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. 2008 Jul;7(7):1146-56.
doi: 10.4161/cbt.7.7.6208. Epub 2008 Apr 29.

Genome-wide profiling of methylated promoters in pancreatic adenocarcinoma

Noriyuki Omura  1 , Chung-Pin Li, Ang Li, Seung-Mo Hong, Kimberly Walter, Antonio Jimeno, Manuel Hidalgo, Michael Goggins
Affiliations

Genome-wide profiling of methylated promoters in pancreatic adenocarcinoma

Noriyuki Omura et al. Cancer Biol Ther. 2008 Jul.

Abstract

Many genes undergo aberrant methylation in human cancers, and microarray platforms enable more comprehensive profiling of aberrant DNA methylation patterns.

Results: 1,010 of 87,922 probes on the 88 K promoter array (606 genes) had a higher signal (log(2) > 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 1/2 of the genes aberrantly hypermethylated in Panc-1 were not expressed in the pancreatic duct (HPDE) by expression array analysis. Using the 244 K 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 10 ng of DNA were highly correlated with those obtained using 5 ug 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 97% of normal pancreata, respectively.

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.

Conclusion: Promoter and CpG island array analysis finds aberrant methylation of hundreds of promoters and CpG islands in pancreatic cancer cells.

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Figures

Figure 1
Figure 1
An overview of experimental strategy to evaluate differential DNA methylation in pancreatic cancer cells using MCA microarrays.
Figure 2
Figure 2
(A and B) Magnitude versus amplitude (MA) plot of an MCA microarray hybridization. The x-axis represents the log2 intensity of the Panc-1 (Cy5) and HPDE (Cy3) channel, and the y-axis represents the log2 ratio of Panc-1/HPDE. The red and green line represents the cut-off of the methylation probability (log2 ratio ± 2). (A) Chip A (B) chip B. (C) DNA methylation profiles of chromosomes X. The plotted signal indicates individual probes (green circle). Probes showing signal log2 ratio < -2 represent hypermethylation in HPDE relative to Panc-1. The probes marked by red triangles represent genes which have been identified as subject to X chromosome inactivation.
Figure 3
Figure 3
(A) Reproducibility of the MCA microarray. The scatter plots represent the correlation of probe intensities on replicated arrays for Panc-1 (Cy5) and HPDE (Cy3). (B) DNA methylation profiles of chromosome 1 using the MCA Agilent array. Each circle indicates the signal of an individual probe (blue circle, array 1; red circle, array 2). The black triangles represent the location of TNFRSF18, PAX7, hsa-miR-9-1, PKP1, ACTA1 and LIN28, genes whose methylation was verified by bisulfite sequencing. (C) Replicate experiments show individual probe signals of these genes’ promoters. Each circle indicates the signal of an individual probe (blue circle, array 1; red circle, array 2).
Figure 4
Figure 4
MSP analysis of select genes in pancreatic and periampullary cancers and normal pancreata. Black box indicates completely methylated, white box indicates completely unmethylated, gray box indicates partially methylated genes.
Figure 4
Figure 4
MSP analysis of select genes in pancreatic and periampullary cancers and normal pancreata. Black box indicates completely methylated, white box indicates completely unmethylated, gray box indicates partially methylated genes.
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