The Role Of APE1 In Promoting DNA Double-Strand Break Formation And Repair Following Ionizing Radiation In Cervical Cancer And Its Molecular Mechanism

BackgroundRadiotherapy is one of the cornerstones of tumor treatment.About 50%-70%of patients with malignant tumors need to use radiotherapy in the process of tumor therapy,and nearly half of them are radical radiotherapy.Radiotherapy kills tumor mainly by causing DNA damage through ionizing radiation（IR）.IR-mediated DNA damage can be divided into direct DNA damage and indirect DNA damage.Direct damages represent a minor portion（20%-30%）of the IR-induced DNA damage,whereas damages that are generated via indirect effect represent 70–80%of the total DSB damage.Hence,70-80%IR-mediated DNA damage is oxidative DNA damage,which is composed of various types of DNA damage forms.Oxidative DNA damage can be broadly classified into isolated DNA damage and clustered DNA damage based on the distance between the different damages.Isolated damage mainly includes single abnormal base damage lesions,abasic sites（also known as AP sites）,single strand break（SSB）,etc.Because there is correct template on the complementary DNA strand,isolated DNA damage could be precisely repaired without threatening the integrity of genome.Conversely,clustered DNA damages,consisting of closely spaced isolated DNA damage（generally defined as within 10 bp）,have long been postulated to be the lethal damage induced by IR due to its complex composition and the lack of a specific and matched repair pattern.The repair mechanism of clustered damage remains unclear to date,and further explore its repair mechanism is important to overcome radio-resistance.AP site deserves special attention among the various forms of IR-mediated DNA damages.Firstly,IR can directly attack DNA molecules to generate AP sites.Secondly,IR can indirectly attack DNA molecules through reactive oxygen species（ROS）to cause abnormal base modifications,and the latter could be converted into AP sites via DNA glycosylase.Therefore,AP sites are crucial part of DNA damage after IR exposure.Base excision repair（BER）pathway is the main pathway for repairing isolated AP sites.Apurinic/apyrimidinic endo-deoxyribonuclease 1（APE1）plays a key role in this pathway via initiating AP site repair.APE1 could recognize AP site in isolated DNA damage and cleave AP site’s DNA backbone to produce an SSB intermediate,which in turn promotes the recruitment of downstream molecules of BER pathway to complete the isolated DNA damage repair.Our group has long been committed to explore the effect of DNA damage repair on multiple biological behaviors of tumor and its related molecular mechanisms,especially the role of APE1 on tumor development and drug resistance.APE1 is the key enzyme and lack of an effective back-up repair mechanism,it performs about 95%endonuclease activity in cells and plays an indispensable role in maintaining genomic stability.Previously,we found APE1 was significantly upregulated in tumor samples in comparison to paired peri-tumor tissue,and it was also correlated with the tumor malignancy.Moreover,other researches also demonstrated that the APE1 overexpression was related to the therapeutic resistance of tumor cells.Combined,APE1 is considered to be a promising antitumor molecular target.Nowadays,whether APE1 plays a role in the repair process of clustered DNA damage containing AP sites remains unknown.Unlike isolated DNA damage after processed by APE1 will produce a SSB intermediate,when two adjacent DNA damages containing AP sites and locating on opposite DNA strands in clustered DNA damage,if APE1 is able to recognize and cleave these two adjacent AP sites,theoretically,a double strand break（DSB）intermediate will be induced.DSB is the most lethal DNA damage lesion.Thus,if APE1could convert clustered DNA damage into DSB,it may increase radio-sensitivity of tumor cells.However,several previous studies have shown that APE1 promotes radio-resistance in tumor cells,which is contradicted to our hypothesis.Therefore,whether APE1 promotes DSB formation post-IR exposure and the effect of this process on the therapeutic sensitivity for tumor cells still needs further investigation.The aim of this study was to explore the role of APE1 in repairing clustered DNA damage under IR stress and the related molecular mechanisms,expecting to providing new ideas to overcome therapeutic resistance of radiotherapy.Methods1.APE1 promoted early phase DSB formation following IR exposure.Lentivirus mediated short hairpin RNA（sh RNA）against APE1 were used to knockdown its expression in the human cervix carcinoma cell line He La and Si Ha.He La APE1 lentiviral negative control and He La APE1 knockdown cell lines are abbreviated as NC and KD,respectively.After IR exposure,time-course and dose-dependent immunoblotting assays were utilized to detect the activation ofγH2AX（hallmark of DSB）.Comet assay was utilized to measure the SSB and DSB yield.Small molecule inhibitors of APE1 were used to block its redox activity or endonuclease activity,then immunoblotting assay were performed to determinate which activity was involved in DSB formation after IR stress.2.The early phase DSBs induced by APE1 underwent efficient repair.Clonogenesis were performed to examine the effect of APE1 on cervical cancer cells’survival.Micronuclei（MN）assays were used to assess the