Ubiquitin Ligase Cbl-b/c-Cbl Regulate The Expression And Function Of PD-L1 In Lung Cancer Cells

Objective:Lung cancer is one of the most common malignancies worldwide and the major cause of cancer death,notably in Asia.Most patients with non-small cell lung cancer(NSCLC)present with advanced or metastatic disease at diagnosis and lose the chance of operation.Therefore,it is necessary and urgent to explore the new strategies for this subpopulation.In the past decade,one of the most important advances has been the individualized therapy under the guidance of biomarker testing.Selective use of tyrosine kinase inhibitors(TKI)for patients who harbour EGFR mutation has dramatically improved the survival rate in the metastatic setting for specific population.Even so,the 5-year survival rate of the majority is very low.Recently,noval immunotherapy approaches,including a number of checkpoint inhibitors,mainly targeting programmed cell death 1(PD-1)and programmed cell death 1ligand 1(PD-L1)pathway have made a great breakthrough in NSCLC treatment.PD-1 is expressed on activated T cell and B cell whereas PD-L1 is typically expressed on many types of immune cells and also expressed in various of tissue and cell types,including tumor cells.Under physiological conditions,bingding of PD-1 to PD-L1 inhibits T cell activation,induces T cell apoptosis and downregulates T cell functions,thus,involves in negative regulation of immune responses.This mechanism used by tumor cells enable them to evade host immune surveillance and responsible for immune tolerance.Antibodies against the PD-1/PD-L1 pathway can reverse T cell tolerance of tumor cells and induce tumor regression.In the setting of secondline treatment,these novel treatments have demonstrated clinical activity and superiority compared with standard chemotherapy by increasing 3 months of median OS both for squamous cell carcinoma and non-squamous cell carcinoma.A predictive biomarker to select patients most likely to benefit from anti-PD-1/PD-L1 therapies is needed.PD-L1 expression is the most commonly used.However,the predictive role of it is controversial,some PD-L1 positive patients are still ineffective against treatment and approximately 15%of PD-L1–negative patients respond to the anti–PD-1 therapy.Thus,the regulation of PD-L1 expression requires further investigation.EGFR is the most important driver gene of NSCLC.While patients whose tumors harbor activating mutations in EGFR have benefited from the therapies of TKIs,specific therapeutic strategies for patients of wild-type EGFR are limited and poorly developed.Recently,several phase III clinical trials have revealed anti-PD-1/PD-L1 inhibitors are more effective in patients with wild-type EGFR.Moreover,it has been demonstrated that EGFR mutations up-regulate PD-L1 expression,and PD-L1 status serve as a predictor of TKIs therapy.However,there is no evidence about the regulation of PD-L1 in EGFR wild type.Ubiquitination is a post-translational modification that targets cellular proteins for degradation.Casitas B-lineage Lymphoma(Cbl)family of ubiquitin ligases is an important regulator.The Cbl family of proteins consists of three homologues known as c-Cbl,Cbl-b and Cbl-c.It has been reported Cbl family is involved in regulation of STAT3/AKT/ERKsignal pathways,which are also associated with PD-L1 expression.However,whether Cbl family could regualte PD-L1 expression and involve in the process of PD-L1 function requires further investigation.In this study,we investigated the mechanisms Cbl family protein regulating PD-L1expression in lung cancer cell lines A549 and H460 which both are EGFR wild type.The function of PD-L1 involvs in the process of invasion and metastasis of tumor cell was also simply exploreed.Methods:1.Expression of STAT3,p-STAT3,AKT,p-AKT,ERK,p-ERK,Cbl-b,c-Cbl,PD-L1,E-cadherin,Vimentin and GAPDH proteins were analyzed by Western blot.2.Transwell assay was used to measure cell migration ability.3.Relative levels of miR-181a and miR-940 were measured by Real-time PCR.4.Relative levels of human miRNAs were measured by GeneChip miRNA Array.5.The expression of PD-L1,PD-1,Cbl-b,c-Cbl and their relationship was analyzed in primary lung cancer patients using immunohistochemistry.6.Relative levels of PD-L1 and PD-1 on cell surface were measured by Flow cytometry.7.Statistical analysis.All statistical analyses were performed using the SPSS(16.0)software program.All values are expressed as means±SD.The differences of the results between two groups were evaluated by Student's t-test.Association of clinicopathological parameters with PD-L1 expression was assessed with?