The Effect Of Nucleolar Protein PICT-1on Regulating MDM2-p53Pathway In Response To Ribosomal Stress And On The Growth Of Cancer Cells And Its Underlying Mechanisms

Part Ⅰ:PICT-1is a Mdm2acidic domain binding nucleolar protein,It undergoes rapid proteasome degradation upon ribosomal stress as a Mdm2E3ligase substrateObjectives:To explore the effects of Mdm2acidic domain in controlling Mdm2mediated negative regulation of P53by screening the new mdm2acidic domain interacting proteins.To study the mutual interaction between MDM2and candidate protein PICT-1Methods:Yeast two hybrid screening method using acidic domain of mdm2as a bait and immunoprecipitation were to screen new MDM2binding proteins. we analysised the potential functional domain of PICT-1by genetyx ver9.1softwere. the localization of PICT-1was identified by immunofluorescence.Western blotting and immunofluorescence were used for studying PICT-1protein level comparison in normal vs tumor cell lines from different organ origins. The translocation and subsequent degradation of PICT-1and the role of MDM2in PICT-1degradation by applying MDM2c464a E3ligase mutant and P53&MDM2double knockout MEFs upon low dose Actinomycin D treatment was also detected with immunofluorescence and western blotting.Results:PICT-1is one of the20Mdm2acidic domain binding proteins,It’s a PEST sequence rich protein that colocalized with necleolar marker protein B23,and its protein level is constantly lower in cancer cells than in nontransformed cells in non-small cell lung cancer,prostate cancer and breast cancer cell lines. PICT-1translocated from nucleolar to nucleus followed by rapid degradation in a dose and time dependent manner upon low dose of Actinomycin D treatment, this effect can be blocked by cycloheximide(CHX),which is a proteasome inhibitor.We also found that PICT-1can’t be degradated by introducing MDM2c464a,a E3ligase mutant, in lung cancer cell line or in MDM2&p53double knockout MEFs.Conclusions:(1) PICT-1is a nucleolar protein which can bind Mdm2acidic domain;(2) PICT-1serves as a substrate of MDM2E3ligase activity, it translocate into nucleus follow by rapid proteasome degradation upon external ribosomal stress; Part Ⅱ:study the impact of PICT-1overexpression in cancer cell growth and the underlying mechanismObjectives:To study the cellular effect caused by PICT-1overexpression and the underlying mechanismMethods:We overexpressed PICT-1in various cancer cell lines including non small cell lung cancer,osteosarcoma,breast cancer cell and prostate cancer cell line.Colony formation assay and alarmo blue assay were performed to detect whether there was a growth inhibitory effect in those cell lines.Western blotting was used for checking interactions of mdm2-p53pathway and PICT-1overexpression as well as PTEN&PICT-1interaction. Extopic Mdm2translocation upon PICT-1overexpression and cellular colocalization of these two were detected by immunofluorescence. Integration of GFP fluorescence tagged PICT-1in A549cells and then subject to fluorescence microscope was a direct method to check cell viability.Results:P53wt&pten wt NSCLC A549and P53wt&pten wt breat cancer MCF7showed growth inhibitory effect, PICT-1overexpression caused cells growth slower than the negative control group(p<0.01,or p<0.05). But P53null&pten mutant NSCLC H1299cells; P53wt&pten null osteosarcoma U2OS cells and P53null&pten null PC3cells didn’t show any positive cellular effect.(p>0.05). Extopic Overexpression of PICT-1can stabilize MDM2in the presence or absence of P53, suggesting a p53transcription independent effect. Pict-1inhibit MDM2-mediated p53polyubiquitination and degradation.Extopic PICT-1can also colocalize with Mdm2in the nucleoli,thus sequenstering mdm2from meeting P53in the nucleus.Interestingly,PICT-1overexpression can prolong the harf life of ARF from6hours to approximately10hours and stabilize tumor suppressor PTEN,PTEN and PICT-1colocalized in the nucleus in the presence of low dose of Actinomycin D. Cells integrated of GFP-tagged PICT-1also showed compromised viability.Conclusions:(1) Overexpression of pict-1inhibits MDM2-mediated p53polyubiquit-ination and degradation, leading to growth inhibitory effect in A549and MCF7cells;(2) PICT-1exerts its tumor suppressive function also by sequestering MDM2in the nucleoli,prolonging the half life of ARF and stabilizing PTEN(3) the cellular effect caused by pict-1overexpression is cell type dependent,the mechanism is complex which may attribute to P53and PTEN status Part Ⅲ:The effect of lentivirus mediated PICT-1silence in cell growth and proliferation and its mechanismObjectives:To study the effect of knocking down PICT-1in cancer cell growth and proliferation, and explore the underlying mechanism.Methods:Two different shRNA sequences were applied to construct PLKO1lenti-virus vector and mouse pict-1"add back" experiment was done in order to make sure the knock down effect was specific.For every experiment,sh GFP serves as a negative control, luciferase assay was used to screen the potential molecular pathways involved in PICT-1knockdown, RT-PCR was used to check P53and PTEN mRNA level western blotting was used for detecting the impact of PICT-1knockdown on MDM2-p53pathway and PTEN,B23protein level. We performed immunofluorescence to check nucleoli integrity and B23protein cellular localization after PICT-1knockdown,;Colony formation assay and alarmo blue assay were performed to detect cell growth and proliferation,while "wound-healing"assay test the ability of cells to invade and metastasis.Flow cytometry was utilized for cell cycle analysis.Results:Lenti virus mediated PICT-1silence caused cell inhibitory effect was not"off target" phenomenon,Pict-1knockdown cells grew slower than the negative control cells(P<0.01or p<0.05)and both the cell colony number formed and colony size are less than the negative control cells(P<0.01or p<0.05) in NSCLC and various cell lines.After PICT-1 silencing,B23protein level was reduced and diffusively distributed in the nucleus.More interestingly,the nucleoli integrity was disrupted,suggesting an intrinsic ribosomal stress was triggered.There is no significant change in P53and PTEN mRNA level after PICT-lknockdown,indicating post-translational modification was crucial for P53activation.further experiment demonstrated that PICT-1knockdown inhibits MDM2-mediated p53polyubiquitination and degradation and induces a p53-dependent cell cycle arrest.PICT-1knockdown alleviate P53induction caused by extrinsic ribosomal stress. Surprisingly,although there is inhibitory effect caused by PICT-1knockdown,we detected that MCF10A cells and A549cells gained better capability of invasiveness and metastasis,suggesting different pathways were involved in those two distinct cellular effect observed in PICT-1knockdown cells.Conclusions:(1) PICT-1knockdown disrupts the nucleoli integrity,thus causes an intrinsic ribosomal stress;(2) PICT-1knockdown triggers intrinsic ribosomal stress,thus inhibites mdm2mediated p53ubiquitination and degradation and induces a p53dependent cell cycle arrest;(3) PICT-1is crucial for the pharmacological effect of low dose actinomycin D,down regulation of PICT-1abolishes the induction of P53caused by low dose actinomycin D;...