Abstract
Cytotoxic necrotizing factor 1 (CNF1), a protein toxin produced by extraintestinal pathogenic Escherichia coli, activates the Rho-family small GTPases in eukaryotic cell, thereby perturbing multiple cellular functions. Increasing epidemiological evidence suggests a link between CNF1 and human inflammatory bowel disease and colorectal cancer. At the cellular level, CNF1 has been hypothesized to reprogram cell fate towards survival due to the role in perturbing cell cycle and apoptosis. However, it remains undetermined how cells survive from CNF1 intoxication. In this work, we show that CNF1 treatment blocks mitosis/cytokinesis, elicits endoreplication and polyploidisation in cultured human colon cancer cells, and drives them into reversible senescence, which provides a survival route for cells via depolyploidisation. Senescence in CNF1-treated cells is demonstrated with upregulation of several senescence markers including senescence-associated β-galactosidase activity, p53, p21 and p16, and concomitant inhibition of the retinoblastoma protein phosphorylation. Importantly, progeny derived from CNF1 treatment exhibit genomic instability exemplified by increased aneuploidy and become more resistant to CNF1, but not to 5-fluorouracil and oxaliplatin, the two agents commonly used in chemotherapeutic treatment for colorectal cancer. These observations display survival features of the cell after CNF1 treatment that may have implications for the potential role of CNF1 in carcinogenesis.
Highlights
Many pathogenic bacteria produce virulence factors to target host GTP-binding proteins, especially Rho-family small GTPases[1,2]
When cells were plated at low density and treated with different concentrations of Cytotoxic necrotizing factor-1 (CNF1) for 10 days, the colony formation of HCT-116 decreased with increasing CNF1 concentration
The results indicate that CNF1 induces cell polyploidisation in dosage- and time-dependent manner
Summary
Many pathogenic bacteria produce virulence factors to target host GTP-binding proteins, especially Rho-family small GTPases[1,2]. Earlier studies demonstrated multiple roles of CNF1 in cell signaling, such as counteracting apoptosis, and inducing production of pro-inflammatory cytokines, COX2 expression, and NF-kB activation[34,35,36,37]. Based on these findings, CNF1 is proposed to reprogram the cell fate towards survival[22,23,25,38]. We show that CNF1 blocks cell mitosis/cytokinesis in human colon cancer cell line, triggers endoreplication and destines cells to multinucleation, polyploidy and reversible senescent arrest These events are followed by depolyploidisation-associated survival to generate genomically unstable progeny
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