Regulation Of TAP1 Expression In EBV-Associated Gastric Carcinoma And Impact On Immunotherapy Response

Objective:Epstein-Barr Virus-associated gastric carcinoma(EBVaGC)is a special subtype of gastric cancer.Patients with EBV infection in gastric cancer have characteristics of high expression of Programmed Cell Death-Ligand 1(PD-L1)and high sensitivity to immune therapy.However,the potential molecular mechanisms of PD-L1 amplification and good immune therapy response in EBVaGC are still unclear.Our previous research found that Transporter 1,associated with antigen processing(TAP1)is significantly upregulated in EBVaGC,and is an important molecule involved in tumor cell antigen presentation response.However,the specific regulatory mechanism of TAP1 and its impact on immune therapy response in gastric cancer need further elucidation.Methods:First,we obtained the transcriptome sequencing data and survival information of Epstein-Barr virus(EBV)infected and uninfected gastric cancer patients from the TCGA-STAD project(The Cancer Genome Atlas,TCGA)and the Gene Expression Omnibus(GEO)database,both belonging to the National Cancer Institute(NCI)in the United States.We retrieved the differentially expressed genes between EBVassociated gastric carcinoma(EBVaGC)and EBV-negative gastric carcinoma(EBVnGC)patients from each data set and obtained the intersection of these genes.Survival analysis was performed on the intersected genes,and TAP1,a gene associated with patient prognosis,was identified as the key gene of interest for further study.We verified the expression levels of TAP1 and other immune-related molecules by q-PCR,western blot,and immunohistochemistry using EBV-infected gastric cancer cell lines and pathological sections from clinical EBVaGC patients.Gene Set Enrichment Analysis(GSEA)was used to identify the signal pathways and transcription factors associated with TAP1,which were further validated by q-PCR and western blot after overexpression or inhibition of these factors in vitro,and specific binding sites of transcription factors on the TAP1 promoter were further verified by a dual-luciferase reporter assay.Protein molecular docking,protein conformation,and in vitro co-immunoprecipitation experiments were performed to simulate the protein-protein interactions between the EBV latent membrane protein 1(LMP1)and transcription factors.After determining the downstream signaling pathways and immune molecules associated with TAP1,we overexpressed or knocked out TAP1 in gastric cancer cell lines to detect the expression levels of downstream predicted pathway-related immune molecules.We also used downstream pathway inhibitors to detect the expression levels of immune molecules.Finally,we constructed stable TAP1-knockout AGS and MKN45 cell lines using CRISPR Cas9 technology,as well as a mouse-derived gastric cancer cell line MFC with stable knockout of the target gene,and analyzed the apoptosis level of tumor cells using in vitro T-cell killing assays.Tumor growth rate was compared in BALB/c and BALB/c nude mice,and the effect of combined immunotherapy was observed after treatment with PD-1 monoclonal antibodies.Results:1.Through searching in the TCGA-STAD and GEO datasets,we selected three independent cohorts,TCGA-STAD,GSE51575,and GSE135644.By intersecting the differentially expressed genes between EBV-negative and EBV-positive patients in these cohorts,we obtained nine intersecting genes,among which only TAP1 was significantly associated with the prognosis of gastric cancer patients and thus was chosen as the target gene for further study.We also detected high expression of TAP1 in both in vitro cell line AGS/EBV and EB virus-positive patient tissues.2.GSEA analysis revealed that TAP1 expression was associated with activation of the NF-κB signaling pathway,particularly with the expression of P65.We also found abnormal activation of NF-κB in AGS/EBV and further validated the activation of TAP1 expression through the NF-κB pathway in AGS and MKN45 cells.3.JASPAR predicted that there was a binding site for the P65 protein in the TAP1 promoter region,which was further confirmed by a dual luciferase reporter gene experiment showing specific recognition of the TAP1 promoter region by P65.4.In vitro molecular docking simulation and COIP experiments showed that the EB virus protein LMP1 could bind to the P65 protein,activating the translocation of P65 into the nucleus,and this result was further confirmed by a rescue experiment.5.Correlation analysis results showed that TAP1 expression was related to JNK,STAT1,and PD-L1 expression.The specific mechanism was that TAP1 activated the JNK/STAT1 pathway,promoting the phosphorylation activation of STAT1,P-STAT1 translocated into the nucleus,and promoted the transcription of PD-L1.6.Finally,through T-cell in vitro killing experiments and animal models,we found that TAP1 upregulated PD-L1 to affect the killing activity of T cells,and that target TAP1 combined with PD-1 monoclonal antibody could significantly inhibit tumor growth.Conclusion1.This study first elucidated the specific mechanism by which the EB virus protein LMP1 regulates the expression level of PD-L1 by regulating TAP1 in EBVaGC.2.This study first proposed that the combined use of TAP1-targeted drugs and PD-1monoclonal antibodies can significantly inhibit gastric cancer growth,and TAP1 can serve as a new immunotherapy target for gastric cancer.