Saveria Aquila

Estrogen Receptor α Binds to Peroxisome Proliferator–Activated Receptor Response Element and Negatively Interferes with Peroxisome Proliferator–Activated Receptor γ Signaling in Breast Cancer Cells

Purpose: The molecular mechanisms involved in the repressive effects exerted by estrogen receptors (ER) on peroxisome proliferator–activated receptor (PPAR) γ–mediated transcriptional activity remain to be elucidated. The aim of the present study was to provide new insight into the crosstalk between ERα and PPARγ pathways in breast cancer cells. Experimental Design: Using MCF7 and HeLa cells as model systems, we did transient transfections and electrophoretic mobility shift assay and chromatin immunoprecipitation studies to evaluate the ability of ERα to influence PPAR response element–mediated transcription. A possible direct interaction between ERα and PPARγ was ascertained by coimmunoprecipitation assay, whereas their modulatory role in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway was evaluated by determining PI3K activity and AKT phosphorylation. As a biological counterpart, we investigated the growth response to the cognate ligands of both receptors in hormone-dependent MCF7 breast cancer cells. Results: Our data show for the first time that ERα binds to PPAR response element and represses its transactivation. Moreover, we have documented the physical and functional interactions of ERα and PPARγ, which also involve the p85 regulatory subunit of PI3K. Interestingly, ERα and PPARγ pathways have an opposite effect on the regulation of the PI3K/AKT transduction cascade, explaining, at least in part, the divergent response exerted by the cognate ligands 17β-estradiol and BRL49653 on MCF7 cell proliferation. Conclusion: ERα physically associates with PPARγ and functionally interferes with PPARγ signaling. This crosstalk could be taken into account in setting new pharmacologic strategies for breast cancer disease.

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.

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.

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.

Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism

BackgroundA wider biological role of 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, in tissues not primarily related to mineral metabolism was suggested. Recently, we evidenced the ultrastructural localization the 1,25(OH)2D3 receptor in the human sperm. However, the 1,25(OH)2D3 action in human male reproduction has not yet been clarified.Methods and ResultsBy RT-PCR, Western blot and Immunofluorescence techniques, we demonstrated that human sperm expresses the 1,25(OH)2D3 receptor (VDR). Besides, 25(OH)D3-1 alpha-hydroxylase, evidenced by Western blot analysis, indicated that in sperm 1,25(OH)2D3 is locally produced, highlighting the potential for autocrine-paracrine responses. 1,25(OH)2D3 through VDR, increased intracellular Ca2+ levels, motility and acrosin activity revealing an unexpected significance of this hormone in the acquisition of fertilizing ability. In sperm, 1,25(OH)2D3 through VDR, reduces triglycerides content concomitantly to the increase of lipase activity. Rapid responses stimulated by 1,25(OH)2D3 have been observed on Akt, MAPK and GSK3 implying that this secosteroid is involved in different sperm signalling pathways.ConclusionOur data extended the role of 1,25(OH)2D3 beyond its conventional physiological actions, paving the way for novel therapeutic opportunities in the treatment of the male reproduction disorders.

Human sperm anatomy: ultrastructural localization of 1α,25-dihydroxyvitamin D3 receptor and its possible role in the human male gamete

Previous studies have suggested that 1α,25‐dihydroxyvitamin D3[1,25(OH)2D3] has a role in reproductive function. Gonadal insufficiencies were observed as a result of 1,25(OH)2D3 deficiency and in 1,25(OH)2D3 receptor (VDR) null mutant mice. To study human sperm anatomy at the molecular level, we first evaluated the ultrastructural localization of VDR by immunogold electron microscopy using a monoclonal antibody against amino acids 344–424 of human VDR, in normozoospermic samples. Intriguingly, VDR was associated predominantly with the cell nucleus. In fact, it is known that VDR is a transcription factor, and that in vitamin‐D‐depleted animals, VDR is largely localized in the cell nucleus. To assess the significance of VDR in the male gamete, we investigated the role of 1,25(OH)2D3/VDR in sperm survival and capacitation. Our results revealed that the action of 1,25(OH)2D3 depended on its concentration because although lower doses induced cholesterol efflux, protein phosphorylation and sperm survival, a higher concentration seemed to be ineffective or did not show an increased effect. These results increase our knowledge of human sperm anatomy at the molecular level and suggest that 1,25(OH)2D3/VDR may have an important role in sperm survival and the acquisition of fertilizing ability.

