PRSS22 promotes the immune evasion of gastric cancer via inhibiting ANXA1-mediated degradation of PD-1

Abstract

Purpose: Gastric cancer (GC) ranks as the third most prevalent cause of mortality among malignant tumors globally. This study endeavors to uncover the protective function and underlying mechanism of PRSS22 on the immune evasion of GC.

Methods: A total of 24 GC patients and 7 normal volunteer were recruited. The gene chip data GSE291766 in GC was downloaded to analyze the differentially expressed genes (DEGs). DAVID database was used to perform the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. TIMER database was used to quantify PRSS22 in different cancers as well as the correlation between the PRSS22 expression and the abundance of immune infiltration by gene module. GEPIA2 was used to analyze the prognostic significance of PRSS22 in GC patients. GC cells (NCI-N87, MKN-45, MKN-28, and AGS) were used to compare the PRSS22 expression with GES-1. Jurkat cells transfected with negative, PRSS22, si-NC, or si-PRSS22 were cultivated with AGS to establish the in vitro coculture system. All mice were inoculated with AGS and injected with negative or sh-PRSS22 lentivirus. qPCR was used to analyze the PRSS22, TP53, Cox2, MYC, TNF-α, CD28, GLUT1, Granzyme B, TCF-1, LAG-3, NR4A, and TIM-3 mRNA levels. Western blotting was used to measure the PRSS22, ANXA1, and PD-1 proteins levels. The commercial kits were used to determine the caspase-3/7/9, LDH, TGF-β, TNF-α, IFNγ, and IL-10 levels. Cell viability, migration, and proliferation were determined using CCK-8, PI fluorescent stain, transwell, and EdU assays. TNFα/CD8 and CD4⁺CD25⁺FoxP3⁺ levels were measured by flow analysis. In vivo imaging and immunohistochemical analysis were used to detect the ANXA1 expression. Immunofluorescence analysis was used to determine the PRSS22 and ANXA1 expressions. IP assay was used to analyze the interaction of PRSS22 protein with ANXA1 protein along with PD-1 ubiquitination.

Results: PRSS22 was highly expressed in GC patients. The overall survival in GC patients (high-PRSS22) was lower than GC patients (low-PRSS22). PRSS22 mRNA and protein expressions were significantly upregulated in AGS cells. In the GC mouse model, PRSS22 downregulation decreased tumor volume and weight. PRSS22 was manifested in T cells of GC patients. In coculture with AGS and Jurkat cell, PRSS22 could significantly promote cell proliferation, migration, and EdU positivity but reduced LDH activity and PI levels. We discovered that the PRSS22 promoted the immune evasion in T cells of GC. ANXA1 and PD-1 were DEGS in sh-PRSS22 GC samples, and ANXA1/PD-1 pathway might be important for the functions of PRSS22 on immune evasion in the GC model. PRSS22 knockout upregulated ANXA1 but downmodulated PD-1 in in vitro and in vivo experiments. PRSS22 WT protein could interact with the ANXA1 WT protein, and ANXA1 upregulation could promote the ubiquitination of PD-1 protein. In coculture with AGS and Jurkat cell, si-PRSS22 treatment could reduce the tumor cell growth, migration, Edu positivity, and the immune evasion ability, which would be reversed by suppressing ANXA1.

Conclusion: PRSS22 suppressed ANXA1-mediated degradation of PD-1 in T cell to promote the immune evasion of GC. Targeting PRSS22 is thus a potentially effective therapeutic strategy for GC.