TGF-β1-mediated PD-L1 Glycosylation Contributes To Immune Escape Via C-Jun/STT3A Pathway In Nasopharyngeal Carcinoma

BackgroundNasopharyngeal carcinoma(NPC)is a kind of epithelial squamous cell carcinoma that originates from the nasopharyngeal mucosa with a high incidence in South China and Southeast Asia.With deeper cognition about etiology mechanisms and the progress of diagnosis and treatment technology,the survival rate of NPC patients has significantly improved.However,metastasis and recurrence are still the biggest challenges in the clinical treatment of NPC,which are the main death causes of NPC patients.Immunotherapy targeting programmed cell death protein-1/programmed death ligand-1(PD-1/PD-L1)has achieved great success in multiple cancers,but only 20-30%NPC patients showed clinical responses.Recent evidences have shown that post-translational modification of PD-L1 protein could regulate its protein stability and interaction with cognate receptor PD-1,thereby regulating immunosuppressive microenviroment in tumor and affecting anti cancer immunotherapy in several solid tumors.However,the molecular mechanisms underlying how PD-1/PD-L1 expression is regulated still remain unclear in NPC.Moreover,our previous study has revealed that enrichment of TGF-β1 in NPC exerts immunosuppressive effect.In this study,we verified the N-glycosylation of PD-L1 in NPC and further investigated whether TGF-β1 induced glycosylation of PD-L1 to promote immune escape,which may contribute to develop more effective immunotherapy strategies and improve the survival rate of NPC patients.Materials and MethodsPD-L1 expression in NPC tissues and different NPC cell lines was detected by Western blot,and N-glycosylation of PD-L1 in NPC was confirmed by further experiments.In vitro cell experiments,we mechanistically showed that TGF-β1 could positively regulate PD-L1 glycosylation via c-Jun/STT3A pathway by qRT-PCR and Western blot.Dual-luciferase assay and CHIP were performed to confirm that c-Jun directly binds to STT3A promoter.Functional assays showed that inhibition of TGF-β1 resulted in a decrease of glycosylated PD-L1 and enhanced cytotoxic T-cell function against NPC cells.Analysis of clinical specimens revealed that the expression of STT3A was positively correlated with TGF-β1 and c-Jun,and high STT3A expression was positively correlated with a more advanced clinical stage.Results1.PD-L1 was N-glycosylated in NPC tissues and cell lines,and blockade of TGF-β1 by SB43 1542 reduced PD-L1 glycosylation in vitro.2.SB431542 downregulated glycosylation of PD-L1 via c-Jun/STT3A pathway in vitro,and c-Jun was a direct transcriptional regulator of STT3 A.3.STT3A expression was positively correlated with TGF-β1 and c-Jun in NPC Tissues,and high STT3A expression was associated with a more advanced stage in NPC.4.Inhibition of TGF-β1 by SB431542 suppressed T cells activity by inhibiting PD-L1 glycosylation in Vitro.ConclusionsIn this study,we confirmed glycosylation of PD-L1 and investigated the potential mechanisms underlying PD-L1 glycosylation.Our data showed that TGF-β1-mediated PD-L1 glycosylation contributed to immune escape via c-Jun/STT3A pathway in NPC.Taken together,this study suggests that targeting TGF-β1 pathway might be a promising approach to enhance immune checkpoint blockade,and simultaneous blockade of PD-L1 and TGF-β1 pathways might elicit potent and superior antitumor activity relative to monotherapies.