USP2 Promotes Tumor Immune Evasion Via Deubiquitination And Stabilization Of PD-L1 And The Anti-tumor Immunity Role Of Salvianolic Acid B

Background:Malignant tumor is one of the diseases that seriously endanger human life and health.There are 4 million new cancer cases in China every year,accounting for about a quarter of the new cases worldwide.About 3 million residents in China are killed by cancer every year,and the number of cancer incidence and death in China has continued to rise in the past decade,which has brought a great burden to the healthy life of people.Therefore,finding new and effective cancer treatments is a key issue in the current medical research field.In recent years,immunotherapy has been developing rapidly and has achieved unprecedented clinical efficacy.Programmed death ligand 1(PD-L1)and programmed death receptor 1(PD-1)are a pair of T-cell activation co-suppressor molecules.High expression of PD-L1 in tumor cells and inhibition of T-cell activation are the main reasons for the generation of suppressive tumor immune microenvironment.The regulation of PD-L1 expression in tumor cells involves multiple levels including genomic level,transcriptional level,post-transcriptional level,translational and post-translational levels,and systematic study of PD-L1 expression regulation mechanisms can help improve the clinical efficacy of therapies targeting PD-1/PD-L1.Post-translational modifications of PD-L1,such as glycosylation,ubiquitination and palmitoylation,have been reported to affect the expression level of PD-L1 in tumor cells and its localization in the cells.The ubiquitin-proteasome system is one of the major protein degradation systems in eukaryotes,and it has now been shown that PD-L1 is regulated by deubiquitinating enzymes at the post-translational level and influences its occurrence of endoplasmic reticulum-associated protein degradation.Deubiquitinating enzymes are a large family of proteases,and there is a lack of systematic studies on the mechanism of regulation of PD-L1 protein levels by deubiquitinating enzymes.We screened the deubiquitinating enzyme family proteins and found that the deubiquitinating enzyme USP2 could significantly affect the expression of PD-L1 protein in tumor cells.Previous studies have shown that USP2 can regulate the ubiquitination of MDM2 and indirectly promote the degradation of p53/TP53 to inhibit its activity to play a pro-cancer role,but its role as a positive regulator of PD-L1 has not been reported,so we investigated the mechanism of USP2 regulation of PD-L1 protein and its role and mechanism on tumor immune microenvironment.In Immune checkpoint therapy(ICTs),blocking PD-1 or PD-L1 with antibodies has achieved remarkable therapeutic effects and improved the overall survival of patients with a variety of solid tumors,but there are also problems such as low clinical response rates and serious immune-related adverse effects.The development of small molecule tumor immunotherapy drugs is currently a hot issue in immunotherapy.Compared with antibody drugs,small molecule drugs have the advantages of multiple routes of administration,low production cost and better permeability to the tumor microenvironment.Salvianolic acid B is a small molecule compound of natural product origin isolated from the dried roots and rhizomes of Salvia miltiorrhiza,a plant of the family Labiatae.Previous studies have shown that salvianolic acid B has antioxidant effects,protection against myocardial ischemia-reperfusion injury and prevention of atherosclerosis,but its antitumor effects by affecting the tumor microenvironment have not been reported.Methods:Using human colon cancer cells RKO as the target,the effect of different deubiquitinating enzymes on PD-L1 protein expression in RKO cells was detected by flow cytometry using a CRISPR-CAS9-based screening method;the effect of USP2 knockdown on PD-L1 and other immune checkpoint protein levels in different tumor cells was detected by western blot and flow cytometry.The effect of USP2 on PD-L1 protein stability was detected by actinomycin(CHX)assay;the effect of USP2 knockdown on PD-L1 mRNA expression level was detected by qRT-PCR;the interaction of USP2 with PD-L1 protein was detected by immunoprecipitation(Co-IP)and DuoLink in situ fluorescence assay;the ubiquitination assay,inhibitor assay and immunofluorescence to analyze the mechanism of USP2-regulated PD-L1 ubiquitination;detecting changes in PD-1 protein binding by tumor cells after USP2 knockdown by flow cytometry and laser confocal;cell impedance analysis,crystalline violet staining,and flow cytometry to detect the killing activity of co-cultured T cells against tumor cells after USP2 knockdown in tumor cells;in C57BL/6 mice with prostate cancer RM-1 and MC38 transplantation tumor