Role of PMNs in inhibition of DNA repair and induction of genomic instability

Abstract

PMNs play key roles in host defense and tissue homeostasis, however, en‐masse tissue infiltration by PMNs often leads to aberrant inflammation and tissue injury, and is a hallmark of inflammatory bowel diseases (IBD). High numbers of mucosa infiltrating PMNs have been correlated with the severity of IBD, whereas IBD patients were found to be at significantly higher risk of developing colorectal cancer (CRC). While the link between inflammation and cancer is well established, little is known about PMN contribution to carcinogenesis. The accumulation of DNA damage is one of the leading causes of carcinogenesis. Activated PMNs can induce DNA damage via generation of reactive oxygen species (ROS). However, in the current work we describe a novel mechanism whereby the release of membrane‐derived microparticles (MPs) by tissue infiltrating/activated PMNs resultes in potent, ROS‐independent accumulation of double stranded DNA breaks (DSBs). PMN‐MP treatment of intestinal epithelial cells (IECs) induced persistent activation of DNA damage responses, including post‐translational modification of protein 53BP1, activation of DNA‐dependent protein kinase (DNA‐PKcs) and phosphorylation of histone H2AX, indicating increased generation of DSBs. Moreover, PMN‐MP treatment (96 hours) resulted in cell cycle arrest in S‐phase, deficient elongation of replication fork and persistent accumulation of γH2AX/53BP1 nuclear foci, suggesting inhibition of Homologous Recombination (HR)‐dependent DSB repair. Importantly, pre‐treatment of PMN‐MPs with RNAse significantly attenuated PMN‐MP‐mediated inhibition of DSB repair suggesting a role for micro‐RNAs (miRNAs) in this process. Indeed, a screen for potential miRNAs candidates with a known role in the regulation of DNA damage/repair responses revealed a significant upregulation of miR‐155 in PMNs following activation with IFNγ or TNFα, and its release in PMN‐MPs. MiR‐155 has been previously suggested to target a key regulator of HR, RAD51, which was found to be downregulated following PMN‐MP treatment. Importantly, an addition of miR‐155 antagonists reversed the loss of RAD51, partially recovered HR and decreased accumulation of DSBs induced by PMN‐MPs. Finally, in an in vitro model simulating PMN induced inflammation (continued co‐culture of IECs with PMN‐MPs over 12 population doublings) we detected an increase in aneuploidy and a high frequency of chromosomal aberrations indicating induction of genomic instability. Thus, our studies show for the first time that PMNs, via secretion of miRNA‐rich MPs, promotes genomic instability through the inhibition of HR, which may lead to mutagenesis and inactivation of tumor suppressor genes, and cellular transformation.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (NIH) DK101675 as well as American Cancer Society IRG‐9303718