Data from proteomic characterization of the role of Snail1 in murine mesenchymal stem cells and 3T3-L1 fibroblasts differentiation.

The transcription factor (TF) Snail1 is a major inducer of the epithelial-mesenchymal transition (EMT) during embryonic development and cancer progression. Ectopic expression of Snail in murine mesenchymal stem cells (mMSC) abrogated their differentiation to osteoblasts or adipocytes. We used either stable isotopic metabolic labeling (SILAC) for 3T3-L1 cells or isobaric labeling with tandem mass tags (TMT) for mMSC stably transfected cells with Snail1 or control. We carried out a proteomic analysis on the nuclear fraction since Snail is a nuclear TF that mediates its effects mainly through the regulation of other TFs. Proteomics data have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifiers PXD001529 and PXD002157 (Vizcaino et al., 2014) [1]. Data are associated with a research article published in Molecular and Cellular Proteomics (Pelaez-Garcia et al., 2015) [2].

Regulation of the protein stability of EMT transcription factors.

The epithelial to mesenchymal transition (EMT) consists of a rapid change of cell phenotype, characterized by the loss of epithelial characteristics and the acquisition of a more invasive phenotype. Transcription factors regulating EMT (Snail, Twist and Zeb) are extremely labile proteins, rapidly degraded by the proteasome system. In this review we analyze the current mechanisms controlling degradation of EMT transcription factors, focusing on the role of new E3 ubiquitin-ligases involved in EMT. We also summarize the regulation of the stability of these EMT transcription factors, specially observed in different stress conditions, such as hypoxia, chemotherapeutic drugs, oxidative stress or γ-irradiation.

Snail1 controls TGF-ß responsiveness and differentiation of mesenchymal stem cells.

The Snail1 transcriptional repressor plays a key role in triggering epithelial-to-mesenchymal transition. Although Snail1 is widely expressed in early development, in adult animals it is limited to a subset of mesenchymal cells where it has a largely unknown function. Using a mouse model with inducible depletion of Snail1, here we demonstrate that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to transforming growth factor (TGF)-ß1 that shows a strong Snail1 dependence. Snail1 depletion in conditional knockout adult animals causes a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiate to osteoblasts or adipocytes and, in contrast to controls, are resistant to the TGF-ß1-induced differentiation block. These results demonstrate a new role for Snail1 in TGF-ß response and MSC maintenance.

Akt2 interacts with Snail1 in the E-cadherin promoter.

Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action.

Expression of Snail protein in tumor-stroma interface.

The product of Snail gene is a repressor of E-cadherin transcription and an inductor of the epithelial-to-mesenchymal transition in several epithelial tumor cell lines. In order to examine Snail expression in animal and human tissues, we have raised a monoclonal antibody (MAb) that reacts with the regulatory domain of this protein. Analysis of murine embryos shows that Snail is expressed in extraembryonic tissues and embryonic mesoderm, in mesenchymal cells of lungs and dermis as well as in cartilage. Little reactivity was detected in adult tissues as Snail was not constitutively expressed in most mesenchymal cells. However, Snail expression was observed in activated fibroblasts involved in wound healing in mice skin. Moreover, Snail was detected in pathological conditions causing hyperstimulation of fibroblasts, such as fibromatosis. Analysis of Snail expression in tumors revealed that it was highly expressed in sarcomas and fibrosarcomas. In epithelial tumors, it presented a more limited distribution, restricted to stromal cells placed in the vicinity of the tumor and to tumoral cells in the same areas. These results demonstrate that Snail is present in activated mesenchymal cells, indicate its relevance in the communication between tumor and stroma and suggest that it can promote the conversion of carcinoma cells to stromal cells.

Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription.

Beta-catenin undergoes both serine and tyrosine phosphorylation. Serine phosphorylation in the amino terminus targets beta-catenin for proteasome degradation, whereas tyrosine phosphorylation in the COOH terminus influences interaction with E-cadherin. We examined the tyrosine phosphorylation status of beta-catenin in melanoma cells expressing proteasome-resistant beta-catenin, as well as the effects that perturbation of beta-catenin tyrosine phosphorylation had on its association with E-cadherin and on its transcriptional activity. Beta-catenin is tyrosine phosphorylated in three melanoma cell lines and associates with both the ErbB2 receptor tyrosine kinase and the LAR receptor tyrosine phosphatase. Geldanamycin, a drug which destabilizes ErbB2, caused rapid cellular depletion of the kinase and loss of its association with beta-catenin without perturbing either LAR or beta-catenin levels or LAR/beta-catenin association. Geldanamycin also stimulated tyrosine dephosphorylation of beta-catenin and increased beta-catenin/E-cadherin association, resulting in substantially decreased cell motility. Geldanamycin also decreased the nuclear beta-catenin level and inhibited beta-catenin-driven transcription, as assessed using two different beta-catenin-sensitive reporters and the endogenous cyclin D1 gene. These findings were confirmed by transient transfection of two beta-catenin point mutants, Tyr-654Phe and Tyr-654Glu, which, respectively, mimic the dephosphorylated and phosphorylated states of Tyr-654, a tyrosine residue contained within the beta-catenin-ErbB2-binding domain. These data demonstrate that the functional activity of proteasome-resistant beta-catenin is regulated further by geldanamycin-sensitive tyrosine phosphorylation in melanoma cells.