models of colon cancer,the growth of transplantation tumors with USP2 knockdown,PD-L1 overexpressing mice after USP2 knockdown,and PD-L1 overexpressing mice was examined;changes in CD8+T cell function and the activity of immunosuppressive cells Tregs and MDSCs in transplantation tumors were also examined using multicolor flow cytometry;tissue microarray experiments were performed to study The correlation between USP2 protein and PD-L1 protein expression in colon cancer clinical samples;the effect of USP2 expression level on tumor immune infiltration and patient survival was analyzed by TCGA data;the effect of tannic acid B on cell proliferation of RKO cells and PC3 cells was examined by MTT assay using colon cancer and prostate cancer cells;the effect of tannic acid B on cell proliferation of RKO cells and PC3 cells was examined by western blot using The effect of salvianolic acid B on intracellular PD-L1 protein level was examined by western blot;the binding region of salvianolic acid B in USP2 was analyzed by Discovery Studio 4,5 software;the binding constant of salvianolic acid B on USP2 recombinant protein was determined by surface plasmon resonance technique;the in vivo tumor suppressive activity of salvianolic acid B was examined in MC38 mouse transplantation tumor model of colon cancer.Result:Aberrant upregulation of programmed death ligand-1(PD-L1)on tumor cells impedes tumor cell killing by cytotoxic T cells through binding to PD-1,thus further exploration of PD-L1 regulatory mechanisms in tumors has the opportunity to improve the clinical efficacy of PD-L1 blockade therapies.Using the(sgRNAs)screening system,we identified ubiquitin carboxy-terminal hydrolase 2(USP2)as a novel regulator of PD-L1 stability promoting tumor immune escape.USP2 increases PD-L1 expression abundance in tumor cells by directly interacting with PD-L1 by removing the K48-linked polyubiquitin chain from PD-L1 protein,and knockdown of USP2 would lead to endoplasmic reticulum(ER)-associated degradation of PD-L1,thereby attenuating the PD-L1/PD-1 interaction and sensitizing tumor cells to T-cell-mediated killing.Meanwhile,USP2 knockdown-induced PD-L1 reduction was able to enhance anti-tumor immunity in mice by enhancing CD8+T cell function and reducing immunosuppression of bone marrow-derived suppressor cells(MDSCs)and regulatory T cells(Tregs),while PD-L1 overexpression reversed the tumor growth suppression caused by USP2 knockdown.Furthermore,analysis of clinical tissue samples also suggested a potential role for USP2 in upregulating PD-L1 expression in tumors.Taken together,our data reveal the existence of a key role of USP2 in regulating PD-L1 stability in tumor cells and reveal USP2 protein as a potential therapeutic target for tumor immunotherapy.Subsequently,we investigated the anti-tumor effect of salvianolic acid B(SAB)on PD-L1 expression in tumor cells and its mechanism.The results showed that SAB downregulated PD-L1 levels in RKO and PC3 cells and on the cell membrane surface in a concentration-dependent and time-dependent manner,respectively,which was associated with the inhibition of the activity of deubiquitinating enzyme USP2 in tumor cells by SAB,Mechanistic studies revealed that SAB directly interacted with USP2 and inhibited its deubiquitinating enzyme activity,thereby promoting the degradation of PD-L1 by the ubiquitin-proteasome pathway.The binding constant of SAB to USP2 recombinant protein was 42.6 μmol-L-1.In addition,SAB promoted the killing effect of co-cultured PBMC on RKO cells.SAB significantly inhibited the growth of MC38 transplanted tumors in C57BL/6 mice.20 mg-kg-1 SAB treatment in tumor-bearing mice resulted in a 63.2%reduction in tumor volume.Conclusion:In this study,we identified USP2,a member of the ubiquitin-specific proteases(USPs)subfamily of DUBs,as a novel regulator of PD-L1 stabilization for cancer cell evasion of immune surveillance.We found USP2 specifically interacts with PD-L1 and removes the K48-linked polyubiquitin chains of PD-L1,thus prevents its proteasomal degradation.USP2 depletion lead to ERAD-dependent degradation of PD-L1,thereby functionally blocking PD-1 binding and enhancing T cell cytotoxicity as well as reprogramming the tumor microenvironment.Our findings suggest targeting USP2 may potentially improve PD-L1-dependent cancer immunotherapy.Our results proved that USP2 specifically interacts with PD-L1 on the ICD region and regulates PD-L1 stability through its deubiquitination activity.=Our results demonstrate that SAB exerts its anti-tumor activity by direct binding and inhibiting the activity of USP2 and reducing the PD-L1 level.Our study provides an important material basis and scientific basis for the potential application of SAB in tumor immunotherapy drug targeting USP2-PD-L1 axis.