Mutated K-ras(Asp12) promotes tumourigenesis in Apc(Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways

Abstract

Summary K-ras mutations are found in 40-50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K-ras mouse model (K-ras(Asp12)/Cre), with transient intestinal Cre activation by beta-Naphthoflavone (beta-NF) treatment, displayed transgene recombination and K-ras(Asp12) expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with Apc(Min/+) mice, the K-ras(Asp12)/Cre/Apc(Min/+) offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 +/- 5.4 weeks from 20.9 +/- 4.7 weeks (control Apc(Min/+) mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5-fold and 5.7-fold, respectively, in K-ras(Asp12)/Cre/Apc(Min/+) mice compared with Apc(Min/+) mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K-ras(Asp12) and Apc(Min) co-operation, the Mitogen-activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K-ras(Asp12) increased activation of Mapk and Akt signalling pathway targets phospho-extracellular signal-regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo-oxygenase 2 (Cox2) and T-cell lymphoma invasion and metastasis 1 (Tiam1) in K-ras(Asp12)/Cre/Apc(Min/+) adenomas compared with that of Apc(Min/+) adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator-activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c-myc remained unchanged. In conclusion, intestinal expression of K-ras(Asp12) promotes mutant Apc-initiated intestinal adenoma formation in vivo more in the large intestine than the small intestine, with evidence of synergistic co-operation between mutant K-ras and Apc involving increased expression of some Wnt-pathway target genes.

Figure 1

K-ras^Asp12 construct, PCR genotyping and construct copy number assay. (a) Structure of the K-ras^Asp12 transgene: cytomegalovirus (CMV) immediate early promoter; neomycin (NEO), resistance gene; 1, 2, 3 and 4B, K-ras exons 1, 2, 3 and 4B; encephalomyocarditis virus internal ribosome entry site (IRES); enhanced green fluorescent protein (EGFP); pA, polyadenylation signal; ▸, 34 bp LoxP sites; the positions and orientations of the PCR primers used for genotyping analyses are depicted by arrows (→). (b) Typical results of genotyping PCR assays from offspring of transgenic mice and control (B6) mice. Polymerase chain reaction (PCR) amplification of an Apc gene fragment was used as the control for the quality of genomic DNA samples. + represents mouse tail DNA from mice positive for the K-ras^Asp12 transgene; − represents mouse tail DNA from control B6 mice negative for the transgene constructs. (c) Genotyping Min mice by allele-specific primers binding either the wild-type or the mutant Apc gene sequence. Upper two panels: use of Apc1a/Apc1b (wild-type Apc primer pair) and Apc1a/Apc2b (mutant Apc primer pair) to amplify wild-type B6 genomic DNA, shows that at 66 °C and 67 °C annealing temperatures, wild-type Apc can be distinguished from mutant Apc. Middle two panels: use of Apc1c/Apc1d (wild-type Apc primer pair) and Apc1c/Apc2d (mutant Apc primer pair) to amplify wild-type B6 genomic DNA, shows that at 72 °C annealing temperature wild-type Apc can be distinguished from mutant Apc. Lower two panels: an example of the use of both sets of wild-type and mutant Apc primer pairs Apc1a/Apc1b (W) and Apc1a/Apc2b (M) at 66.5 °C (upper) and Apc1c/Apc1d (W) and Apc1c/Apc2d (M) at 72 °C (lower) to amplify genomic tail DNA samples from known negative control B6 (wild-type only) and known Min (M1 to M3, with both wild-type and mutant Apc alleles) mice. (d) Bar chart of relative copy numbers of K-ras exon 3 sequences (human and mouse) by Real-time quantitative DNA PCR (qPCR), showing the mean (error bar = SD) relative copy number of K-ras exon 3 in B6 control mice was 2.00 ± 0.37 (n = 6), and that of K-ras^Asp12 was 3.80 ± 0.55 (n = 6), indicating the presence of two endogenous and two transgenic copies of K-ras.

