Dynamic epigenetic regulation of the microRNA-200 family mediates epithelial and mesenchymal transitions in human tumorigenesis

Epithelial-mesenchymal (EMT) and mesenchymal-epithelial (MET) transitions occur in the development of human tumorigenesis and are part of the natural history of the process to adapt to the changing microenvironment. In this setting, the miR-200 family is recognized as a master regulator of the epithelial phenotype by targeting ZEB1 and ZEB2, two important transcriptional repressors of the cell adherence (E-cadherin) and polarity (CRB3 and LGL2) genes. Recently, the putative DNA methylation associated inactivation of various miR-200 members has been described in cancer. Herein, we show that the miR-200ba429 and miR-200c141 transcripts undergo a dynamic epigenetic regulation linked to EMT or MET phenotypes in tumor progression. The 5′-CpG islands of both miR-200 loci were found unmethylated and coupled to the expression of the corresponding miRNAs in human cancer cell lines with epithelial features, such as low levels of ZEB1/ZEB2 and high expression of E-cadherin, CRB3 and LGL2, while CpG island hypermethylation-associated silencing was observed in transformed cells with mesenchymal characteristics. The recovery of miR-200ba429 and miR-200c141 expression by stable transfection in the hypermethylated cells restored the epithelial markers and inhibited migration in cell culture and tumoral growth and metastasis formation in nude mice. We also discovered, using both cell culture and animal models, that the miR-200 epigenetic silencing is not an static and fixed process but it can be shifted to hypermethylated or unmethylated 5′-CpG island status corresponding to the EMT and MET phenotypes, respectively. In fact, careful laser microdissection in human primary colorectal tumorigenesis unveiled that in normal colon mucosa crypts (epithelia) and stroma (mesenchyma) already are unmethylated and methylated at these loci, respectively; and that the colorectal tumors undergo selective miR-200 hypermethylation of their epithelial component. These findings indicate that the epigenetic silencing plasticity of the miR-200 family contributes to the evolving and adapting phenotypes of human tumors.