Zeb1 and Snail1 engage miR-200f transcriptional and epigenetic regulation during EMT

Cell plasticity is emerging as a key regulator of tumor progression and metastasis. During carcinoma dissemination epithelial cells undergo epithelial to mesenchymal transition (EMT) processes characterized by the acquisition of migratory/invasive properties, while the reverse, mesenchymal to epithelial transition (MET) process, is also essential for metastasis outgrowth. Different transcription factors, called EMT-TFs, including Snail, bHLH and Zeb families are drivers of the EMT branch of epithelial plasticity, and can be post-transcriptionally downregulated by several miRNAs, as the miR-200 family. The specific or redundant role of different EMT-TFs and their functional interrelations are not fully understood. To study the interplay between different EMT-TFs, comprehensive gain and loss-of-function studies of Snail1, Snail2 and/or Zeb1 factors were performed in the prototypical MDCK cell model system. We here describe that Snail1 and Zeb1 are mutually required for EMT induction while continuous Snail1 and Snail2 expression, but not Zeb1, is needed for maintenance of the mesenchymal phenotype in MDCK cells. In this model system, EMT is coordinated by Snail1 and Zeb1 through transcriptional and epigenetic downregulation of the miR-200 family. Interestingly, Snail1 is involved in epigenetic CpG DNA methylation of the miR-200 loci, essential to maintain the mesenchymal phenotype. The present results thus define a novel functional interplay between Snail and Zeb EMT-TFs in miR-200 family regulation providing a molecular link to their previous involvement in the generation of EMT process in vivo.Spanish Ministry of Economy and Competitiveness; Grant number: SAF2010-21143, SAF2013-44739-R, Consolider CDS07/0007; Instituto de Salud Carlos III (ISCIII); RETIC-RD12/0036/0007 and RD12/0036/0064; PI13/02477; PI13/00132; AICR; 12-1057; AVON Foundation 2011; AECC; GEIS; Tv3-Telemarato; Comunidad de Madrid; S2010/BMD-2303; Consejerıa de Salud, Junta de Andalucía; PI0581/2009; ADL was funded by a Sara Borrell contract from ISCIII. EPC and VS are founded by the AICR grant (12-1057).Peer Reviewe