The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells.

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.

Regulation of E-cadherin/Catenin association by tyrosine phosphorylation.

Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120- and beta-catenins. We have examined the role of this modification in these proteins in the control of beta-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 beta-catenin and p120-catenin, respectively. pp60(c-src) phosphorylation had opposing effects on the affinities of beta-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of beta-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of beta-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) beta-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 beta-catenin to E-cadherin in vivo is controlled by phosphorylation of beta-catenin Tyr-654.

Adenomatous polyposis coli protein (APC)-independent regulation of beta-catenin/Tcf-4 mediated transcription in intestinal cells.

Alterations in the transcriptional activity of the beta-catenin-Tcf complex have been associated with the earlier stages of colonic transformation. We show here that the activation of protein kinase C by the phorbol ester PMA in several intestinal cell lines increases the levels of beta-catenin detected in the nucleus and augments the transcriptional activity mediated by beta-catenin. The response to PMA was not related to modifications in the cytosolic levels of beta-catenin and was observed not only in cells with wild-type adenomatous polyposis coli protein (APC) but also in APC-deficient cells. Binding assays in vitro revealed that PMA facilitates the interaction of the beta-catenin with the nuclear structure. Our results therefore show that beta-catenin-mediated transcription can be regulated independently of the presence of APC.

Independent regulation of adherens and tight junctions by tyrosine phosphorylation in Caco-2 cells.

To study the role of tyrosine phosphorylation in the control of intercellular adhesion of intestinal cells, we have generated several clones of Caco-2 cells that express high levels of pp60v-src only after addition of butyrate. Expression of this oncogene in cells 5 days after confluence induced beta-catenin and p120-ctn tyrosine phosphorylation, redistribution of E-cadherin to the cytosol and disassembly of adherens junctions. However, tight junctions of Caco-2 cells at 5 days after confluence were not altered by expression of pp60v-src. Similar results were obtained when Caco-2 cells were incubated with phosphotyrosine phosphatase inhibitor orthovanadate. Although addition of this compound to postconfluent cells disrupt adherens junctions, tight junctions remain unaltered, as determined measuring monolayer permeability to mannitol or hyperphosphorylation of Triton-insoluble occludin. Modifications in tight junction permeability of Caco-2 were only observed at high concentrations of orthovanadate (1 mM). Interestingly, this tyrosine phosphorylation-refractory state was achieved after confluence since early postconfluent cells (day 2) showed a limited but significant response to low doses of orthovanadate. These results suggest that tight junctions of differentiated Caco-2 cells are uncoupled from adherens junctions and are insensitive to regulation by tyrosine phosphorylation.

Gender verification in sports by PCR amplification of SRY and DYZ1 Y chromosome specific sequences: presence of DYZ1 repeat in female athletes.

OBJECTIVE: To perform genetic sex typing during the Barcelona Olympic Games using polymerase chain reaction (PCR) amplification of Y chromosome specific sequences. METHODS: The assay consisted of the amplification of a specific sequence corresponding to the repeat DYZ1 element from buccal smears samples of 2406 female competitors. Positive samples were reanalysed for the presence of another Y chromosome specific gene, SRY. RESULTS: The expression of these two elements did not always correlate; six samples were found where the presence of DYZ1 but not SRY was detected. This presence of DYZ1 sequence in female athletes is higher than in unselected females, where no DYZ1 amplification was observed in any of the 1629 samples analysed. CONCLUSIONS: Amplification of DYZ1 repeat should not be used as the only index for determining genetic sex, at least in sporting events.

p120-catenin expression in human colorectal cancer.

