Azoxymethane induces KI-ras activation in the tumor resistant AKR/J mouse colon

Abstract

A differential susceptibility phenotype to the organotropic colon carcinogen azoxymethane (AOM) has been described in mice. The following studies were undertaken to test the hypothesis that intraspecific susceptibility can be accounted for by the specific complement of genetic alterations acquired by precancerous colon lesions referred to as aberrant crypt foci (ACF). As an initial approach to this question, mutations in codons 12 and 13 of the Ki-ras proto-oncogene were assessed in ACF, normal-appearing AOM-treated colonic epithelium, and tumors from A/J and SWR/J (susceptible) as well as AKR/J (resistant) mice. Four-week-old male mice were injected intraperitonealy, with AOM once a week for a total of 6 wk and killed 4 and 24 wk after the last injection. DNA was isolated from microdissected tissue, and polymerase chain reaction (PCR)-amplified products of Ki-ras exon 1 (codons 12 and 13) were directly sequenced from microdissected tissues. At 4 wk after AOM exposure, there was no significant difference in the frequency of Ki-ras activation (20-33%) between the three strains. Ki-ras mRNA expression was also evaluated by reverse transcription (RT)-PCR analysis and was comparably reduced (40-50%) in all three strains at the 4 wk time point. However, Ki-ras expression returned to normal by 24 wk after treatment. Finally, to gain further insight into the molecular pathogenesis underlying this experimental tumor model, analysis of the adenomatous polyposis coli (APC) protein within the colonic epithelium was undertaken by using an immunohistochemical approach. Although the APC protein was lost to a varying extent in tumors from A/J and SWR/J mice, the full-length form of the protein was still present in precancerous ACF isolated from each of the three strains, regardless of the degree of dysplasia of the lesion. A further molecular genetic analyses of ACF will be required to gain a more complete understanding of the molecular basis of tumor susceptibility phenotype in this murine model.

Metabolic Profiling, a Noninvasive Approach for the Detection of Experimental Colorectal Neoplasia

“…The observed expression changes in PHGDH, PSAT, PSPH and DMGDH, SARDH, GNMT within the tumor tissue can explain the increases in serine and sarcosine levels, respectively (Supplementary Table 1). We also note that AOM-induced tumors contain β-catenin and K- ras mutations which may contribute to these metabolomic changes (31–34). …”

Mouse models for the study of colon carcinogenesis

“…Interestingly, this group further showed that only 2.5% of MNU-generated adenomas had this mutation, whereas 33% of carcinomas had a G to A transition in K-Ras (118). In AOM-induced lesions, K-Ras mutations are frequently observed, whereas Apc and p53 mutations are less frequently found (20,119,120). These differences cannot be explained simply by species differences since p53 mutations have been detected in MNU-induced rat colon tumors and Apc mutations have been found in PhIP-induced tumors (121,122).…”

“…Since then, several thousand studies using AOM have been published (17)(18)(19). We have used this mouse model extensively for 15 years and have compiled considerable data on sensitivity and lesion characteristics across a number of mouse lines (20)(21)(22)(23)(24)(25)(26)(27).…”

Suppression of HSF1 activity by wildtype p53 creates the driving force for p53 loss-of-heterozygosity, enabling mutant p53 stabilization and invasion

“…In contrast, we posit that baseline AOM/DSS-induced tumors have insufficient stress levels to drive p53LOH spontaneously, explaining the missing spontaneous p53LOH in the AOM/DSS model (Figures 1F, S1B), compared to KRAS-driven mouse models of pancreas and lung cancer 42, 45 . Notably, in human CRCs strong constitutive oncogenic stress from K-RAS/ EGFR/ TGFβR/ PDGFR mutations are preeminent 65, 69, 75, 76 , while AOM/DSS tumors undergo predominantly CTNNB1 but no K-Ras and other proliferative driver mutations 68, 77 which promote proliferative stress 65 . Thus, baseline murine CRCs might not be stressed enough to spontaneously activate WTp53 and suppress HSF1 (Figure S2G).…”