Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1+/- Mice

Abstract

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world and interactions between genetic and environmental factors, including diet, are suggested to play a critical role in its etiology. We conducted a long-term feeding experiment in the mouse to address gene expression and methylation changes arising in histologically normal colonic mucosa as putative cancer-predisposing events available for early detection. The expression of 94 growth-regulatory genes previously linked to human CRC was studied at two time points (5 weeks and 12 months of age) in the heterozygote Mlh1 +/- mice, an animal model for human Lynch syndrome (LS), and wild type Mlh1 +/+ littermates, fed by either Western-style (WD) or AIN-93G control diet. In mice fed with WD, proximal colon mucosa, the predominant site of cancer formation in LS, exhibited a significant expression decrease in tumor suppressor genes, Dkk1, Hoxd1, Slc5a8, and Socs1, the latter two only in the Mlh1 +/- mice. Reduced mRNA expression was accompanied by increased promoter methylation of the respective genes. The strongest expression decrease (7.3 fold) together with a significant increase in its promoter methylation was seen in Dkk1, an antagonist of the canonical Wnt signaling pathway. Furthermore, the inactivation of Dkk1 seems to predispose to neoplasias in the proximal colon. This and the fact that Mlh1 which showed only modest methylation was still expressed in both Mlh1 +/- and Mlh1 +/+ mice indicate that the expression decreases and the inactivation of Dkk1 in particular is a prominent early marker for colon oncogenesis.

DNA hypermethylation appears early and shows increased frequency with dysplasia in Lynch syndrome-associated colorectal adenomas and carcinomas

“…LS mutation carriers are enrolled in lifelong colonoscopy surveillance with 2–3-year intervals [ 10 ], and we took advantage of the regular surveillance to obtain consecutive specimens. Furthermore, a previous mouse study implicated a number of candidate genes in association with MLH1 mutation and diet [ 11 ], prompting us to evaluate the respective genes as methylation targets in human LS.…”

Mlh1 haploinsufficiency induces microsatellite instability specifically in intestine

“…As we used targeted approach to test the promoter methylation, we cannot negate the possibility of methylation of other CpG sites of the Mlh1 promoter region of Mlh1 +/+ allele leading to tissue-specific MLH1 depletion. Additionally, heterozygosity of Mlh1 is known to provoke promoter methylation and as consequence lower expression of multiple tumor suppressor genes in normal GI tract affecting crucial signaling pathways[25]. It is likely, that the lower tissue-specific Mlh1 expression we observed is consequence of altered signaling pathways in Mlh1 +/intestine.…”

“…Genotyping PCR was performed using Platinum green hot start PCR master mix (Invitrogen, Carlsbad, CA). Lysis buffer components, PCR program and primers [25] used for genotyping are listed in Supplementary table 1. In total 58 mice (39 males and 19 females) were used in this study, the number of mice for each experiment along with the genotype and the age is indicated in the respective figures in the results section.…”

Reduced MAD2 levels dampen the apoptotic response to non-exchange sex chromosomes and lead to sperm aneuploidy

“…No significant interindividual differences were found within the groups. Details of the genotyping procedure, as well as PCR primers and cycling conditions for Mad2, endogenous Spo11, the Tg(Xmr-Spo11β B ) transgene and Mlh1 alleles have been published previously (Baudat et al, 2000;Dobles et al, 2000;Kauppi et al, 2011;Pussila et al, 2013) and are listed in the supplementary Materials and Methods. All applicable national and institutional guidelines (Animal Experiment Board in Finland and Laboratory Animal Centre of the University of Helsinki, respectively) for the care and use of animals were followed.…”