Research On The Mechanism Of Dna Damage Induced PD-L1 Expression And Its Molecular Regulation In Tumor

Background:PD-L1 is the ligand of PD-1 and could activate PD-1 pathway to execute immune-suppressive function.The aim of our project is to investigate the mechanism of radiation induced PD-L1 expression in tumor cells and the impact of radiation induced PD-L1 expression on T cells.Finally,our purpose is to find the new clinical target regulating PD-L1.Methods:1.We chose the proper tumor cell lines after detecting the expression of PD-L1 in different tumor cell lines treated with or without radiation through Western Blot.PD-L1 subceculler level expression after being radiated was investigated through cell fraction experiment.Time course and dose response of radiation induced PD-L1 protein expression were also detected by WB and Immunofluoresence.PD-L1 mRNA expression as indicated time point was also detected by Taqman qPCR.We also used cycloheximide to block mRNA translational process and detected radiation induced PD-L1 expression.2.PD-1/SHP-2 pathway activation and apoptosis of Jurkat cells were detected by WB and flow cytometery after co-culturing with PC-3 cells which were treated with radiation or not previously.PD-L1 siRNA and PD-1 antibody which could abrogate PD-L1/PD-1 interaction were used in this co-culture system.3.Radiation(5Gy,8h)induced PD-L1 mRNA expression was detected by qPCR after treated by ATM,ATR and PI3 K inhibitors.PD-L1 and its interactive protein were pulled down after treated by radiation through co-immunoprecipitation to do mass spectrum analysis.4.From mass spectrum results,potential protein targets related to PD-L1 post-translational modification regulation were selected and this target protein was knocked down with siRNA to investigate PD-L1 expression by qPCR and WB.BCLAF1 was supposed to be PD-L1 post-tranlational modification regulator after validation.Then we knocked down ATM,BCLAF1,CMTM6 and PD-L1 separately and detected their expression under radiation.The relationship between CMTM6 and radiation was examined additionally in different cell lines.The ubiquitination of PD-L1 was also detected after knowing down BCLAF1.5.Using American TCGA database(33 kinds of cancer),we analyzed the relevance between BCLAF1 and PD-L1.Among 1081 breast cancer of TCGA patients,we analyzed the relationship between expression of BCLAF1 and survival rate.The relationship between BCLAF1 expression and PD-L1 expression and clinical characteristic factors were investigated in 132 esophageal squamous carcinoma patient tissues which stained by immunohistochemistry.Results:1.Radiatoin(5Gy,2h)could upregulate PD-L1 protein expression in MDA-MB-231,PC-3 and HT1080 but not PD-L1 mRNA.After radiation(5Gy),PD-L1 protein expression could be upregulate at 2h and achieve the highest level at 8h.But the PD-L1 mRNA expression didn't change 2-4h after radiation.At 8h after radiation,PD-L1 mRNA expression began to incresase.Radiation induced PD-L1 protein continued to increase at 2-4h even treated with cycloheximide.Under different dose of radiation,PD-L1 protein expression could be upregulated at 0.1Gy.Cell fraction experiment showed the upregulated PD-L1 expression protein by radiation located at cell membrane.2.After co-cultured with radiated PC-3 cells,P-SHP-2 and Caspase-3 expression of Jurkat were upregulated.The viability rate of Jurkat cells,which was detected by flow cytometery,also decreased compared with non-radiated group in co-culture system(55.2% vs.37.9%,P<0.05).The P-SHP-2 and Caspase-3 expression and viability rate of Jurkat cells in radiation group didn't change after using PD-L1 siRNA and PD-1 antibody to abolish the PD-L1/PD-1 interaction compared with non-radiation group.(viability rate 60.96% vs.55.78%,P>0.05).3.The upregulated PD-L1 mRNA expression by radiation(8h)could be abolished by ATR and PI3 K inhibitors but not ATM inhibitors.4.From 102 proteins indentified by mass spectrum,BCLAF1 were selected.Knocking down BCLAF1 could decrease PD-L1 protein expression but not mRNA.ATM,BCLAF1,CMTM6 and PD-L1 were knocked down separately and each protein expression was analyzed by WB with treated radiation or not.ATM knocking down could block BCLAF1 upregulation and substantially abolish CMTM6 upregulation and finally influence radiation induced PD-L1 expression.Knocking down BCLAF1 coulde down-regulate PD-L1 expression and increase the ubiquitination level of PD-L1.5.In TCGA database,BCLAF1 expression was positively correlated with PD-L1 in 24 kinds of cancer.In esophageal squamous carcinoma,BCLAF1 expression rate was 76.5% and positively related with PD-L1(r=0.495,P=0.000).There were no relationship between BCLAF1 and clinical characteristic factors and 5 years survival rate of esophageal squamous carcinoma.But in breast cancer,patients with higher BCLAF1 expression showed worse survival.Conclusion:1.Radiation could upregulate PD-L1 protein expression on tumor cell membrane at transcriptional and post-translational modification level.The PD-L1 expression was radiation time dependent and dose independent.2.Radiation induced PD-L1 expression in tumor cells could activate PD-1 pathway and apoptosis of T cells.3.Radiation induced PD-L1 expression at transcriptional level might relate to ATR-PI3 K pathway.4.Radiation might induce PD-L1 expression at post-modification level via ATM-BCLAF1-CMTM6-PD-L1 pathway.BCLAF1 upregulated PD-L1 expression through inhititing the ubiquitination of PD-L1.5.BCLAF1 expression was positively correlated with PD-L1 in 24 kinds of cancer in TCGA database and esophageal cancer.In breast cancer,high BCLAF1 expression was associated with poor survival.