The Study On Expression Of E2F3 And PD-L1 In Invasive Bladder Cancer And Its Relationship With Tumor-infiltrating CD8~+T Cells

BackgroundUrinary bladder cancer is an aggressive disease associated with high morbidity and mortality across the world.The development of bladder cancer is a multistep process characterized by the accumulation of genetic and epigenetic alterations that drive or reflect tumor progression.Tumor invasion and metastasis are the leading causes of death in patients with bladder cancer,and tumor immune escape is an important factor in tumor invasion and metastasis.There have been no major advances for the treatment of urothelial bladder cancer(UBC)in the last 30 years.In recent years,treatment target anti-PD-1or anti-PD-L1antibodies has produced encouraging results in heavily patients with advanced cancers.PD-L1 is frequently found to be over expressed in various cancers,it is a T-cell regulatory molecule expressed on the surface of tumor and tumor-infiltrating immune cells.PD-1 is expressed on the surface of T cells upon activation.Tumors expressing PD-L1 can render cytotoxic T lymphocytes inactivated or nonfunctional by binding to PD-1 in order to evade the host immune response preventing tumors from cytotoxic T lymphocyte-induced killing.Antibody drugs that target PD-L1 pathway have produced encouraging results in a rapidly increasing number of solid tumors,including melanoma and lung cancer.MPDL3280A(also known as Atezolizumab,Tecentriq),an anti-PD-L1antibody,with an objective response rate of 40-50%,shown to be active towards bladder cancer,as a monoclonal antibody drug,it became the first PD-L1 inhibitor post FDA approval of the treatment for bladder cancer.However,many patients fail to respond to anti-PD-1/PD-L1 treatment,and the underlying mechanism of the PD-1/PD-L1 signaling pathway remains poorly understood.Recent studies revealed that higher response rates to anti-PD-L1 therapy might correlate with PD-L1 expression levels in tumor cells.Hence,it is important to well understand the pathways controlling PD-L1 protein expression,which can supply a molecular basis to improve the efficacy of PD-1/PD-L1 blockade and clinical response rate in cancer patients.E2F transcription factor 3(E2F3)belongs to E2F family,is one of the fundamental drivers in the cell cycle and is critical for the initiation and progression of various malignancies.Latest reports demonstrated that E2F3 is involved in regulating the balance ofmanybiochemicaleventsinvariouscancers,includingproliferation,metastasis,autophagy,apoptosis,as well as drug resistance.In bladder cancer,E2F3signaling pathway activation,contributing to bladder cancer cell proliferation,apoptosis and autophagy suppression mainly through Caspase-3 and p53 inhibition in vitro and in vivo.E2F3 is rich in biological functions.Up to now,the research on the relationship between E2F3 and tumor immunity is still far from in-depth,especially the relationship between E2F3 and tumor immune escape in bladder cancer has not been reported.Objective1.To investigate the expression profile of E2F3 and PD-L1 as well as the infiltration of CD8~+T cells in bladder cancer tissues,and analyze their correlation between them.2.To observe the effect of E2F3 on PD-L1 expression in bladder cancer cell lines,in order to explore the possible regulation mechanism of E2F3 on PD-L1 expression.3.To observe the correlation between the expression of E2F3 and PD-L1 in bladder cancer tissues and the malignant biological behavior of bladder cancer,and the relationship between the expression levels of them and the clinical prognosis of bladder cancer patients.Methods1.The expression level of E2F3 and the infiltration characteristics of CD8~+T cells in 110cases of bladder cancer tissues with different histological stage and grade as well as in the corresponding normal bladder tissues were detected by immunohistochemistry staining,Western blot was used to detect E2F3,CD8 and PD-L1 protein levels in 82 cases of bladder cancer tissues and the corresponding normal bladder tissues with different histological stage and grade,qRT-PCR was used to detect E2F3 and PD-L1 mRNA levels in 60 cases of bladder cancer tissues and the corresponding normal bladder tissues,and then the correlation between E2F3 expression and CD8~+T cell infiltration was analyzed statistically by linear correlation analysis,the correlation between E2F3 expression and PD-L1 expression was also analyzed statistically by linear correlation analysis.2.The expression of E2F3 and PD-L1 protein in bladder cancer cell lines J82,RT4,UMUC3,HT1376 and TCCSUP were detected by Western blot;ChIP-seq was performed to detect the binding sites of E2F3 protein in the PD-L1 DNA region in the cell lines J82,RT4,UMUC3,HT1376 and TCCSUP;E2F3 gene was knocked out in E2F3high-expressing HT1376 and TCCSUP cell lines,the protein level of PD-L1 after knockout was detected by Western blot,and the mRNA level of PD-L1 after knockout was detected by qRT-PCR.3.Statistical analysis of the relationship between E2F3 expression in bladder cancer tissues and cancer clinical invasive indexes including tumor size,lymph node metastasis and distant metastasis was performed.Survival analysis and Cox proportional hazard regression model were used to analyze the relationship between the expression levels of E2F3 and PD-L1 and the survival time of patients within 5 years after operation.Results1.E2F3 and PD-L1 were expressed in bladder cancer tissues but not in normal bladder tissues.E2F3 expression in invasive bladder cancer was higher than that in non-invasive bladder cancer,and its expression level in invasive bladder cancer increased synchronously with the increase in histological stage and grade of bladder cancer,while the number of tumor-infiltrating CD8~+T cells in the corresponding tumor tissues reduced synchronization.The expression of PD-L1 in bladder cancer tissues was positively correlated with E2F3 expression level in the corresponding tumors,while the number of tumor-infiltrating CD8~+T cells in tumor tissues was negatively correlated with the expression level of E2F3.2.In the bladder cancer cell lines J82,RT4,UMUC3,HT1376 and TCCSUP,the transcription factor E2F3 was enriched in the PD-L1 DNA promoter region;after knocking out the E2F3 gene in the E2F3 high expressing cell line HT1376 and TCCSUP,PD-L1 was down-regulated both at mRNA and protein levels.3.E2F3 positive expression in bladder cancer tissues and the decrease of CD8~+T cell infiltration in bladder cancer tissues are closely related to the tumor invasiveness indexes such as tumor stage,grade,tumor size,lymph node metastasis or distant metastasis;High level of E2F3 and PD-L1 protein were significantly associated with a reduced overall survival time following radical cystectomy for 5 years in invasive disease.ConclusionsThe expression level of E2F3 in bladder cancer is closely related to tumor invasiveness.E2F3 up-regulates PD-L1 expression through transcriptional activation to involve in tumor immune escape.