Research On Mechanism Of DNA Base Damage Sites As Novel Epigenetic Markers In EGFR-TKI Treatment Resistance In Non-small Cell Lung Cancer

BackgroundDNA nucleotedes mainly contain thymine（T）,cytosine（C）,guanine（G）and adenine（A）.DNA base damage is common in tumor cells,and the damage factors mainly include reactive oxygen species（ROS）,oxidants,alkylation agents and ultraviolet radiation,DNA oxidative damage was the most frequent.8-oxo-7,8-dihydroguanine（8-oxo-G）formed due to the high susceptibility to oxidative attack of the carbon atom at position 8 of guanine,8-oxo-G is the most oxidative damage base in the cell^[1].Base Excision Repair（BER）is a highly conserved mechanism to Repair oxidative damage of cellular DNA,and BER pathway contains more than 30 proteins^[2].The 8-oxoguanine DNA glycosylase（OGG1）recognizes and removes the 8-oxo-G to form apurinic-apyrimidinic site（AP）,then apurinic/apyrimidinic endonuclease1（APE1）binds and collects downstream repair enzymes to perform indirect resection and repair of AP site,and DNA polymerase beta（DNA Polβ）restores its correct base pairing^[3,4].Non-small cell lung cancer（NSCLC）is the main subtype of lung cancer^[5,6],epidermal growth factor receptor-Tyrosine kinase inhibitor（EGFR-TKI）targeting therapy is now effectively used to drive mutation-positive patients.However,in advanced metastatic NSCLC,patients developed drug resistance and tumor metastasis recurrence.Epithelial-mesnchymal transition（EMT）and Cancer Stem cells（CSCs）in lung cancer cells are important causes of cancer drug resistance.EMT is regulated at different levels through epigenetic modification,transcriptional regulation of variable splicing and protein subcellular localization,during which 2,000-3,000 genes are altered in expression^[7].CSCs are one of the most difficult subsets of cancer cells to treat in tumors.They have the ability of self-renewal and DNA damage resistance,and can maintain primary and secondary tumor growth,CSCs have a strong gene of DNA repair and protein activation network system,so cancer stem cell transformation is an important cause of EGFR-TKI resistance in addition to secondary mutations of EGFR gene（T790M mutation）and EMT^[8,9].There is also a strong link between EMT and CSCs^[10,11].APE1 is a key rate-limiting enzyme of BER pathway,and it has endonuclease and redox bifunctional protease.OGG1 is also a bifunctional enzyme^[2].Since AP and 8-oxo-G sites are enzyme substrates of APE1 and OGG1 respectively,it is assumed that APE1 and OGG1 enrichment regions on the genome represent the distribution of AP and 8-oxo-G sites.BER protein has been shown to have beyond DNA repair,among which there are new discoveries in epigenetics.The weakened repair activity of APE1 to non-B DNA structure leads to the existence of base damage hotspots in the genome,and the 8-oxo-G sites also presents the characteristics of non-random distribution.Combined with a large number of literatures,it has been reported that APE1 and OGG1 are associated with tumor recurrence,metastasis and drug resistance.The main purpose of this study was to explore the distribution of 8-oxo-G/AP sites as a novel epigenetic marker in the whole gene.APE1 and OGG1 proteins repair the base damage sites（8-oxo-G/AP sites）while coupling gene expression regulation.This bifunctional feature may promote cancer cell resistance to EGFR-TKI therapy through epigenetic reprogramming that regulates abnormal expression of EMT and CSC-related genes.Methods1.Western Blot assay was used to verify the efficient and specific binding of anti-APE1（Abcam,ab194,USA）and anti-OGG1（Novus,NB100-106,USA）with APE1protein and OGG1 protein expressed in A549 cells.2.Agarose gel electrophoresis was used to test whether ultrasound fragmentation of chromatin into DNA fragments of 200bp-1500bp length met the requirements of Ch IP-seq,60%DNA fragments in the range of 200bp-1500bp.5%polyacrylamide gel electrophoresis with ultra-sensitive gold nucleic acid dye（Invitrogen,S11494,USA）Nucleic acid staining showed that there were obvious nucleic acid bands between 200bp-1500bp between APE1,positive,2%input and negative control IP sample,so as to verify the success of Ch IP-seq experimental system.3.DNA fragments adjacent to the AP and 8-oxo-G sites specifically bound to APE1 and OGG1 proteins in A549 cells were obtained by Ch IP-seq assay,and the reliability of the whole experimental system was verified by Ch IP-PCR assay.4.Bioinformatics analysis of Ch IP-seq data,MACS2（Version 2.1.0）peak call software was used to identify background IP enrich areas.The q-value threshold of all data sets is0.05.The significance level of the peak width pleat abundance and the distribution of each peak number after the peak call are displayed.Use Homer software to detect the new sequence module and the known matching module,Ch IPseeker can confirm the peak related genes,and then conduct Gene body Ontology（GO）enrichment analysis,KOBAS software was used to detect the statistical enrichment of KEGG pathway peak related genes.5.Use GEO and ENCODE data to mine the Ch IP-seq data of histone H3K4me3,H3K4me1,H3K27ac,H3K27me3 and H3K36me3 compared with the Ch IP-seq data of APE1 and OGG1 proteins in this study,so as to explore the possibility of 8-oxo-G/AP sites as epigenetic marker molecules.6.The acquired EGFR-TKI resistant cells HCC827ER and PC9ER were