~2 or Fisher's exact tests as appropriate.Spearman correlation coefficients were obtained to explore the relationships between between PD-L1 and Cbl-b/C-Cbl expression.Kaplan–Meier analysis and log-rank test were used for survival analysis.P<0.05 was considered as statistically significant.Results:1.STAT3/AKT/ERK pathways involve in up-regulation of PD-L1 expression in NSCLC of EGFR wide type.Cultured A549 and H460 lung adenocarcinoma cell lines without treatment demonstrated the significant difference of PD-L1 expression between the two,both on cell surface and in cytoplasm.The relative expression of H460 was 30times of A549,while A549 with little expression.Western blot also revealed significantly high expression of p-STAT3,p-AKT,p-ERK protein in H460 cells.To explore the role of STAT3/AKT/ERK pathways,H460 cells was respectively treated with STAT3/AKT/ERK inhibitor STATTIC(2?M)?LY294002(25?M)and PD98059(20?M)for 24h or 48h,PD-L1 was down-regulation compared with negtive control groups.Meanwhile,transient knockdown of STAT3/AKT/ERK genes with two different sequences showed the similar results.These data confirm that STAT3/AKT/ERK pathways involve in up-regulation of PD-L1 expression.2.Cbl-b and c-Cbl down-regulated PD-L1 expression via STAT3/AKT/ERK pathway.To investigate the effect of Cbl-b and c-Cbl,siRNA targeting Cbl-b or c-Cbl and non-silencing control were transiently transfected into A549 cells which were high expression of Cbl-b and c-Cbl,Cbl-b or c-Cbl siRNA-transfected groups showed up-regulation of p-STAT3,p-AKT,p-ERK protein and PD-L1 expression compared with the non-silencing controls.Further,co-transfected with Cbl-b and c-Cbl siRNA into A549 cells,PD-L1 increased more obviously.On the other hand,when transfected Cbl-b and c-Cbl overexpression plasmid into H460 cells,p-STAT3,p-AKT,p-ERK protein and PD-L1 protein were down-regulated.These results indicate that Cbl-b and c-Cbl negatively control PD-L1 expression via STAT3/AKT/ERK pathway.3.PD-L1expression of lung cancer cells are regulated via miR-181a and miR-940 respectively targeting Cbl-b and c-Cbl.We performed a microarray analysis to detect the differentially expressed miRNAs between A549 and H460 cells.We identified several miRNAs that were differentially expressed between A549 and H460 cells and could potentially targeting Cbl-b and c-Cbl using miRDB predictions(http://www.mirdb.org/miRDB/).Using quantitative real-time PCR,the expression levels of these mi RNAs were quantitated as fold change normalized to U6 snRNA.The expression of miR-181a and miR-940 were stable and different between the two cells.A 2.5 times higher expression of miR-181a and miR-940 were observed in H460 cells compared with A549 cells.The expression of others were neither inconsistent with chip,nor were they stable.To confirm the regulation of Cbl-b and c-Cbl by mi RNAs,A549 cells were transfected with either miR-181a or miR-940 mimics or the negative control miRNA,we observed down-regulation of Cbl-b and c-Cbl,and up-regulation of p-STAT3,p-AKT,p-ERK protein and PD-L1 expression.When inhibiting the endogenous mi R-181a or miR-940 in H460 cells using inhibitors increased the Cbl-b and c-Cbl protein and decreased downstream p-STAT3,p-AKT,p-ERK protein and PD-L1.These results indicate that PD-L1expression of lung cancer cells are regulated via miR-181a and miR-940 respectively targeting Cbl-b and c-Cbl.5.PD-L1 induce metastasis in lung cancer cells.H460 cells were transfected with PD-L1 siRNA,western blot revealed significant up-regulation of the epithelial marker E-cadherin and down-regulation of the mesenchymal marker Vimentin.Additionally,PD-L1 siRNA treatment reduced cellular migration with0.39±0.07 of control group undergoing migration.More interesting,when there were Jurka T cells in bottom well,H460 cells in up well migrated obviously more than the negative control.Moreover,Jurkat cells induced up-regulation of migration rate was reduced after transient knockdown of PD-L1 gene in H460 cells.However,up-regulation of migration induced by Jurkat cells was not found on A549 cells.Taken these findings together indicate that PD-L1 on lung cancer cells is asscociated with metastasis.6.Correlations between PD-L1 and clinicalpathological characteristics and evaluation correlations of Cbl-b/c-Cbl and PD-L1 in primary lung cancer patients.Immunohistochemical analysis was used to detecte PD-L1,PD-1 and Cbl-b/c-Cbl expressions and clinical data were collected.49 out of the 133 patients(36.8%)showed PD-L1 positive staining in tumor cells and 71(53.4%)of patients had Cbl-b positive expression,61(45.9%)of patients had c-Cbl positive expression.There was a significant negative correlation between the expression of PD-L1 and Cbl-b(r=-0.349,p<0.001)and c-Cbl(r=-0.234,p=0.007).We also analyzed the correlations between the expression of PD-L1 and the clinicopathological characteristics of NSCLC.The expression of PD-L1was significantly associated with smoking status and histological type(P<0.001).PD-L1was also correlated with differentiation degree(P=0.040)and pathologic node(pN)staging(P=0.046).To determine the prognostic value of PD-L1,Kaplan-Meier analysis was used with a log-rank test.The result showed high level of PD-L1 expression indicated poor prognosis(P=0.023)and this effect was more obvious in non-SCC subtype(P=0.019).However,in SCC subtype,PD-L1 expression did not demonstrate any significant effect(P=0.526).Coexpression of PD-L1 and PD-1 indicated the worst prognosis in the entire population(p=0.001)as well as in subgroup analysis(p=0.033 in non-SCC and p=0.037 in SCC).Conclusion:1.STAT3/AKT/ERK pathways up-regulated PD-L1 expression in NSCLC of EGFR wide type.2.Cbl-b and c-Cbl down-regulated PD-L1 expression via inhibiting STAT3/AKT/ERK pathway.3.PD-L1 expression of lung cancer cells were regulated via miR-181a and miR-940 respectively targeting Cbl-b and c-Cbl.4.PD-L1 induced EMT in lung cancer cells.5.There was a significant negative correlation between the expression of Cbl-b/c-Cbl and PD-L1 in primary non-small lung cancer patients and PD-L1 maybe used as a potential prognostic marker in lung cancer.