studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1169A
1973
PPPDE1 is a deubiquitinase that promotes HCC development
by stabilizing a 25kd MDM2 N terminal fragment
and subsequently suppressing the p53 pathway
Xing-Wang Xie 1,2 , Yang-Jing Zhao 1,2 , Xue-Yan Wang 1,2 , Ran
Fei 1,2 , Heng-Hui Zhang 1,2 , Wei-Jia Liao 3 , Lai Wei 1,2 , Yu Wang 4 ,
Hong-Song Chen 1,2 ; 1 Peking University People’s Hospital, Peking
University Hepatology Institute, Beijing, China; 2 Beijing Key Laboratory
of Hepatitis C and Immunotherapy for Liver Diseases, Beijing,
China; 3 Guilin Medical College, Guilin, China; 4 Chinese
Center for Disease Control and Prevention, Beijing, China
Introduction: In our previous study, PPPDE1 (DESI2) was identified
and validated as a HCC driver gene, but the underlying
mechanism was unclear. The PPPDE1 protein belongs to a protein
family named PPPDE (Permuted Papain fold Peptidases of
Ds-RNA viruses and Eukaryotes) that was predicted to be a
deubiquitinating enzyme (DUB) family by protein homology
search. Hence, this study aims to validate the DUB activity of
PPPDE1 and to elucidate how it is involved in the HCC development.
Method: Lentvirus expressing wild type PPPDE1 and
PPPDE1 C108S mutant (the cysteine 108 was replaced by serine)
were used for over-expression and protein purification. Lentivirus
expressing PPPDE1 specific shRNAs were used to silencing
PPPDE1. The DUB activity of PPPDE1 and PPPDE1 C108S
were determind by in vitro and in vivo de-ubiquitination assay.
Co-immunoprecipitation (Co-IP) was used in combination with
Mass Spectrometry and Western blot to identify and validation
protein binding partners of PPPDE1. Colony formation
assay and subcutaneous tumor model were used to evaluate
the clonogenic and tumorigenic ability of HCC cells. Results:
Purified PPPDE1 protein cleaved K48-linked and K63-linked
Tetra-Ubiquitin into monoubiquitin in vitro. Transfected PPPDE1
also markedly reduced the K48-linked and K63-linked protein
ubiqutination level in cultured cells. These results showed that
PPPDE1 is a novel DUB and the PPPDE protein family is a new
DUBs family with cysteine proteases activity. The C108S substitution
of PPPDE1 C108S destroyed the deubiquitination activity
of PPPDE1. Moreover, PPPDE1 C108S lost the ability to promote
the clonogenic growth of HCC cell lines compared to wild
type PPPDE1, which suggested that PPPDE1 promote the HCC
development mainly through its DUB activity. We also found
that PPPDE1 stabilized a 25kd N termal MDM2 protein fragment
(MDM2 p25), a degradation fragment from MDM2-FL
(full length) and MDM2 p60 (60kd) protein, by binding and
deubiquitinated this protein fragment. When over-expressed
in HCC cell lines, PPPDE1 significantly promoted the degradation
of p53 through a MDM2 p25 dependent manner and
down regulated the protein level of BAX, a downstream effector
of p53-induced apoptosis. Reversely, knockdown of PPPDE1
in HCC cell lines considerably increased the p53 and BAX
protein level while greatly down-regulated the MDM2 p25
level. Most importantly, PPPDE1 silencing with lentiviral shRNA
tremendously inhibited the in vivo growth of subcutaneous
xenografts tumor of HCC cell lines in nude mice. Conclusion:
PPPDE1 is a novel DUB that can promote the development of
HCC by stabilizing a 25kd N terminal MDM2 fragment and
subsequently suppressing the p53 pathway.
Disclosures:
Lai Wei - Advisory Committees or Review Panels: Gilead, AbbVie; Grant/
Research Support: BMS
The following authors have nothing to disclose: Xing-Wang Xie, Yang-Jing Zhao,
Xue-Yan Wang, Ran Fei, Heng-Hui Zhang, Wei-Jia Liao, Yu Wang, Hong-Song
Chen
1974
Mevalonate pathway targets FoxM1 transcription factor
via protein geranylgeranylation in human hepatocellular
carcinoma
Satoshi Ogura, Yuichi Yoshida, Mayumi Egawa, Tomohide Kurahashi,
Kunimaro Furuta, Shinichi Kiso, Yoshihiro Kamada, Tetsuo
Takehara; Department of Gastroenterology and Hepatology,
Osaka University, Suita, Osaka, Japan
Background & Aims: Mevalonate (MV) pathway, which is
required for cholesterol biosynthesis, has been implicated in
the pathogenesis of hepatocellular carcinoma (HCC). Inhibition
of HMG-CoA reductase (HMGCR), a rate limiting enzyme for
MV pathway, has been also reported to reduce incidence of
HCC in animal and clinical studies. The Forkhead box M1
(FoxM1) is a proliferation-specific transcription factor and is
over-expressed in a variety of cancers, including HCC. In addition,
loss of FoxM1 is shown to reduce hepatocarcinogenesis
in mouse models. However, to date, it remains unclear whether
FoxM1 is involved in MV pathway of HCC. In this study, we
aimed to elucidate this issue using in vitro culture systems.
Method: Human hepatoma cell lines HepG2, Huh7, and HLF,
were treated with chemical inhibitors of enzymes in MV pathway
for 24 hours and the effect of these inhibitors on FoxM1
protein expression was examined by Western blot analysis.
Results: Administration of statin (pitavastatin), HMGCR inhibitor,
induced an about 50 % reduction of FoxM1 expression
in hepatoma cells (p