Activation of K-RAS by co-mutation of codons 19 and 20 is transforming
© Naguib et al. 2011
Received: 5 November 2010
Accepted: 3 March 2011
Published: 3 March 2011
The K-RAS oncogene is widely mutated in human cancers. Activating mutations in K-RAS give rise to constitutive signalling through the MAPK/ERK and PI3K/AKT pathways promoting increased cell division, reduced apoptosis and transformation. The majority of activating mutations in K-RAS are located in codons 12 and 13. In a human colorectal cancer we identified a novel K-RAS co-mutation that altered codons 19 and 20 resulting in transitions at both codons (L19F/T20A) in the same allele. Using focus forming transformation assays in vitro , we showed that co-mutation of L19F/T20A in K-RAS demonstrated intermediate transforming ability that was greater than that of individual L19F and T20A mutants, but less than that of G12D and G12V K-RAS mutants. This demonstrated the synergistic effects of co-mutation of codons 19 and 20 and illustrated that co-mutation of these codons is functionally significant.
Mutations in RAS family genes occur in approximately 20-30% of all human cancers, with mutations in the K-RAS gene comprising ~80% of these mutations . K-RAS mutations have been documented in the majority of human cancer types with pancreatic (~90% of these cancers) and colorectal (~40%) cancers demonstrating the highest incidence of mutations in this gene [2, 3]. K-RAS codons 12 and 13 are the most common sites of oncogenic activation with over 90% of documented mutations being found in these codons . Amino acid alterations at these codons, which encode amino acids adjacent to the GDP/GTP binding pocket, reduce or abolish GTPase activity of K-RAS after GAP binding and lock the protein in an active, GTP-bound state . Codons 12 and 13 in wildtype K-RAS both encode glycine residues. The incorporation of other amino acids, most commonly aspartate and valine at codon 12 and aspartate at codon 13 , brings about projection of larger amino acid side chains into the GDP/GTP binding pocket of the protein, interfering with the geometry of the transition state in which GTP hydrolysis is catalysed . Mutations in codons 61 and 146 have also been described to be oncogenic in K-RAS, although mutations at these positions occur at a much lower prevalences (<5% of total K-RAS mutations) than codon 12 and 13 mutations . Activation of RAS has several effects on rodent cells in vitro, including the establishment of a transformed phenotype with anchorage independent growth in soft agar, transformed focus formation, as well as tumour formation following injection into animals [8–10]. Our previous mouse model studies have shown that induced expression of mutant K-RAS accelerates intestinal adenoma formation in vivo on both a mutant Apc background and a Msh2-null background, increases proliferation, modulates apoptosis and alters gene expression patterns [11–15]. In this study, we identify a novel double mutation outside of codons 12 and 13, recoding K-RAS codon 19 from leucine to phenylalanine (L19F) and codon 20 from threonine to alanine (T20A), in a colorectal cancer and we demonstrate that it causes transformation in vitro and is therefore a rare, but functionally significant co-mutation of K-RAS.
Missense mutations of K-RAS codon 20 in human cancers have not been previously reported, however, mutations giving rise to phenylalanine incorporation into K-RAS codon 19 have been described previously in seven individual human colorectal cancers, a single human lung adenocarcinoma sample and a single lymphoblastic leukaemia [17–20]. No observations of a double mutation giving rise to both L19F and T20A amino acid alterations have been described previously. As such, the observation in our study of double mutant peaks giving rise to L19F/T20A missense co-mutations at codons 19 and 20 of K-RAS describes a previously unreported change in this gene.
It is not surprising that the T20A substitution alone was not sufficiently oncogenic to cause focus formation as this amino acid alteration has never been found previously in human cancers. However, the L19F substitution has been documented, albeit in a limited number of studies, and may be expected to have some transforming potential. One study described analysis of L19F in the C. elegans RAS homologue let-60. C. elegans carrying this mutation showed a temperature-sensitive multivulval phenotype. In a mammalian system, at body temperature, H-RAS (L19F) protein had a reduced rate of GTP hydrolysis relative to wildtype H-RAS, suggesting that H-RAS L19F conferred an increased level of activation . However, transfection of NIH3T3 cells with human H-RAS with the incorporated L19F mutation failed to demonstrate increased focus formation above that of controls, a similar observation to that made here using L19F K-RAS. A second report, however, describes L19F as causing increased cell proliferation, anchorage-independent growth, increased tumourigenicity in nude mice and elevated levels of RAS-GTP . Additionally, a recent analysis describing K-RAS mutations outside of codons 12 and 13 also described mildly increased focus formation by L19F mutants . In this report the L19F K-RAS mutation was described to induce formation of ~5 foci whereas positive controls (G12V and G12D) were observed to develop ~80 - 90 foci. These data are not mutually exclusive with our observations, as the mean focus count observed here with the G12D mutant was 35 colonies per plate, thus a mild focus forming ability of the L19F mutation may not have been detected. The observed differences between the number of colonies formed in the study by Smith and colleagues  and those in our own assays may be due to variations in protocols used, such as the different expression vectors used in the two analyses giving rise to different levels of transcription following transfection into NIH3T3 cells and different tissue culture plating conditions.
Our identification and analysis of the L19F/T20A double mutation in K-RAS is the first description of this genetic change, which confers a greater transforming ability than individual mutations in these codons. The rare observed frequency of this double amino acid change giving rise to oncogenic K-RAS in human cancers, compared with that of the individual L19F amino acid change, despite its increased oncogenic potential, may be due to the lower likelihood of 2 base changes occurring at these positions in the same allele during cancer development.
K-RAS mutations affecting codons 15 and 22 have also been reported in human colorectal cancers [23, 24]. Although the transforming ability of these specific codon changes is yet to be confirmed, the observation of sequence changes in human cancers affecting codons 15 and 22, as well as at co-mutation of codons 19 and 20, as described here, strongly suggests that alteration of this region in the K-RAS protein provides a selective growth advantage for cells which contributes to neoplastic transformation.
We thank Professor RY Ball for help in obtaining the human tumour samples for analysis. This work was supported by Cancer Research UK, Medical Research Council and Wellcome Trust.
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