Oncogenic HRas^G12V Cooperated With P53^N236S Mutation To Escape Ras Induced Cellular Senescence Via PGC-1α

Tumor is the number one killer of human health and an important subject in the field of human disease research.Although there are a variety of factors that cause tumors,usually tumorigenesis is accompanied by activation of oncogenes and inactivation of tumor suppressor genes.However,due to the presence of the oncogene induced cellular senescence（OIS）,a natural anti-tumor barrier,the activation of a single oncogene or the inactivation of a tumor suppressor gene does not always lead to tumorigenesis,however OIS is often found in animaltumor models and precancerous lesions in human tumor patients,but is absent in advanced cancer,suggesting a mechanism for escape from OIS in malignant cells.Therefore,it is necessary to study the mechanism of tumor cell escape from aging and the synergy between oncogene activation and tumor suppressor gene mutation.Many oncogenes have been shown to induce OIS,such as Ras,Raf,Myc,etc.,among which Ras is one of the most susceptible oncogenes in human cancers.About33%of human tumors have activated Ras.The activated Ras gene up-regulates the expression of senescence-inducing factors,p19^ARF and p16^INK4A,mainly through the Ras-Raf-MEK-ERK signaling pathway,causing activation of p53 and Rb to trigger OIS.Our preliminary work found that the fifth generation of WS（WRN Premature Syndrome）mouse embryonic fibroblasts entered aging during in vitro culture very soon,but some of them could escape aging and immortalization.Furthermore,these senescence-bypassed cells could form tumors when subcutaneously injected into SCID mouse,and in these tumor cells we found the same p53 mutation,p53N236S（p53N239S in humans,hereinafter referred to as p53S）.Studies on p53S gene knock-in mice have revealed that p53S mutant protein not only loses the wild-type p53function to regulate cell cycle and apoptosis,but also has the potential of oncogene through gain of function,suggesting that p53S may be the key to cell escape senescence and subsequent tumorigenesis.Our study shows that exogenous expressed HRas^G12Vcould induce Mouse Embryonic Fibroblasts（MEFs）with heterozygous p53N236S(p53^S/+)background undergo OIS process.After a period of aging（after about ten generations,hereinafter referred to as late stage cells）,the cells will escape senescence and these escape-senescent cells could form tumors when subcutaneously injected into SCID mouse.In order to understand the mechanism of these cells escaping from OIS.Firstly,we detected the localization of wild-type p53 and p53S in cells by immunofluorescence,and found that wild-type p53 is concentrated in the nucleus in senescent cells（hereinafter referred to as early stage cells）,while p53S is mainly expressed in the cytoplasm.In late stage cells,the localization of p53S and wild-type p53 is opposite to that of early stage cells.The results of western blot showed that the protein level of total p53 increased in late stage cells,but the level of p21 did not increase,suggesting that p53S in late stage cells regulated different downstream pathways from wild-type p53.Subsequently,through differential gene network interaction analysis of Ch IP-on-chip microarray results,we found that PGC-1α（peroxisome proliferator-activated receptorγcoactivator 1α）was significantly up-regulated in p53S background cells,and we also found the expression of PGC-1αin late stage cells was up-regulated.Further Ch IP experiments showed that p53S protein can bind to the promoter of PGC-1α,suggesting that PGC-1αis a downstream factor regulated by p53S.After that,we detected the localization of PGC-1αby immunofluorescence assay,and found that the expression of PGC-1αin the late stage cell nucleus increased,indicating that PGC-1αmay regulate its downstream signals.However,PGC-1a mainly regulates the synthesis of mitochondria and the expression of antioxidant enzymes,and the ROS level of senescent cells is usually higher.Therefore,we first detected ROS levels in early stage and late stage cells,and found that ROS significantly decreased in late stage cells.Real-time PCR result revealed that the level of antioxidant enzymes in late stage cells was up-regulated,suggesting that PGC-1a may reduce ROS levels by increasing the expression of antioxidant enzymes in late stage cells.Then we examined the quality and quantity of mitochondria and found that in late stage cells,regardless of mitochondrial membrane potential,the number of ATP and mitochondria,were significantly higher than early stage cells.It has been reported that after overexpression of HRas^G12Vin cells,ROS level and autophagy increased.Therefore,we examined the expression of autophagy-associated proteins and found that expression of both LC3-II,Atg-5,Atg-7,and Atg-12 was up-regulated in early stage cells and the expression of autophagy in early stage cells was significantly increased by Lyso Tracker^TM Red DND-99 staining.It is suggested that PGC-1αcan increase the number of mitochondria by reducing ROS level and inhibiting autophagy indirectly in late escaping senescent cells under the background of p53S heterozygous cells.After that,we increased the ROS by adding H₂O₂stimulation to the late stage cells of escape aging.We found that the level of autophagy increased,cell cycle arrest,indicating that ROS induced the up-regulation of autophagy and early stage cells senescence.The increase of mitochondrial quantity and quality can enhance mitochondrial activity,oxidative phosphorylation,ATP production,improve energy metabolism,restart cell viability and restart cell cycle,which is conducive to cell escape from growth arrest induced by oncogene.Taken together,this study demonstrates that p53S up-regulates the expression of PGC-1α,enhances the expression of antioxidant enzymes,decreases ROS,attenuates autophagy,and increases the quality and quantity of mitochondria,thereby escaping HRas^G12V-induced cellular senescence.The significance of this study is that in the context of p53S mutant heterozygous cells,elucidate the mechanism of cell escape from H-Ras^G12V-induced OIS,to further understand the molecular mechanism of malignant transformation of cells,and to provide new ideas and targets for the treatment of this type of cancer.