Colibactin causes genomic instability and promotes Wnt independence in primary colon epithelial cells

Colorectal cancer is driven by a sequential cascade of mutations known as the adenoma-carcinoma sequence. Recent studies have revealed that specific bacterial species present in the colonic microbiota can induce mutations and contribute to this malignancy. Specifically, genotoxic colibactin-producing pks+ Escherichia coli strains can induce DNA double strand breaks (DSBs) and promote tumor development in mouse models of colorectal cancer. Here, we investigated the transformation potential of colibactin by using organoids and polarized monolayers derived from primary murine colon epithelial cells and reveal striking phenotypic changes upon short-term infection. E. coli-induced DNA DSBs resulted in an increased mutational burden in primary cells, as revealed by copy number variations and chromosomal instability. This was associated with enhanced proliferative activity and impaired differentiation. Moreover, organoids that recovered from infection grew independently of exogenous Wnt ligands, mimicking transformed cells with mutations in the Wnt signaling pathway. Although we did not find classic Wnt signaling mutations, such as in APC or ß-catenin, we identified several mutations in genes related to p53 signaling, including Arid1a, miR-34, and p21. Further analysis revealed that Wnt-independent clones accumulated nuclear p53 and exhibited Nutlin-3a resistance. Concomitantly, we show that knockout of Tp53 in organoids also results in Wnt independence, corroborating a functional interplay between the two pathways. Our data demonstrates for the first time that cellular transformation resulting from pks+ E. coli-induced DNA damage in normal primary cells can recapitulate phenotypic changes observed in the erly stage of malignant transformation. It also highlights the cooperation of early colitis-associated cancer-driving mutations, such as Tp53, in facilitating Wnt independence in colon cells.