Kras regulatory elements and exon 4A determine mutation specificity in lung cancer

Abstract

KRASis the most frequently mutatedrasfamily member in lung carcinomas 1,2 , whereasHRASmutations are common in tumours from stratified epithelia such as bladder or skin (www.sanger.ac.uk/genetics/CGP/cosmic/). Using a mouse model (HrasKI) 3 in which theHrascoding sequence was inserted into theKraslocus, we demonstrate that specificity forKrasmutations in lung andHras mutations in skin tumours is determined by local regulatory elements in the targetrasgenes. We further show that, whileKras-4Ais dispensable for mouse development 4,5 , it is necessary both for lung carcinogenesisin vivoand for the previously reported 6,7 inhibitory effect of wild-type (WT)Krason the transforming properties of the mutant allele. Kras-4A expression is detected in a sub-population of normal lung epithelial cells, but at very low levels in lung tumours, suggesting a role in tumour initiation rather than in tumour maintenance. The two Kras isoforms undergo different post-translational modifications 8 , therefore these findings can have important implications for the design of therapeutic strategies for inhibiting oncogenic Kras activity in the prevention and treatment of cancer.Kras deficiency in mice leads to embryonic lethality 4,5 , thus it has not been possible to determine whether the selection for Kras mutations in lung tumours reflects a specific oncogenic function that is required for lung carcinogenesis and cannot be compensated for by mutant Hras or Nras. The HrasKI mouse provides a viable model that completely lacks Kras expression 3 (Fig. 1), but carries a "knock-in" Hras cDNA expressed under the control of Kras regulatory elements. We reasoned that HrasKI mice should be resistant to lung tumour development if Kras protein is essential for lung carcinogenesis. To control for the possibility of structural alterations affecting experimental outcomes, we used KrasKI mice in which the coding sequence corresponding to the Kras-4B isoform was knocked back into the Kras locus in the exact manner used to generate the HrasKI mouse 3 . HrasKI mice expressed elevated levels of Hras protein, consistent with the increase in Hras gene dosage from two copies to four in these animals ( Fig. 1). KrasKI mice express similar levels of Kras protein as WT mice, as expected since Kras-4B is the major splice isoform 9,10 . Levels of Akt and Erk activation in these animals were similar to WT mice (Fig. 1). There was also no difference in levels of p38α, recently shown to affect ras mediated lung carcinogenesis 11 . The elevated level of Hras We further pursued the specificity of ras gene mutations in lung and skin tumours by inducing both tumour types in the same HrasKI homozygous mice. A single dose of urethane was injected intraperitoneally to induce lung tumours, followed by a single topical treatment on the skin with urethane and subsequent biweekly application of tetradecanoyl-phorbol acetate (TPA) to induce papilloma development. All lung tumours (n = 28) analyzed have the CAA>CTA mutation in the HrasKI allele. Of the papillomas (n = ...

K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif

“…However, a similar study using a mouse genetic model to induce colon carcinomas showed no such effect of K-Ras4A deficiency (27). Another study using a different mouse transgenic at the KRAS locus revealed a role for K-Ras4A in tumorigenesis: when the cDNA sequence of K-Ras4B was knocked into the KRAS locus, such that only K-Ras4B could be expressed (designated KRAS KI ), homozygous KRAS KI mice were resistant to urethane-induced lung carcinogenesis (14). However, the same study called into question the protumor effects of WT K-Ras4A by showing that, when the KRAS KI allele was heterozygous with the KRAS LA2 allele that spontaneously recombines to generate oncogenic K-Ras, more lung tumors developed in KRAS KI/LA2 than KRAS wt/LA2 mice, consistent with a tumor suppressor effect of WT K-Ras4A (14).…”

“…Another study using a different mouse transgenic at the KRAS locus revealed a role for K-Ras4A in tumorigenesis: when the cDNA sequence of K-Ras4B was knocked into the KRAS locus, such that only K-Ras4B could be expressed (designated KRAS KI ), homozygous KRAS KI mice were resistant to urethane-induced lung carcinogenesis (14). However, the same study called into question the protumor effects of WT K-Ras4A by showing that, when the KRAS KI allele was heterozygous with the KRAS LA2 allele that spontaneously recombines to generate oncogenic K-Ras, more lung tumors developed in KRAS KI/LA2 than KRAS wt/LA2 mice, consistent with a tumor suppressor effect of WT K-Ras4A (14). Thus, the literature on K-Ras4A-deficient mice provides little clarity and no mechanistic understanding of any biological differences that may distinguish the two highly conserved splice variants of the KRAS locus.…”

“…A recent study suggested that the presence of oncogenic K-Ras4A was necessary to initiate tumor formation and growth in a mouse model of induced lung carcinogenesis through KRAS mutation (14). Importantly, because the mutations found in human cancer that render KRAS constitutively active are located in exons 1 and 2, both splice variants expressed from a mutated allele will encode oncogenic proteins.…”

Endogenous expression of Hras G12V induces developmental defects and neoplasms with copy number imbalances of the oncogene

“…The molecular mechanism for cell-type specificity for activating mutations of particular RAS genes has recently been proposed to be determined, at least in part, by the expression levels of the individual RAS genes. Thus, urethane induces lung cancers that are associated with Kras mutations in mice, but when an Hras cDNA is knocked into the Kras gene locus, and is thus expressed under the control of the Kras gene promoter, the urethane-induced tumors harbor Hras mutations instead (5). However, there is also evidence that distinct membrane targeting of the different Ras proteins may confer them with specific functions (6).…”

N-Arachidonoyl Dopamine Inhibits NRAS Neoplastic Transformation by Suppressing Its Plasma Membrane Translocation

“…It has been shown that KRAS4A plays an essential role in the development of carcinogen-induced lung cancer in mice and that oncogenic KRAS4A is widely expressed in human cancers (46,47). Suppression of palmitoylated RAS signaling is, therefore, likely to be an effective therapeutic strategy for a broad range of cancers.…”