TFCP2c/LSF/LBP-1c is required for Snail1-induced fibronectin gene expression.

Fibronectins are cell-secreted glycoproteins that modulate cell attachment, spreading, migration, morphology, differentiation and oncogenic transformation. Fibronectin expression is activated during EMT (epithelial-mesenchymal transition) and is a hallmark of mesenchymal cells. It is shown in the present study that a transcription factor previously unrelated with EMT, TFCP2c/LSF/LBP-1c, was translocated to the nucleus and bound to the fibronectin promoter upon EMT induction by Snail1. Consequently, the interference of TFCP2c/LSF/LBP-1c's activity prevented fibronectin expression. Moreover, TFCP2c/LSF/LBP-1c was detected in nuclei of embryonic dermal mesenchymal cells adjacent to the hair bud, a cell population that expresses endogenous nuclear Snail1 and fibronectin. Therefore we indicate a new molecular role for TFCP2c/LSF/LBP-1c in fibronectin expression.

Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition.

We report that the activity of glycogen synthase kinase-3 (GSK-3) is necessary for the maintenance of the epithelial architecture. Pharmacological inhibition of its activity or reducing its expression using small interfering RNAs in normal breast and skin epithelial cells results in a reduction of E-cadherin expression and a more mesenchymal morphology, both of which are features associated with an epithelial-mesenchymal transition (EMT). Importantly, GSK-3 inhibition also stimulates the transcription of Snail, a repressor of E-cadherin and an inducer of the EMT. We identify NFkappaB as a transcription factor inhibited by GSK-3 in epithelial cells that is relevant for Snail expression. These findings indicate that epithelial cells must sustain activation of a specific kinase to impede a mesenchymal transition.

Snail1 transcriptional repressor binds to its own promoter and controls its expression.

The product of Snail1 gene is a transcriptional repressor of E-cadherin expression and an inductor of the epithelial-mesenchymal transition in several epithelial tumour cell lines. Transcription of Snail1 is induced when epithelial cells are forced to acquire a mesenchymal phenotype. In this work we demonstrate that Snail1 protein limits its own expression: Snail1 binds to an E-box present in its promoter (at -146 with respect to the transcription start) and represses its activity. Therefore, mutation of the E-box increases Snail1 transcription in epithelial and mesenchymal cells. Evidence of binding of ectopic or endogenous Snail1 to its own promoter was obtained by chromatin immunoprecipitation (ChIP) experiments. Studies performed expressing different forms of Snail1 under the control of its own promoter demonstrate that disruption of the regulatory loop increases the cellular levels of Snail protein. These results indicate that expression of Snail1 gene can be regulated by its product and evidence the existence of a fine-tuning feed-back mechanism of regulation of Snail1 transcription.

Tyrosine phosphorylation of plakoglobin causes contrary effects on its association with desmosomes and adherens junction components and modulates beta-catenin-mediated transcription.

Plakoglobin is a protein closely related to beta-catenin that links desmosomal cadherins to intermediate filaments. Plakoglobin can also substitute for beta-catenin in adherens junctions, providing a connection between E-cadherin and alpha-catenin. Association of beta-catenin with E-cadherin and alpha-catenin is regulated by phosphorylation of specific tyrosine residues; modification of beta-catenin Tyr654 and Tyr142 decreases binding to E-cadherin and alpha-catenin, respectively. We show here that plakoglobin can also be phosphorylated on tyrosine residues, but unlike beta-catenin, this modification is not always associated with disrupted association with junctional components. Protein tyrosine kinases present distinct specificities on beta-catenin and plakoglobin, and phosphorylation of beta-catenin-equivalent Tyr residues of plakoglobin affects its interaction with components of desmosomes or adherens junctions differently. For instance, Src, which mainly phosphorylates Tyr86 in beta-catenin, modifies Tyr643 in plakoglobin, decreasing the interaction with E-cadherin and alpha-catenin and increasing the interaction with the alpha-catenin-equivalent protein in desmosomes, desmoplakin. The tyrosine kinase Fer, which modifies beta-catenin Tyr142, lessening its association with alpha-catenin, phosphorylates plakoglobin Tyr549 and exerts the contrary effect: it raises the binding of plakoglobin to alpha-catenin. These results suggest that tyrosine kinases like Src or Fer modulate desmosomes and adherens junctions differently. Our results also indicate that phosphorylation of Tyr549 and the increased binding of plakoglobin to components of adherens junctions can contribute to the upregulation of the transcriptional activity of the beta-catenin-Tcf-4 complex observed in many epithelial tumor cells.

Phosphorylation regulates the subcellular location and activity of the snail transcriptional repressor.

