amplification of glyceronephosphate o-acyltransferase and recruitment … · 2018. 8. 24. ·...
TRANSCRIPT
Amplification of glyceronephosphate O-acyltransferase and recruitment of
USP30 stabilize DRP1 to promote hepatocarcinogenesis
Li Gu #, 1, 2
, Yahui Zhu #, 1, 2
, Xi Lin 1, 2
, Yajun Li 1, 2
, Kasa Cui 1, 2
, Edward V.
Prochownik 3, Youjun Li
1, 2,*
1 Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan
University, Wuhan 430072, China
2 Medical Research Institute, School of Medicine, Wuhan University, Wuhan 430071,
China
3Division of Hematology/Oncology, Children's Hospital of Pittsburgh of UPMC, The
Department of Microbiology and Molecular Genetics and The Hillman Cancer Center
of UPMC The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
15224, USA
#These authors contributed equally.
Running title: GNPAT and USP30-mediated DRP1 stabilization in HCC.
*Correspondence to:
Youjun Li, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences,
Wuhan University, Wuhan 430072, China; Medical Research Institute, School of
Medicine, Wuhan University, Wuhan 430071, China. Tel.: (86-27) 6875-2050;
Fax: (86-27) 6875-2560; E-mail: [email protected]
Conflict of interest: The authors declare no conflicts of interest.
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Abbreviations: AGPS, alkylglycerone phosphate synthase; CCl4, carbon tetrachloride;
CCLE, cancer cell line encyclopedia; CHIP, chromatin immunoprecipitation; CHX,
cycloheximide; DEN, diethylnitrosamine; DMSO, dimethyl sulfoxide; DRP1,
dynamin-related protein 1; ER, endoplasmic reticulum; ERK2,mitogen-activated
protein kinase 1; GNPAT, glyceronephosphate O-acyltransferase; HBV, hepatitis B
virus; HCC, heptocellular carcinoma; HCV, hepatitis C virus; H&E, hematoxylin and
eosin; H3K4me2, histone H3 lysine 4 dimethylation; LSD1, lysine demethylase 1A;
3-MA, 3-methyladenine; MAPK, mitogen-activated protein kinase; MAPL,
mitochondrial E3 ubiquitin protein ligase 1; MARCH5, membrane associated
ring-CH-type finger 5; MEF, Mouse Embryonic Fibroblast; MG132,
Z-Leu-Leu-Leu-CHO; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide; NEFA, nonesterified free fatty acids; NH4Cl, ammonium chloride; PDK1,
pyruvate dehydrogenase kinase 1; PEX14,peroxisomal biogenesis factor 14; PKC,
protein kinase C; TG, triglyceride; RPKM, Reads Per Kilobase Million; RT-qPCR,
reverse transcription quantitative polymerase chain reaction; SENP3, sentrin specific
peptidase 3; SENP5, sentrin specific peptidase 5; SNP, single nucleotide
polymorphism; SKL, peroxisomal targeting sequence serine–lysine–leucine; SUMO,
small ubiquitin-like modifier; TFAM, transcription factor A, mitochondrial; USP30,
ubiquitin specific peptidase 30; USP48, ubiquitin specific peptidase 48.
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Abstract
Hepatocellular carcinoma is a leading cause of cancer-related death worldwide
and the underlying pathophysiology of HCC is highly complex. In this study, we
report that, in a bioinformatic screen of 2783 genes encoding metabolic enzymes,
GNPAT, which encodes the enzyme glyceronephosphate O-acyltransferase, is
amplified, upregulated, and highly correlated with poor clinical outcome in human
HCC patients. High GNPAT expression in HCC was due to its amplification and
transcriptional activation by the c-Myc/KDM1A complex. GNPAT compensated the
oncogenic phenotypes in c-Myc-depleted HCC cells. Mechanistically, GNPAT
recruited the enzyme USP30, which deubiquitylated and stabilized dynamin-related
protein 1 (DRP1), thereby facilitating regulation of mitochondrial morphology, lipid
metabolism, and hepatocarcinogenesis. Inhibition of GNPAT and DRP1 dramatically
attenuated lipid metabolism and hepatocarcinogenesis. Furthermore, DRP1 mediated
the oncogenic phenotypes driven by GNPAT. Taken together, these results indicate
that GNPAT and USP30-mediated stabilizaiton of DRP1 play a critical role in the
development of HCC.
