Genome-wide analysis of promoter methylation associated with gene expression profile in pancreatic adenocarcinoma

Abstract

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 27,800 CpG islands covering 21 MB of the human genome in nine pairs of pancreatic cancer versus normal pancreatic epithelial tissues and in three matched pairs of pancreatic cancer versus lymphoid tissues from the same individual.

Results: This analysis identified 1,658 known loci that were commonly differentially methylated in pancreatic cancer compared with 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 expected that the identification of aberrantly hypermethylated and silenced genes will have diagnostic, prognostic, and therapeutic applications.

Figure 1

An overview of experimental strategy to evaluate differential DNA methylation in pancreatic cancer cells compared to normal pancreas. NP: Normal Pancreas.

Figure 2

Distribution of Agilent Human CpG island 244K probes covering known genes A, global distribution. B, Distribution of hypermethylated probes. C, Distribution of hypomethylated probes.

Figure 3

Correlation between gene methylation and gene expression levels in pancreatic cancer samples (A32-1, A38-5, Panc215, Panc2.5, Panc2.8 and Panc3.014). A, Boxplots stratifying mean gene expression (intensities values from Affymetrix Exon Array ST1.0) by mean DNA methylation values (NLR from 244K CpG Island Microarrays). For genes with multiple promoter probes the NLRs for each probe were averaged within sample before calculating the promoter region mean at each across the six sample pairs. B, Scatterplot representing for each gene the mean expression fold-change compared to their methylation profile. The line represents the statistically significant association between mean differential DNA methylation and gene expression.

Figure 4

An overview of experimental strategy to evaluate differential DNA methylation in pancreatic cancer cells compared to normal lymphoid tissue. NP: Microdissected normal pancreatic ductal epithelium; ly: peripheral blood lymphocytes.