The Functions And Mechanisms Of Mutant P53 In The C-terminal With Extension

Background: The tumor suppressor p53 protein is a sequence-dependent nuclear transcription factor,as it can activate the expression of a multitude of genes that encode proteins crucial for cell-cycle control,DNA repair,senescence,ferroptosis,autophagy,and apoptosis,in response to a variety of cellular stress signals.Also,it is the mostly mutated gene or inactivated protein in all types of human cancers,as ～50% of human tumors harbor mutated TP53.Approximately 80% of the missense mutations occur in the p53’s DBD,because it is essential for sequence-specific DNA binding.Recently,we observed a clinical patient harbor p53 mutation in the C terminus with extension(p53-374*48).After analyzing the c Bioportal database,many p53 mutations in the C terminus with extension have been found,such as p53-393*78,and this kind of p53 mutation in the C-terminus with extension present in a variety of cancer types,including lung,colorectal,prostate and breast cancers,but there is no relevant reports.Therefore,figuring out the functions and mechanisms of this kind of p53 mutations are valuable to clinical therapeutic designs and also offer new molecule targets for future development of personalized anti-cancer drugs against these cancers.Objectives: To explore the functions and mechanisms of mutant p53 with C-terminal extension(p53 mutants with long C-terminus,p53LCs).Methods:(1)After expressing p53 LCs in p53 null cells,cell proliferation assay and colony formation assay was used to investigate the functions of p53 LCs in cancer cells;Western blot and q CR were used to detect the effect of p53 LCs on the protein and m RNA expression of downstream target genes;(2)In the absence or presence of p53 LCs,cell colony formation assay was used to investigate the the effect of p53 LCs on the function of wild type p53;In the absence or presence of p53 LCs,western blot and q PCR were used to investigate whether p53 LCs inhibit activation of wild type p53 which activated by reagents to exert dominant negative effect;(3)To detect the distribution of p53 LCs in cells and whether it can affect the localization of wild type p53 in the nucleus via Subcellular fractionation and immunofluorescence assay;(4)Determine if the p53 LCs could bind to the p53-response DNA elements and if they could affect the ability of wild type p53 to bind the DNA elements in cells;(5)To detect the half-life of p53 LCs and whether it could be degraded by MDM2-mediated ubiquitination;(6)To explore whether p53 LCs could confer chemoresistance to p53 activating drugs through DNE in p53-positive H460 cancer cells.Results:(1)Comparing to the cells which expressing wild type p53,cell proliferation and colony formation assay showed that p53 LCs could not inhibit cell growth and proliferation;meanwhile,Western blot and q PCR results showed that p53 LCs significantly lost the ability to activate downstream target genes;those results indicated that p53 LCs lost the function of wild type p53.(2)In the cell colony formation assay,the results showed that wild type p53 can inhibit cell growth and proliferation,and p53 LCs could not inhibit cell growth and proliferation;furthermore,the ability of wild type p53 to inhibit cell growth and proliferation was significantly decreased after co-expressing with p53LCs;meanwhile,Western blotting and q PCR showed that the ability of wild type p53 to induce the expressing of downstream target genes which activated by reagents,was inhibited by p53LCs;those results proved that p53 LCs could exert DNE effect.(3)The subcellular fractionation and immunofluorescence assay showed that the p53 LCs was mainly located in the cytoplasm and combined with wild type p53 to form a tetramer,so that the wild type p53 was retained in the cytoplasm and could not enter into the nucleus to play the role of transcription factor;those results unveil the underlying mechanisms how p53 LCs exert DNE.(4)The chromatin immunoprecipitation assay also showed that the ability of p53 LCs binding to the promoter of downstream target gene was significantly decreased,which may be related to the decreasement of acetylation ability caused by C-terminal mutation;at the same time,p53 LCs also could inhibit wild type p53 to bind to the promoter of downstream target gene,which further demonstrated how p53 LCs exert DNE.(5)Western blotting results showed that the half-life of p53 LCs was significantly prolonged and could not be degraded by MDM2,even though it still could be ubiquitinated by MDM2.(6)The cell proliferation and colony formation assay showed that the cells which stably expressing p53 LCs exhibited significant advantage in cell growth and proliferation comparing to control group after treated with actinomycin D;the results indicated that p53 LCs confered the chemoresistance to cancer cells on actinomycin D through DNE.Conclusions: Collectively,our results unveil a kind of longer form of p53 mutants in C-teiminal with extension that possess a dominant negative effect on its wild type counterpart,besides losing its wild type activity.The DNE effects could confer chemoresistance to p53 activating drugs in p53-positive cancer cells.The mechanism underlying this phenomenon is related with the subcellular distribution of p53 LCs,that the p53 LCs is mainly located in cytoplasm and could form a tetramer with wild type p53,then retain wild type p53 in cytoplasm.