Genetic and Epigenetic Modification of MLH1 Accounts for a Major Share of Microsatellite-Unstable Colorectal Cancers

Abstract

Microsatellite instability (MSI) is a hallmark of hereditary nonpolyposis colorectal cancer, and in these patients, results from inherited defects in DNA mismatch repair genes, mostly MSH2 and MLH1. MSI also occurs in 15% of sporadic colorectal cancers, but in these tumors, its basis is less well characterized. We investigated 46 sporadic MSI+ colorectal cancers for changes in MSH2 and MLH1 protein expression, followed by the analysis of somatic mutation, loss of heterozygosity (LOH), and promoter hypermethylation as possible underlying defects. Most cases (36/46, 78%) showed lost or reduced MLH1 expression. Among these, a majority (83%) was associated with MLH1 promoter hypermethylation, whereas the rates of LOH and somatic mutation of MLH1 were 24% and 13%, respectively. Hypermethylation and LOH were inversely correlated, suggesting that they had alternative functions in the inactivation of MLH1. MSH2 expression was lost in 7/46 (15%), and of these, 2 (29%) showed LOH and/or somatic mutation of MSH2. We conclude that most sporadic MSI+ colorectal cancers have an MLH1-associated etiology and that epigenetic modification is a major mechanism of MLH1 inactivation. Moreover, we found a significantly lower prevalence for MLH1 promoter hypermethylation in hereditary nonpolyposis colorectal cancer tumors with MLH1 germline mutations (12/26, 46%), which might explain some differences that are known to occur in the clinicopathological characteristics and tumorigenic pathways between sporadic and hereditary MSI+ colorectal cancers.

Mechanisms of inactivation of MLH1 in hereditary nonpolyposis colorectal carcinoma: a novel approach

“…MLH1 (MutL homolog 1) is the most important susceptibility gene for HNPCC (Peltomaki and Vasen, 2004) and also accounts for a major share of sporadic colorectal cancers (CRC) with microsatellite instability (MSI) (Kuismanen et al, 2000). In HNPCCrelated CRCs, the residual wild-type (wt) allele is often silenced by loss of heterozygosity (LOH) (Tannergard et al, 1997;Kuismanen et al, 2000;Potocnik et al, 2001;Yuen et al, 2002), whereas (biallelic) methylation of the MLH1 promoter primarily underlies sporadic microsatellite-unstable CRCs (Cunningham et al, 1998;Veigl et al, 1998;Kuismanen et al, 2000). Considerably less data are available on the mechanisms of inactivation of the wt allele in HNPCC spectrum tumors other than CRC, notably endometrial cancer (EC) that is the most common extracolonic malignancy in HNPCC.…”

“…Sporadic EC resembles sporadic CRC, in that MLH1 promoter methylation appears to be the primary mechanism of MSI (Esteller et al, 1998;Simpkins et al, 1999). In general, somatic point mutations rarely explain MMR gene inactivation in CRC (Tannergard et al, 1997;Kuismanen et al, 2000;Potocnik et al, 2001;Yuen et al, 2002) or EC (Chadwick et al, 2001).…”

No evidence for dual role of loss of heterozygosity in hereditary non-polyposis colorectal cancer

“…Somatic mutations as second hits have been found in both MLH1-and MSH2-deficient tumors, albeit at low frequencies Konishi et al, 1996;Lu et al, 1996;Cunningham et al, 2001). MLH1 promoter hypermethylation has been detected in 17-46% of the HNPCC tumors (Cunningham et al, 1998;Herman et al, 1998;Kuismanen et al, 2000;Potocnik et al, 2001). Loss of the wild-type allele in HNPCC tumors was proposed by Hemminki et al (1994) and it has been found to be the major mechanism for somatic second hits in most of the studies.…”

“…Loss of the wild-type allele in HNPCC tumors was proposed by Hemminki et al (1994) and it has been found to be the major mechanism for somatic second hits in most of the studies. Frequency of LOH in published studies varies between 33 and 86% (Hemminki et al, 1994;Lu et al, 1996;Tannerga˚rd et al, 1997;Kuismanen et al, 2000;Potocnik et al, 2001;Yuen et al, 2002), although the lost allele has not always been specified. In contrast, sporadic MMR-deficient tumors are most commonly due to bi-allelic inactivation of MLH1 by promoter hypermethylation, and these tumors rarely show LOH or somatic mutations in MLH1 (Kane et al, 1997;Cunningham et al, 1998;Herman et al, 1998;Veigl et al, 1998;Wheeler et al, 1999;Kuismanen et al, 2000).…”

“…No evidence for dual role of LOH in HNPCC tumors S Tuupanen et al Kuismanen et al (2000) studied the role of LOH and promoter hypermethylation in 27 HNPCC tumors with a known mutation in MLH1 or MSH2, and because these two phenomenons were never seen in the same patient, they suggested that the wild-type rather than the mutant allele was the target for somatic modification. However, that work as well as many others does not specify whether indeed the wild-type allele was the target of observed losses, leaving some space for ambiguity.…”