Neutrophils Deregulate Tumoral DNA Damage Response and Contribute to Non‐Homologous End‐Joining (NHEJ)‐dependent Survival of Colorectal Cancer

Abstract

Infiltration of polymorphonuclear neutrophil (PMNs) is a pathological feature of colorectal cancer (CRC). PMN accumulation in the cancer lesions and the tumor microenvironment is prominently associated with advanced‐stage CRC. While PMN presence has been a powerful predictor of poor clinical outcomes, the molecular basis underlying PMN contribution to cancer progression and its relationship to genomic instability remains elusive. We recently identified a novel mechanism by which PMNs contribute to CRC progression through the deregulation of DNA Damage Response (DDR) pathways. Using human tumor xenograft and colitis‐induced AOM/DSS murine models, we found that infiltration by PMNs promoted CRC growth and survival. We demonstrate that PMNs, by persistently inhibiting Homologous Recombination (HR)‐mediated double‐strand break (DSB) repair, contributed to upregulation of Non‐Homologous End‐Joining (NHEJ)‐mediated DSB repair. NHEJ activity was shown to be elevated in developing CRC tumors infiltrated by PMNs and was further accompanied by PMN‐dependent alteration in DDR transcriptional profile. PARP1 inhibition by Olaparib or NHEJ inhibition by Mirin and NU‐7741 prior to NHEJ upregulation significantly reduced tumor growth and increased tumor cell apoptosis, while had no effect on NHEJ‐upregulated CRC tumors at later stages. Our data thus suggest that while acutely, PMN infiltration leads to HR suppression and accumulation of DSBs, in longer‐term, PMN‐driven NHEJ upregulation leads effective DSB resolution, reduced replication stress, and increased CRC survival. In conclusion, our findings reveal a novel mechanistic underlying the biphasic effects of PMNs on DSB induction/resolution and its subsequent contribution to CRC carcinogenesis.Support or Funding InformationThis work was supported by grants from the American Cancer Society Research Scholar Award, the Crohn’s & Colitis Foundation Senior Research Award, and the Chicago Baseball Cancer Charities (CBCC)/ H Foundation Fellowship.Neutrophils promote survival and growth of HR‐inhibited CRC tumorsPMNs promote survival and growth of HR‐inhibited CRC tumors. A. Comet assay confirmed similar amount of DSB‐bearing nuclei between PMN+ and PMN− tumors cells. B. Quantification of Annexin V/PI‐stained CRC tumors cells confirmed that PMN+ tumor cells were significantly less apoptotic. C. Caliper measures and D. Images of CRC tumors. PMN+ tumors were of significantly greater volumes than PMN− tumors. E. HR activity was analyzed in tumor cells extracted from CRC xenograft tumors by plasmid‐based EGFP reconstitution reporter system. HR activity was significantly lower in PMN+ tumor cells. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, significantly different between PMN+ and PMN−; #, p < 0.05; ##, p < 0.01, significantly different compared to tumor onset.Figure 1Double‐strand break (DSB) repair by NHEJ is upregulated in neutrophilin‐filtrated CRC tumorsDSB repair by NHEJ is upregulated in PMN‐infiltrated CRC tumors.NHEJ activity was analyzed using plasmid‐based EGFP reconstitution NHEJ reporter system in A. in vitro long‐term co‐culture of HCT116 with PMN‐MPs and in B. extracted tumor cells from PMN+ and PMN− xenograft tumors. Both models confirmed significant upregulation of NHEJ DSB repair activity in the presence of PMNs or PMN‐MPs. C. Transcriptional profiling of 11 NHEJ‐specific genes indicated a PMN‐dependent shift toward NHEJ gene signature in late CRC tumors. D. Immunofluorescence (40x) of tumor sections from PMN+ and PMN− xenograft tumors indicated an induction of Ku70 and 53BP1 foci in the presence of PMNs. ##, p < 0.01; ####, p < 0.0001, significantly different from non‐treated HCT116; *, p < 0.05; **, p < 0.01; ***, p < 0.001, significantly different between PMN+ and PMN−.Figure 2