The bacterial toxin CNF1 drives CRC progression though the induction of cellular senescence and stemness features

Background: The Cytotoxic Necrotizing Factor 1 (CNF1), produced by pathogenic E. coli, has been associated with inflammatory diseases and colorectal cancer (CRC). It permanently activates Rho-GTPases, promoting actin polymerization, cell motility, release of inflammatory factors, and apoptosis protection. CNF1 induces epithelial-mesenchymal transition, a crucial step in malignant tumor progression, and giant multinucleated cells, strongly resembling dormant cancer cells known to be at the basis for metastatic cascades and disease recurrence. Aim: The aim of this study is to investigate whether CNF1 may fuel metastases formation in vitro and in vivo. Methods: Human CRC cell lines exposed to CNF1 were analyzed for: i) Rac-1 activation by ELISA; ii) cell viability by MTT; iii) migration by wound healing and Boyden chambers assay; iv) cellular senescence by beta-galactosidase staining. Expression of genes associated with cancer cells stemness was analyzed by real-time PCR. Results: CNF1 was able to activate its intracellular target Rac1 and to polymerize the actin cytoskeleton. All the neoplastic cell lines tested (HT29, SW480, HCT116, Caco-2), except for the metastatic SW620, proved susceptible to the antiproliferative effect of CNF1. CNF1 stimulated the migration of transformed cells, both in wound-healing and Boyden Chamber assays. In particular, exposure of Caco-2 and HT29 cells to CNF1 induced giant multinucleated cells that acquired a senescent phenotype. Multinucleation and senescent phenotype were reversible upon toxin removal. In this context, the cells resumed the morphology of control cells and restarted growth. They acquired the ability to grow in an anchorage-independent culture model and expressed genes associated with the cancer stemness. Conclusions: Our results indicate that CNF1 may promote cell motility and appearance of cancer cell stemness traits, typical features associated with CRC progression and metastasis. Ongoing in vivo experiments with luciferase-expressing cells will confirm the impact of CNF1 on tumor progression and metastasis formation.