The Role And Mechanism Of PARP1 In Alternative End-joining Of Dna Double-strand Break Repair In Triple-negative Breast Cancer Cells

ObjectiveAmong various types of DNA damage,double-strand breaks(DSBs)is the most serious form of damage that affects the stability and integrity of the genome.It will further cause cell death if not correctly repaired in time.In normal cells,classical/canonical non-homologous end-joining(C-NHEJ)is the main pathway of DSB repair.When the key factor of C-NHEJ Ku70 is deficient,alternative end-joining(A-EJ)occurs as a backup repair pathway.Because it's an error-prone pathway which leads to genomic instability,the A-EJ pathway is highly carcinogenic and closely related to the tumorigenesis and tumor progression.Recent studies have shown that poly(ADP-ribose)polymerase 1(PARP1)may be an important molecule that regulates the A-EJ pathway.In this study,model of A-EJ pathway in DSB repair is established in human triple-negative breast cancer(TNBC)cells to explore the recruitment of PARP1 to chromatin and clarify its interaction with possible downstream repair protein,meiotic recombination11(MRE11).This study helps to further understand the role of PARP1 in A-EJ pathway of DSB repair,the effective mechanism of PARP1 inhibitors in breast cancer treatment,and finally provide new experimental basis for expanding its clinical indications for breast cancer treatment.Methods:1.Ku70 deficiency was achieved by Ku70 shRNA adenovirus in human TNBC cell lines,MDA-MB-231 and BT-549,and DSBs were induced by several agents,such as neocarzinostatin(NCS),calicheamicin(CLM),bleomycin(BLM)and methyl methanesulfonate(MMS).Fraction protein was extracted according to its affinity of the damaged chromatin by fractionation.The distribution of PARP1 and other repair proteins in each fraction was identified by western blot(WB).After removing the loosely-bound chromatin proteins by in situ extraction of cell coverslips,immunofluorescence was applied to detect the expression and location of PARP1,which intended to observe the change of its chromatin affinity in A-EJ and C-NHEJ pathway.2.PARP1 inhibitor was added,followed by DSB induction.WB was applied to detect the expression of its active product poly(ADP-ribose).The effect of PARP1 inhibition on A-EJ repair was also determined by the dephosphorylation level of ?-H2 AX after treatment of DSB inducer for short time and recovery.Stable strains in TNBC cells were obtained by infecting with lentivirus carrying endonuclease I-PpoI to induce DSBs atspecific site.After Ku70 deficiency and treatment with PARP1 inhibitor,quantitative real-time PCR(qPCR)was performed by primers flanking the cutting site to accurately reflect the function of PARP1 on the repair efficiency of specific site DSB via A-EJ pathway.3.After the treatment of PARP1 inhibitors at different concentrations in TNBC cells,insoluble fraction during A-EJ pathway was extracted by fractionation.The affinity to chromatin of PARP1 downstream candidate,MRE11 and NBS1,was assessed by WB.After inhibition of PARP1 in I-PpoI cells,chromatin immunoprecipitation(ChIP)was performed with MRE11 antibody and DNA fragments,followed by qPCR via primers of 3different DNA sites to reflect the effect of PARP1 inhibition on the interaction between MRE11 and chromatin and the specific binding site of MRE11 at DNA ends.Finally,PARP1 and MRE11 were subjected to co-immunoprecipitation(co-IP)to verify their interaction and connection in A-EJ pathway in TNBC cells.Results1.Ku70 deficiency in TNBC cells promotes recruitment of PARP1 and MRE11 to chromatin with treatment of efficient DSB inducers(CLM and NCS)in contrast with treatment of inefficient DSB inducer BLM and single-strand break inducer MMS.Immunofluorescence confirmed that PARP1 and ?-H2 AX co-localize in the damaged chromatin and that PARP1 became extraction-resistant in Ku70 deficient cells,in contrast with itsinfrequent retention after in situ extraction from Ku-normal cells.2.In Ku-deficient TNBC cells,the dephosphorylation process of?-H2 AX after DSB was suppressed and was going to be blocked by addition of PARP1 inhibitor.PARP1 inhibition resulted in deficiency of ligation of DNA ends induced by I-PpoI.3.In A-EJ repair in TNBC cells,recruitment of MRE11 and NBS1 to chromatin decreases with the increased concentration of PARP1 inhibitor.MRE11 mainly binds at the DNA ends(50 bp from the I-PpoI cutting site),and small amount binds near the DNA ends.PARP1 Inhibitor can reduce the MRE11-chromatin interaction.PARP1 was detected by IP with MRE11 and vice versa.ConclusionIn A-EJ repair pathway in response to DSB of human TNBC cells,PARP1 is recruited bound to chromatin with high affinity.DSB repair efficiency and recruitment of MRE11 to chromatin depend on PARP1 activity,and MRE11 may act as o potential downstream protein of PARP1 in A-EJ pathway.