K‐ras exon 4A has a tumour suppressor effect on carcinogen‐induced murine colonic adenoma formation

Abstract

K-ras encodes two isoforms, K-ras 4A and 4B, that are jointly affected by K-ras activating mutations, which are prevalent in colorectal cancer (CRC). CRC shows alterations in the expressed K-ras 4A : 4B isoform ratio in favour of K-ras 4B, in tumours both with and without K-ras mutations. The present study evaluated whether K-ras 4A expression can suppress colonic adenoma development in the absence of its oncogenic allele. Mice with homozygous targeted deletions of K-ras exon 4A (K-ras(tmDelta4A/tmDelta4A)) that can express the K-ras 4B isoform only, along with heterozygous K-ras(tmDelta4A/+) and wild-type mice, were given ten weekly 1,2-dimethylhydrazine (DMH) treatments to induce colonic adenomas. There was a significant increase in both the number and the size of colonic adenomas in DMH-treated K-ras(tmDelta4A/tmDelta4A) mice, with reduced survival, compared with heterozygous and wild-type mice. No K-ras mutations were found in any of the 30 tumours tested from the three groups. Lack of expression of K-ras 4A transcripts was confirmed, whereas the relative expression levels of K-ras 4B transcripts were significantly increased in the adenomas of K-ras(tmDelta4A/tmDelta4A) mice compared with K-ras(tmDelta4A/+) and wild-type mice. Immunohistochemical studies showed that adenomas of K-ras(tmDelta4A/tmDelta4A) mice had significantly increased cell proliferation and significantly decreased apoptosis with evidence of activation of MapKinase and Akt pathways, with increased phospho-Erk1/2 and both phospho-Akt-Thr308 and phospho-Akt-Ser473 immunostaining, compared with adenomas from K-ras(tmDelta4A/+) and wild-type mice. In conclusion, following DMH treatment, K-ras exon 4A deletion promoted increased number and size of colonic adenomas showing increased K-ras 4B expression, increased proliferation, decreased apoptosis, and activation of MapKinase and Akt pathways, in the absence of K-ras mutations. Therefore, K-ras 4A expression had a tumour suppressor effect on carcinogen-induced murine colonic adenoma formation, explaining the selective advantage of the altered K-ras 4A : 4B isoform ratio found in human colorectal cancer.

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

“…injection (25). Similarly, when colonic adenomas were induced in these mice by treatment with 1,2-dimethylhydrazine, mice lacking K-Ras4A had an increase in number and size of colonic adenomas that revealed increased markers of proliferation and decreased markers of apoptosis (26), suggesting that, despite the highly counterintuitive nature of these findings, WT K-Ras4A can behave like a tumor suppressor. However, a similar study using a mouse genetic model to induce colon carcinomas showed no such effect of K-Ras4A deficiency (27).…”

Synergism between K-rasVal12 and mutant Apc accelerates murine large intestinal tumourigenesis

“…previous studies have shown that expression of mutant K-ras Val12 in either mouse embryonic stem (es) cells or mutant K-ras transgenic mice was associated with changes in expression of a range of gene transcripts and proteins, including stem cell associated genes and components of the Mapk and Akt pathway genes (10,18,(20)(21)(22). Quantitative rt-pcr and immunohistochemical expression analysis of the intestinal tumours Val12 has been shown to stimulate Wnt signaling in colonic cancer through inhibition of GSK-3β (32), which is regulated by the activation of the pI3K/Akt pathway.…”

“…the pMApK Family rabbit mAb sampler Kit and the pAKt pathway sampler Kit (cell signaling, usA) were used for MApK and AKt pathway protein analysis by Western blotting. Immunohistochemical analysis was performed with the goat anti-gastrin polyclonal antibody (santa cruz Biotechnology, usA), the rabbit anti-cox-2 monoclonal antibody and the rabbit anti-vascular endothelial growth factor (VegF) polyclonal antibody (both from novus Biologicals, usA) as described previously (21,22).…”

“…tumours were cut in two and half was used for extraction of dnA, rnA and protein and the other half for formalin fixation and histopathological analysis. the tumour samples from the small and large intestines were processed for paraffin embedding following fixation in 10% neutral-buffered formalin solution and sections were prepared for H&e staining and immunohistochemistry as described previously (21,22).…”

“…Real-time quantitative reverse transcription PCR (real-time RT-qPCR) was carried out to measure the relative expression levels of selected target genes (table I shows PCR primer sequences) using the comparative Ct method that has been described previously (18,(20)(21)(22). gene expression levels were normalized against β-actin (using the following formulas: ∆∆ct = ∆ct test-∆ct control, fold change = 2 -∆∆ct ).…”