The Snail gene product is a transcriptional repressor of E-cadherin expression and an inducer of the epithelial-to-mesenchymal transition in several epithelial tumor cell lines. This report presents data indicating that Snail function is controlled by its intracellular location. The cytosolic distribution of Snail depended on export from the nucleus by a CRM1-dependent mechanism, and a nuclear export sequence (NES) was located in the regulatory domain of this protein. Export of Snail was controlled by phosphorylation of a Ser-rich sequence adjacent to this NES. Modification of this sequence released the restriction created by the zinc finger domain and allowed nuclear export of the protein. The phosphorylation and subcellular distribution of Snail are controlled by cell attachment to the extracellular matrix. Suspended cells presented higher levels of phosphorylated Snail and an augmented extranuclear localization with respect to cells attached to the plate. These findings show the existence in tumor cells of an effective and fine-tuning nontranscriptional mechanism of regulation of Snail activity dependent on the extracellular environment.

Snail1 is required for the maintenance of the pancreatic acinar phenotype.

The Snail1 transcriptional factor is required for correct embryonic development, yet its expression in adult animals is very limited and its functional roles are not evident. We have now conditionally inactivated Snail1 in adult mice and analyzed the phenotype of these animals. Snail1 ablation rapidly altered pancreas structure: one month after Snail1 depletion, acinar cells were markedly depleted, and pancreas accumulated adipose tissue. Snail1 expression was not detected in the epithelium but was in pancreatic mesenchymal cells (PMCs). Snail1 ablation in cultured PMCs downregulated the expression of several ß-catenin/Tcf-4 target genes, modified the secretome of these cells and decreased their ability to maintain acinar markers in cultured pancreas cells. Finally, Snail1 deficiency modified the phenotype of pancreatic tumors generated in transgenic mice expressing c-myc under the control of the elastase promoter. Specifically, Snail1 depletion did not significantly alter the size of the tumors but accelerated acinar-ductal metaplasia. These results demonstrate that Snail1 is expressed in PMCs and plays a pivotal role in maintaining acinar cells within the pancreas in normal and pathological conditions.

Regulation of Snail transcription during epithelial to mesenchymal transition of tumor cells.

Expression of Snail transcriptional factor is a determinant in the acquisition of a mesenchymal phenotype by epithelial tumor cells. However, the regulation of the transcription of this gene is still unknown. We describe here the characterization of a human SNAIL promoter that contains the initiation of transcription and regulates the expression of this gene in tumor cells. This promoter was activated in cell lines in response to agents that induce Snail transcription and the mesenchymal phenotype, as addition of the phorbol ester PMA or overexpression of integrin-linked kinase (ILK) or oncogenes such as Ha-ras or v-Akt. Although other regions of the promoter were required for a complete stimulation by Akt or ILK, a minimal fragment (-78/+59) was sufficient to maintain the mesenchymal specificity. Activity of this minimal promoter and SNAIL RNA levels were dependent on ERK signaling pathway. NFkappaB/p65 also stimulated SNAIL transcription through a region located immediately upstream the minimal promoter, between -194 and -78. These results indicate that Snail transcription is driven by signaling pathways known to induce epithelial to mesenchymal transition, reinforcing the role of Snail in this process.

Snail1 expression is required for sarcomagenesis.

Snail1 transcriptional repressor is a major inducer of epithelial-to mesenchymal transition but is very limitedly expressed in adult animals. We have previously demonstrated that Snail1 is required for the maintenance of mesenchymal stem cells (MSCs), preventing their premature differentiation. Now, we show that Snail1 controls the tumorigenic properties of mesenchymal cells. Increased Snail1 expression provides tumorigenic capabilities to fibroblastic cells; on the contrary, Snail1 depletion decreases tumor growth. Genetic depletion of Snail1 in MSCs that are deficient in p53 tumor suppressor downregulates MSC markers and prevents the capability of these cells to originate sarcomas in immunodeficient SCID mice. Notably, an analysis of human sarcomas shows that, contrarily to epithelial tumors, these neoplasms display high Snail1 expression. This is particularly clear for undifferentiated tumors, which are associated with poor outcome. Together, our results indicate a role for Snail1 in the generation of sarcomas.

Snail1 protein in the stroma as a new putative prognosis marker for colon tumours.

Over-expression of Snail1 gene transcriptional repressor promotes an epithelial-to-mesenchymal transition in epithelial tumour cell lines. Expression of Snail1 RNA has been associated to the pathogenesis of a number of malignancies; however, the lack of good monoclonal antibodies against this protein has precluded a definitive analysis of Snail1 protein. In this study, we aimed to determine the expression of this transcriptional factor in colorectal tumours. Using a Snail1 well-characterized monoclonal antibody developed in our laboratories we have analyzed by immunohistochemistry a cohort of 162 human colorectal tumours. Ninety tumours (56%) showed nuclear expression in the tumoral tissue and the adjacent stroma; in 34 (21%), Snail1 was detected just in the stroma, whereas in only 4 the expression of Snail1 was detected in the tumoral tissue and the stroma was negative. No correlation was found between the presence of Snail1 in the tumour and tumour stage; however, a trend (p = 0.054) was detected when the expression of this factor in the stroma was considered. Snail1 immunoreactivity in this compartment was associated with presence of distant metastasis (p = 0.006). Moreover, expression of Snail1 in the tumor stroma correlated with lower specific survival of cancer patients (p = 0.011). Interestingly, this correlation was also detected in stage I and II tumors. Therefore, our results indicate that the presence of nuclear Snail1 immunoreactive cells in the stroma may be an informative indicator of prognosis of colon tumours especially useful in those corresponding to lower stages and identify a new marker suitable to label activated stroma in colon tumours.

