Unravelling the role of cytotoxic necrotizing factor-1 from E. coli in colorectal carcinogenesis: a preclinical study

Colorectal cancer (CRC) is one of the most commonly diagnosed and deadliest neoplasms in Italy and worldwide. Over 15% of newly diagnosed cancers are attributable to bacterial infections (1). Recent data highlight that specific toxins from E. coli have a higher incidence rate in CRC (2). However, a definitive causal link between E. coli toxins and CRC has not been established yet. Among E. coli virulence factors, cytotoxic necrotizing factor 1 (CNF1) deserves considerable attention due to its ability to subvert key cellular pathways known to be involved in neoplastic transformation. Upon cell entry, CNF1 permanently activates Rho-GTPases and induces cell motility, apoptosis resistance, NF-kB-dependent inflammatory cytokine release and epithelial mesenchymal transition in vitro (3). In the present study, we investigated the possible genotoxic effect of CNF1 on intestinal non-neoplastic epithelial cells in vitro and evaluated the carcinogenic potential of the toxin in vivo. We observed that CNF1 induces DNA damage, multinucleation and cell cycle dysregulation in non-malignant epithelial cells, and stimulates the release of reactive oxidizing species after 24h and 48h, as compared to controls. In addition, treatment with CNF1 toxin blocks cell proliferation and induces apoptosis in HT-29 CRC cells, but not in non-neoplastic intestinal cells. Interestingly, this effect was reversible as cells recovered their proliferating capabilities after CNF1 removal from the culture medium. The analysis of key cell cycle proteins in neoplastic and non-neoplastic epithelial cells showed that CNF1 increases the expression of the cyclin-dependent kinase inhibitors p19, p21 and p27 and, at the same time, rises Cyclin D1, a protein required for progression through the G1 phase of the cell cycle in both models, suggesting that cells exposed to CNF1 react in an aberrant way. In vivo, repeated exposure to CNF1 toxin induces an increase of leucocyte infiltrates in the colon tissue, as observed by hematoxylin/eosin staining of tissue biopsies, and the accumulation of intraepithelial TCR-gamma/delta T lymphocytes, which are known to support the growth and progression of CRC. In addition, the intrarectal administration of CNF1 in mice with experimental colitis induces morphological modifications in the intestinal mucosa and abnormal protrusions rising from the lining of the large intestine and invading the intestinal lumen, as observed by video-colonoscopy three months after treatment initiation. Histological classification of colon lesions as well as molecular and immune infiltrate characterization of mice with experimental colitis exposed to CNF1, as compared to controls, are ongoing. Taken together, our results suggest that CNF1 toxin may exert cell-intrinsic and cell-extrinsic (i.e. immune-mediated) effects promoting malignant transformation and can concur to colorectal carcinogenesis in vivo.