膜联蛋白A2对PD-L1蛋白稳定性的影响及其在食管鳞癌免疫逃逸中的作用OA

OBJECTIVE:To investigate effect of annexin A2(ANXA2)on PD-L1 protein stability and immune evasion in esophageal squamous cell carcinoma(ESCC).METHODS:ANXA2 knockdown and knockout experiments were performed in ESCC cells.Western blotting and immunofluorescence were used to detect PD-L1 protein levels and their localization changes on the cell membrane,respectively.RT-qPCR was used to analyze the CD274 mRNA level.Cells were treated with cycloheximide(CHX)and MG132 respectively,and the half-life and degradation of PD-L1 protein were detected by Western blotting.The immune co-culture systems of tumor cells with CD8+T cells were utilized,and the CCK-8 assay was used to evaluate effects of ANXA2 knockout alone or in combination with PD-1 inhibitor(nivolumab)or PD-L1 inhibitor(avelumab)on proliferation of CD8+T cells.RESULTS:Compared with the negative control group,PD-L1 protein expression levels in the ANXA2 knockdown and knockout groups were significantly lower(P<0.05),and their membrane localization also decreased.However,there was no significant difference in the level of CD274 mRNA(P>0.05).In the CHX assays,the degradation rate of PD-L1 protein in ANXA2 knockdown cells was faster than that in control cells.MG132 treatment reversed the reduction of PD-L1 protein caused by ANXA2 knockdown,while bafilomycin A1 did not.In the co-culture experiments,the proliferation rate of CD8+T cells in the group with ANXA2 knockout combined with PD-1 inhibitor or PD-L1 inhibitor was significantly higher than that in the group with only ANXA2 knockout(P<0.05).CONCLUSION:ANXA2 affected the stability of the PD-L1 protein,and its degradation mainly occurred through the proteasome pathway.Combined targeting of ANXA2 and the PD-1 pathway enhanced the effector function of T cells,suggesting that it has application value in overcoming immunotherapy resistance of ESCC.