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 and leptin receptor in pig spermatozoa: evidence of their involvement in sperm capacitation and survival

Several studies have recently investigated the role of leptin, the adipocyte-secreted hormone, in the growth and reproduction of rodents, humans, and domestic animals. The present study was designed to explore the expression of leptin and its receptor in pig spermatozoa. Successful Western blot evidenced a 16 kDa band for leptin and six isoforms, ranging from 120 to 40 kDa, for the leptin receptor. Both leptin and leptin receptor were interestingly located at sperm acrosomal level, suggesting their involvement in the oocyte fertilization events. In fact, both capacitation indexes and acrosin activity were enhanced by leptin, and these effects were reduced by the anti-leptin receptor antibody. Afterwards, we investigated the main transduction pathways regulated by the hormone. Our results showed that, in pig sperm, leptin can trigger the signal transducer and activator of transcription 3, a classical component of cytokine signal transduction pathways, whose expression has not been previously reported in male gamete; in addition it was found constitutively activated. Besides, leptin was able to induce the activation of phosphatidylinositol phosphate kinase 3 and MAP kinase pathways as well as of BCL2, a known antiapoptotic protein. These data address to a role of leptin and its receptor on pig sperm survival. The presence of leptin and its receptor in pig sperm suggests that they, through an autocrine short loop, may induce signal transduction and molecular changes associated with sperm capacitation and survival.

The Role of Oxidative Stress and Autophagy in Atherosclerosis

Atherosclerosis is a multifactorial, multistep disorder of large- and medium-sized arteries involving, in addition to age, gender and menopausal status, a complex interplay between lifestyle and genetic risk factors. Atherosclerosis usually begins with the diffusion and retention of atherogenic lipoproteins into the subendothelial space of the artery wall where they become oxidized by local enzymes and accumulate, leading to the formation of a cushion called atheroma or atheromatous or fibrofatty plaque, composed of a mixture of macrophages, lymphocytes, smooth muscle cells (SMCs), cholesterol cleft, necrotic debris, and lipid-laden foam cells. The pathogenesis of atherosclerosis still remains incompletely understood but emerging evidence suggests that it may involve multiple cellular events, including endothelial cell (EC) dysfunction, inflammation, proliferation of vascular SMCs, matrix (ECM) alteration, and neovascularization. Actually, a growing body of evidence indicates that autophagy along with the chronic and acute overproduction of reactive oxygen species (ROS) is integral to the development and progression of the disease and may represent fruitful avenues for biological investigation and for the identification of new therapeutic targets. In this review, we give an overview of ROS and autophagy in atherosclerosis as background to understand their potential role in this vascular disease.

Peroxisome proliferator‐activated receptor (PPAR)γ is expressed by human spermatozoa: Its potential role on the sperm physiology

The peroxisome proliferation‐activated receptor gamma (PPARγ) is mainly expressed in the adipose tissue and integrates the control of energy, lipid, and glucose homeostasis. The present study, by means of RT‐PCR, Western blot, and immunofluorescence techniques, demonstrates that human sperm express the PPARγ. The functionality of the receptor was evidenced by 15‐deoxy‐12,14‐prostaglandin J2 (PGJ2) and rosiglitazone (BRL) PPARγ‐agonists that were tested on capacitation, acrosome reaction, and motility. Both treatments also increase AKT phosphorylations and influence glucose and lipid metabolism in sperm. The specificity of PGJ2 and BRL effects through PPARγ on human sperm was confirmed by an irreversible PPARγ antagonist, GW9662. Our findings provide evidence that human sperm express a functional PPARγ whose activation influences sperm physiology. In conclusion, the presence of PPARγ in male gamete broadens the field of action of this nuclear receptor, bringing us to look towards sperm as an endocrine mobile unit independent of the systemic regulation. J. Cell. Physiol. 209: 977–986, 2006.