Exploring the tumor-promoting potential of the Escherichia coli toxin CNF1: a preclinical study”

Colorectal cancer (CRC) is one of the most commonly diagnosed and deadliest neoplasms in Italy and worldwide. Gut microbiota imbalance has been proposed as one of the etiologic factors in CRC. Recent evidence suggests that some toxicogenic bacteria are correlated to CRC and that specific toxins from Escherichia coli have a higher incidence rate in CRC, thus suggesting that these toxins may play a role in CRC onset and development. Based on these premises, we aim to investigate the CRC-promoting ability of the cytotoxic necrotizing factor 1 (CNF1), a protein toxin produced by pathogenic E. coli that permanently activates Rho-GTPases, highly conserved intracellular factors regulating a variety of key cellular processes. It has been previously shown that CNF1 stimulates cell motility, apoptosis resistance, NF-kB-dependent inflammatory cytokine release and epithelial mesenchymal transition in several in vitro models. In the present study, we analyzed the induction of DNA damage, multinucleation and cell cycle dysregulation in colon cancer cell lines (e.g. HT-29, SW480) and normal epithelial cells (IEC-6) exposed to CNF1 or to the enzymatically-inactive CNF1 toxin. We observed that CNF1 induces the phosphorylation of Histone 2AX in both neoplastic and non-neoplastic cell lines, and stimulates the release of ROS species only in IEC-6 cells. CNF1 blocks cell proliferation and induces apoptosis in HT-29 and SW480 cells, but not in IEC-6 cells, as evidenced by flow cytometry. 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 by western blot showed that in normal or transformed cells, CNF1 increased the expression of cyclin-dependent kinase inhibitors p19, p21 and p27 and, at the same time, rose Cyclin D1, a protein required for progression through the G1 phase of the cell cycle, suggesting that cells react in an aberrant way. Preliminary evidence from animal studies indicate that in vivo exposure to CNF1 induces the accumulation of intraepithelial TCR-gamma/delta T lymphocytes, which are known to stimulate the growth and progression of CRC. Taken together, our results suggest that CNF1 may exert tumor-intrinsic and tumor-extrinsic effects promoting malignant transformation and tumor progression.