RAS signaling in colorectal carcinomas through alteration of RAS, RAF, NF1, and/or RASSF1A

Abstract

More than half of all colorectal carcinomas are known to exhibit an activated mitogen-activated protein kinase pathway. The NF1 gene, a negative regulator of KRAS, has not previously been examined in a series of colorectal cancer. In the present study, primary colorectal carcinomas stratified according to microsatellite instability status were analyzed. The whole coding region of NF1 was analyzed for mutations using denaturing high-performance liquid chromatography and sequencing, and the copy number alterations of NF1 were examined using multiple ligation-dependent probe amplification and real-time polymerase chain reaction. The mutational hot spots in KRAS and BRAF were sequenced, and promoter hypermethylation status of RASSF1A was assessed with a methylation-specific polymerase chain reaction. One sample had two missense mutations in NF1, whereas nine additional tumors had intronic mutations likely to affect exon splicing. Interestingly, 8 of these 10 tumors were microsatellite-unstable. Four other tumors showed a duplication of NF1. Mutations in KRAS and BRAF were mutually exclusive and were present at 40% and 22%, respectively. RASSF1A was hypermethylated in 31% of the samples. We show that the RAS signaling network is extensively dysregulated in colorectal carcinomas, because more than 70% of the tumors had an alteration in one or more of the four examined components.

Figure 1

Site distribution of mutations within each gene. The mutations for the respective gene are placed according to their sequence position. In (a) and (b), only the exons in blue have been analyzed. In (c), all exons are analyzed, and the exons in orange indicate those that are only expressed in isoforms. To the right, representative sequencing results of mutant samples are presented.

Figure 2

Alterations across the sample series. The pie chart indicates the four analyzed components and the percentage of tumors which showed alterations among these. Clockwise from the wild type pie, we see alterations in RASSF1A and BRAF; BRAF; BRAF and NF1; NF1; NF1 and KRAS; KRAS; KRAS and RASSF1A; RASSF1A; RASSF1A, NF1 and BRAF.