The Deubiquitinating Enzyme PPPDE1 Stabilizes The N-terminal MDM2 Protein Fragment And Promotes The Progression Of Primary Hepatocellular Carcinoma

lq44 was one of the most frequently amplified genomic regions in Hepatocellular carcinoma(HCC),with an occurrence rate of 51.7%in some report.This phenomenon suggests that this genomic region may harbor potent oncogenes or tumor driver genes.But none of these coding genes in this region was reported to play a role in the carcinogenesis or development of HCC.By integrated analysis of available data from public cancer genomics data base,a gene on lq44,PPPDE1,was identified as a candidate driver gene of HCC.In the HCC cohort that was deeply analyzed by RNAseq and CNVs(copy number variations)analysis in Cancer Genome Atlas(TCGA)project,PPPDE1 coding region was found to be amplified in about 32%of analyzed patient(42/133)with fold change cutoff of≥1.5.In two independent cohorts of HCC patient(TCGA and Guilin cohort),PPPDE1 was also found to be oveexpressed in tumor tissue as compared to nontumor tissue,and be significantly correlated to bad prognosis.PPPDE1 mRNA also overexpressed in Brain Low Grade Glioma(LGG),Colon&Rectum Adenocarcinoma(COADREAD)and Renal Clear Cell Carcinoma(KIRC)and linked to the bad prognosis of patients in all these three kind of tumors.Focal positive staining of PPPDE1 protein was seen in 41%(9/22)of analysed paraffin tissue section from 22 HCC patients by IHC analysis,while only 14%(3/22)of analysed adjacent non-tumor tissue showed positive staining.The clonogenicity potential and subcutaneous tumor growth of HCC cell lines with PPPDE1 coding region amplification(Huh7,MHCC97H and PLC/PRF/5)was significantly inhibited by transducted with lentivirus expressing shRNA targeting PPPDE1.Data and results mentioned above showed that PPPDE1 is novel HCC driver gene.Invitro and in vivo de-ubiquitination assay showed that PPPDE1 is a Deubiquitinase(DUB)with specific DUB activity toward both K48-linked and K63-linked polyubiquitin chains.Co-Immunoprecipitation(Co-IP)combined with Mass Spectrometry and western blot analysis show that PPPDE1 can bind a 25kd N termal MDM2 protein fragment(MDM2 p25).PPPDE1 can promote the deubiquitination of the K63-linked ubiquitin of MDM2 p25 and consequently stabilize MDM2 p25.Importantly,PPPDE1 can promote the degaradation of p53 through a MDM2 p25 dependent manner.Furthermore,expression silencing of PPPDE1 can greatly downregulated the MDM2 p25 level and lead to increased p53 and BAX protein level,and inhibite the xenografts tumor growth of HCC cell lines in nude mice.