The Expression Of APE1 In Breast Cancer And Its Effect On Drug Sensitivity Of Olaparib

PART1 The expression of APE1 and its significance in breast cancer tissuesObjective:Triple negative breast cancer(TNBC)is one of the worst prognosis types in breast cancers,which refers to the negative expression ofER,PR and HER2.TNBC has a very special biological behavior and lacks of potential endocrine targets and HER2 therapy.TNBC is becoming a bottleneck in currently clinical breast cancer treatment study.The presence of nearly 15%in patients with TNBC has pathogenic mutations ofDNA repair genes,suggesting the existence of different molecules clonal origin in TNBC.APE1 plays an important role in the base excision repair pathway.In this study,APE 1 was detected in breast cancer.The relationship of APE1 and clinicopathological featuresand its prognostic value in breast cancerwas analyzed.Methods:APE1 was annalyzed by immunohistochemical annalysis(Envision two-step)in323 cases of breast cancers and 108 of them were TNBC.SPSS 21.0 was used as statistics software.Chi-square test was used in count data,as well as Fisher's exact test.Kaplan-Meier curve and log rank test analysiswas usedin survivaloutcomes.Cox regression model was used in multivariate analysis.Three times tests was made to confirm the resultsand statistical significance was confirmed when P<0.05.Results:Compared with adjacent tissues,APE 1 was higher in breast cancer tissued,whichwas 99.3%Vs 0.7%in non-TNBCand 85.6%Vs 14.4%inTNBC(P<0.0001).The expression of APE1 was significantly correlated with tumor size,HER2,axillary lymph node status,ER and clinical stage(P<0.05).Even inTNBC patients with axillary lymph node-positive,low APE1 group had a better prognosis(P = 0.0427).Conclusion:APE1 has a higher expression inbreast cancer than normal tissue,suggesting its relationship withoccurrence of breast cancer.COX results suggest that APE1 is an independent prognosis factor of patients with TNBC,which may be a useful clinic target.PART 2 APE1 affects susceptibility of Olaparib in TNBC cellObjective:PARP-1 is an important gene in DNA single strand repair,as well as APE1.BRCA1 is an important repair enzyme in DNA double strand damage repair.When the BRCA1 is defected and PARP1 is inhibited,syntheticlethality effect may happen.However,the clinical study of PARP1 inhibitors did not show a significant "high efficiency" in BRGA1 mutated breast cancer and the reason is not cdear until now.As the role of APE1 and PARP1 in the DNA base excision repair pathway is not clear,whether there is a competition between the two genes,which affects the efficacy of PARP1 inhibitors,needs to be studied.Therefore,we knockdown APE1 in HCC1937 cell line(BRCA1 defected,TNBC cell line)to observe APE1's effect on PARP1 inhibitor and find out the relationship between PARP1 and APE1.Methods:The stable cell line HCC1937 with APE1 knockout was established by CRISPR/Cas9 system.APE1 was tested before and after using Olaparib by western blot,as well as PARP1 level,in wild-type HCC1937 and APE1 knockout HCC1937.The inhibition rate of Olaparib was measured in wild type HCC1937 and APE1 knockout HCC1937by CCK8 experiment.Cell cycle and apoptosis of HCC1937 and APE lknockoutHCC1937 after Olaparib treatment were tested by flow cytometry.Results:When wild-type HCC1937treated with 10?M Olaparib,APE1 expression decreased.What is more,when increased the concentration of Olaparib to 15?M,APE1 reduced more obviously.In APE1 knockout HCC1937,endogenous PARP1 expression was also less than that of HCC1937.HCC1937-APE1-KO's IC50 increased significantlythanHCC1937.On thefifth day of using PARP1 inhibitor,the drug27%APE1 knockout HCC1937 and 68%wild type inhibited,indicating that wild-type HCC 1937 was more sensitive to PARP1 inhibitor.The results of flow cytometry suggested that HCC1937 with 40?M Olaparib treatment causes a more pronounced G2/M arrestin mitosis,and HCC1937-APE1-KO showed no significant changes.The apoptosis of HCC1937 also increased significantly than HCC1937-APE1-KO.Conclusion:The data shows that PARP1 inhibitor is more sensitive to wild-type HCC 1937,while knocking out APE1 may produce drug resistance.APE1 expression is an important basis for maintaining the function of PARP1 and the loss of APE1 may be related to the drug resistance of PARP1 inhibitor.