Figure 2

Analysis of conditional K-ras^Asp12 transgene recombination. (a) Schematic representations of the K-ras^Asp12 construct undergoing recombination (after treatment with β-NF to induce Cre expression) at the two LoxP sites to bring about expression of K-ras^Asp12 transcripts. Without Cre recombinase expression, the K-ras^Asp12 transgenes remain silent due to the presence of the neomycin (NEO) containing ‘STOP’ cassette. Upon Cre-mediated recombination of the LoxP sites (arrowheads), the K-ras^Asp12 transgene is placed directly under the control of the cytomegalovirus (CMV) promoter. Position and orientation of the polymerase chain reaction (PCR) primers used for analysis are depicted by arrows (→). (b) PCR amplification of the DNA fragment between the CMV promoter and K-ras^Asp12 exon 3 generated a 500-bp fragment from genomic DNA extracted from the intestines and some other tissues. B6, Black6 wild-type; C, Cre-only genotype; K/C, K-ras^Asp12/Cre genotypes; SI, small intestine; LI, large intestine; Sp, spleen; St, stomach; Pan; pancreas; Sk, skin; Liv, liver; H, heart. (c) DNA sequencing traces of the 500-bp amplified products showed that the ‘STOP’ cassette had been deleted and there was only one LoxP site (flanked by two NotI restriction enzyme sites) between the sequences of the CMV promoter and K-ras exon 1, with the appropriate codon 12 mutation for the transgene construct: codon 12 GAT in the K-ras^Asp12 mouse intestine.

Figure 3

Expression of K-ras^Asp12 in intestinal tissues and tumours. (a) Reverse transcription polymerase chain reaction (RT-PCR) analysis of the expression of K-ras^Asp12 4B transcripts in different tissues of K-ras^Asp12/Cre transgenic mice, 10 days after completion of the β-NF treatment. C, Cre-only genotype, K/C, K-ras^Asp12/Cre genotypes; LI, large intestine; SI, small intestine; St, stomach; Sp, spleen; Sk, skin and Liv, liver as described previously. (b) PCR amplification of a 500-bp DNA fragment between the recombined CMV promoter and K-ras^Asp12 exon 3 from genomic DNA of large intestinal tumours (LIT) from K-ras^Asp12/Cre/Apc^Min/+ transgenic mice treated with β-NF (Apc amplification as control). (c) RT-PCR analysis of the expression of K-ras^Asp12 4B transcripts in two small intestinal tumours (SIT) and two LIT from K-ras^Asp12/Cre/Apc^Min/+ (K/C/M) mice after treatment with β-NF (β-actin RNA expression as normalization reference). (d) Western blot analysis of the expression of mutant K-Ras^Asp12 protein in a large intestinal tumour of a K-ras^Asp12/Cre/Apc^Min/+ (K/C/M-LIT) mouse after β-NF treatment (β-actin protein expression as control) compared with a Cre-only mouse large intestine (C-LI).

Figure 4

Intestinal tumour prevalence and lifespan in Apc^Min/+ mice and K-ras^Asp12/Cre/Apc^Min/+ mice. (A & B) Average numbers of small intestinal tumours (a) and large intestinal tumours (b) in K-ras^Asp12/Cre/Apc^Min/+ mice (speckled bars), compared with control Apc^Min/+ and Apc^Min/+/Cre mice (open bars). (c) Kaplan–Meier survival curves of Cre mice (n = 29), Apc^Min/+ (n = 29 in total, including 21 Apc^Min/+ and 8 Apc^Min/+/Cre mice), and K-ras^Asp12/Cre/Apc^Min/+ mice (n = 25). Ages of the animals at death (or when killed, if moribund) are given in weeks (X-axis).

Figure 5

Immunohistochemical analysis of intestinal adenomas. Large intestinal tumours from Apc^Min/+ mice (Min-LIT) and from K-ras^Asp12/Cre/Apc^Min/+ transgenic mice (K-ras-Min-LIT) were analysed immunohistochemically for expression of pAkt, pErk 1, 2, pGSK, gastrin, VEGF, β-catenin, Tiam1 and Cox2.

Figure 6

Hierarchical cluster analysis of the differential expression patterns of 24 genes in normal colon and large intestinal tumours. Normal large intestine (LI) tissues from Cre (C), K-ras^Asp12/Cre (K-C) and Apc^Min/+ (M) mice and large intestinal tumours (LIT) from Apc^Min/+ (M) and K-ras^Asp12/Cre/Apc^Min/+ (K-C-M) mice were analysed for the relative expression levels of 24 selected genes by real-time quantitative reverse transcription polymerase chain reaction (see Table 2): red represents marked overexpression; dark red/black represents mild over-expression and green represents unchanged or mildly decreased expression levels, with the fold-change shown according to the colour key of the row-Z score. T numbers refer to individual samples. There is a pattern of clustering broadly together of LI tissues and LITs from mice of the same genotypes indicating mostly consistent gene expression changes.