The Role Of HDAC In IFN-γ Induced B7-H1 Expression In Gastric Cancer Cells

Part I The role of HDAC in IFN-γ induced B7-H1 expression in gastric cancer cellsAim:To investigate the role of histone deacetylase(HDAC)in the regulation of IFN-γ induced B7-H1 expression in gastric cancer cells and the related mechanism.Methods: B7-H1,HDAC1,HDAC2 and HDAC3 expression and possible correlation were evaluated through analysis of gastric cancer data in TCGA.Clinical gastric cancer tissues and adjacent tissues were collected to evaluate the expression of B7-H1 and HDAC using RT-q PCR and western blot.Following pretreatment of HDAC inhibitors(TSA,SAHA and Butyrate),or si RNA knockdown of HDAC1,HDAC2 or HDAC3 gene,IFN-γ induced B7-H1 expression in HGC27 cels was determined by RT-q PCR,western blot and flow cytometry.Expression of IFN-γ signaling pathway components including JAK-1,JAK2 and STAT1 and their phosphorylated forms was evaluated though western blot.STAT1 nuclear translocation was evaluated through immunofluorescence staining.Ch IP-q PCR was performed to evaluate histone H3K9 acetylation status.Mouse model of subcutaneous grafted gastric cancer was constructed and 12 mice were randomly assigned to the SAHA treatment group or vehicle treatment group.The volume of the tumors and mouse body weight changes were measured daily.To evaluate B7-H1 expression of the tumor cels and percentage of tumor infiltrating CD8+ T cells,flow cytometry was performed.Results: RNA-seq data from TCGA revealed that expression of B7-H1,HDAC1,HDAC2 and HDAC3 was significantly higher in gastric cancer compared to those in normal gastric tissues;that HDAC3 and HDAC1 were significantly correlated with B7-H1 expression in gastric cancer with a respective r value of 0.42(p<0.001)and 0.21(p<0.001).HDAC inhibitor(TSA,SAHA and Butyrate)pretreatment suppressed IFN-γ induced B7-H1 expression on HGC-27 cells.HDAC1 and HDAC3 gene knockdown had the same effect.SAHA pretreatment or HDAC1 and HDAC3 knockdown resulted in impaired IFN-γ signaling,demonstrated by reduction of JAK2,p-JAK1,p-JAK2 and p-STAT1 expression and inefficient STAT1 nuclear translocation.Furthermore,SAHA pretreatment compromised IFN-γ induced upregulation of histone H3 lysine 9 acetylation level in B7-H1 gene promoter.In the transplanted mouse gastric cancer model,SAHA treatment suppressed tumor growth,inhibited B7-H1 expression and elevated the percentage of tumor infiltrating CD8+ T cells.Conclusion: HDAC is indispensable for IFN-γ induced B7-H1 in gastric cancer.The study suggests the possibility of targeting B7-H1 using small molecular HDAC inhibitors for cancer treatment.Part II B7-H1 expression and gastric cancer prognosis: A meta-analysisAim:To evaluate the relationship between B7-H1 expression and prognosis in gastric cancer systemically and quantitatively,a meta-analysis was performed.Methods: Search in the Pub Med,Embase,Web of Science,Cochrane Library database with the key words including “gastric cancer”,“B7-H1”,“prognosis” and “immunohistochemistry” to obtain literatures about the relationship between B7-H1 expression status on gastric cancer cels and overall survival(OS).For included studies,Revman 5.3 software was used to calculate the pooled hazard ratio and 95% confidence interval(HR,95% CI).Subgroup analysis based on TNM stage,region and EBV status was performed.Results: 23 studies covering 5037 patients from China,Korea,Japan,USA and Germany were included in the meta-analysis.Positive B7-H1 expression on gastric cancer cels was associated with poorer OS in the patients(HR 1.40,95% CI: 1.14-1.72).Furthermore,positive B7-H1 expression on gastric cancer cels was associated with poorer OS in the following subgroups: TNM stage I-IV(HR 1.31,95% CI: 1.04-1.65),TNM stage I-III(HR 2.15,95% CI: 1.37-3.37)and Asia(HR 1.42,95% CI: 1.15-1.74).A correlation between B7-H1 positivity and OS was not observed in the following subgroups: TNM stage II-III(HR 1.70,95% CI: 0.98-2.94),western countries(HR 2.15,95% CI: 0.22-20.98),EBV positive group(HR 0.91,95%CI: 0.31-2.66)and EBV negative group(HR 1.27,95% CI 0.83-1.96).Conclusion: Positive B7-H1 expression was associated with poorer prognosis in gastric cancer.B7-H1 may serve as a useful biomarker to predict prognosis and the treatment efficacy in immune therapy in gastric cancer.