Recent data suggest that p120-catenin plays a role in the regulation of functionality of E-cadherin, a protein essential for the establishment and maintenance of cell-cell contacts. Since dysfunction of intercellular adhesiveness is an alteration frequently observed in colon cancer we have studied the expression and distribution of p120-catenin in human colorectal tumors. In normal colon, p120-catenin was observed in the crypt and surface epithelium; the cells showed reactivity both in the membrane and in the cytosol. Thirteen primary tumors were examined for p120-catenin expression: they were graded as uniformly positives (+) (4); heterogeneous (+/-) (6), with a diminished expression, detected mainly in the cytosol; and negatives (-) (3). Although the number of tumors was low, the reduction in p120-catenin correlated with a larger size of the tumors (p = 0.038). Association of p120-catenin to the cytoskeleton was also determined in 5 tumors by detergent extraction and Western blot; this analysis shows that lack of reactivity in the membrane was accompanied by absence of p120-catenin in the cytoskeleton-associated fraction. Analysis of E-cadherin was performed in order to compare the distribution of this protein and p120-catenin. Although no complete correlation was found between the expression of both proteins (p = 0.077), our results showed that alterations in the level or distribution of p120-catenin were accompanied by lack of E-cadherin reactivity in the membrane, whereas absence of p120-catenin in the cytoskeleton fraction was associated with important decreases in the amount of E-cadherin in this same fraction. These results show that alterations in p120-catenin levels are a common event in colorectal tumors, and suggest that the distribution of this protein and E-cadherin is coordinately regulated.

Intestinal HT-29 cells with dysfunction of E-cadherin show increased pp60src activity and tyrosine phosphorylation of p120-catenin.

1. HT-29 M6 cells are a subpopulation of HT-29 cells that, contrarily to the parental cells, establish tight cell contacts and differentiate. Cell-to-cell contacts in HT-29 M6 cells are also regulated by protein kinase C; addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) decreases the homotypic contacts of these cells. We show here that HT-29 cells or HT-29 M6 cells treated with PMA contain lower levels of functional E-cadherin, determined by analysing the association of this protein with the cytoskeleton. No significant differences in the localization of alpha-, beta-, or p120-catenins were detected under the three different conditions. 2. Dysfunction of E-cadherin can be reversed by incubation of HT-29 cells with the tyrosine kinase inhibitor herbimycin A. On the other hand an augmentation of c-src activity in HT-29 cells or HT-29 M6 cells treated with PMA was observed with respect to control HT-29 M6 cells. The phosphorylation status of catenins was also investigated; in HT-29 or in HT-29 M6 cells treated with PMA, dysfunction of E-cadherin was accompanied by an increased phosphorylation of p120-catenin and by an elevated association of this protein to E-cadherin. These results suggest a role for pp60src and the pp60src substrate p120-catenin in the control of E-cadherin function in HT-29 cells.

Evidence for a role of conventional protein kinase-C alpha in the control of homotypic contacts and cell scattering of HT-29 human intestinal cells.

Incubation of HT-29 M6 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) induces cell scattering, loss of cellular contacts and inactivation of E-cadherin. We have investigated the involvement of different protein kinase C (PK-C) isoforms in these processes using specific activators. Thymeleatoxin, a derivative of mezerein that activates conventional PK-Cs (cPK-Cs) but not novel PK-Cs (nPK-Cs), promoted effects that were similar to those of PMA, i.e. at concentrations of 200 nM it induced scattering of HT-29 M6 colonies, loss of homotypic contacts and dissociation of E-cadherin from the cytoskeleton. Among the isoforms activated by this compound, only cPK-C alpha was detected in HT-29 M6 cells by Western blot. The specificity of this compound with respect to the rest of the PK-C isoforms present in these cells was determined; thymeleatoxin induced, as did PMA, the translocation of cPK-C alpha from the cytosol to the membrane and the cytoskeleton, and its partial down-regulation. On the other hand, thymeleatoxin did not modify the cellular levels or localization of nPK-C epsilon or atypical PK-C zeta. "In vitro' assays also showed that thymeleatoxin did not activate nPK-C epsilon at the concentrations added to the cell cultures. These results indicate that thymeleatoxin is selective for cPK-C alpha over nPK-C epsilon and show a role for the former enzyme in the regulation of cell-cell contacts and the inactivation of E-cadherin in HT-29 M6 cells.

Isolation of tissue-type plasminogen activator, cathepsin H, and non-specific cross-reacting antigen from SK-PC-1 pancreas cancer cells using subtractive hybridization.

We have used subtractive hybridization to isolate cDNAs overexpressed in SK-PC-1 pancreas cancer cells. Forty-five independent clones corresponding to 11 genes were identified. Their expression in cultured pancreas cancer cells, normal pancreas tissue, and normal exocrine pancreas cultures was examined by Northern blotting. cDNA clones can be grouped into two broad categories: (1) those corresponding to genes expressed at high levels both in tumor cell lines and in primary cultures of normal pancreas, but not in normal tissue (i.e. thymosin beta4(3), cytokeratin 18, beta-actin, pyruvate kinase and mitochondrial genes); and (2) those corresponding to genes expressed at high levels in pancreas cancer cultures but not in normal pancreas tissue or cultured cells (i.e. tissue-type plasminogen activator and cathepsin H). The overexpression of these proteases in pancreas cancers suggests that they play a role in the aggressive biological behavior of this tumor.

