Neddylation Inhibitor MLN4924 Suppresses DNA Double-strand Break Repair Through Blocking The Recruitment Of BRCA1 Complex

DNA double strand breaks is the severest type of DNA damage induced by environmental harmful factors.To repair the DSBs,eukaryotic cells employ two main pathways: the non-homologous end joining(NHEJ)and the homologous recombination(HR).HR plays a role only in late S phase and G2 phase of the cell cycle because it needs the homologous chromatid as a template.Therefore,it is an error free repair.NHEJ can occur throughout the cell cycle,particularly in G1 phase,but it is error prone.Breast cancer related gene 1(breast cancer associated gene 1,BRCA1)is a tumor suppressor gene and plays an important role in the activation of cell cycle checkpoint and DNA repair process.It is also a key protein of HR repair pathway.According to previous reports,neddylation can regulate the DNA damage repair process possibly though affecting BRCA1.To better understand the mechanism,we used the small molecule inhibitor MLN4924 in our study.MLN4924 is an effective and specific small molecule inhibitor of NEDD8,which can form a complex with NEDD8 activating enzyme and inhibit neddylation modification effectively and specifically.As we all know,NEDD8 is an ubiquitin-like protein,it may modify the BRCA1 protein and then regulates the HR pathway.So we began by Pull-down experiments to verify this hypothesis.The result showed that NEDD8 failed to modify the BRCA1 protein.However,did not mean that there is no effect of NEDD8 on the BRCA1.Therefore,we used MLN4924 to detect the effect of neddylation on BRCA1 complex.The results showed that upon neddylation being inhibited,the recruitment of components of BRCA1 complex to the DNA damage sites is significantly inhibited,suggesting that MLN4924 is able to inhibit the HR.It is well known that PARP inhibitors can inhibit HR repair,so we considered that MLN4924 and PARP inhibitor Olaparib may inhibit DNA damage repair synergistically,and then inhibit the growth of tumor cells.Therefore,we performed the proliferative and toxicity test using cell counting kit-8(CCK8)to detect the effect of combination of MLN4924 and Olaparib on two non-small cell lung cancer cell lines,A549 and H1299.Cells were pretreated with combination of MLN4924 and Olaparib.As we expected,compared with control groups,the combination treatment inhibited cell growth more severely.Since BRCA1 is so important in HR pathway,we propose combinational treatment of MLN4924 and PARP1 inhibition will increase the sensitivity of cells to DNA damage.So we detected the repair of IR-induced DSBs using DSB biomarker ?H2AX.In both A549 and H1299 cells,we scored the ?H2AX foci before and after 2 Gy irradiation..The results showed that higher level of residual ?H2AX foci persistently existed in the MLN4924 and Olaparib treated cells till 24 h after IR,suggesting that the DNA repair process was greatly hindered.Next,we wondered if NEDD8 or BRCA1 or PARP1/2 correlated with the prognosis of lung cancer patients.The data were searched from the Kaplan-Meier database,the Kaplan-Meier survival plots for each genes showed that high expression either NEDD8 or BRCA1 or PARP1/2 predicted a worse prognosis for the lung cancer patients.These phenomena suggest that the combination of MLN4924 and Olaparib may be a new strategy for the treatment of non-small cell lung cancer.Objective1 To understand the effect of MLN4924 pretreatment on the recruitment of components of BRCA1 complex to DNA damage sites in U2 OS cell line.2 To investigate the effect of combinational treatment of MLN4924 and Olaparib on cell growth of A549 and H1299 cells.3 To determine the impairment of combination of MLN4924 and Olaparib on DNA double-strand repair.4 To reveal the correlation between expression of NEDD8,PARPs,BRCA1 and prognosis of lung cancer patients.MethodsSFB-VEC,wild type SFB-NEDD8 and SFB-NEDD8 mutant plasmids were transfected into 293 T cells.After 24 h,cells were irradiated with 10Gyg-rays.One hour after treatment,cells were collected.Then we performed Pull-down assay to detect neddylation of BRCA1.U2 OS cells were pretreated with 3 ?M MLN4924 for 1h,then treated with 8Gy irradiation.Six hours later,cells were processed to immunofluorescence assay to detect the BRCA1 foci.In the laser micro-irradiation experiment,GFP-BARD1 and GFP-XRCC1 were transfected into U2 OS cells.After24h,cells were subjected to laser microirradiation,and then the fluorescence intensity of GFP was detected.U2 OS cells were pretreated with MLN4924 for 1h,and then 8Gy IR,cells were collected at different time points(1,2,4,8 and 12h),then chromatin fraction was extracted with 0.2M HCL and subjected to Western Blot with Lamin-C as reference.A549 and H1299 cells were treated with MLN4924 and Olaparib(range from 0.01?M to10?M)respectively,then 0.1?M was chosen for further study since half number of the cells were dead.Then we treated two cell lines with combination of MLN4924 and Olaparib to detect the growth.A549 and H1299 cells were pretreated with 0.1?M MLN4924 and 0.1?M Olaparib for 1h,then 2Gy IR.After 24 h,?H2AX was detected by immunofluorescence staining.The relationship between the prognosis of patients with lung cancer and the expression levels of NEDD8,BRCA1 and PARPs in patients with lung cancer was illustrated by Kaplan-Meier database.Results1 Pull-down experiment showed that NEDD8 cannot modify BRCA1;2 After inhibition of neddylation,the recruitment of BRCA1,BRAD1 and RAP80 proteins to DNA damage sites was significantly inhibited as compared to the control group;3.Cell proliferative toxicity test showed that the combination of MLN4924 and Olaparib synergistically inhibited the growth of A549 and H1299 cells;4.A higher level of ?H2AX foci persistently existed in the MLN4924 and Olaparib treated cells 24 h after IR,suggesting that the DNA repair process was greatly hindered5.Kaplan-Meier database analysis showed that the expression levels of NEDD8,PARPs and BRCA1 in patients with lung cancer were negatively correlated with the prognosis of patients with lung cancer.Conclusions1 NEDD8 regulates the HR pathway not directly through modifying BRCA1 protein,but when neddylation was inhibited by MLN4924,the recruitment of DNA repair proteins BRCA1 BARD1 and RAP80 to DNA damage sites was inhibited significantly;2 The combination of MLN4924 and Olaparib significantly hindered the process of DNA DSB repair,which led to growth inhibition of A549 and H1299,two non-small cell lung cancer cell lines;3 The expression of NEDD8,PARPs and BRCA1 gene were negatively correlated with the prognosis of patients with lung cancer,suggesting that the combination of MLN4924 and Olaparib may be a new strategy for the treatment of non-small cell lung cancer.