The Molecular Mechanism Of PP2A Participating In DNA Damage Response And Oxidative Stress

Cancer is the number one killer that affects human health all over the world,which causes great burden to patients’ families and society.Chemotherapy,as the main clinical means to treat various primary and metastatic tumors after surgery,is often accompanied by great side effects and drug resistance.They reduce the sensitivity and efficacy of chemotherapy.Studies have shown that chemotherapy drugs not only induce specific hypersensitivity and gene dysfunction,but also lead to DNA damage response(DDR),apoptosis and epigenetic changes in normal cells.Gene mutations caused by DNA damage are the major source responsible for oncogene activation,silencing cancer suppressors,and malfunctioning of proteins.These factors would suppress chemotherapy sensitivity and lead to tumor recurrence.Therefore,understanding the toxic effects underlying chemotherapy and the mechanism of drug resistance is of great interest for improving chemotherapy efficacy,as well as providing new ideas for developing targeted therapeutic drugs.This thesis consists the following three parts:(a)DNA repair-induced oxidative stressDNA damage chemotherapy drugs such as Cisplatin(CIS)and Doxorubicin(Dox)are commonly used to treat a variety of malignancies,including non-small-cell lung carcinoma(NSCLC),lymphoma and ovarian cancer.However,they often cause serious organs toxicity and the molecular mechanisms are still in debate.Using human dermal fibroblasts(HDF)as a cell model,we demonstrated that oxidative stress was stimulated following CIS and DOXinduced bulky DNA damages,and addressed the signaling mechanisms between DNA repair and the modulation of cytoplasmic energy metabolism.A single treatment of the HDF cells with CIS or DOX led to prolonged production of endogenous reactive oxygen species(ROS),which was positively correlated with the formation of DNA double strand breaks(DSB).Knockdown of BRCA1,a key protein involved in the homologous recombination(HR)repair pathway,further increased ROS level.Knockdown of DNA-PKcs,a key enzyme of the non-homologous terminal junction(NHEJ)pathway,or overexpression of BRCA1 could partially inhibit ROS level.These results indicated that ROS production was a downstream event of DNA repair and was related to NHEJ-mediated DSB repair.Further study showed that CIS and DOX induced-DNA damage resulted in up-regulation of repair regulatory factor protein phosphatase 2A(PP2A)and ATM.Knockdown of PP2 A but not ATM was found to partially reduce ROS production,suggesting that PP2 A may play a role in DNA damage-induced ROS production.In addition,the increase of ROS was accompanied by changes in cell energy metabolism,and the oxidative stress caused high expression of proinflammatory cytokines.These results prove that the repair of certain DNA damages could cause cellar redox system dysfunction,and leading to inflammation.This finding provides a new perspective for solving the side effects of chemotherapy and giving potential target in developing more efficient drugs.(b)PP2A-a potential target for enhancing chemosensitivity toward tumorsLung cancer is the second leading cause of cancer morbidity and mortality worldwide,among which NSCLC accounts for about 85% according to the latest statistics.Despite the rapid development of therapeutic drugs targeting different mutations in NSCLC,drug resistance has greatly limited the therapeutic efficacy on NSCLC patients.Through bioinformatics analysis,it was found that PP2 A gene was highly expressed in NSCLC tissues and cell lines,and showed a trend of high expression in NSCLC cells(H1299)after CIS and DOX treatment,indicating that PP2 A may be potentially related to chemosensitivity and drug resistance.Using small molecular compound LB100(PP2A inhibitor),it was found that it can enhance the cytotoxicity of CIS and DOX on H1299 cells.Immunofluorescence co-localization and Co-IP assay showed that the recruitment of γ-H2 AX was accompanied by the aggregation of PP2 A to the damaged site during DSB formation and repair,and there was a protein-protein interaction between them.Inhibition of PP2 A resulted in down-regulation of BRCA1 expression and phosphorylation of ATM and DNA-PKcs,leading to DSB repair inhibition.Further study found that LB100 significantly affected the level of 8-hydroxy-2’-deoxyguanosine(8-OHDG)in mitochondria,leading to increased mitochondrial ROS accumulation and oxidative stress.Detection of Nrf2,the key transcription factor of antioxidant reaction,showed that LB100 could inhibit its expression and transportation to nuclear,and down-regulated the expression of heme oxygenase(HO-1)involved in antioxidant activity.Silencing PP2 A by small interfering RNA and overexpression of PP2 A further proved that PP2 A directly regulated the expression of Nrf2 and downstream target genes.Ch IP analysis showed that Nrf2 regulated the expression of BRCA1 and 53BP1 by binding to the TGACnnn C region of BRCA1 and 53BP1 promoters.The above results revealed that PP2 A inhibitor LB100 could inhibit DNA repair and improve chemotherapy sensitivity by regulating Nrf2/HO-1 pathway,and also lead to sustained oxidative stress in cancer cells,thus promoting cancer cell death.The enhanced anti-tumor effect of CIS and DOX by LB100 was also confirmed in the xenograft model of NSCLC.Furthermore,LB100 also showed an impact on intestinal flora,thus further enhancing its therapeutic efficacy on NSCLC.In conclusion,these results revealed that PP2 A inhibitor LB100 played an important role in enhancing the chemosensitivity of CIS and DOX by regulating Nrf2/HO-1 pathway,inhibiting DNA damage repair and promoting the continuous increase of oxidative stress.(c)Establishment of TMAO-induced AD cell model in vitro and preliminary study on the effect of PP2AIn recent years,some researchers have suggested that there is a close relationship between neurodegenerative diseases and tumor gene mutations.The study of PP2 A and tumor therapy and neurodegenerative diseases has gradually become a new hotspot.In this part,we found that PP2 A was low expressed in the model of TMAO-induced AD cells.The above functions of PP2 A on DNA damage repair and oxidative stress have some relations with the regulation of Tau hyperphosphorylation.