The Transcription And Expression Profile Of P53^N236S Induced By Environmental DNADamage Mimic And Its Regulation In P19^ARF And PI3K Pathway

Carcinogenesis is highly related to environmental pollution.According to a WHO global environment report in 2017,about 20%of cancer patients are attributed to various environmental pollution,such as air pollution,water pollution and toxic substance exposure.In developed or developing country,caner has already become the one of the most mortality diseases.In china,the incidence rate of cancer has increased since 2000.Basic research has made a great contribution to treat cancer.It has been past 2000years since Hippocrates named cancer by“carcinoma”,and now we have accomplished the cancer genome atlas（TCGA）.The definition of cancer has also become a complex multi-gene mutant disease instead of crab-like lump.With the establishment of more fine technologies,caner research has entered into a new field which combines high throughput sequencing technologies with single cell research.In this era,with new theories such as the tumor heterogeneity,the subclonal tumor cells,the cancer stem cells and the clonal evolution of cancer cells,the view of cancer scientists have been broaden greatly.They not only investigate into tumor microenvironment but also focus on the tumor biology of the entire tumor.This revolution of technology leads to the big data era of basic cancer research and consequently reforms the streategy of diagnosis and treatment in clinical oncology.Personalized diagnosis and treatment regime with high throughput array/sequencing is on its way to applied in clinic.Combined bioinformatics with high throughput array or sequencing technology,researchers have successfully drawn the regulation landscape of whole genome and whole proteome.The regulation landscape has revealed the relationship between gene mutation and carcinogenesis.The study in the regulation mechanism of mutant p53 is the essential part of this field.According to the mutation statistic of International Agency for Research on Cancer（IARC）and TCGA database,more than 50%of cancer incidences are closely related to the p53 mutation;and over 80%cancer patients occurred the p53 mutation in the end stage of disease.Thus,the p53 mutation is still the most significant carcinogenic risk factor in big data era.We previously indentified a mutant p53,p53^N236S（p53^N239S239S in human,p53S）in three independent tumorigenic mouse cell lines that used the alternative lengthening of telomeres（ALT）mechanism to maintain cell proliferation.The following study has demonstrated that the p53S can promote the tumor cell invasion,migration and anti-apoptosis.Two recently researches confirmed the p53S as one of the hot recurrent mutations,which contained true driver mutation but at low mutation frequency.The recurrent gene mutation is the driver force for cancer phenotype,and is a clonal mutation that occurred early in tumor.The recurrent mutation,although rare,is related with specific phenotypes,and it can endow subpopulation of cacer cells with de novo drug resistance by specific regulation of cancer pathways.This present study is based on the deep data mining of high throughput arrays results of p53^N236S.By combining transcriptional profile and gene expression profile,,we have revealed the gain of function of p53S and its mechanism in carcinogenesis.First,we analyzed the transcriptional profile of p53S,and creatively used ss GSEA to analyze transcriptional data,we transformed the enrichment scores of p53S and WT to a precent fold change scores for keeping the information of decreased or increased change,as well as the percentage of change.Based on these results,we drew the whole transcriptional profile of p53S.At the transcription level,we found that 77.8%target genes of p53S are different from wild-type p53.And the result of whole genome transcriptional profile drawn by Circos suggested that p53S is still a transcriptional factor,its target genes are distributing on chromosomes,however,the transcriptional mechanism of p53S is far different from WT p53.Then the PANTHER system was used for gene ontology（GO）enrichment analysis.We found that target genes of p53S in biological process（BP）term,are related to regulation of transcription and promote transcription,suggests p53S may promote the transcription of pro-transcriptional genes.This function can endow p53S with more aggressive transcriptional ability.In cell component（CC）term,target genes of p53S are enriched terms:membrane,nucleus,mitochondrion and nucleoplasm,suggests p53S may regulate genes transcription which are involved in those terms.In molecular function（MF）term,the results show a consistent with BPs’.Next,we performed p53S functionally grouped network by using Clue GO and found that p53S may regulate embryonic development genes,also regulate cell migration,cell localization,leukocyte proliferation and mesenchymal cell differentiation,et.al.,these results fitted well on the phenotype of p53S mice.And then,we combined ss GSEA with R for drawing the p53S transcription and expression profile.In that way,we found p53S can regulate the genes involved in DNA damage response,immune response and estrogen response.For the DNA damage,doxorubicin（Dox）was used for treating p53S MEFs.Compared with none-treatment p53S MEFs,the Dox treated one can deregulate the genes involved in DNA repair,apoptosis,G2/M check point and mitotic spindle check point.Simultaneously,p53S can regulate genes involved in glycolysis pathway,hypoxia,up-regulation K-Ras signaling pathway,TGF-beta signaling pathway and reactive oxygen species.This result suggests that under DNA damage stress,p53S plays loss of function with DNA damage response related genes and makes relationship with cell growth,cell proliferation and glycolysis.It reflects the oncogenic function of p53S.By combined transcription and expression