Role And Mechanism Of USP7-regulating PD-L1 Expression In Multiple Cancers

Background and objectives: The binding of programmed cell death1(PD-1)and programmed cell death ligand 1(PD-L1),two classic immune checkpoints,leads to tumor immune escape by inhibiting the activity and function of T lymphocytes and preventing cytotoxic T cells from killing tumor cells.The PD-1/PD-L1 immune checkpoint is one of the main targets of tumor immunotherapy,and research indicates that patients with higher PD-L1 expression will respond better to anti-PD-1/PD-L1 therapy.Thus,it has significant theoretical and clinical significance in revealing the mechanism regulating PD-L1 expression and stability in tumor cells.In this study,we screened a USP deubiquitinase plasmid library to identify deubiquitinases that regulate PD-L1 expression and stability.Ultimately,we found that ubiquitination-specific proteinase7(USP7)is a deubiquitinase that regulates PD-L1.To this end,this study investigated the role and mechanism of USP7 in regulating PD-L1 expression in breast cancer,non-small cell lung cancer,and melanoma cells and analyzed the correlation between USP7 and PD-L1 expression and the clinicopathological features of these tumors.Methods: To identify the deubiquitinase of PD-L1,HEK 293 cells were transfected with expression plasmids of PD-L1 and a series of USP deubiquitinase family,and the protein level of PD-L1 was examined by Western blot.The interaction between USP7 and PD-L1 was detected by immunoprecipitation(CO-IP)and Duolink Proximity Ligation Assay(PLA)in breast cancer,non-small cell lung cancer cell,and melanoma cell lines.The specific interaction domain of USP7 and PD-L1 was analyzed by molecular simulations and deletion mutations.Three USP7 knockdown tumor cell lines and each control cell line were treated with the protein synthesis inhibitor CHX,and the protein half-life of PD-L1 was determined by Western blot.To analyze whether proteasome inhibitor could restore the expression level of PD-L1,these cells were also treated with MG132,and changes in PD-L1 levels were determined by Western blot.To investigate whether USP7 regulated PD-L1 expression and stability via the ubiquitin-proteasome pathway,CO-IP was performed to detect the expression and ubiquitination levels of PDL1 after USP7 knockdown in three tumor cell lines.The correlation between USP7 expression level and immune cell infiltration in the tumor microenvironment was analyzed by bioinformatics.Immunohistochemistry was used to detect the expression levels of USP7 and PD-L1 in surgically resected tissues of 195 breast cancer,188 nonsmall cell lung cancer,and 163 melanoma patients and clarify the correlation between USP7 and PD-L1 expression levels and patients’ clinical characteristics or prognosis.Results:The deubiquitinase plasmid library was screened for the deubiquitinase of PD-L1,and we found that USP7 significantly upregulated PD-L1 expression.CO-IP and Duolink PLA assay confirmed that USP7 interacted with PD-L1 in breast cancer,non-small-cell lung cancer,and melanoma cells.Molecular simulations and deletion mutations analysis revealed that the TRAF domain of USP7 bound to the ICD domain of PD-L1.USP7 regulated the expression and stability of PD-L1 through the ubiquitin-proteasome pathway in breast cancer,nonsmall-cell lung cancer,and melanoma cells.Bioinformatics analysis showed that the USP7 expression level was associated with less CD8+ T cell infiltration in the microenvironment of breast cancer,non-small cell lung cancer,and melanoma.The expression levels of USP7 and PD-L1 were positively correlated in breast cancer,non-small cell lung cancer,and melanoma.The expression levels of USP7 and PD-L1 were positively associated with the malignant pathological features of the three tumors.The disease-free survival(DFS)and overall survival(OS)of patients with high expression of USP7 and PD-L1 were worse than those with low expression.Multivariate Cox regression analysis showed that the combination of USP7 and PD-L1 was an independent predictor of patient survival.Conclusion:(1)USP7 is a deubiquitinase of PD-L1 in breast cancer,non-small cell lung cancer,and melanoma cells;(2)USP7 regulates the expression level and stability of PD-L1 in breast cancer,non-small cell lung cancer,and melanoma cells through the ubiquitin-proteasome pathway;(3)Breast cancer,non-small cell lung cancer and melanoma patients with higher USP7 and PD-L1 expression had a worse prognosis than patients with lower protein expression,and the combination of the two proteins was an independent predictor of the patient survival.