Conditional expression of mutated K-ras accelerates intestinal tumorigenesis in Msh2-deficient mice

Abstract

K-ras mutation occurs in 40-50% of human colorectal adenomas and carcinomas, but its contribution to intestinal tumorigenesis in vivo is unclear. We developed K-ras(V12) transgenic mice that were crossed with Ah-Cre mice to generate K-ras(V12)/Cre mice, which showed beta-naphthoflavone-induction of Cre-mediated LoxP recombination that activated intestinal expression of K-ras(V12) 4A and 4B transcripts and proteins. Only very occasional intestinal adenomas were observed in beta-naphthoflavone-treated K-ras(V12)/Cre mice aged up to 2 years, suggesting that mutated K-ras expression alone does not significantly initiate intestinal tumourigenesis. To investigate the effects of mutated K-ras on DNA mismatch repair (MMR)-deficient intestinal tumour formation, these mice were crossed with Msh2(-/-) mice to generate K-ras(V12)/Cre/Msh2(-/-) offspring. After beta-naphthoflavone treatment, K-ras(V12)/Cre/Msh2(-/-) mice showed reduced average lifespan of 17.3+/-5.0 weeks from 26.9+/-6.8 (control Msh2(-/-) mice) (P<0.01). They demonstrated increased adenomas in the small intestine from 1.41 (Msh2(-/-) controls) to 7.75 per mouse (increased fivefold, P<0.01). In the large intestine, very few adenomas were found in Msh2(-/-) mice (0.13 per mouse) whereas K-ras(V12)/Cre/Msh2(-/-) mice produced 2.70 adenomas per mouse (increased 20-fold, P<0.01). Over 80% adenomas from K-ras(V12)/Cre/Msh2(-/-) mice showed transgene recombination with expression of K-ras(V12) 4A and 4B transcripts and proteins. Sequencing of endogenous murine K-ras showed mutations in two out of 10 tumours examined from Msh2(-/-) mice, but no mutations in 17 tumours from K-ras(V12)/Cre/Msh2(-/-) mice. Expression of K-ras(V12) in tumours caused activation of the mitogen-activated protein kinase and Akt/protein kinase B signaling pathways, demonstrated by phosphorylation of p44MAPK, Akt and GSK3beta, as well as transcriptional upregulation of Pem, Tcl-1 and Trap1a genes (known targets of K-ras(V12) expression in stem cells). Thus, mutated K-ras cooperates synergistically with MMR deficiency to accelerate intestinal tumorigenesis, particularly in the large intestine.