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

Abstract

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.

Reactivation of epigenetically silenced miR-512 and miR-373 sensitizes lung cancer cells to cisplatin and restricts tumor growth

“…Changes in DNA methylation have been correlated with altered miR expression in cancer. 11,12 To examine the effect of genomewide reversal of DNA hypermethylation and histone deacetylation on miR expression patterns, we treated A549 lung cancer cells for 72 h with a combination of 5′aza and TSA. Cisplatin was then added for an additional 48 h, at which time cells were harvested for FACS-based cell cycle analysis and miR microarray profiling.…”

“…9,10 Changes in DNA methylation status have been implicated in cancer-associated miR deregulation. [11][12][13] As a single miR often inhibits numerous mRNAs within a defined biological pathway, understanding the epigenetic regulation of miRs in cancer might facilitate the development of new cancer therapies.…”

Specificity Protein 1 (Sp1) Maintains Basal Epithelial Expression of the miR-200 Family

“…Sp1 and p53 have also been reported to interact (62,63) and may cooperatively enforce miR-200 expression in epithelial cells. Sp1 also plays roles in the maintenance of methylation-free CpG islands (53,64) and may prevent DNA hyper-methylation of the miR-200 promoters, which contributes to their repression during EMT (3,65,66). Furthermore, the expression of the miR200bϳ200aϳ429 transcript can also be stimulated through an alternative downstream promoter and an upstream enhancer element (67,68), which may cooperatively bind Sp1 to enhance the activity of the epithelial-specific miR-200b promoter.…”

Epithelial-mesenchymal Transition and Its Role in the Pathogenesis of Colorectal Cancer

“…Davalos et al (2012) showed that the 5'-CpG island hypermethylation-associated silencing of the miR-200b/200a/429 and miR-200c/141 polycistronic transcripts was a dynamic process that mediated the shifts between EMT and MET phenotypes and contributed to cancer progression and metastasis formation in tumorigenesis through laser micordisection of human primary colorectal cancer. In addition, they found that the hypermethylation of miR-200 and up-regulation of ZEB1/ZEB2 were associated with down-regulation in CDH1, CRB3 and LGL2, and they demonstrated that miR-200 hypermethylation-associated inactivation in TGF-β induced EMT was accompanied by increased expression of ZEB1, loss of E-cadherin and the acquisition of a spindle-like shape, a mesenchymal phenotype marked by Vimentin (Kenney et al, 2011).…”