Genetic Profiling of Epithelial Cells Expressing E-Cadherin Repressors Reveals a Distinct Role for Snail, Slug, and E47 Factors in Epithelial-Mesenchymal Transition

Abstract

The transcription factors Snail, Slug, and bHLH E47 have been recently described as direct repressors of E-cadherin and inducers of epithelial-mesenchymal transition (EMT) and invasion when overexpressed in epithelial cells. Although a role of those factors in tumor progression and invasion has been proposed, whether the different repressors play distinct or redundant roles in the tumorigenic process has not been established. To further investigate this important issue, we have analyzed the gene expression profiling of MadinDarby canine kidney (MDCK) epithelial cells expressing the different repressors (MDCK-Snail, MDCK-Slug, and MDCK-E47 cells) versus control MDCK cells by cDNA microarrays. A total of 243 clones (228 genes and 15 expressed sequence tags) were found to be differentially expressed between either of the three MDCK-derived cell lines and control MDCK cells. Twenty two of the candidate genes were validated by Northern blot, Western blot, immunofluorescence, and promoter analyses in cell lines and by immunohistochemistry in xenografted tumors. Gene clustering analysis indicated that about a third of the 243 candidate genes were common to MDCK cells expressing Snail, Slug, or E47 factors, whereas the rest of the genes were regulated in only one or two cell types. Differentially regulated genes include those related to EMT (45 genes), transcriptional regulation (18 genes), cell proliferation and signaling (54 genes), apoptosis (12 genes), and angiogenesis (9 genes). These results indicate that Snail, Slug, and E47 transcription factors induce common and specific genetic programs, supporting a differential role of the factors in tumor progression and invasion. (Cancer Res 2006; 66(19): 9543-56)

Transcriptional regulation of cell polarity in EMT and cancer

“…Indeed, direct binding of SNAI1 to proximal E-boxes of the CRB3 promoter was demonstrated in MDCK-SNAI1 cells (Whiteman et al, 2008), in contrast to previous suggestions from breast carcinomas (Aigner et al, 2007). As mentioned above, other core polarity genes like DLG3 are thought to be downregulated by several EMT inducers, including SNAI1/SNAI2 and bHLH factors (E47/TCF3, E2-2/ TCF4), both in MDCK and mouse carcinoma cells (Moreno-Bueno et al, 2006;Peinado et al, 2008;Sobrado V, Moreno-Bueno G et al, unpublished), although no functional promoter studies are yet available. Taken together, these observations suggest that different EMT transcriptional inducers act cooperatively to repress factors involved in determining polarity, which could reinforce the suppression of apical-basal polarity that is required for cells to undergo EMT.…”

“…In fact, direct binding of SNAI1/SNAI2 to conserved E-box elements in the corresponding promoters has been reported for occludin, claudin-1 and claudin-7 (Ikenouchi et al, 2003;Martı´nez-Estrada et al, 2006). Interestingly, downregulation of claudin-4, the junctional adhesion molecule-1 (JAM-1/JAM-A) and Dlg3 has been detected in gene profiling studies of carcinoma cells, as well as in Madin-Darby canine kidney (MDCK) cells undergoing EMT after expression of SNAI1, SNAI2, E47 or other EMT regulators (De Craene et al, 2005b;Moreno-Bueno et al, 2006;Peinado et al, 2008). Because of the close relationship between tight junctions and the organization of epithelial cell polarity, these observations suggest that components of the core cell polarity complexes could also be direct targets of EMT transcriptional regulators.…”

“…SCRIB complex is reciprocally inactivated by aPKC-dependent phosphorylation of Lgl protein. Vandewalle et al, 2005;Bermejo-Rodrı´guez et al, 2006;Moreno-Bueno et al, 2006;Peinado et al, 2007;Escrivaè t al., 2008). These different EMT regulators share a similar basic molecular mechanism of repression, binding to conserved E-box sequences (mainly of the CAGGTG type) in the proximal promoter of E-cadherin and other epithelial genes (Peinado et al, 2004(Peinado et al, , 2007.…”

Phenotypic plasticity of neoplastic ovarian epithelium: unique cadherin profiles in tumor progression

“…Interestingly, a recent gene profiling study of the E-cadherin repressors Snail, Slug, and e47 in MDCK cells found more divergence than similarity in their gene expression profiles. This led the authors to conclude that there are different "EMT programs" corresponding to different states of epithelial plasticity and that specific pathways contribute to distinct aspects of EMT [37]. Thus, tumor phenotype would likely reflect the particular complement of EMT regulatory factors expressed in cells or within the tumor microenvironment [30,31,38] and account for the divergence from a "classical" EMT that is frequently observed in cancer.…”

Tissue transglutaminase links TGF-β, epithelial to mesenchymal transition and a stem cell phenotype in ovarian cancer

“…Downregula-tion of E-cadherin is a critical disruptor of epithelial homeostasis, leading to the adoption of a mesenchymal behavior (Nagafuchi et al, 1987;Thiery, 2002). Loss of E-cadherin is modulated by transcriptional repressors like Snail, Slug, Zeb 1/2 and the basic helix-loop-helix factors E47 and Twist (Moreno-Bueno et al, 2006). Activation of SMADs by TGF-b recruits Snail (Vincent et al, 2009), Zeb (Comijn et al, 2001) or Twist (Thuault et al, 2006), which repress E-cadherin.…”