profile data,we found that gene number of only can be up regulated by p53S is 893,45.8%of all genes,and the gene number of only can be down regulated by p53S is 676,34.6%of all genes.This result proved p53S is a functionable transcription factor again.We consistently identified 12 gene clusters,which revealed a new function of p53S,the alteration and enhancement of function（AEF）.It means,in a specific condition,the genes up-regulated by WT p53 can be down regulated by p53S,vice versa.In a traditional theory,mutant p53 maintains its protein stabilization by losing the transactivation of MDM2,or blocks its MDM2 binding domain through protein structure modified,or interacts with chaperone for suppressing the MDM2 mediate ubiquitination.According to the expression profile results of p53S primary,we performed a experimental research in cells and in tissues.It revealed a new stabilization mechanism of mutant p53.Here,we discovered a new mechanism of mutant p53 stabilization by performed a series of tissue and cell level experiments,and revealed the dual role of p19^ARFRF in tumor promotion or suppression.P53S is some sort of DNA damage signaling,and it can promote the instability of genome.P53S induces p19^ARFRF high expression allowed by these two functions,high expression p19^ARFRF promotes MDM2 degradation through ubiquitination,thus,p53S and p19ARF can form a self-activation loop.In a clinical diagnosis,considering the positive of p19^ARFRF should be depended on p53 mutation status.In a traditional theory,mutant p53 maintains its protein stabilization by losing the transactivation of MDM2,or blocks its MDM2 binding domain through protein structure modified,or interacts with chaperone for suppressing the MDM2 mediate ubiquitination.According to the high throughput array results of p53S tumor tissue,we found that p19^ARFRF encoding gene,CDKN2A expression fold-change is 270 times to control team.This result is inconsistent with previously researches.So,we performed a experimental research in cells and in tissues to verify this result and it revealed a new stabilization mechanism of mutant p53 and the dual role of p19^ARFRF in tumor promotion or suppression.Firstly,we found that p19^ARFRF and p53S are both high expression in p53S tumors on a protein level.Subsequently,we found that the count of DMs in p53S primary cultured tumor cell lines is more than that in p53^-/-tumor cells,p53^+/+MEFs and p53^S/S/S MEFs.DMs can be used as a biomarker of genome instability.Secondly,we investigated the DNA damage signaling in p53S tumor cells and found thatγ-H2AX can be up-regulated by p53S and its high expression level can be restored through knocking down p53S.γ-H2AX is a biomarker of DNA damage.These results suggest that p53S is some sort of DNA damage signaling,and it can promote the instability of genome.We subsequently investigated the p19^ARFRF in p53S tumor cells,and found that high expression p19^ARFRF on protein level can be dramatic down-regulated through know-down the p53S,simultaneously,the MDM2 can be up-regulated,vice versa.Finally,we analyzed the human sarcoma data from TCGA,and verified the correlation of mutant p53 and high expression p19^ARFRF in human.Together,these results demonstrate that p53S induces p19^ARFRF high expression allowed by its DNA damage signaling,and high expression p19^ARFRF promotes MDM2degradation through ubiquitination,thus,p53S and p19^ARFRF can form a self-activation loop.In a clinical diagnosis,considering the positive of p19^ARFRF should be depended on p53 mutation status.Due to the PI3K and related gene sets are high expression in transcriptional and expressional profile after Dox treated,we also investigated the mechanism of AKT-m TOR axis up-regulated by p53S.The reports about interaction between mutant p53 and PI3K pathway is rare,especially the mechanism of directly interaction is unclear.We investigated deeply the interaction between p53S and PI3K pathway on tissue,tumor primary cultured cells and in vivo levels.Firstly,we found that p53S protein high expression is related to AKT phosphorylation and high expression of m TOR protein in p53S tumors.Secondly,we found that p53S protein promote the phosphorylation of AKT and m TOR expression only in p53S tumor,not even in p53S MEFs.These results strongly suggest that the up-regulation of AKT phosphorylation and m TOR protein can be correlated with protein expression level in p53S tumors,and this relationship may be an essential part of carcinogenetic mechanism in p53S tumor.Thirdly,we found that knocking down the p53S protein expression can induce the down-regulate phosphorylation of AKT and m TOR in vitro.And The colony formation assay demonstrated that knocking down p53S tumor cells formed significantly fewer colonies than p53S tumor cells,indicating that high level of AKT phosphorylation,m TOR and p53S protein have an important role in promoting the proliferation of p53S tumor cells.Subsequently,we injected p53S tumor cells,with or without interfering p53S expression into nude mice subcutaneously.Our data revealed that the p53S tumor cells grew rapidly,while knocking down p53S by introducing sh RNA in p53S tumor cells inhibited the tumor growth significantly.Together,these result demonstrated that p53S can directly interact with AKT by a protein-protein interaction,p53S can transactivate m TOR expression.Knocking down the p53S expression can reduce tumor volume in vivo.Finally,this present research can promote subsequent p53S studies;and we present the alteration and enhancement of function of mutant p53;and we uncovered a new stabilization mechanism of mutant p53,may it shed a light in clinical diagnosis.At the end of paper,we proved p53S can transactivate m TOR,and also can directly phosphorylate AKT at S473 by binding with another kinase.These result may inspire the PI3K targeted therapy should consider the regulation of mutant p53.