The Effect Of Tumor Cell APE1 On The Immune Microenvironment Of Lung Adenocarcinoma And Its Mechanism

Background:Lung cancer is the leading cause of morbility and mortality of malignant tumor in China.Although the treatment of lung cancer has made great progress in the past 20 years,the overall survival rate of lung cancer is still poor.Statistics from 2012 to 2015 showed that the5-year survival rate of male patients with non-small cell lung cancer was 16.8%,while that of female patients was 25.1%,which showed that the treatment of lung cancer still has a long way to go,especially the treatment of advanced lung cancer.Non-small cell lung cancer(NSCLC)is the most common type of lung cancer and lung adenocarcinoma(LUAD)is the majority of NSCLC.Immune therapy significantly improved the treatment plight of the lung cancer,and provided new insights into the treatment,but the overall effective rate was 25%.The immune therapy efficacy depends on the tumor immune microenvironment,therefore,in order to improve the efficacy of immunotherapy,tumor immune microenvironment of patients with lung cancer need to be analysed in detail.The 5-year survival rate of advanced lung cancer is less than 5%in the era of chemotherapy.With the continuous development of immunotherapy,the treatment strategy of lung cancer has changed dramatically,the 5-year survival rate of advanced lung cancer with the immunotherapy(KEYNOT-024)has increased to 31.9%.The powerful efficacy of immunotherapy is limited by its low efficiency.How to improve the efficacy of immunotherapy and how to accurately screen the people suitable for immunotherapy are the hot issues of tumor immunotherapy.Current studies have shown that tumor-infiltrating lymphocytes are important factors affecting the prognosis,and a large number of infiltrating lymphocytes are associated with good prognosis.In lung cancer,there are many studies demonstrating that patients with more infiltrating lymphocytes have better prognosis and immunotherapy response.Tumor cells play an important role in regulating the tumor immune microenvironment,but how tumor cells affect the infiltration of lymphocytes in the tumor immune microenvironment is still unclear.The I-IFN signaling pathway is essential for production of cytokines,death of immunogenic tumor cells,antigen presentation and maturation of dendritic cells.The STING pathway,which is considered to be the main source of the production of I-IFN in the TME,is activated by cyclic dinucleotides(CDN)in the cytosol.Cytosolic CDN can be recognized by cyclic GMP-AMP synthase(c GAS)to form cyclic GMP-AMP(c GAMP),which then binds to STING.The complex subsequently facilitates the phosphorylation of interferon regulatory factor 3(IRF3)by TANK-binding kinase 1(TBK1)and then upregulates the production of I-IFN.The STING pathway is responsible for the activation of innate immunity after virus infection through the production of I-IFN.Apurinic/Apyrimidinic Endonuclease/Reduction Oxidation Factor 1(APE1)is an enzyme with multiple functions that plays an important role in DNA repair and redox signaling pathways.APE1,as a rate-limiting enzymes in BER,which is initiate by glycosylation enzyme cutting off N-glycosidic bond to remove damaged bases,leaving apurinic/apyrimidinic site(AP).And then APE1 cut the locus of DNA dihydrogen phosphate ester skeleton,then DNA polymerase and DNA ligases participate in the BER to keep the integrity of the DNA strand.The incision of AP site is the crucial step in BER,therefore,APE1 regulates the speed of the entire repair pathway in the BER.In mammals,APE1 also has the function of redox by regulating the activity of many transcription factors,mainly including the NF-kappa B,ERG,AP-1,p53,PEBP2,Myb,HIF-1 alpha,Pax5/8 and so on,which thus involved in many crucial cellular processes including cell proliferation,differentiation,transformation and apoptosis,etc.Due to the dual function,APE1 is widely involved in the occurrence and development of tumors and drug resistance to chemotherapy,radiotherapy and targeted therapy.However,whether APE1 of tumor cells can regulate the tumor immune microenvironment by STING pathway is still unclear.Objective1.To investigate the effect of APE1 on the number and population of infiltrating lymphocytes in tumor microenvironment,to further expand the biological function of APE1;2.To further clarify the mechanism of the effect of APE1 on tumor-infiltrating lymphocytes,so as to provide a research basis for improving the efficacy of immunotherapy.3.To further explore the correalation of DNA damage repair proteins in BER pathway and TILs,and to explore the value of the proteins of BER pathway and STING pathway in predicting prognosis.Materials and methodsFirstly,the possible genes and pathways affected by APE1 were identified by bioinformatics method and RNA sequencing data,then effects of APE1 on the number and population of TILs were analysed.Secondly,The results based on our own RNA sequencing were verified by TCGA and GEO public databases.At the same time,the single cell sequencing data of lymphocytes from GEO database was used to investigate whether APE1 has a regulatory effect on lymphocytes themselves.Thirdly,some basic experiments including WB,IF,CO-IP,RT-PCR,ELISA and so on were used to clarify the regulation mechanism of APE1 on STING pathway.At last,The correlation between BER pathway,STING pathway and TILs and their influence on the prognosis of patients were investigated by using clinical data.Results1.RNA sequencing data showed that the abundance of CD4~+T cells was significantly negative correlated with the expression of APE1.2.TCGA and GEO database data suggested that abundance of CD4~+T cell was negative correlated with APE1 expression,and the large number of CD4~+T cells were associated with good prognosis.3.The single cell sequencing analysis of GEO database showed there was no difference in the expression of APE1 in different types of lymphocytes,which indicated that APE1 did not affect the differentiation and maturation of lymphocytes.4.The results of GEO database analysis showed that APE1 was correlated with the number of CD4~+T and CD8~+T cells of different subtypes,specifically,the higher expression of APE1,the lower number of T cells with killing activity and the more number of depleted T cells.5.Basic experiment proved that the increased cytoplasm ds DNA could be caused by low expression of APE1,which activate the STING pathway and then up-regulate the level of I-IFN.6.APE1 competitively binded to STING and further inhibited the activation of downstream pathway.7.The nomogram model based on BER pathway,STING pathway and TILs could accurately predict the recurrence of lung cancer in early stage.8.Data from tissue specimens showed that BER pathway,STING pathway and TILs were correlated.ConclusionAPE1 regulates the number of lymphocytes in the immune microenvironment of lung cancer through the STING pathway and participates in the acclimation of the tumor immune microenvironment.