Insights into the importance of the microbiome from mouse models

Individuals born in 1990 have twice the risk of developing colon cancer and four times the risk of developing rectal cancer as those born in 1950. The gut microbiome is being proposed as a potential contributor to this difference because of the surge in obesity in the US, the link between obesity and gut dysbiosis, and the growing number of studies which have associated a dysbiotic gut microbiome with CRC. Enterotoxigenic Bacteroides fragilis (ETBF) is one of the bacteria most studied in relation to CRC development; it is found at a higher frequency in both the stool and mucosa of CRC patients, and it rapidly induces tumour formation in an Apcmin/+ mouse model of CRC. In this model, tumour formation typically occurs via loss of heterozygosity of the Apc gene, the genetic mutation found in approximately 80% of sporadic CRC cases. Initial forays into the mechanisms behind ETBF-induced tumor formation show that the bacterium produces a potent exotoxin (BFT) which induces E-cadherin cleavage, beta-catenin nuclear localisation and colonic epithelial cell proliferation. But we still do not understand how these downstream effects cause lasting changes in the genome of colon epithelial cells that then initiate tumor formation and growth. We hypothesise that ETBF induces DNA mutations via BFT that encourage tumor formation, and enhance tumor growth. To test this hypothesis, we performed whole-exome sequencing on tumours and normal tissue isolated from Apcmin/+ mice after ETBF or sham inoculation. Additionally, we isolated colon organoids from Apcmin/+ mouse normal tissue after ETBF or sham inoculation. We performed in vitro DNA damage and intracellular reactive oxygen species (ROS) generation assays.

 

Our data indicate that BFT alone can induce ROS generation and double-stranded DNA breaks in normal colon organoids as soon as 1 hour and 3 hours after cell treatment respectively. Furthermore, all tumors which arise in Apcmin/+ mice possess Apc LOH, but the severity of LOH varies unexpectedly by mouse. In mice with an additional mutation in the Msh2 gene, an important member of the mismatch repair pathway, Apc LOH is absent in all tumours examined. The majority of the tumours in these mice show higher rates of mutation across the genome, and possess an independent mutation in the second copy of the Apc gene. These data suggest that in vivo, ETBF induces DNA damage, potentially via intracellular ROS production, which is normally repaired by the mismatch repair pathway. In individuals with a germline or acquired mutation in this pathway, colonisation with ETBF may further promote tumour formation and growth via accumulation of additional oncogenic mutations.

 

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