Polycomb complex 2 is required for E-cadherin repression by the Snail1 transcription factor.

The transcriptional factor Snail1 is a repressor of E-cadherin (CDH1) gene expression essential for triggering epithelial-mesenchymal transition. Snail1 represses CDH1, directly binding its promoter and inducing the synthesis of the Zeb1 repressor. In this article, we show that repression of CDH1 by Snail1, but not by Zeb1, is dependent on the activity of Polycomb repressive complex 2 (PRC2). Embryonic stem (ES) cells null for Suz12, one of the components of PRC2, show higher levels of Cdh1 mRNA than control ES cells. In tumor cells, interference of PRC2 activity prevents the ability of Snail1 to downregulate CDH1 and partially derepresses CDH1. Chromatin immunoprecipitation assays demonstrated that Snail1 increases the binding of Suz12 to the CDH1 promoter and the trimethylation of lysine 27 in histone H3. Moreover, Snail1 interacts with Suz12 and Ezh2, as shown by coimmunoprecipitation experiments. In conclusion, these results demonstrate that Snail1 recruits PRC2 to the CDH1 promoter and requires the activity of this complex to repress E-cadherin expression.

Repression of PTEN phosphatase by Snail1 transcriptional factor during gamma radiation-induced apoptosis.

The product of the Snail1 gene is a transcriptional repressor required for triggering the epithelial-to-mesenchymal transition. Furthermore, ectopic expression of Snail1 in epithelial cells promotes resistance to apoptosis. In this study, we demonstrate that this resistance to gamma radiation-induced apoptosis caused by Snail1 is associated with the inhibition of PTEN phosphatase. In MDCK cells, mRNA levels of the p53 target gene PTEN are induced after gamma radiation; the transfection of Snail1 prevents this up-regulation. Decreased mRNA levels of PTEN were also detected in RWP-1 cells after the ectopic expression of this transcriptional factor. Snail1 represses and associates to the PTEN promoter as detected both by the electrophoretic mobility shift assay and chromatin immunoprecipitation experiments performed with either endogenous or ectopic Snail1. The binding of Snail1 to the PTEN promoter increases after gamma radiation, correlating with the stabilization of Snail1 protein, and prevents the association of p53 to the PTEN promoter. These results stress the critical role of Snail1 in the control of apoptosis and demonstrate the regulation of PTEN phosphatase by this transcriptional repressor.

Presenilin-1 interacts with plakoglobin and enhances plakoglobin-Tcf-4 association. Implications for the regulation of beta-catenin/Tcf-4-dependent transcription.

Alzheimer disease-linked Presenilin-1 (PS1) is a negative modulator of beta-catenin/Tcf-4 activity. However, the mechanism underlying this effect is not well understood. We show here that the effects of PS1 on the activity of this complex in epithelial cells are independent of its gamma-secretase activity and its interaction with beta-catenin. As presented in this report PS1 also binds plakoglobin with similar affinity as beta-catenin, although this interaction does not involve equivalent residues in the two catenins. Moreover, PS1 association with plakoglobin enhances the interaction of this molecule with Tcf-4 and prevents its binding to DNA. These effects were observed with the unprocessed form of PS1, which has higher affinity for plakoglobin and beta-catenin than processed PS1. These results provide a new explanation for the effects of PS1 on gene transcription mediated by beta-catenin in epithelial cells.

Snail induction of epithelial to mesenchymal transition in tumor cells is accompanied by MUC1 repression and ZEB1 expression.

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.

Upstream determinants of estrogen receptor-alpha regulation of metastatic tumor antigen 3 pathway.

Although recent studies have shown a role of estrogen receptor-alpha (ER) in the regulation of epithelial-to-mesenchymal transition via MTA3, the role of upstream determinants of ER regulation of MTA3 and the underlying molecular mechanism remains unknown. Here we show that MTA3 gene regulation by ER is influenced by dynamic changes in levels of nuclear coregulators. MTA3 promoter has a functional ER element half-site with which MTA1 and HDACs interact under basal conditions. Upon estrogen stimulation, these corepressors are derecruited with concomitant recruitment of ER, leading to increased MTA3 transcription and expression. Genetic inactivation of MTA1 pathway promotes the ability of ER to up-regulate MTA3 expression, whereas knockdown of ER enhances MTA1 association with MTA3 gene. Modulation of ER functions, by corepressors (i.e. MTA1 and MTA1s) or coactivators (i.e. AIB1 and PELP1/MNAR), alters ER recruitment to MTA3 chromatin, MTA3 transcription, and expression of downstream epithelial-to-mesenchymal transition components. These studies provide novel insights into the transregulation of the MTA3 gene and reveal novel roles of upstream determinants in modifying the outcome of MTA3 axis and cell differentiation.