Inhibition of E-cadherin is associated with down-regulation of members of the miR-200 family but not with promoter methylation in human breast cancer cell lines.

Abstract

Abstract Abstract #6017 Background: Increasing evidence indicates the fundamental role of epithelial to mesenchymal transition (EMT) in breast cancer progression and this is viewed as an essential early step in tumor cell invasion and metastasis. One of the pivotal events of EMT is down-regulation of E-cadherin. Recent findings have demonstrated the importance of the miR-200 microRNA family in the regulation of E-cadherin and EMT. In view of these findings, we examined the relationship between expression of members of the miR-200 family and status of E-cadherin levels in various human breast cancer cell lines.
 Materials and Methods: Using quantitative real-time PCR analysis, we analyzed the expression of miR-200b, miR-200c, and miR-141 in 15 human breast cancer cell lines. Additionally, we measured the level of ZEB1, ZEB2, and E-cadherin proteins using Western blot analysis and E-cadherin promoter methylation by methylation-specific PCR.
 Results: We found a substantial down-regulation of miR-200b, miR-200c, and miR-141 in the breast cancer cell lines. The extent of down-regulation was correlated with metastatic potential of cells. The down-regulation of members of the miR-200 family was associated with up-regulation of ZEB1 and ZEB2, transcriptional repressors of E-cadherin, and a subsequent decrease in the E-cadherin levels. More importantly, inhibition of E-cadherin expression was not due to its promoter hypermethylation. Treatment of breast cancer cells with demethylating agent 5-aza-2'-deoxycytidine resulted in over-expression of miR-200b and miR-200c, increase of the level of E-cadherin, and diminished metastatic properties of cancer cells.Discussion: Previous studies have revealed the important role of promoter methylation-related inhibition of E-cadherin in EMT in human breast cancer. However, the transcriptional silencing of E-cadherin is not always related to promoter hypermethylation, which indicates the existence of other mechanisms in E-cadherin down-regulation. In the present study, we demonstrated the crucial role of members of the miR-200 family in up-regulation of ZEB1 and ZEB2 proteins leading to subsequent E-cadherin down-regulation in human breast cancer cell lines. The extent of down-regulation of the miR-200 family was dependent of cancer stage and was associated with metastatic potential of cancer cell lines. This suggests that expression of miR-200 may be a powerful diagnostic marker determining breast cancer stage and cancer invasive phenotype. More importantly, the results indicate the great potential for reversing the invasive phenotype of cancer cells by up-regulation of miR-200 microRNAs. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6017.