effect of APE1 on genome instability following IR exposure.To assess the effect of APE1 on the dynamics of DSB repair,immunoblotting assay was utilized to detect the expression ofγH2AX during 0-64 hours following IR exposure.TBHP（tert-butyl hydroperoxide）,a commonly mimetic of IR effects and inducer of cellular oxidative stress,was used to further verify whether the effect that APE1 promoted clustered DNA damage repair via initiating DSB formation following IR exposure were widespread existed following oxidative stress.Time-course immunofluorescence and comet assay were used to measure the dynamics of DSB repair after TBHP treatment.3.APE1 promoted DSB repair through activating NHEJ（Non-homologous end-joining）repair pathway.Following IR or TBHP exposure,immunoblotting and immunoprecipitation assay were used to evaluate the effect of APE1 on the activation of key kinases of DNA damage response（DDR）pathway,DNA-PK_cs and ATM.Immunofluorescence and chromatin fractionation assay were utilized to detect the formation of 53BP1（p53 binding protein）foci and the recruitment of NHEJ pathway proteins in DSB lesions to evaluate the activation of NHEJ pathway.4.APE1 promoted oxidative therapeutic resistance by maintaining the stability of Artemis protein of the NHEJ pathway.q RT-PCR and immunoblotting assay was used to evaluate the effect of APE1 on the m RNA expression and the protein expression and the stability of Artemis,respectively.Immunoprecipitation was utilized to assess the effect of APE1 on Artemis’s ubiquitination.Immunohistochemistry was used to analysis the correlation of protein expression between APE1 and Artemis in cervical cancer samples,and the relationship between protein expression of APE1 or Artemis and the curative effect in cervical cancer patients treated by radical radio-chemotherapy.5.Combinatorial inhibition of APE1 and DBS repair activity promoted synergistic sensitization after oxidative therapeutic.In vitro and in vivo assays were performed to assess the synergistic sensitizing effect via combinatorial inhibition of APE1 and ataxia-telangiectasia mutated（ATM）under oxidative stress.Results1.APE1 promoted early phase DSB formation following IR exposure.In the early phase（about within 1 h）post-IR exposure,APE1 significantly increased the DSB level.Inhibiting its endonuclease activity markedly downregulated the activation ofγH2AX.2.The early phase DSBs induced by APE1 underwent efficient repair.Post-IR exposure,APE1 promoted cervical cancer cells survival and the early-phase DSBs mediated by APE1 were not converted into more micronuclei after 96 hours repair.The analysis of DSB repair process showed that APE1 significantly promoted the DSB formation at early phase,but this part DSB markedly decreased in the late phase（after 24 h）.Conversely,the DSB level in APE1 KD cells was significantly increased 24 hours after the treatment of IR and TBHP.The above results indicate that APE1-mediated early phase DSBs undergo effective repair.3.APE1 promoted DSB repair through activating NHEJ repair pathway.Following oxidative stress,APE1 significantly promoted the activation of DNA-dependent protein kinase catalytic subunit(DNA-PK_cs),the key kinase of NHEJ pathway.APE1 also facilitated the recruitment of core proteins of the NHEJ pathway,including Ku70,Ku80,DNA-PK_cs,Artemis,DNA Ligase 4 to chromatin and the formation of 53BP1 foci at DSB damage lesions.4.APE1 promoted oxidative therapeutic resistance by maintaining the stability of Artemis protein of the NHEJ pathway.Knockdown APE1 in cells and mice significantly downregulated the protein expression of Artemis,but the m RNA expression was not affected.Knockdown APE1 increased protein ubiquitination of Artemis,and inhibited proteasome activity could markedly rescue the Artemis protein expression in APE1deficiency cells.A strong positive correlation between APE1 and Artemis protein expression in cervical cancer patient samples also was observed,with a correlation coefficient of 0.747（P<0.01）.The protein expression level of Artemis correlated with the prognosis of cervical cancer patients treated with radical radio-chemotherapy,with a median survival time of 47 months versus 24 months for patients with high versus low Artemis protein expression（P=0.002）.Overexpression Artemis in APE1 knockdown cells could partially rescue the cellular sensitivity to oxidative stress.5.Combinatorial inhibition of APE1 and DBS repair activity impart a synergistic sensitizing effect after the oxidative stress exposure.Combinatorial inhibition of APE1 and ATM can induce more apoptosis in vitro and inhibit tumor proliferation in vivo after the oxidative stress exposure.Conclusions1.The process by which APE1 facilitated the conversion of clustered damage into DSB through its endonuclease activity is a critical step to provide a recognizable DSB ends for the NHEJ pathway in early phase following IR exposure.Thus,APE1 activated NHEJ signaling and resulted in radio-resistance by promoting early DSB formation.2.APE1 promotes NHEJ pathway repairing DSB by maintaining the stability of Artemis proteins.3.APE1 deficiency resulted in numerous replication-related late-phase DSB accumulation,The key kinase that promotes DSB repair during DNA replication is ATM.Combinatorial inhibition of APE1 and DBS repair activity impart a synergistic sensitizing effect under the oxidative stress...