Correlation between hypermethylation of the RASSF2A promoter and K‐ras/BRAF mutations in microsatellite‐stable colorectal cancers

Abstract

Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p 5 0.033) and distal CRC (p 5 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC. ' 2006 Wiley-Liss, Inc.Key words: RASSF2A; methylation; K-ras; BRAF; microsatellite stability Accumulation of genetic alterations is associated with the development of cancer. The multistep carcinogenesis model in colon cancer serves as the classical model of genetic alterations in cancer.1 Colorectal cancer (CRC) is initiated by alterations in molecules in the Wnt, TGF-b signaling pathways and K-ras and TP53.2 Together with the accumulation of genetic alterations, epigenetic alterations also result in the transformation of normal colon epithelium into adenocarcinoma.3 Analysis of the methylation profiles of colon cancer cell lines and primary tumors show that multiple tumor suppressor genes are methylated and participate in the development of CRC. 4 The Ras signaling pathway plays an important role in tumorigenesis, and mutation of the ras gene is found frequently in many tumor types including CRC.5 The Ras-GTPase controls cell fate through the binding of effector molecules such as Raf and PI3 kinase. Mutations of K-ras and BRAF that activate the Ras/Raf/ MAPK pathway are frequent oncogenic events in sporadic CRC. V600 is a hotspot for mutation of BRAF, and the V600E mutation accounts for more than 80% of the BRAF mutations in CRC.6 In addition, the K-ras mutation is detected in up to 50% of large bowel adenocarcinomas. 7,8 The BRAF V600E and K-ras mutations are known to be mutually exclusive. 9,10Recently, Khokhlatchev et al. identified another Ras effector pathway, wherein active K-ras initiates apoptosis through the direct recruitment of the effector NORE.11 NORE1 (RASSF5) interacts with RASSF1, another Ras effector, and has tumor suppressor activity.12 Methylation of the RASSF1 gene has been reported ...

Genetic and epigenetic alterations of Ras signalling pathway in colorectal neoplasia: analysis based on tumour clinicopathological features

“…The majority of reports adopted MSP as the experimental procedure, and reported a higher prevalence of methylation (70 -73% in CRCs and 88 -100% in adenomas) (Hesson et al, 2005;Park et al, 2006) than in our study (46% in CRCs and 25% in adenomas) or Akino et al's study (42% in CRCs and 43% in adenomas). However, the methylation frequency of RASSF2 in colorectal neoplasia was much higher than that of RASSF1 even in our results.…”

“…Meanwhile, Sakamoto et al (2004) have shown that the early flat-type colorectal tumours exhibited a much higher frequency of RASSF1 methylation (81.3%). The frequency of RASSF2 methylation in CRC was reported to be 42 -73% (Akino et al, 2005;Hesson et al, 2005;Park et al, 2006).…”

“…In addition, clinicopathological features of colorectal tumours that carry the methylation of either RASSF1 or RASSF2 are still largely unknown in adenomas as well as in cancers. Moreover, the association between K-ras/BRAF mutations and RASSF methylation, particularly whether or not they work synergistically or are mutually exclusive, is controversial in CRCs (Akino et al, 2005;Hesson et al, 2005;Park et al, 2006), and has not been sufficiently investigated in adenomas.…”

“…We then examined the correlation between these mutations and methylation based on the clinicopathological features of the adenomas. In particular, we focused on locational differences in combinations of these genetic and epigenetic alterations because a locational imbalance in each of these alterations has been indicated (Samowitz et al, 2000;Luchtenborg et al, 2005;Park et al, 2006). Moreover, we compared these alterations in adenomas with those in CRCs, and examined when these alterations are likely to occur during colorectal carcinogenesis.…”

Promoter hypermethylation of the ADAM23 gene in colorectal cancer cell lines and cancer tissues

“…Our laboratory have previously investigated the clinicopathological features, microsatellite instability status and mutations of K-ras and BRAF genes in colorectal cancer tissues. 17 A total of 70 cases in this study were same with the colorectal cancer tissues of previous study. There was no significant association between ADAM23 methylation and other clinicopathological features (Table II).…”