Significance: This study identifies and establishes the role of the enzyme
glyceronephosphate o-acyltransferase in liver cancer progression which may serve as
a potential therapeutic target for liver cancer.
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Introduction
World-wide, hepatocellular carcinoma (HCC) is among the most lethal of human
cancers. Although numerous inherited and acquired conditions can predispose to the
development of HCC, infection by hepatitis viruses B and C (HBV and HCV) account
for more than 50% of all cases (1-3). Other risk factors include food contamination
with aflatoxin, alcoholic and non-alcoholic cirrhosis and non-alcoholic fatty liver
disease. Excessive alcohol consumption and smoking further increase the overall risk
(1-3). Gene mutations involving TP53 and CTNNB1, numerous copy number
variations, epigenetic alterations and dysregulation of several microRNAs also likely
play key roles in HCC initiation and/or progression (4).
Glyceronephosphate O-acyltransferase (GNPAT), which is mainly localized in
peroxisome membranes, is essential for ether lipid (EL) synthesis. Defects in the
biosynthetic pathways of EL are responsible for rhizomelic chondrodysplasia punctata
(RCDP), a serious peroxisomal disorder characterized by proximal limb shortening,
recurrent respiratory tract infections, congenital cataracts and seizures (5). In mice
GNPAT deficiency is associated with male infertility, defective eye development and
impaired neurotransmission (6). GNPAT deficiency in mice also seriously affects
thymic maturation of semi-invariant natural killer T (iNKT) cells and iNKT cell
antigen production (7). It has also been reported that the GNPAT variant p.D519G is
associated with higher serum iron and serum ferritin levels following an iron
challenge in healthy women and that it significantly worsens the iron overload in
individuals with the p.C282Y+/+
variant of the HFE gene (1-3). In addition, ectopic
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expression of AGPS (alkylglycerone phosphate synthase), which is also essential for
EL synthesis and which associates with GNPAT during the synthesis of plasmalogens
(5), alters the balance of structural and signaling lipids to fuel cancer growth.
However, there is little information as to how GNPAT functions in liver
tumorigenesis.
Dynamin-related protein 1 (DRP1) plays an important role in mitochondrial and
peroxisome membrane fission (8). DRP1 contains an N-terminal GTPase domain, a
bundle signaling element (BSE), a middle domain (MD) and a GTPase effector
domain (GED) (9). Extensive investigations have revealed that defects in DRP1 can
cause neurodegenerative disorders such as Alzheimer's disease (AD), Huntington's
disease (HD) and neonatal lethality (10, 11). Other studies indicate that the C452F
mutant of DRP1 may lead to energy deficiency and cardiomyopathy. Inhibition of
DRP1-mediated mitochondrial fission by metformin efficiently attenuates
diabetes-induced atherosclerosis (12, 13). Finally enforced expression of DRP1 in
HCC cells leads to mitochondrial fission but also enhances HCC growth by promoting
G1/S phase transition via pathways that involve p53 and NF-kB (14). Enforced
over-expression of Drp1 in rat fibroblasts, leads to a state of chronic increased
mitochondrial fission, decreased mitochondrial mass and a concurrent attenuation of
oxidative phosphorylation and ATP production. This in turn is accompanied by the
activation of AMP-dependent protein kinase that presumably represents an abortive
attempt to restore a normal energy balance (15). In Saccharomyces cerevisiae Drp1 is
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also involved in peroxisome fission, especially under peroxisome-inducing growth
conditions (12, 13).
DRP1 undergoes multiple post-translation modifications including
phosphorylation, SUMOylation, S-nitrosylation and ubiquitination. Phosphorylation
of DRP1 at Ser616 by PDK1 in mitosis and PKCδ under stress conditions increases
mitochondrial fragmentation, and phosphorylation of DRP1 at the same site by ERK2
promotes MAPK-driven tumor growth and cell programming (16-19). Similarly
phosphorylation of DRP1 at Ser637 by c-AMP and PKA inhibits its GTPase activity,
whereas its de-phosphorylation by calcineurin and PP2A modulates the translocation
to mitochondria and dynamic balance (20-22). DRP1 SUMOylation by MAPL
stabilizes the mitochondrial/ER platform required for apoptosis while
de-SUMOylation by SENP5 and SENP2 blocks cell death (23-25). In contrast DRP1
de-SUMOylation by SENP3 facilitates DRP1 localization at mitochondria, thereby
promoting fragmentation and cytochrome c release (26). Previous studies have shown
that MARCH5 and PARK2 degrade DRP1 protein and maintain normal mitochondrial
morphology (9, 27, 28).
