Wild-type K-ras has a tumour suppressor effect on carcinogen-induced murine colorectal adenoma formation

Abstract

K-ras mutations are found in ~40% of human colorectal adenomas and carcinomas and contribute to colorectal tumour formation at an early stage. Wild-type K-ras has been reported to be deleted in some tumours, but the consequences of changes in wild-type K-ras copy number for experimental colorectal carcinogenesis have not been investigated. To characterize the effects of K-ras copy number changes on formation of carcinogen-induced colorectal neoplasms in mice, wild-type (K-ras(+/+) ) and heterozygous K-ras exon 1 knockout (K-ras(+/-) ) mice were given 10 weekly treatments of 1, 2-dimethylhydrazine (DMH) to induce colorectal tumours. Colorectal expression levels of K-ras 4A and 4B transcripts in K-ras(+/-) mice were ~50% decreased compared with K-ras(+/+) mice. One year after DMH treatment, survival of K-ras(+/-) mice decreased from 88 to 82% compared with wild-type mice. Colorectal adenomas significantly increased from 0.52 ± 0.15 in K-ras(+/+) mice to 0.87 ± 0.14 in K-ras(+/-) mice (mean ± SEM per mouse, P < 0.01); total tumour volume increased 2.13-fold (P < 0.05). Comparing K-ras(+/+) with K-ras(+/-) murine adenomas, Ki-67-positive proliferating tumour cells significantly increased from 7.77 ± 0.64% to 9.15 ± 0.92% and cleaved caspase-3-positive apoptotic tumour cells decreased from 1.40 ± 0.37% to 0.80 ± 0.22% (mean ± SEM, P < 0.05 for both). No K-ras or B-raf mutations were detected in the adenomas. Immunohistochemical studies showed no significant changes in extracellular signal regulating kinase/mitogen-activated protein kinase (Erk/MapK) or PI3K/Akt pathway activation in the adenomas. In conclusion, the data collectively show that a 50% reduction in K-ras gene dosage and RNA expression promoted experimental colorectal tumourigenesis, consistent with wild-type K-ras having a tumour suppressor effect on carcinogen-induced murine colorectal adenoma formation.

Keywords: 1, 2-dimethylhydrazine; K-ras; adenoma; carcinogen; colorectal; mouse; wild-type.

Figure 1

Relative expression levels of K-ras 4A and 4B transcripts in the normal colonic tissues from control wild-type K-ras^+/+ and K-ras^+/− mice by real-time RT-qPCR (mean ± SEM). The relative expression levels of both K-ras 4A and 4B transcripts were significantly decreased in colons of K-ras^+/− vs. wild-type mice (P < 0.01).

Figure 2

Differences in number, incidence and volume of colorectal adenomas and survival of DMH-treated control wild-type K-ras^+/+ mice compared with DMH-treated K-ras^+/− mice. (a) Mean numbers (±SEM) of large intestinal adenomas per mouse in DMH-treated mice were significantly higher in the K-ras^+/− mice (n = 28; P < 0.01) than in control K-ras^+/+ mice (n = 25). (b) Incidence of adenomas in the large intestine in DMH-treated control mice (11/25 mice) and K-ras^+/− mice (17/28 mice). (c) Mean relative values of tumour volume in DMH-treated mice were significantly higher in the K-ras^+/− mice (n = 28; P < 0.05) than in control mice (n = 25). (d) Survival of DMH-treated control and K-ras^+/− mice, given as proportions of mice surviving at various time periods in weeks, up to one year after starting DMH treatment.

Figure 3

Immunohistochemical staining of the proliferation marker Ki-67 and the apoptosis marker cleaved caspase-3 in colorectal adenomas from age-matched DMH-treated wild-type and K-ras^+/− mice (a–d, all at magnification ×200): Ki-67 expression in a colorectal adenoma from a DMH-treated wild-type mouse (a) and a K-ras^+/− mouse (b). Ki-67 positivity in adenomas was significantly higher in the K-ras^+/− mice than in wild-type mice (P < 0.05). Immunohistochemical staining of cleaved caspase-3 detection in a colorectal adenoma from a DMH-treated wild-type mouse (c) and a K-ras^+/− mouse (d). Cleaved caspase-3 positive cells in adenomas were significantly lower in frequency in the K-ras^+/− mice than in wild-type mice (P < 0.05).

Figure 4

Relative RNA expression levels in colorectal adenomas from DMH-treated wild-type K-ras^+/+ and K-ras^+/− mice, of pRB, p107/RBL1, p130/RBL2, cyclin D1 and c-myc transcripts (mean ± SEM), determined by real-time RT-qPCR. Adenomas from K-ras^+/− mice showed significantly lower levels of p107/RBL1 and p130/RBL2; (P < 0.05 for both) than wild-type mice.