Epigenetic Inactivation of a Cluster of Genes Flanking MLH1 in Microsatellite-Unstable Colorectal Cancer

Megan P Hitchins,Deborah Packham,Chau-To Kwok,Su Ku,Alan Meagher,Vita Ap Lin,Andrew Buckle,Nimita Halani,Susan Clark,Catherine M Suter,Clare Stirzaker,Nicholas J Hawkins,Robyn L Ward,Kayfong Cheong

doi:10.1158/0008-5472.can-07-0869

Abstract

Biallelic promoter methylation and transcriptional silencing of the MLH1 gene occurs in the majority of sporadic colorectal cancers exhibiting microsatellite instability due to defective DNA mismatch repair. Long-range epigenetic silencing of contiguous genes has been found on chromosome 2q14 in colorectal cancer. We hypothesized that epigenetic silencing of MLH1 could occur on a regional scale affecting additional genes within 3p22, rather than as a focal event. We studied the levels of CpG island methylation and expression of multiple contiguous genes across a 4 Mb segment of 3p22 including MLH1 in microsatellite-unstable and -stable cancers, and their paired normal colonic mucosa. We found concordant CpG island hypermethylation, H3-K9 dimethylation and transcriptional silencing of MLH1 and multiple flanking genes spanning up to 2.4 Mb in microsatellite-unstable colorectal cancers. This region was interspersed with unmethylated genes, which were also transcriptionally repressed. Expression of both methylated and unmethylated genes was reactivated by methyltransferase and histone deacetylase inhibitors in a microsatellite-unstable colorectal carcinoma cell line. Two genes at the telomeric end of the region were also hypermethylated in microsatellite-stable cancers, adenomas, and at low levels in normal colonic mucosa from older individuals. Thus, the cluster of genes flanking MLH1 that was specifically methylated in the microsatellite-unstable group of cancers extended across 1.1 Mb. Our results show that coordinate epigenetic silencing extends across a large chromosomal region encompassing MLH1 in microsatellite-unstable colorectal cancers. Simultaneous epigenetic silencing of this cluster of 3p22 genes may contribute to the development or progression of this type of cancer.

Highlights

CpG islands span the promoters of f60% of genes, and in normal somatic cells, are usually unmethylated, allowing active transcription from the associated genes
In microsatellite instability (MSI) cancers from females older than 65 years (n = 10), CpG island methylation was found to extend across 2.4 Mb to include the ARPP-21, STAC, AB002340, EPM2AIP/MLH1, ITGA9, PLCD1, and DLEC1 genes in the majority of tumors (C1–9; Fig. 2A)
We have shown that a cluster of genes within 3p22 is epigenetically silenced in the majority of MSI colorectal cancers which display MLH1 promoter hypermethylation

Summary

Introduction

CpG islands (defined as >200 bp, CpG:GpC >0.6) span the promoters of f60% of genes, and in normal somatic cells, are usually unmethylated, allowing active transcription from the associated genes. Cancer cells often exhibit hypermethylation of CpG islands, and commonly affect tumor suppressor. CpG methylation acts synergistically with repressive histone modifications, such as dimethylation or trimethylation of the histone 3 lysine 9 (H3-K9) residue, to consolidate transcriptional silencing [2]. CpG island methylation is a common epigenetic event in colorectal neoplasia, with MLH1 promoter methylation representing a classic example of this phenomenon. It is well established that biallelic somatic methylation of MLH1 is seen in f15% of sporadic colorectal cancers, and these tumors display alterations at microsatellite repeat sequences due to loss of DNA mismatch repair function [3, 4]. Colorectal tumors demonstrating this microsatellite instability (MSI) phenotype occur most frequently in elderly women, and have a distinctive pathologic appearance [5, 6]

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Conclusion