Mutations of BRAF are associated with extensive hMLH1 promoter methylation in sporadic colorectal carcinomas

Abstract

In the development of colorectal cancer (CRC), it is now widely accepted that some forms of genetic instability lead to the sequential accumulation of genetic alterations and consequently develop carcinomas. 1 RAS activation in the MAP kinase cascade is supposed to constitute a part of the primary events in colorectal carcinogenesis, and the KRAS gene mutations have been found in about 30 -40% cases of sporadic CRCs. [2][3][4] Recently, activating BRAF mutations have been found almost invariably in melanoma cells and sometimes in other types of carcinoma, including CRCs, 5-7 implying a function of BRAF as a protooncogene. The RAF genes are members of MAPK pathway, encoding serine/threonine kinases that integrate the upstream input signals. 8,9 Once recruited at the cell membrane by GTP-loaded RAS, RAF becomes activated and subsequently phosphorylates the downstream kinases, MEKs, which eventually induce transcriptional activation of the target genes. 9 More recently, frequent BRAF mutations and infrequent KRAS mutations have been reported in DNA-mismatch repair (MMR)-deficient CRCs. 10 Inactivation of MMR genes incurs instability of genomic microsatellite sequence (microsatellite instability, or MSI), which is found in the majority of patients with hereditary nonpolyposis colorectal cancer syndrome (HNPCC) and in 10 -15% of cases of sporadic CRCs. 11-13 Moreover, it was also reported that 70 -90% of sporadic CRCs with MSI (MSI ϩ CRCs) are associated with hypermethylation of hMLH1, one of DNA-MMR genes, and have distinct clinical and pathologic characteristics, i.e., occurrence in older females, location in the proximal colon and histopathology of mucious or poor differentiation. 14 -20 We have previously examined the methylation status of hMLH1 gene in sporadic CRCs by use of 5 sets of primer spanning the whole CpG sites within its promoter region and have classified the methylation status into 3 subtypes: full methylation, partial methylation and nonmethylation. 21,22 We reported that an extensive methylation, or full methylation, of hMLH1 promoter was found in about 80% of MSI ϩ CRC cases and was highly associated with loss of expression of its gene product. Interestingly, this type of CRC cells are rarely associated with KRAS mutations and loss of heterozygosity (LOH) of TP53 gene. 22 It is therefore possible that extensive methylation of hMLH1 promoter region may contribute to the carcinogenesis of the right-sided sporadic CRCs, independently of KRAS/p53 alterations.From these results, 2 questions may arise. First, does the activation of BRAF, instead of KRAS, take part in the carcinogenesis of CRCs with extensive hMLH1 methylation? Second, if so, does the BRAF activation have any relationship with the CRCs with partial methylation, although most of which are microsatellite stable (MSI Ϫ ), maintain MMR gene expression and show a relatively high incidence of KRAS and p53 alterations? 22 Additionally, in the melanoma cells, high frequency of mutations of ␤-catenin and BRAF has been recognized. 23 Some resea...

Low frequency of BRAF and CDKN2A mutations in endometrial cancer

“…BRAF, which encodes a RAF family member that functions downstream of RAS, has been reported to be somatically mutated in a number of human cancers. 3,5,25 Recently, activating mutations of BRAF have been frequently observed in microsatellite unstable (MSI+) colorectal carcinomas, in which mutations of BRAF and K-ras have been reported to be mutually exclusive. 5 Another recent study has suggested that alterations of the RAS pathway, both by RAS and BRAF mutations, contribute to the pathogenesis of stomach cancer.…”

“…3,5,25 Recently, activating mutations of BRAF have been frequently observed in microsatellite unstable (MSI+) colorectal carcinomas, in which mutations of BRAF and K-ras have been reported to be mutually exclusive. 5 Another recent study has suggested that alterations of the RAS pathway, both by RAS and BRAF mutations, contribute to the pathogenesis of stomach cancer. 25 Also, mutations in BRAF and K-ras have been found to characterise the development of lowgrade ovarian serous carcinoma.…”

“…The median follow-up period for the survivors was 9 years (range [5][6][7][8][9][10][11][12][13][14]. None of the patients were lost due to insufficient follow-up data.…”

“…3,4 These mutations of BRAF have also been linked to microsatellite instability (MSI) in colorectal carcinoma. 5 We wanted to investigate the frequency of BRAF mutations in endometrial cancer and in particular their relation to MSI and Ras mutations, especially since mutations in Ras are relatively few in this group of tumours.As evidenced from studies on melanoma, the mutations in the BRAF gene, though early, are not sufficient for tumour formation, for which an additional genetic hit is required. This is supported by characterization of overlapping BRAF mutations and CDKN2A aberrations in melanomas 6 and also occurrence of a high frequency of BRAF mutations in tumours from families with germline CDKN2A mutations.…”

“…3,4 These mutations of BRAF have also been linked to microsatellite instability (MSI) in colorectal carcinoma. 5 We wanted to investigate the frequency of BRAF mutations in endometrial cancer and in particular their relation to MSI and Ras mutations, especially since mutations in Ras are relatively few in this group of tumours.…”