A Novel Functional Domain of Tab2 Involved in the Interaction with Estrogen Receptor Alpha in Breast Cancer Cells.

Tab2, originally described as a component of the inflammatory pathway, has been implicated in phenomena of gene de-repression in several contexts, due to its ability to interact with the NCoR corepressor. Tab2 interacts also with steroid receptors and dismisses NCoR from antagonist-bound Estrogen and Androgen Receptors on gene regulatory regions, thus modifying their transcriptional activity and leading to pharmacological resistance in breast and prostate cancer cells. We demonstrated previously that either Tab2 knock-down, or a peptide mimicking the Estrogen Receptor alpha domain interacting with Tab2, restore the antiproliferative response to Tamoxifen in Tamoxifen-resistant breast cancer cells. In this work, we map the domain of Tab2 responsible of Estrogen Receptor alpha interaction. First, using both co-immunoprecipitation and pull-down with recombinant proteins, we found that the central part of Tab2 is primarily responsible for this interaction, and that this region also interacts with Androgen Receptor. Then, we narrowed down the essential interaction region by means of competition assays using recombinant protein pull-down. The interaction motif was finally identified as a small region adjacent to, but not overlapping, the Tab2 MEKK1 phosphorylation sites. A synthetic peptide mimicking this motif efficiently displaced Tab2 from interacting with recombinant Estrogen Receptor alpha in vitro, prompting us to test its efficacy using derivatives of the MCF7 breast carcinoma cell lines that are spontaneously resistant to Tamoxifen. Indeed, we observed that this mimic peptide, made cell-permeable by addition of the TAT minimal carrier domain, reduced the growth of Tamoxifen-resistant MCF7 cells in the presence of Tamoxifen. These data indicate a novel functional domain of the Tab2 protein with potential application in drug design.

E2 Regulates Epigenetic Signature on Neuroglobin Enhancer-Promoter in Neuronal Cells.

Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one of the estrogen target genes involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERß) that bind to specific regulatory genomic regions. We focused our attention on 17ß-estradiol (E2)-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2). Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ERα) binding suggesting that ERα may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERα binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative diseases.

Luminal long non-coding RNAs regulated by estrogen receptor alpha in a ligand-independent manner show functional roles in breast cancer.

Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells.

Genome-wide activity of unliganded estrogen receptor-α in breast cancer cells.

Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.

Genomic lens on neuroglobin transcription.

Neuroglobin is a brain globin with neuroprotective effects against ischemia and related pathological processes, acting as O2 sensor and antiapoptotic pathway transducer. Here, we survey data on neuroanatomical coexpression of transcription factors, epigenetic signature and predictive transcription factor binding sites at the neuroglobin gene locus to find hints of pathways to neuroglobin transcriptional regulation. These data provide a glimpse of how neuroglobin expression may translate into neuronal diversity and function, as well as disease.

Membrane lipid rafts coordinate estrogen-dependent signaling in human platelets.

The impact of estrogens on the viability of cardiovascular system and their ability to regulate platelet function is still an open and debated question. We have previously shown that estrogen is able to significantly potentiate the aggregation induced by low doses of thrombin and to initiate a rapid and reversible signaling pathway mediated by ERbeta-directed activation of the tyrosine kinases Src and Pyk2 at the level of the plasma membrane. Lipid rafts are critical, cholesterol-enriched membrane domains, which play a major role in blood platelet activation processes. In this work, we investigated the role of lipid rafts in 17beta-estradiol signaling in human platelets. We observed that membrane rafts were essential for both 17beta-estradiol-dependent potentiation of platelet aggregation induced by subthreshold concentrations of thrombin and 17beta-estradiol-induced phosphorylation of Src. 17beta-estradiol caused the reversible translocation of ERbeta to the raft fractions and promoted the rapid and transient recruitment to, and activation within the membrane raft domains of the tyrosine kinases Src and Pyk2. The raft integrity was essential with this respect, as these effects of 17beta-estradiol were completely inhibited by cholesterol depletion. This paper provides evidence for the first time that membrane lipid rafts coordinate estrogen signaling in human platelets.

Nongenomic effects of 17beta-estradiol in human platelets: potentiation of thrombin-induced aggregation through estrogen receptor beta and Src kinase.

The impact of estrogens on the cardiovascular system and their ability to regulate platelet function are matters of controversy. The recent finding that estrogen receptors are expressed in human platelets renders these cells an excellent model for studying the nongenomic effects of these hormones. In this work, we investigated 17beta-estradiol-dependent signaling in platelets from adult healthy men. 17beta-estradiol caused the rapid phosphorylation of the tyrosine kinases Src and Pyk2 and the formation of a signaling complex, which included Src, Pyk2, and the phosphatidylinositol 3-kinase. Both these events were dependent on estrogen receptor beta engagement. We found that estrogen receptor beta was membrane-associated in platelets. On treatment with 17beta-estradiol, Src and Pyk2 activation occurred in the membrane fraction but not in the cytosol. In contrast, no significant activation of phosphatidylinositol 3-kinase was detected in estrogen-treated platelets. 17beta-estradiol did not induce any platelet response directly, but it strongly potentiated the activation of integrin alpha(IIb)beta3 and the platelet aggregation induced by subthreshold concentrations of thrombin. These effects were dependent on estrogen receptor beta recruitment and were associated with a strong synergistic effect with thrombin on Src activation. Taken together, these results indicate that 17beta-estradiol can modulate platelet function by exercising a proaggregating role.

A role for p38 MAP kinase in platelet activation by von Willebrand factor.

Platelet activation induced by von Willebrand factor (VWF) binding to the membrane GPIb-IX-V receptor involves multiple signal transduction pathways. Among these, recruitment and activation of the FCgammaRIIA and stimulation of phospholipase A2 represent independent events equally essential to support a complete platelet response. Phospholipase A2 is activated by calcium and by phosphorylation through MAP kinases. In this work, we found that VWF stimulated the rapid and sustained phosphorylation of p38 MAP kinase (p38MAPK). In vitro kinase assay revealed that VWF-stimulated phosphorylation of p38MAPK was associated with increased kinase activity. Binding of VWF to GPIb-IX-V, but not to integrin alphaIibbeta3, was required to support phosphorylation of p38MAPK. Neither the blockade of the membrane FCgammaRIIA by a specific monoclonal antibody or the prevention of thromboxane A(2) synthesis by cyclooxygenase inhibitors affected VWF-induced p38MAPK activation. How-ever, phosphorylation of p38MAPK was prevented by the tyro-sine kinase Syk inhibitor piceatannol. Treatment of platelets with the p38MAPK inhibitor SB203580 totally prevented VWF-stimulated platelet aggregation. Moreover, release of arachidonic acid induced by VWF was strongly impaired by inhibition of p38MAPK. We also found that VWF induced phosphorylation of cytosolic phospholipase A(2), and that this process was prevented by the p38MAPK inhibitor SB203580. These results demon-strate that p38MAPK is a key element in the FCgammaRIIA-independent pathway for VWF-induced platelet activation, and is involved in the stimulation of phospholipase A(2) and arachidonic acid release.