DNA Damage Repair Gene Ape1 Contributes To The Inhibition Of Immune Microenvironment In Urothelial Carcinoma

BackgroundUrothelial carcinoma,including bladder cancer and upper urothelial carcinoma,is a multi-source malignant tumor originated from the urothelium.It is the most common immune related malignant tumor.In recent years,immunocheckpoint inhibitors have made great progress in the treatment of urothelial carcinoma,but the overall effective rate is not high.In addition to PD-L1 expression and tumor mutation load,immune microenvironment is also an important factor affecting the therapeutic effect.The imbalance or defect of DNA damage and repair of tumor cells can affect the production of new tumor antigens and changes to immune microenvironment,consequently affecting the immunotherapy of tumors.The aim of this study was to investigate the relationship between DNA damage repair gene APE1 and the immune microenvironment of urothelial carcinoma,to provide evidence for further elucidating the mechanism of immunosuppressive microenvironment in urothelial carcinoma,and to provide research evidence for individualized and combined treatment of patients with ICI.Materials and Methods1.Use of public clinical database: TCGA database2.Cell models: different human bladder tumor cell lines T24 and BIU87,mouse bladder tumor cell line MB49,inflammatory cell line THP-1 and RAW264.7 were used.3.Animal model:(1)C57 mice were subcutaneously implanted with MB49 cells at the age of 4 weeks.They were evenly divided into 4 groups and treated: control group,e3330 group,PD-1 group and e3330 combined with PD-1 group to evaluate the effect of APE1 inhibitor on bladder tumor.4.Patient specimens: A.collect the paraffin block marks of the surgical focus of patients with upper urinary tract epithelial carcinoma diagnosed in our hospital,Immunohistochemistry was used to evaluate the relationship between BER pathway,sting pathway related proteins,TILs and prognosis of UTUC;B.paraffin embedded specimens of surgical lesions and patients with bladder urothelial carcinoma diagnosed in our hospital were collected,and the relationship between APE1,VEGFA,CD163 and prognosis of bladder cancer was evaluated by immunohistochemistry.5.Experimental methods: immunohistochemistry,Western blot,multiple immunofluorescences,cell transfection,RNA sequencing and flow cytometry were used in this study.Results1.TCGA database analysis showed that APE1 expression was an independent prognostic factor of PFI in patients with MIBC.High expression of APE1 was significantly associated with immunosuppression in bladder cancer,including immune score,immune cells and various inflammatory factors.APE1 combined with M2 macrophages can better divide MIBC into low-risk group and high-risk group.There were significant differences in gene expression between the two groups.In high-risk group,high APE1 expression and M2 macrophage abundance were found.Yet the expression of all the other anti-tumor factors were significantly decreased,especially M1 macrophage,cytotoxic T cell abundance and IFN-?bisection,IFN-? gene expression level and ICC.2.In the GO analysis,the top three factors were receptor regulatory activity,In the top 10 pathways of KEGG enrichment analysis,cytokine receptor interaction,rheumatoid arthritis,IL-17 signaling pathway and TNF signaling pathway are closely related to cytokines.3.In UTUC,high expression of IRF3 is associated with better prognosis,while APE1 and pol ? are associated with poor prognosis;CD8 + T cell infiltration contributes to better prognosis.pol ? is correlated with T cell infiltration.4.In bladder cancer,the high ratio of CD163 + TAMs in bladder cancer tissues was confirmed by immunohistochemistry and multiple immunofluorescence staining.The expression of APE1 was positively correlated with the expression of VEGFA and the increase of CD163 + Tam infiltration,suggesting that APE1 may participate in the regulation of macrophage polarization through VEGFA.In addition,the high expression of APE1 is related to the presence of LVI in bladder cancer.LVI positive,high expression of APE1 and high ratio of CD163 + TAM in bladder cancer may shorten the survival time of patients.5.Furthermore,it was found that APE1 inhibitor e3330 and PD-1 ab had synergistic antitumor effect.E3330 can significantly inhibit tumor angiogenesis and M2 macrophage infiltration;pd-1ab can significantly promote the infiltration of CD8 cells.Immunohistochemistry and flow cytometry have complementary significance in evaluating tumor immune cell infiltration.ConclusionDNA damage repair gene APE1 may play an important role affecting the immunosuppressive environment and prognosis of patients with urothelial carcinoma;inhibition of APE1 has synergistic effects on the immunotherapy of bladder cancer;APE1 as a marker or target of synergistic therapy has important scientific significance and potential application value in future immunotherapy of urothelial cancer.