Here we report that GNPAT is upregulated in HCC patients and is recruited by
USP30 to deubiquitinate and stabilize DRP1, thus regulating mitochondrial
morphology and hepatocarcinogenesis. USP30, is a deubiqutinylase that normally
localizes to the outer mitochondrial membrane and acts as a PARK2 inhibitor, thus
preventing autophagy (27, 28). These findings identify a relationship between
GNPAT and USP30-mediated DRP1 stabilization that contributes to HCC
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development. Further, they reveal a previously unappreciated cross-talk between
mitochondrial and peroxisomal biogenesis and function that may be important in both
normal and neoplastic cell metabolism.
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Materials and Methods
Clinical human HCC specimens
HCC samples were used as previously described (29). Written informed consent
was obtained from all patients at the Union Hospital in Wuhan, China. The studies
were conducted in accordance with Declaration of Helsinki and approved by the
review board of Wuhan University. The diagnoses of all samples were confirmed by
histological review.
Cell culture and reagents
Human embryonic kidney HEK293 cells and human retinal pigmented epithelium
(RPE) cells immortalized with hTERT were obtained from
American Type Culture Collection (Rockville, MD, USA) in 2009 while normal
human hepatocytes HL-7702, human liver tumor-derived cell lines BEL7402,
FHCC98, Hep3B, Huh7, HepG2 and HCCLM9, MHHC97L were obtained from
China Center for Type Culture Collection (Wuhan, China) and Cell Bank of Type
Culture Collection of Chinese Academy Sciences (Shanghai, China) between 2014
and 2015, respectively. All cells, regularly authenticated by short tandem repeat
analysis and tested for absence of Mycoplasma contamination, were used within 5
passages after thawing and cultured as previously described (29). Cycloheximide
(R750107), proteasome inhibitor MG132 (M8699) and autophagy inhibitor
3-methyladenine (3-MA) (M9281) were purchased from Sigma-Aldrich. Antibodies
used in this study are listed in Supplementary Table S1.
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Plasmids and stable cell lines
The human GNPAT, DRP1, KDM1A, Myc and Ubiquitin coding sequences were
amplified and cloned into pHAGE-CMV-MCS-PGK-3×Flag and pCMV-HA vectors.
Mutations in the Ubiquitin and DRP1 cDNA sequences were generated by overlap
extension PCR. Human GNPAT, DRP1, USP30, Myc and KDM1A shRNA vectors
were obtained from GENECHEM (Shanghai, China). siRNAs and short hairpin
DNAs to inhibit murine Gnpat were designed and synthesized by GENEWIZ
(Souzhou, China). The latter were annealed and then inserted into pLKO.1-puro
vector. The DRP1-luciferase vector was generated by cloning the human DRP1
coding sequences to the luciferase gene tail of pcDNA3.1(+)-5’flag Luc (a kind gift
from Dr. Ping Wang, Life Sciences of East China Normal University, Shanghai, China)
(30). To detect GNPAT and DRP1 localization in peroxisomes, the peroxisomal
targeting sequence serine–lysine–leucine (SKL) was inserted into the pmCherry-N1
vector (Clontech) (29). GNPAT and DRP1 sgRNAs were designed by an online tool
(http://www.genome-engineering.org) and cloned into the Lenti-CRISPER v2 vector
(Addgene) (31). Primers used in this study are listed in Supporting information, Table
S2. Transfection and the establishment of stable cell lines were performed as
previously described (29). Cell viability was measured by MTT assay according to the
manufacturer’s instructions (Promega, Madison, WI, USA).