The protein kinase C activator TPA modulates cellular levels and distribution of E-cadherin in HT-29 human intestinal epithelial cells.

The importance of E-cadherin protein in the establishment and maintenance of homotypic contacts in epithelial cells has already been determined. We report here that the association of E-cadherin to cytoskeleton, required for the functionality of this protein, increases progressively after seeding; in HT-29 M6 cells 4-5 days were required for detecting most of E-cadherin in the Triton X-100-insoluble (cytoskeleton-associated) fraction. The phorbol ester TPA differently affected E-cadherin levels in HT-29 M6 cells; at day 2-3, when most E-cadherin was found not-associated to the cytoskeleton, very important decreases (90%) in the total levels of this protein were detected as soon as 6 h after the addition of this compound. However, on later days (day 5), the predominant effect by 6 h was a translocation from the Triton-insoluble to the soluble fraction. The E-cadherin-associated proteins alpha-catenin and beta-catenin were not significantly affected by treatment with TPA.

Antipeptide antibodies directed against the C-terminus of protein kinase C zeta (PKC zeta) react with a Ca(2+)- and TPA-sensitive PKC in HT-29 human intestinal epithelial cells.

We have studied the PKC isoforms present in HT-29 M6 colon cancer cells, the differentiation of which to mucus-secreting cells is blocked by TPA. In addition to a major 72 kDa band, a 77 kDa PKC isoform was recognized by two different antibodies raised against a C-terminus-specific peptide for the TPA-insensitive isoform, PKC zeta. By different criteria (association to the membrane, down-regulation, PKC activity in immunoprecipitates) we conclude that, contrary to the 72 kDa band, the 77 kDa band corresponds to a Ca(2+)- and TPA-sensitive PKC. These results suggest that antipeptide antibodies directed against the C-terminus of PKC zeta react in human cells with a member of the conventional PKC subfamily besides PKC zeta. Therefore, the data indicating that PKC zeta is sensitive to different agents in various cell lines should be carefully re-evaluated.

Phorbol ester-induced scattering of HT-29 human intestinal cancer cells is associated with down-modulation of E-cadherin.

The effects of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth characteristics of the colon cancer cell line HT-29 M6 were studied. TPA induced the scattering of proliferative HT-29 M6 cells: in the presence of the phorbol ester, HT-29 M6 colonies scattered and the cells acquired a flatter aspect with diminished cell-cell contacts. This effect of TPA required a persistent activation of PK-C and was accompanied by a slight decrease (30%) in the growth rate. Modifications by TPA of two scattering associated properties of these cells were also detected: TPA decreased cell-to-cell aggregation and enhanced the cellular attachment to matrix substrata (collagen, laminin). The decrease in cell-to-cell adhesion was correlated with a loss of cellular E-cadherin as evidenced by immunofluorescence or immunoblotting with a specific monoclonal antibody. Cell scattering was dependent on the extracellular concentration of Ca2+; an increase from 1.6 to 10 mM in the concentration of this ion completely blocked the morphological effects of TPA as well as its action on cell aggregation. This high concentration of Ca2+ also prevented the down modulation of E-cadherin as determined by immunofluorescence. However, the TPA-induced increase in cell attachment to the matrix was not affected by high calcium. These findings support the importance of altered cell-cell adhesion in the process of scattering and provide a good system for the study of down modulation of E-cadherin, a protein involved in the control of cell growth, differentiation and invasion of epithelial cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate blocks differentiation of HT-29 human colon cancer cells.

Recently developed HT-29-derived cell lines, which display variable differentiated phenotypes provide an invaluable opportunity to analyze the mechanism by which cell differentiation is regulated in the intestine. We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in the differentiation phenotype of mucus-secreting (HT-29 M6) and absorptive (HT-29 M3) cells. TPA prevented the accumulation of differentiation markers such as dipeptidylpeptidase IV, villin or mucins, down-regulated the expression of these molecules in post-confluent differentiated cell cultures and induced the loss of the functional integrity of the tight junction in the monolayer (i.e. decreased transepithelial resistance and inhibited dome formation). These effects were mediated by activation of protein kinase C (PK-C), as demonstrated using the specific inhibitor GF109203x. Analysis by immunoblotting of the PK-C isoforms present in HT-29 M6 cells revealed that the most abundant TPA-sensitive isoform was PK-C epsilon, although low levels of cPK-C were also detected. Further studies are necessary to elucidate the role of the different PK-C isoforms in the differentiation of HT-29 cells.