title: 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.
authors.affiliation: Department of Pathology, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK.
authors.forename: F
authors.initials: F
authors.lastname: Luo
authors.forename: D G
authors.initials: DG
authors.lastname: Brooks
authors.forename: H
authors.initials: H
authors.lastname: Ye
authors.forename: R
authors.initials: R
authors.lastname: Hamoudi
authors.forename: G
authors.initials: G
authors.lastname: Poulogiannis
authors.forename: C E
authors.initials: CE
authors.lastname: Patek
authors.forename: D J
authors.initials: DJ
authors.lastname: Winton
authors.forename: M J
authors.initials: MJ
authors.lastname: Arends
mesh_terms: D000818:Animals; D053843:DNA Mismatch Repair; D011905:Genes, ras; D019426:Integrases / Q000502:physiology; D007414:Intestinal Neoplasms / Q000453:epidemiology / Q000209:etiology* / Q000235:genetics; D020935:MAP Kinase Signaling System; D051379:Mice; D008822:Mice, Transgenic; D051718:MutS Homolog 2 Protein / Q000172:deficiency / Q000502:physiology*; D009154:Mutation; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D013945:Thymoma / Q000209:etiology; D013953:Thymus Neoplasms / Q000209:etiology
chemical_list: D051057:Proto-Oncogene Proteins c-akt; C045073:Cre recombinase; D019426:Integrases; C497173:Msh2 protein, mouse; D051718:MutS Homolog 2 Protein