Subcellular fractionation
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Subcellular fractionation was performed as described previously (32, 33). In brief,
cells were trypsinized, collected in PBS, re-suspended in HEPES-mannitol-sucrose
buffer and homogenized through a 25G needle. Nuclei and unlysed cells were pelleted
from the homogenate and the mitochondria were recovered from the supernatant by
centrifugation. The crude mitochondrial pellet was resuspended and washed for
further experiments.
Co-immunoprecipitation and Western blotting
Transfected cells were lysed in 1 ml lysis buffer [20 mM Tris (pH 7.4), 300 mM
NaCl, 1% Triton, 1 mM EDTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM
PMSF]. Sepharose beads were washed three times with 1 ml lysis buffer containing
150 mM NaCl. Co-IP and immunoblot analysis was performed as described elsewhere
(34).
CHIP assays and qRT-PCR
Chromatin immuno-precipitation (CHIP) was performed as described previously
(34, 35). In brief, intracellular protein-DNA complexes were cross-linked with 1%
formaldehyde, sonicated and subjected to chromatin-conjugated IP using specific
antibodies. After reversal of cross-links, precipitated DNA was purified and analyzed
by qPCR with the primers indicated in Supplementary Table S2.
For qRT-PCR, total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA)
followed by DNase (Thermo Scientific) treatment. Reverse transcription was
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performed with a cDNA Synthesis Kit (Promega) and qPCR were performed using
SYBR Green master mix (Bio-Rad, Hercules, CA) using standard protocols. All
primer sequences used are shown in Supplementary information, Table S1. β-actin
was used as an internal control.
Lipid uptake and synthesis
For lipid uptake, 106 cells were trypsinized, collected in PBS, and stained at 37°C
for 30 min in the dark using BODIPY-493/503 (Sigma, D3922). Flow cytometry was
performed using a FACStar flow cytometer (BD Biosciences) (36-38).
For lipid synthesis, cells were cultured in a 6-well dish in growth medium. For
labeling, 3H-acetic acid (1.59 Ci/mmol, Perkin Elmer, 37 KBq/ml) was added to the
wells and incubates at 37°C overnight. The next day, cells were disrupted with lysis
buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 2% SDS, 10% glycerol, 12.5 mM
EDTA, 1% Triton X-100 and 0.5% NP-40) per well and incubated at 37°C for 30 min.
Organic extraction was performed in chloroform following the protocol of Bligh and
Dyer and lipid-soluble products were measured by scintillation counting (Tri-Carb
2910 TR, Perkin Elmer) (36-38).
Other metabolic assays
Cellular non-esterified fatty acids (NEFA) levels were determined with a
commercially available ELISA kit (AO42-2, Jiancheng Bio., Nanjing, China)
according to the manufacturer’s instruction. Serum TG levels were measured using
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the Infinity Triglycerides Reagent (Thermo Scientific).
Reporter Assays
Human GNPAT promoter sequences were inserted into pGL3-basic luciferase
vector (Promega). Luciferase assays were performed as described previously (34).
Animal studies
All animal studies were approved by the Animal Care Committee of Wuhan
University. For xenograft experiments, four-week-old male BALB/c nude mice were
purchased from SLAC Laboratory Animal Corporation (Changsha, China) and
maintained in microisolator cages. Detailed procedures were described previously
(29).
For the DEN/CCl4-induced HCC model, C57/B6 mice were intraperitoneally
administrated 25 mg/kg of diethylnitrosamine (DEN) (Sigma) on Day 15 of life.
Following this, CCl4 (Sigma) was then injected thrice weekly for four weeks (n=10
per group). Concentrated shRNA-lentivirus were injected by tail vein. After 28 weeks,
mice were sacrificed for analysis.
IHC assays and immunofluorescent confocal microscopy
Liver sections were fixed in 10% formalin and embedded in paraffin according to
standard protocols. Livers were sectioned and stained with H&E. Ki-67 staining was
performed according to the supplier’s protocol (mouse monoantibody, 1:200,
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Sc-25280, Santa Cruz).
Confocal microscopy was performed as previously described (35). Briefly, cells
were fixed with 4% paraformaldehyde at room temperature, permeabilized and then
stained with the primary antibody against DRP1 (1:100), GNPAT (1:100) or PEX14
(1:100) for 1 hour. After exhaustive removal of the primary antibody by washing, a
second incubation was performed with HRP-conjugated goat anti-mouse (A-11005 or
A-21050, Invitrogen) or goat anti-rabbit-488 antibodies (A-11008, Invitrogen) (1:500).
