Genome-wide profiling at methylated promoters in pancreatic adenocarcinoma

Abstract

Many genes undergo aberrant methylation in human cancers, and microarray platforms enable more comprehensive profiling of aberrant DNA methylation patterns. Methods: We used methylated CpG island amplification (MCA) and Agilent promoter and CpG island microarrays to identify differential DNA methylation patterns in pancreatic cancer vs. normal pancreas. We examined MCA array reproducibility, compared it to methylation profiles obtained using a cocktail of methylation-sensitive restriction enzymes and examined gene expression of methylated genes Results: 1,010 of 87,922 probes on the 88K promoter array (606 genes) had a higher signal (log2>2) in the pancreatic cancer line, Panc-1 compared to the non-neoplastic pancreatic duct line, HPDE. Using this cut-off, bisulfite sequencing and/or MSP confirmed differential methylation of all 27 genes (66 probes) predicted to be methylated by the MCA array. More than ½ of the genes aberrantly hypermethylated in Panc-1 were not expressed in the pancreatic duct (HPDE) by expression array analysis. Using the 244K CpG island array, 1,968 CpG islands were differentially methylated in MiaPaca2 compared to normal pancreas. The MCA method was more likely to identify hypermethylation within CpG islands than a cocktail of methylation sensitive restriction enzymes. DNA methylation profiles using 10ng of DNA were highly correlated with those obtained 4 using 5ug of DNA (R2=0.98). Analysis of 57 pancreatic cancers and 34 normal pancreata using MSP identified MDFI, hsa-miR-9-1, ZNF415, CNTNAP2 and ELOVL4 as methylated in 96%, 89%, 86%, 82% and 68% of the cancers vs. 9%, 15%, 6%, 3% and 9% of normal pancreata, respectively. Conclusion: Promoter and CpG island array analysis finds aberrant methylation of hundreds of promoters and CpG islands in pancreatic cancer cells.