Cell Type-Preferential Expression of Peptidylarginine Deiminase 4 and p53-Dependent Therapeutic Vulnerabilities in Gastric Cancer.

BACKGROUND AND AIMS

Previous studies have demonstrated that peptidylarginine deiminase 4 (PAD4) functions as a suppressor, promoter, or both in cancer pathogenesis and therapeutic outcomes. Although PAD4 expression has been proposed to be one of the molecular features of gastric cancer (GC), the biological basis of PAD4 in GC progression and chemotherapy has not been formally established.

METHODS

Cell type-preferential expression of PAD4 was analyzed in both preclinical and clinical models. The colocalization of PAD4 expression and tumor-infiltrating neutrophils in GC patients was evaluated by immunofluorescence assay. The effects of forced expression of PAD4 on GC cell proliferation were evaluated both in vitro and in vivo. The effects of forced expression of PAD4 on the cytotoxicity of 5-fluorouracil and oxaliplatin were performed by EdU, Annexin V/PI, and comet assays. Co-immunoprecipitation assay was used to investigate the endogenous and exogenous interaction between PAD4 and p53. To investigate whether p53 participated in the chemopotentiating effects of PAD4, small interfering RNA (siRNA)-mediated knockdown of p53 was conducted in PAD4-overexpressing GC cells.

RESULTS

Contrary to the previous report, we initially observed that PAD4 was underexpressed in GC patients and presented as a favorable prognostic factor across the TCGA cohort. Interestingly, the normal gastric epithelial cell line GES-1 exhibited low-level expression of PAD4. In comparison, PAD4 was not detected in GC cell lines, including AGS, HGC-27, and MKN-45. Using an orthotopic mouse model of GC, we found that PAD4 was surprisingly abundant in HGC-27 cell line-derived xenografts. Immunofluorescence staining for PAD4 and neutrophil elastase indicated that PAD4 was mainly expressed in neutrophils but not in GC cells. PAD4 expression was upregulated in all-trans retinoic acid-induced neutrophil-like dHL-60 cells, which were used as a positive control for PAD4 expression. Surprisingly, the enforced expression of PAD4 suppressed the proliferation of GC cells in vitro and in vivo. In addition, cells harboring high PAD4 expression were more susceptible to G1/S boundary arrest, apoptotic death, and DNA damage by regulating p53 target proteins. Mechanistically, PAD4 might affect p53 function through physical interaction with p53, and the chemopotentiating effects of PAD4 could be compromised by p53 knockdown.

CONCLUSIONS

PAD4 appeared to be constitutively expressed in tumor-infiltrating neutrophils but not in GC cells. Our findings highlight unique roles of PAD4, in which origin-dependent PAD4 might work complementarily on the progression and treatment vulnerability of GC.