induced by low concentration gradient induction,and the changes of Cell morphology were observed under a microscope.The changes of invasion ability of resistant strains were detected by Transwell,and Cell toxicity tests were performed,CCK-8 were used to detect the IC50value of sensitive and resistant strains to Erlotinib and verify the stable culture resistant strains.Western Blot was used to detect the expression changes of BER,EMT and CSCs related marker proteins of sensitive and resistant strains.7.Limited dilution method was used to separate drug-resistant subsets of different clone subtypes,and whole exon sequencing method and first-generation sequencing method were used to verify whether the selected monoclonal cell line was T790M mutant cell line.8.The APE1 and OGG1 genes of PC9,HCC827 cells and drug-resistant strain PC9ER,HCC827ER cells were silenced and overexpressed by lentivirus transfection experiment,and the expression levels of APE1 and OGG1 proteins were verified by Western Blot.9.Gene expression differences between EGFR-TKI resistant and sensitive strains were screened by RNA-seq data,and gene sets related to EMT and CSCs pathways were screened by difference analysis and GSEA gene set enrichment analysis.10.The enrichment regions of APE1 OGG1 and G4 in HCC827 and HCC827ER cell genomes were obtained by CUT&Tag.11.PC9ER＿APE1＿Ch IP-seq data mining sequence of significant enrichment peak on target gene P21,MYC,KRAS,TMBIM6,POTEH,ANKRD30BL,GUSBP1,design sequence primers,and then compare the enrichment difference of APE1 binding region on target gene of drug-resistant and sensitive strains.Results1.The Chromatin immunoprecipitation（Ch IP）system for BER pathway proteins APE1 and OGG1 was successfully constructed.2.APE1 and OGG1 were widely distributed in the whole genome and non-randomly located in the gene regulatory region.APE1 and OGG1 were distributed in the promoter region of 41.7%and 49.26%respectively.3.APE1 and OGG1 binding motif highly match transcription factors that activate and inhibit cell growth cycle,cancer transcription.The APE1 related motif best matches the transcription factor（Dof2,TCP19,ZNF165,PLAGL1,CDC5）and the OGG1 related motif best matches the transcription factor（MBNL1,CAMTA1,E2F1,ZFP161,GT-1）.4.APE1 and OGG1 binding peak related genes are closely related to cancer pathways.5.The binding regions of APE1 and OGG1 proteins and epigenetic marker histones overlap in the whole genome.6.Both APE1 and OGG1 were upregulated in EGFR-TKI resistance of Non-small cell lung cancer,and the cell morphology of the drug-resistant strain changed to a spindle shape similar to the mesenchymal cells with enhanced invasion ability.The IC₅₀value of Erlotinib of the drug-resistant strain PC9ER and HCC827ER was higher than that of the sensitive strain more than 10 times higher.After overexpression of APE1 and OGG1 genes of susceptible strains,the IC₅₀value of susceptible strains to Erlotinib increased,while the IC₅₀value of resistant strains to Erlotinib decreased after knockdown of APE1 and OGG1genes.7.EGFR-TKI resistance of lung cancer cells was significantly correlated with EMT and CSCs.In HCC827ER,EMT and CSCs marker molecule Vimentin,CD44,c-MYC,FGF2 was increased while e-cadherin,OCT-4A,Nanog,Sox2,KLF4,P21,P16 and EGFR decreased.But EMT and CSCs marker molecule Vimentin,CD44 were decreased and e-cadherin,OCT-4A,Nanog,Sox2,KLF4,c-MYC,P21,P16 and EGFR were increased in PC9 drug resistance.APE1 increased the enrichment degree of promoter region of P21,MYC,KRAS,TMBIM6,POTEH,ANKRD30BL,GUSBP1 gene in PC9ER.8.APE1 and OGG1 can be involved in the mechanism of EGFR-TKI resistance by binding a novel epigenetic marker 8-OXO-G/AP locus to regulate EMT and CSCs;APE1and OGG1 can participate in the mechanism of EGFR-TKI resistance by regulating EMT and CSCs.APE1 and OGG1 genes in overexpressed sensitive strains could promote the molecular transformation of EMT and CSCs into resistant strains.However,knockdown APE1 and OGG1 could restore the sensitivity of the resistant strains to Erlotinib and partially restore the EMT and CSCs molecules to the level of sensitive strains.Conclusions1.BER pathway proteins APE1 and OGG1 were distributed non-randomly throughout the genome and mainly located in gene regulatory regions,AP sites and 8-oxo-G sites are the catalytic substrates of APE1 and OGG1 respectively.According to the specificity of enzyme substrates,we infer that the distribution of 8-oxo-G/AP sites is non-random and located in the gene regulatory region.2.APE1 and OGG1 bind motifs that highly match cancer-related gene transcription factors.APE1 and OGG1 binding peak related genes were significantly associated with cancer and partially overlapped with epigenetic modified histones in the genome-wide binding region.Therefore,it is inferred that the oxidative base damage sites 8-oxo-G/AP of DNA is a novel epigenetic marker involved in gene regulation of tumors.3.The high expression of APE1 and OGG1 promote EGFR-TKI resistance of lung cancer cells PC9 and HCC827,and APE1 and OGG1 may regulate resistance by affecting the expression of EMT,CSCs and aging-related gene sets of tumor cells.Based on the above conclusions,we speculated that 8-oxo-G/AP as a new epigenetic marker,was involved in the regulation of gene expression in the process of EGFR-TKI resistance in lung cancer cells.