Colibactin causes genomic instability and promotes Wnt independence in primary colon epithelial cells. Colibactin action in mouse 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 β-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.

结直肠癌（Colorectal cancer）由被称为腺瘤-腺癌序列（adenoma-carcinoma sequence）的连续性突变级联反应驱动。近期研究表明，结肠菌群中定植的特定细菌物种可诱发突变并促进该恶性肿瘤的发生发展。具体而言，产基因毒性大肠杆菌素的pks+大肠杆菌（pks+ Escherichia coli）菌株可诱发DNA双链断裂（DNA double-strand breaks, DSBs），并在结直肠癌小鼠模型中促进肿瘤发生。本研究利用源自原代小鼠结肠上皮细胞的类器官（organoids）与极化单层细胞（polarized monolayers），探究了大肠杆菌素的细胞转化潜能，并揭示了短期感染后出现的显著表型变化。大肠杆菌诱导的DNA双链断裂会导致原代细胞的突变负荷升高，该现象可通过拷贝数变异与染色体不稳定性得以验证。该改变与细胞增殖活性增强及分化受损密切相关。此外，从感染中恢复的类器官可脱离外源性Wnt配体（exogenous Wnt ligands）独立生长，模拟了Wnt信号通路（Wnt signaling pathway）发生突变的转化细胞。尽管未检出APC或β-连环蛋白（β-catenin）这类经典Wnt信号通路突变，但我们在与p53信号通路（p53 signaling pathway）相关的基因中鉴定出数种突变，包括Arid1a、miR-34与p21。进一步分析显示，不依赖Wnt的克隆会积累核p53，并表现出对Nutlin-3a的抗性。与此同时，我们证实，在类器官中敲除Tp53同样可导致细胞获得Wnt非依赖性生长，这印证了两条通路间存在功能性互作。本研究首次证明，正常原代细胞经pks+大肠杆菌诱导的DNA损伤所引发的细胞转化，可重现恶性转化早期阶段观察到的表型变化；同时也凸显了早期结肠炎相关癌症驱动突变（如Tp53）在协助结肠细胞获得Wnt非依赖性生长过程中的协同作用。