The Expression Of APE1and The Relevance To Drug Susceptibility Of Olaparib In Triple-negative Breast Cancers

BackgroundThere are various damage repair systems including base excision repair（BER）、nucleotides excision repair（NER）、homologous recombination repair（HR）、non-homologous end joining（NHEJ） in mammals. BER is the major pathway repairingspontaneous base damage and exogenous chemical-induced oxidative DNA damage ineukaryotes. Apurinic/apyrimidinic endonuclease1(APE1)，the main rate-limiting enzymein BER，plays an important role by cutting the AP site generated by damaged DNA.As aDNA damage repair related gene，the disfunction caused by gene mutation will eventuallylead cancers.Studies have found that APE1gene mutation was closely associated withprostate cancer,non-small cell lung cancer,ovarian cancer.Poly(ADP-ribose) polymerase1(PARP-1) is a ribozyme widely expressed in highereukaryotes，involving in DNA single/double-strand broken repair. Breast cancersusceptibility gene（BRCA1）is associated with the development of familial breast cancers.BRCA1mutation happens in most TNBCs and is involved not only in HR but also in otherDNA repair pathways, e.g. promoting NHEJ in DNA DSBs repair or NER.WhenBRCA1/2is deficient，the function of PARP-1will be the key factor.Besides that, PARP-1is also involved in long-patch BER though DNA polymerase β（pol β），DNA ligaseⅠ，DNA ligaseⅢ/X-ray cross-species complimenting1（XRCC1）.Preliminary studieshave found that PARP-1inhibition-olaparib could cause selective apoptosis of BRCA1-mutated cells. But the efficiency is only41%with single olaparib in some phase Ⅱclinicalstudies.Considering the importance of APE1in DNA damage repair（DDR），this study isdesigned to investigate the relevance between APE1expression and clinicopathologicalcharacterizations of breast cancer patients.Meanwhile,the we make an assumption that theBER pathway mediated by APE1maybe the main reason of olaparib’ hyposensitivitytreating triple-negative breast cancers. PART1The expression and clinical significance of APE1inbreast cancer tissues and cellsObjective:Detect the expression of APE1in breast cancer(tissues and cells) and explorethe revelance between APE1expression and patients’ clinicopathologicalcharacterizations further.Methods:256breast cancer tissue cases and73adjacent cancer tissue cases were collectedto construct tissue microarrays（TMAs）.IHC was used to detect the expression of APE1in tissues.Western blot was used to detect the expression in cells. Relevant clinical datawas collected to perform statistic analysis.Results:There is significant difference between cancer and adjacent cancer tissues in theexpression of APE1(p＜0.001). The expression of APE1was significantly higher in ER-positive than ER-negative group（p=0.022）.But it was significantly higher in HER-2overexpression group.Besides that,the APE1expression was related to the triple-negativesubtype.The overall survival(OS) of patients with APE1negative-expression was longerthan the positive ones.Except MDA-MB-231，the expression of APE1in two HER-2overexpression cell lines was higher than others.Conclusion:The expression of APE1was significantly high in breast cancer tissues andcells.Besides that, ER-positive、HER-2positive state and cancer subtype was associatedwith it.Survival analysis indicated that APE1was the independent risk factor.All resultsdemonstrated that APE1was a oncogene and could be a prognostic marker in clinic.PART2The influences on the biological function and APE1expression of olaparib in breast cancer cellsObjective:To detect the influence on the biological function and APE1expression ofolaparib in breast cancer cells by MTT and flow cytometry.Methods:Different concentration gradient and time gradient were set to treat the normalbreast cell(MCF-10A) and breast cancer cell lines(MDA-MB-231、MCF-7、SK-BR-3、ZR-75-30、T47D) with olaparib.MTT was used to measure the IC50（half maximalinhibitory concentration）and flow cytometry was used to detect the apoptosis effect.Results:The IC50was range from11.206to32.164μM in breast cancer cells and wasover100μM in normal breast cells.The apoptosis rate was range from13.4%to95.74%in50μM,48hour. Conclusion:The sensitivity of olaparib was various in different kinds of breast cancercells.It was especially sensitive to HER-2-overexpression and triple-negative subtype.PART3Impact on the expression of APE1of olaparib in MDA-MB-231cell and establishment of RNAi method knocking downAPE1Objective:Western blot was used to detect the change of APE1expression level in MDA-MB-231cell.Establish RNAi method to knock down the expression of APE1Methods:Western blot was used to detect the expression of APE1in MDA-MB-231celltreated with olaparib in an appropriate concentration（less then IC50measured in part1）.MDA-MB-231cell was transfected with MLP-shRNA-APE1and the efficiency wasobserved by fluorescence microscope and measured by western blot.Results:The expression level of APE1was higher after treated with olaparib in MDA-MB-231cell.About36~48hours after transfected,60~70%GFP（green fluorescent protein）-positive cells was observed by fluorescence microscope and the expression of APE1wasdistinctly decreased by western blot.Conclusion:The treatment of olaparib will lead the higher expression of APE1inBRCA1/2-mutation related cells.The expression of APE1was significantly knockdown byRNAi method in MDA-MB-231cells.