Fluorescence imaging was performed on a Leica SP8 confocal microscope and
images analyzed with Laika AF Lite software. To determine peroxisome morphology,
cells were randomly selected for quantitative analysis.
Transmission electron microscopy
This was performed as described previously (32, 33). In brief, cells were fixed in
2.5% glutaraldehyde (Sigma, Shanghai, China) in PB buffer. After three washes in PB
buffer, cells were scratched, collected and centrifuged. Then cells were post-fixed in
1% osmium tetroxide (OSO4). After further washes with PB, cells were dehydrated in
50%, 75%, 95% and 100% ethanol, and then further dehydrated in 100% acetone
twice. The samples were then infiltrated in 1:1 acetone/epoxy resin, 1:2
acetone/epoxy resin, 100% epoxy resin and finally 100% epoxy resin at 65 °C for
polymerization. The blocks were cut into micrometer sections with a diamond knife,
picked up on 200-mesh grids, stained, and observed according to the standard
procedures with Transmission Electron Microscope (JEM-1400plus, Tokyo, Japan).
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Bioinformatic analysis
Datasets for HCC were downloaded from the Cancer Genome Atlas (TCGA) data
portal (http://www.tcga-data.nci.nih.gov). GNPAT expression data were assessed and
Kaplan-Meier curves were calculated in human HCC tissues from the TCGA mRNA
data set. GSE datasets were analyzed from NCBI GEO databases
(https://www.ncbi.nlm.nih.gov), which were reported from indicated gene expression
profile array. CCLE datasets (https://portals.broadinstitute.org/ccle/home) were used
to analyze gene expression and DNA copy number in HCC cell lines.
Statistical analysis
Survival curves were plotted by the Kaplan-Meier method with GraphPad Prism 5
software using the log-rank test and correlation with a Spearman rank correlation test.
Other statistical significance (p<0.05) was assessed by the Student’s t test. Data were
presented as the mean ±SD.
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Results
GNPAT gene gain and amplification is predictive of poor clinical outcomes in
human HCC
To identify de-regulated metabolic genes involved in HCC progression, an
unbiased bioinformatic screening of 2783 metabolic enzyme genes was conducted in
the HCC database from the Cancer Genome Atlas (TCGA) (39). Interestingly, 896 of
these genes were commonly upregulated in HCCs (1.5-fold threshold) with the
mRNA abundance from 442 of these genes being >100 RPKM (Reads Per Kilobase
Million) by RNA-sequence analysis (Fig. 1A, B). As tumor cells almost always
harbor unstable genomic DNA (40), we sought candidates from the above group
whose over-expression could be explained by gene amplification. Interestingly, 36
such examples were noted as being highly correlated (R>0.5). 9 of these 36 genes
reside on chromosome 1q, which is one of the most frequently amplified
chromosomal regions in HCCs (Fig. 1C, R>0.62) (1, 4). To evaluate the role of each
of these 9 genes on human HCC cell growth, 3 siRNAs against each were synthesized
and used to inhibit their expression in human HCC cells HepG2, Hep3B and
MHCC97L. MTT assays showed that GNPAT silencing significantly inhibited growth
of each of the three cell lines (Fig. 1D and Supplementary Fig. S1A, B) and hence, we
focused on GNPAT in the subsequent work described below.
To study the clinical significance of GNPAT expression in HCC, the publicly
available TCGA, GSE14520, GSE40367 and CCLE datasets of HCC cases and cell
lines were analyzed. On average, GNPAT mRNA levels were expressed at higher
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levels in HCCs relative to normal hepatic tissues in all tested data bases (Fig. 1E and
Supplementary Fig. S1C-E. Human HCC cell lines also contained similarly elevated
levels of GNPAT transcripts than did non-transformed hepatocyte lines
(Supplementary Fig. S1F). In addition, Western blot assays showed GNPAT protein
levels to be significantly upregulated in HCC tissues and cell lines (Supplementary
Fig. S1G, H). In HCC patients, those with stage II/III/IV had significantly higher
GNPAT expression than those with stage I (Fig. 1F). Also in HCC patients, those with
high GNPAT expression had significantly worse survival than those with low GNPAT
expression (Supplementary Fig. S1I).
