The adhesion protein E-cadherin plays a central part in the process of epithelial morphogenesis. Expression of this protein is downregulated during the acquisition of metastatic potential at late stages of epithelial tumour progression. There is evidence for a transcriptional blockage of E-cadherin gene expression in this process. Here we show that the transcription factor Snail, which is expressed by fibroblasts and some E-cadherin-negative epithelial tumour cell lines, binds to three E-boxes present in the human E-cadherin promoter and represses transcription of E-cadherin. Inhibition of Snail function in epithelial cancer cell lines lacking E-cadherin protein restores the expression of the E-cadherin gene.
Epithelial-mesenchymal transition (EMT) is a cellular process during which epithelial cells acquire mesen chymal phenotypes and behaviour following the down regulation of epithelial features. EMT is triggered in response to signals that cells receive from their micro environment. The epithelial state of the cells in which EMT is initiated is characterized by stable epithelial cell-cell junctions, apical-basal polarity and interac tions with basement membrane. During EMT, changes in gene expression and posttranslational regulation mechanisms lead to the repression of these epithelial characteristics and the acquisition of mesenchymal char acteristics. Cells then display fibroblastlike morphol ogy and cytoarchitecture, as well as increased migratory capacity. Furthermore, these now migratory cells often acquire invasive properties (Fig. 1). EMT was first described by researchers studying early embryogenesis as a programme with welldefined cellular features 1,2. It is now widely accepted that EMT occurs normally during early embryonic development, to enable a variety of morphogenetic events, as well as later in development and during wound healing in adults.
The β-catenin signaling pathway is deregulated in nearly all colon cancers. Nonhypercalcemic vitamin D3 (1α,25-dehydroxyvitamin D3) analogues are candidate drugs to treat this neoplasia. We show that these compounds promote the differentiation of human colon carcinoma SW480 cells expressing vitamin D receptors (VDRs) (SW480-ADH) but not that of a malignant subline (SW480-R) or metastasic derivative (SW620) cells lacking VDR. 1α,25(OH)2D3 induced the expression of E-cadherin and other adhesion proteins (occludin, Zonula occludens [ZO]-1, ZO-2, vinculin) and promoted the translocation of β-catenin, plakoglobin, and ZO-1 from the nucleus to the plasma membrane. Ligand-activated VDR competed with T cell transcription factor (TCF)-4 for β-catenin binding. Accordingly, 1α,25(OH)2D3 repressed β-catenin–TCF-4 transcriptional activity. Moreover, VDR activity was enhanced by ectopic β-catenin and reduced by TCF-4. Also, 1α,25(OH)2D3 inhibited expression of β-catenin–TCF-4-responsive genes, c-myc, peroxisome proliferator-activated receptor δ, Tcf-1, and CD44, whereas it induced expression of ZO-1. Our results show that 1α,25(OH)2D3 induces E-cadherin and modulates β-catenin–TCF-4 target genes in a manner opposite to that of β-catenin, promoting the differentiation of colon carcinoma cells.
Epithelial-mesenchymal transitions (EMT) are essential for organogenesis and triggered in carcinoma progression into an invasive state1. Transforming growth factor-β (TGF-β) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT2-5, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT6, 7. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight junction protein, and E-cadherin during TGF-β-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited the repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a novel mechanism of gene repression during EMT.
Expression of Snail1 in epithelial cells triggers an epithelial-mesenchymal transition (EMT). Here, we demonstrate that the synthesis of Zeb2, a transcriptional repressor of E-cadherin, is up-regulated after Snail1-induced EMT. Snail1 does not affect the synthesis of Zeb2 mRNA, but prevents the processing of a large intron located in its 5-untranslated region (UTR). This intron contains an internal ribosome entry site (IRES) necessary for the expression of Zeb2. Maintenance of 5-UTR Zeb2 intron is dependent on the expression of a natural antisense transcript (NAT) that overlaps the 5 splice site in the intron. Ectopic overexpression of this NAT in epithelial cells prevents splicing of the Zeb2 5-UTR, increases the levels of Zeb2 protein, and consequently down-regulates E-cadherin mRNA and protein. The relevance of these results is demonstrated by the strong association between NAT presence and conservation of the 5-UTR intron in cells that have undergone EMT or in human tumors with low E-cadherin expression. Therefore, the results presented in this article reveal the existence of a NAT capable of activating Zeb2 expression, explain the mechanism involved in this activation, and demonstrate that this NAT regulates E-cadherin expression.
Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120-and -catenins. We have examined the role of this modification in these proteins in the control of -catenin/E-cadherin binding using in vitro assays with recombinant proteins. Recombinant pp60 c-src efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for -catenin and p120-catenin, respectively. pp60 c-src phosphorylation had opposing effects on the affinities of -catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of -catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of -catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) -catenin for E-cadherin. The phosphorylated Tyr residues were identified as Tyr-86 and Tyr-654. Experiments using point mutants in these two residues indicated that, although Tyr-86 was a better substrate for pp60 c-src , only modification of Tyr-654 was relevant for the interaction with E-cadherin. Transient transfections of different mutants demonstrated that Tyr-654 is phosphorylated in conditions in which adherens junctions are disrupted and evidenced that binding of -catenin to E-cadherin in vivo is controlled by phosphorylation of -catenin Tyr-654.
E-cadherin protein plays a key role in the establishment and maintenance of adherent junctions. Recent evidence implicates the transcription factor Snail in the blockage of E-cadherin expression in fibroblasts and some epithelial tumor cells through direct binding to three E-boxes in the E-cadherin promoter. Transfection of Snail into epithelial cells leads to a more fibroblastic phenotype. Cells expressing Snail presented a scattered flattened phenotype with low intercellular contacts. Other epithelial markers like Cytokeratin 18 or MUC1 were also repressed. The effects of Snail on MUC1 transcription were mediated by two E-boxes present in the proximal promoter. Snail also induced expression of the mesenchymal markers fibronectin and LEF1 and the transcription repressor ZEB1. ZEB1 and Snail had a similar pattern of expression in epithelial cell lines, and both were induced by overexpression of ILK1, a kinase that causes the loss of E-cadherin and the acquisition of a fibroblastic phenotype. Snail overexpression in several cell lines raised ZEB1 RNA levels and increased the activity of ZEB1 promoter. ZEB1 could also repress E-cadherin and MUC1 promoters but less strongly than Snail. However, since ZEB1 expression persisted after Snail was down-regulated, ZEB1 may regulate epithelial genes in several tumor cell lines.
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