E47 and Id1 interplay in epithelial-mesenchymal transition.

E12/E47 proteins (encoded by E2A gene) are members of the class I basic helix-loop-helix (bHLH) transcription factors (also known as E proteins). E47 has been described as repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). We reported previously that EMT mediated by E47 in MDCK cells occurs with a concomitant overexpression of Id1 and Id3 proteins. Id proteins belong to class V of HLH factors that lack the basic domain; they dimerise with E proteins and prevent their DNA interaction, thus, acting as dominant negative of E proteins. Here, we show that E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full EMT as well as in mesenchymal breast carcinoma and melanoma cell lines. By conducting chromatin immunoprecipitation assays we demonstrate that E47 binds directly to the endogenous E-cadherin promoter of mesenchymal MDCK-E47 cells in a complex devoid of Id1. Importantly, our data suggest that both E47 and Id1 are required to maintain the mesenchymal phenotype of MDCK-E47 cells. These data support the collaboration between E47 and Id1 in the maintenance of EMT by mechanisms independent of the dominant negative action of Id1 on E47 binding to E-cadherin promoter. Finally, the analysis of several N0 breast tumour series indicates that the expression of E47 and ID1 is significantly associated with the basal-like phenotype supporting the biological significance of the present findings.

Characterization of the SNAG and SLUG domains of Snail2 in the repression of E-cadherin and EMT induction: modulation by serine 4 phosphorylation.

Snail1 and Snail2, two highly related members of the Snail superfamily, are direct transcriptional repressors of E-cadherin and EMT inducers. Previous comparative gene profiling analyses have revealed important differences in the gene expression pattern regulated by Snail1 and Snail2, indicating functional differences between both factors. The molecular mechanism of Snail1-mediated repression has been elucidated to some extent, but very little is presently known on the repression mediated by Snail2. In the present work, we report on the characterization of Snail2 repression of E-cadherin and its regulation by phosphorylation. Both the N-terminal SNAG and the central SLUG domains of Snail2 are required for efficient repression of the E-cadherin promoter. The co-repressor NCoR interacts with Snail2 through the SNAG domain, while CtBP1 is recruited through the SLUG domain. Interestingly, the SNAG domain is absolutely required for EMT induction while the SLUG domain plays a negative modulation of Snail2 mediated EMT. Additionally, we identify here novel in vivo phosphorylation sites at serine 4 and serine 88 of Snail2 and demonstrate the functional implication of serine 4 in the regulation of Snail2-mediated repressor activity of E-cadherin and in Snail2 induction of EMT.

Lysyl oxidase-like 2 (LOXL2), a new regulator of cell polarity required for metastatic dissemination of basal-like breast carcinomas.

Basal-like breast carcinoma is characterized by the expression of basal/myoepithelial markers, undifferentiated phenotype, highly aggressive behaviour and frequent triple negative status (ESR-, PR-, Her2neu-). We have previously shown that epithelial-mesenchymal transition (EMT) occurs in basal-like breast tumours and identified Lysyl-oxidase-like 2 (LOXL2) as an EMT player and poor prognosis marker in squamous cell carcinomas. We now show that LOXL2 mRNA is overexpressed in basal-like human breast carcinomas. Breast carcinoma cell lines with basal-like phenotype show a specific cytoplasmic/perinuclear LOXL2 expression, and this subcellular distribution is significantly associated with distant metastatic incidence in basal-like breast carcinomas. LOXL2 silencing in basal-like carcinoma cells induces a mesenchymal-epithelial transition (MET) associated with a decrease of tumourigenicity and suppression of metastatic potential. Mechanistic studies indicate that LOXL2 maintains the mesenchymal phenotype of basal-like carcinoma cells by a novel mechanism involving transcriptional downregulation of Lgl2 and claudin1 and disorganization of cell polarity and tight junction complexes. Therefore, intracellular LOXL2 is a new candidate marker of basal-like carcinomas and a target to block metastatic dissemination of this aggressive breast tumour subtype.