The Mechanism Of Oncogene Ras And P53^n236sMutation Cooperation In Escape Of Senescence And Tumorigenesis

Cancer is a genetic disease that threatens human health greatly.It is also an important problem in the field of disease research.Although there are many causes of cancer,the accumulation of oncogene mutations is one of the most important causes of tumorigenesis.However,due to the mechanism of Oncogene Induced Senescence(OIS),single oncogene activation rarely leads to tumorigenesis.The dysfunction of tumor suppressor gene signaling pathway is required for malignant transformation.OIS is an innate tumor suppressing mechanism occurred both in vivo and in vitro,which is caused by the activation of oncogene and leads to irreversible cell cycle arrest.Despite the presence of OIS,the oncogene induced cell transformation still occurs frequently,suggesting the existence of senescence-bypass mechanism in oncogene induced carcinogenesis.Therefore,the mechanism of senescence-bypass and the synergistic effect of dysfunction of oncogenes and tumor suppressor genes have been a hot topic in the field of cancer research.Ras family is special among a large number of the oncogenes.Ras gene is a proto-oncogene encoding a small GDP binding protein.The family members of Ras including K-,N-,H-Ras.When activated by its upstream signal through signal transduction,Ras activates its downstream pathway and triggers strong proliferation signal in the cell.According to the literature,nearly one third of human cancers has been found abnormal activation of Ras by point mutation.Therefore.Ras and its signaling pathway have become important targets for antitumor drugs.p53(which encoded by TP53 gene)has been the focus of researchers for a long time.It is a transcription factor which responds to stress signals.The p53 protein can monitor various of stress signals and lead to cell apoptosis,cell cycle arrest and so on.Consequently,it maintains the stability of the genome,thus p53 protein is known as the "guardian of the genome".Studies have shown that about half of all human cancers have been found the deletion or mutation of p53 gene.Around 74%of p53 gene mutation are point mutations.Among these mutations,most hot spot mutations occur in the DNA binding domain,and result in loss of function of wild-type p53.However,studies have shown that some p53 mutations not only lose the function of wild-type p53,but also gain new functions,which is beneficial to tumor progression.In our early works,some mouse embryonic fibroblasts(Mouse Embryonic,Fibroblasts,MEFs)strains of the WS(Werner Syndrome,human presently syndrome),whichoccurred the scenense-bypassprocess,has the tumorigenesis ability in SCID mice.Most importantly,we found a same p53 mutation in all those senescence-bypassed cells,which is know as p53N223S mutation.P53N236S(N239S in human being,denoted as p53S)is a mutation in DNA binding domain,which is caused by a single amino acid substitution of the asparagine to serine.Our previous studies have shown that p53S mutation not only lose the function of wild-type p53.but also has the oncogene potential.According to the p53 mutation database of IARC(International Agency on Research of Cancer,R15),p53S mutation exists in various types of tumors.Moreover,in 2016,an article highlights the p53S mutation to be a hotpot mutation.Our study shows that heterozygous p53N236S(p53S/+)cells will undergo OIS when we introduced overexpressed H-RasG12V in the cells.However,after several passages,those cells will bypass senescence.More importantly,those senescence-bypassed cells could form tumor in the SCID mice.In addition,the phenomenon of loss of heterozygosity was also founded in these senescent cells.To understand the molecular mechanism of OIS bypass induced by Ras.we further detected the Ras associated OIS pathways and found that the activation of Ras-ERK and Ras-p38 MAPK pathways might be the major reason that caused the p53S/+ MEF cell senescence.Remarkably.the peroxisome proliferator-activated receptor y coactivator la(PGC1-a)was upregulated in the p53S/+ MEF cells that bypassed OIS.Consistent with the increase of PGC1-αlevel,the intercellular reactive oxygen species(ROS)level was decreased.Immunostaining data revealed that,when H-RasG12V was overexpressed,mutant p53(p53S)proteins were translocated from the cytoplasm to the nucleus.As a transcriptional activator,p53S protein regulated the expression of PGC1-α by binding to its promoter.Given the fact that PGC1α is a powerful regulator in illuminating ROS,it might play a significant role in bypassing OIS.As a conclusion.p53N236S mutation and H-RasG12V have synergistic effect in the OIS and the bypass process.which might depend on the transcriptional level of PGC-1α regulated by p53S mutant proteins.After a preliminary study of the synergistic effect of Ras and p53S at the cellular level,we continuingly studied the interaction between K-RasG12D and p53S at the animal level.Firstly,we established a mouse model which utilized AD-Cre to induced K-Ras expression in lung of the p53S mice.Through the analysis of WB and pathology of lung tissue in this model,we found that the degree of malignant proliferation of lung in K-Ras p53S/S mice are increasing with time,but not in mice which genotype is K-Ras p53+/+ or K-Ras p53S/+.By immunohistochemical assay,we showed that the percentage of PCNA positive cells was higher in lung tissue of K-Ras p53S/S mice.These results together suggest that K-Ras and p53S may have synergistic effects on cell proliferation.Further study found that,compared with K-Ras and K-Ras p53S/+ mice,lesions in lungs of K-Ras p53S/S mice showed more active p-AKT,decreased DNA damage stress proteins such as p-ATM,CHK2,y-H2AX,decreased heat shock stress,reduced apoptosis proteins,and increased anti-apoptosis proteins.These results suggest that K-Ras can weaken stressresponses pathway with the synergistic effect of p53S.It is worth to note that,although significant apoptosis hasn’t been detected in the whole lung protein level,the TUNEL staining of tissue shows that apoptosis levelin K-Ras p53S/S mice lung lesions were significantly lower than that in K-Ras and K-Ras p53S/+ mice.Altogether,K-Ras and p53S may synergistically reduce the damage stress in the cell transformation and thus anti-apoptosis,so that cells acquire a survival advantage.In summary,this study reveals that p53S may collaborate with H-RasG12V in the OIS by pass process through the regulation of PGC1-α at the cellular level.In mouse level,we have proved that p53S could act as anti-apoptosis factor,weaken the damage stress pathway and cooperate with K-Ras to promote tumor progression.Our study has found an innovate pathways which p53 mutation could promote tumorigenesis through the regulation of mitochondria.Since p53S and Ras has synergetic effects in weaken the DNA damage response,anti-tumor drugs that could induc DNA damage responses may be not be effective for cancers that obtain p53S and Ras mutation.In this case,using anti-tumor drugs for which function is not dependent on DNA damage responses may have better theraputical outcome.Thus,the results of our study may shed light in clinical drug application.