Cancer-educated neutrophils promote lung cancer progression via PARP-1-ALOX5-mediated MMP-9 expression

Objective:Neutrophils are one of the most predominant infiltrating leukocytes in lung cancer tissues and are associated with lung cancer progression.How neutrophils promote lung cancer progression,however,has not been established.Methods:Kaplan–Meier plotter online analysis and tissue immunohistochemistry were used to determine the relationship between neutrophils and overall survival in lung cancer patients.The effect of neutrophils on lung cancer was determined using the Transwell migration assay,a proliferation assay,and a murine tumor model.Gene knockdown was used to determine poly ADPribose polymerase(PARP)-1 function in lung cancer-educated neutrophils.Western blot analysis and gelatin zymography were used to demonstrate the correlation between PARP-1 and matrix metallopeptidase 9(MMP-9).Immunoprecipitation coupled to mass spectrometry(IP/MS)was used to identify the proteins interacting with PARP-1.Co-immunoprecipitation(Co-IP)was used to confirm that PARP-1 interacts with arachidonate 5-lipooxygenase(ALOX5).Neutrophil PARP-1 blockage by AG14361 rescued neutrophil-promoted lung cancer progression.Results:An increased number of infiltrating neutrophils was negatively associated with overall survival in lung cancer patients(P<0.001).Neutrophil activation promoted lung cancer cell invasion,migration,and proliferation in vitro,and murine lung cancer growth in vivo.Mechanistically,PARP-1 was shown to be involved in lung cancer cell-induced neutrophil activation to increase MMP-9 expression through interacting and stabilizing ALOX5 by post-translational protein modification(PARylation).Blocking PARP-1 by gene knockdown or AG14361 significantly decreased ALOX5 expression and MMP-9 production,and eliminated neutrophil-mediated lung cancer cell invasion and in vivo tumor growth.Conclusion:We identified a novel mechanism by which PARP-1 mediates lung cancer cell-induced neutrophil activation and PARylates ALOX5 to regulate MMP-9 expression,which exacerbates lung cancer progression.

DNA-delivered monoclonal antibodies targeting the p53 R175H mutant epitope inhibit tumor development in mice

tThe tumor suppressor p53 is the most common mutated gene in cancer,with the R175H as the most frequent p53 missense mutant.However,there are currently no approved targeted therapies or immunotherapies against mutant p53.Here,we characterized and inves-tigated a monoclonal antibody(mAb)that recognizes the mutant p53-R175H for its affinity,specificity,and activity against tumor cells in vitro.We then delivered DNA plasmids expres-sing the anti-R175H mAb or a bispecific antibody(BsAb)into mice to evaluate their therapeutic effects.Our results showed that the anti-R175H mAb specifically bound to the p53-R175H an-tigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells,with no cross-reactivity with wild-type p53.In cultured cells,the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity.We made a recombinant MC38 mouse cell line(MC38-p53-R175H)that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles.In vivo,administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice.The administration of the anti-R175H BsAb plasmid showed no therapeutic effects,yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody.These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens.