Aberrant DNA methylation and microRNA expression play important roles in the pathogenesis of pancreatic cancer. While interrogating differentially methylated CpG islands in pancreatic cancer, we identified two members of miR-200 family, miR-200a and miR-200b, that were hypomethylated and overexpressed in pancreatic cancer. We also identified prevalent hypermethylation and silencing of one of their downstream targets, SIP1 (ZFHX1B, ZEB2), whose protein product suppresses E-cadherin expression and contributes to epithelial mesenchymal transition. In a panel of 23 pancreatic cell lines, we observed a reciprocal correlation between miR-200, SIP1, and E-cadherin expression, with pancreatic cancer-associated fibroblasts showing the opposite expression pattern to most pancreatic cancers. In Panc-1 cells, which express SIP1, have low E-cadherin expression, and do not express miR-200a or miR-200b, treatment with miR-200a and miR-200b downregulated SIP1 mRNA and increased E-cadherin expression. However, most pancreatic cancers express miR-200a and miR-200b, but this expression does not affect SIP1 expression, as the SIP1 promoter is silenced by hypermethylation and in these cancers E-cadherin is generally expressed. Both miR-200a and miR-200b were significantly elevated in the sera of pancreatic cancer and chronic pancreatitis patients compared with healthy controls (P < 0.0001), yielding receiver operating characteristic curve areas of 0.861 and 0.85, respectively. In conclusion, most pancreatic cancers display hypomethylation and overexpression of miR-200a and miR-200b, silencing of SIP1 by promoter methylation, and retention of E-cadherin expression. The elevated serum levels of miR-200a and miR-200b in most patients with pancreatic cancer could have diagnostic utility. Cancer Res; 70(13); 5226-37. ©2010 AACR.

Purpose The goal of this study was to comprehensively identify CpG island methylation alterations between pancreatic cancers and normal pancreata and their associated gene expression alterations. Experimental Design We employed Methylated CpG island Amplification followed by CpG island Microarray, a method previously validated for its accuracy and reproducibility, to analyze the methylation profile of 27800 CpG islands covering 21MB of the human genome in nine pairs of pancreatic cancer versus normal pancreatic epithelial tissues as well as in three matched pairs of pancreatic cancer versus lymphoid tissues from the same individual. Results This analysis identified 1658 known loci that were commonly differentially methylated in pancreatic cancer compared to normal pancreas. By integrating the pancreatic DNA methylation status with the gene expression profiles of the same samples before and after treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, and the Histone Deacetylase inhibitor, Trichostatin A, we identified dozens of aberrantly methylated and differentially expressed genes in pancreatic cancers including a more comprehensive list of hypermethylated and silenced genes that have not been previously described as targets for aberrant methylation in cancer. Conclusion We expect that the identification of aberrantly hypermethylated and silenced genes will have diagnostic, prognostic and therapeutic applications.