Susceptibility of Snark-deficient mice to azoxymethane-induced colorectal tumorigenesis and the formation of aberrant crypt foci

Abstract

SNF-1/5'-AMP-activated kinase (AMPK)-related kinase (SNARK) is a member of the AMPK-related kinases. Snark(+/-) mice exhibited mature-onset obesity and related metabolic disorders. Obesity is regarded as a risk factor for colorectal cancer. To investigate whether Snark deficiency is involved in tumorigenesis in the large intestine, obese Snark(+/-) mice were treated with a chemical carcinogen, azoxymethane (AOM). The incidences of both adenomas and aberrant crypt foci (ACF) were significantly higher in Snark(+/-) mice than in their wild-type counterparts 28 weeks after the completion of AOM treatment (10 mg/kg/week for 8 weeks). Furthermore, ACF formation was enhanced in Snark(+/-) mice treated with AOM for 2 weeks, suggesting that Snark deficiency contributed to the early phase of tumorigenesis. The total number of ACF was correlated with bodyweight in Snark(+/-) and Snark(+/+) mice, suggesting that obesity was a risk factor for colorectal tumorigenesis in this model. However, the correlation coefficient was higher in Snark(+/-) mice. Moreover, AOM-induced ACF formation was also enhanced in preobese Snark(+/-) mice. Together, these findings suggest that AOM-induced tumorigenesis in Snark(+/-) mice was enhanced via obesity-dependent and -independent mechanisms.

Figure 1

Snark expression in mouse gastrointestinal tract and the generation of Snark‐deficient mice. (a) Relative quantitative (RQ) reverse transcription–polymerase chain reaction (RT‐PCR) quantification of Snark transcripts normalized for Actb in adult C57BL/6 J mouse tissues of the digestive organs. The results are expressed as the mean value ± SD (n = 4) relative to the expression level in the esophagus. (b) Schematic representations of the wild‐type Snark locus, targeting vector, and targeted allele. Exon 1 is indicated by a filled box. The 5′ probe used for Southern hybridization is shown. P, PstI site. (c) Southern blot analysis of genomic DNA from Snark^−/– , Snark^+/– , and Snark^+/+ mouse embryo fibroblasts (MEF) hybridized to the 5′ probe shown (b). (d) RT‐PCR analysis of total RNA from Snark^−/– , Snark^+/– , and Snark^+/+ MEF. (e) RQ RT‐PCR analysis of total RNA from Snark^+/– and Snark^+/+ liver and large intestine. The results are expressed as the mean value ± SD (n = 3) relative to the expression level in the Snark^+/– liver. DTA, diphteria toxin A chain; Gapdh, glyceraldehyde‐3‐phosphate dehydrogenase.

Figure 2

Representative neoplastic and preneoplastic colorectal lesions induced by azoxymethane in Snark^+/– mice. (a) Macroscopic and microscopic views of a tubular adenoma that developed in a Snark^+/– mouse. The microscopic views were acquired using hematoxylin–eosin staining. Original magnification: left bottom, ×4; right, ×100. (b) Representative features of aberrant crypt foci (ACF). ACF were detected using methylene blue staining. The arrowheads indicate the luminal openings of the crypts. Original magnification: ×40.

Figure 3

Distribution of aberrant crypt focus (ACF) size, and correlation between ACF and obesity in Snark^+/– and Snark^+/+ mice. (a) Total number and size distribution of ACF in each mouse. (b) Correlation between ACF numbers and bodyweight or metabolic parameters. Significant correlations between bodyweight and ACF number were observed in both Snark^+/– and Snark^+/+ mice.