ER-beta promoter region, along with an enhanced occupancy of RNA polymerase II. Finally, silencing of ER-beta gene expression by RNA interference is able to partially reverse the effects of mibolerone on cell proliferation, p21 and cyclin D1 expression.ConclusionsCollectively, these data provide evidence for a novel mechanism by which activated AR, through an up-regulation of ER-beta gene expression, inhibits breast cancer cell growth.

Estrogen receptor beta (ERβ) produces autophagy and necroptosis in human seminoma cell line through the binding of the Sp1 on the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) promoter gene.

Testicular germ cell tumors are the most common tumor in male and the least studied. We focused on human seminoma using the TCAM2 cell line. Through ERβ, 10 nM estradiol (E2) was able to induce PTEN gene expression and promoter transactivation. Transient transfections, ChIP and EMSA assays evidenced the 5′-flanking region of PTEN gene promoter E2-responsive. The ERβ binding to the Sp1 on PTEN promoter decreased cell survival. The presence of ERβ or PTEN is necessary to induce the loss of cell survival upon E2, addressing their cooperation in this action. pAKT and AKT expression decreased under E2 and DPN, while known apoptotic markers appeared to be unchanged. The PI3K/AKT pathway inhibition also leads to autophagy: E2 and DPN enhanced the expression of autophagy-related markers such as PI3III, Beclin 1, AMBRA and UVRAG. MDC and TEM assays confirmed E2-induced autophagy. The absence of DNA fragmentation, caspase 9 and PARP1 cleavages suggested that necroptosis and/or parthanatos may occur. FACS analysis, LDH assay and RIP1 expression attested this hypothesis. Our study reveals a unique mechanism through which ERβ/PTEN signaling induces cell death in TCAM2 by autophagy and necroptosis. These data, supporting estrogen-dependency of human seminoma, propose ERβ ligands for therapeutic use in the treatment of this pathological condition.

Leptin increases HER2 protein levels through a STAT3-mediated up-regulation of Hsp90 in breast cancer cells

Obesity condition confers risks to breast cancer development and progression, and several reports indicate that the adipokine leptin, whose synthesis and plasma levels increase with obesity, might play an important role in modulating breast cancer cell phenotype. Functional crosstalk occurring between leptin and different signaling molecules contribute to breast carcinogenesis.

In vitro mechanism for downregulation of ER-α expression by epigallocatechin gallate in ER+/PR+ human breast cancer cells

SCOPE Exposure of the breast to estrogens and other sex hormones is an important cancer risk factor and estrogen receptor downregulators are attracting significant clinical interest. Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, has gained considerable attention for its antitumor properties. Here we aimed to investigate the molecular mechanisms through which EGCG regulates ER-α expression in ER+ PR+ breast cancer cells. MATERIAL AND METHODS Western blotting analysis, real-time PCR, and transient transfections of deletion fragments of the ER-α gene promoter show that EGCG downregulates ER-α protein, mRNA, and gene promoter activity with a concomitant reduction of ER-α genomic and nongenomic signal. These events occur through p38(MAPK) /CK2 activation, causing the release from Hsp90 of progesterone receptor B (PR-B) and its consequent nuclear translocation as evidenced by immunofluorescence studies. EMSA, and ChIP assay reveal that, upon EGCG treatment, PR-B is recruited at the half-PRE site on ER-α promoter. This is concomitant with the formation of a corepressor complex containing NCoR and HDAC1 while RNA polymerase II is displaced. The events are crucially mediated by PR-B isoform, since they are abrogated with PR-B siRNA. CONCLUSION Our data provide evidence for a mechanism by which EGCG downregulates ER-α and explains the inhibitory action of EGCG on the proliferation of ER+ PR+ cancer cells tested. We suggest that the EGCG/PR-B signaling should be further exploited for clinical approach.

Epigallocatechin gallate inhibits growth and epithelial-to-mesenchymal transition in human thyroid carcinoma cell lines

Well‐differentiated papillary and follicular thyroid carcinoma are the most frequent types of thyroid cancer and the prognosis is generally favorable however, a number of patients develops recurrences. Epigallocatechin‐3‐gallate (EGCG), a major catechin in green tea, was shown to possess remarkable therapeutic potential against various types of human cancers, although data on thyroid cancer cells are still lacking. The aim of this study was to investigate the effect of EGCG on the proliferation and motility of human thyroid papillary (FB‐2) and follicular (WRO) carcinoma cell lines. Our results demonstrate that EGCG (10, 40, 60 μM) treatment inhibited the growth of FB‐2 and WRO cells in a dose‐dependent manner. These changes were associated with reduced cyclin D1, increased p21 and p53 expression. Furthermore, EGCG suppressed phosphorylation of AKT and ERK1/2. In addition EGCG treatment results in reduction of cell motility and migration. Changes in motility and migration in FB‐2 were associated with modulation in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. After 24 h EGCG caused an increase of the E‐cadherin expression and a concomitant decrease of SNAIL, ZEB and the basic helix–loop–helix transcription factor TWIST. Besides expression of Vimentin, N‐cadherin and α5‐integrin was down‐regulated. These data well correlate with a reduction of MMP9 activity as evidenced by gelatin zymography. Our findings support the inhibitory role of EGCG on thyroid cancer cell proliferation and motility with concomitant loss of epithelial‐to‐mesenchymal cell transition markers. J. Cell. Physiol. 228: 2054–2062, 2013.