CpG island promoter hypermethylation of a novel Ras-effector gene RASSF2A is an early event in colon carcinogenesis and correlates inversely with K-ras mutations

Abstract

We report in silico identification and characterisation of a novel member of the ras association domain family 1 (RASSF1)/NORE1 family, namely, RASSF2, located at chromosomal region 20p13. It has three isoforms, all contain a ras association domain in the C-terminus. The longest isoform RASSF2A contains a 5 0 CpG island. RASSF2A was cloned from a brain cDNA library and directly sequenced, confirming the genomic gene structure. In previous reports, we and others have demonstrated that RASSF1A is epigenetically inactivated in a variety of cancers, including sporadic colorectal cancer (CRC). In the present report, we analysed the methylation status of RASSF2A promoter region CpG island in sporadic CRC and compared it to K-ras mutation status. RASSF2A promoter region CpG island was hypermethylated in a majority of colorectal tumour cell lines (89%) and in primary colorectal tumours (70%), while DNA from matched normal mucosa was found to be unmethylated (tumour-specific methylation). RASSF2A expression was reactivated in methylated tumour cell lines after treatment with 5-aza 2-deoxycytidine. RASSF2A methylation is an early event, detectable in 7/8 colon adenomas. Furthermore, 75% of colorectal tumours with RASSF2A methylation had no K-ras mutations (codons, 12 and 13) (P ¼ 0.048), Fisher's exact test). Our data demonstrate that RASSF2A is frequently inactivated in CRCs by CpG island promoter hypermethylation, and that epigenetic (RASSF2A) and genetic (K-ras) changes are mutually exclusive and provide alternative pathways for affecting Ras signalling.

Evaluation of the 3p21.3 tumour-suppressor gene cluster

“…RASSF2A expression is frequently lost or downregulated due to tumour-specific promoter CpG island hypermethylation in up to 70% of colorectal carcinomas (CRCs). This is one of the earliest events in CRCs detectable in adenoma polyps (Akino et al, 2005;Hesson et al, 2005). In fact, inactivation of RASSF2A is much earlier and more frequent in CRCs than inactivation of RASSF1A, which occurs at around 15-45% (Akino et al, 2005;Hesson et al, 2005).…”

“…This is one of the earliest events in CRCs detectable in adenoma polyps (Akino et al, 2005;Hesson et al, 2005). In fact, inactivation of RASSF2A is much earlier and more frequent in CRCs than inactivation of RASSF1A, which occurs at around 15-45% (Akino et al, 2005;Hesson et al, 2005). It has recently been shown that methylation in a subset of genes in CRCs and matching faecal tissue may form a basis for early detection (Muller et al, 2004).…”

“…Expression of NORE1A is lost or downregulated in cancer due to promoter hypermethylation (particularly in NSCLC and kidney cancers), while mutation of the NORE1 gene is a rare event (Chen et al, 2003;Hesson et al, 2003;Morris et al, 2003;Irimia et al, 2004). RASSF3A is not methylated in human tumours and RASSF6 is not methylated in colorectal cancer (Hesson et al, , 2005Akino et al, 2005). In addition, RASSF7 and RASSF8 have recently been identified (UCSC Genome Browser; Falvella et al, 2006).…”

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

“…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.…”