The GNPAT gene, located on Chr.1q42.2, was found to be significantly amplified
in both HCCs and most HCC cell lines. As shown in Figure 1G-I and Supplementary
Fig. S1J-L, GNPAT mRNA levels in HCCs also generally correlated with the degree
of GNPAT gene amplification (Log2 SNP>0). Interestingly HCC patients with
amplified GNPAT genes had significantly worse survival than those without gene
amplification (Fig. 1J). As a control, FAM98A, localized next to GNPAT, expression
was not upregulated and not associated with patient survival in HCCs (Supplementary
Fig. S1M). These findings suggest that GNPAT amplification and over-expression are
involved HCC pathogenesis and/or contribute to other clinically relevant features of
the tumor.
GNPAT interacts with DRP1 to regulate mitochondrial morphology
To better study how GNAPT contributes to HCC development, we performed
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SDS-PAGE and mass spectrometry (MS) following ectopic expression of GNPAT to
identify its interacting partner proteins. Interestingly, DRP1, which tends to localize to
mitochondria and peroxisomes, was identified as one of the most prominent
interactors (Fig. 2A and Supplementary Fig. S2A). The interaction between
endogenous GNPAT and DRP1 was also confirmed by co-immunoprecipitation
(Co-IP) experiments in the HepG2 and Hep3B cell lines (Fig. 2B). To identify the
region of each protein responsible for the interaction, various DRP1 and GNPAT
truncation mutants were generated and co-expressed in HEK293 cells. We found the
GTPase domain of DRP1 and the middle domain of GNPAT to be the critical
interacting region of each protein (Fig. 2C, D). Immunofluorescence staining further
indicated that GNPAT and DRP1 were co-localized in mitochondria and peroxisomes
(Fig. 2E and Supplementary Fig. S2B) (41, 42). These results were confirmed by
Subcellular fractionation experiments (Fig. 2F).
Mitochondrial functions are related to mitochondrial dynamics while DRP1 has a
critical role in mitochondrial morphology (8, 43). To investigate whether GNPAT
affects mitochondrial dynamics, we examined mitochondrial morphology in cells with
GNPAT inhibition. As shown in Fig. 2G and Supplemental Fig. S2C, D, GNPAT
inhibition impaired mitochondria fission as indicated by increased numbers of
elongated mitochondria and decreased numbers of fragmented ones. Also these
defects could be rescued with overexpression of shRNA resistant GNPAT and DRP1
but not GNPAT ∆CD or DRP1 ∆GTP. These findings suggest that the regulation of
mitochondrial morphology mediated by the GNPAT-DRP1 interaction may be
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mediated at the level of mitochondria fission.
GNPAT recruits USP30 to deubiquitinate and stabilize DRP1
Next, the effect of GNPAT on DRP1 expression was evaluated. GNPAT
inhibition in Hep3B and HepG2 cells resulted in a marked decrease of DRP1 at the
protein level without affecting mRNA abundance (Supplementary Fig. S2C and S3A).
This indicated that GNPAT likely affects DRP1 protein turnover. GNPAT inhibition
was associated with a significantly shortened DRP1 half-life after treatment with
cycloheximide (CHX), which blocked de novo protein synthesis (Fig. 3A and
Supplementary Fig. S3B). To identify the pathway(s) responsible for
GNPAT-mediated DRP1 degradation, NH4Cl, MG132 and 3-MA were used to inhibit
lysosomal, proteasomal and autophagocytic pathways of protein degradation,
respectively. GNPAT inhibition-mediated degradation of DRP1 was completely
inhibited by the proteasome inhibitor MG132 but not by NH4Cl or 3-MA (Fig. 3B
and Supplementary Fig. S3C). These results suggest that GNPAT inhibition mediated
degradation of DRP1 through the proteasome pathway.
Given that deubiquitinases (DUBs) modulate protein stability, we hypothesized
that GNPAT recruits one or more DUBs to deubiquitinate and stabilize DRP1. As
GNPAT and DRP1 are mainly expressed in mitochondria and peroxisomes, 14 DUBs
known to localize to these organelles were selected for further examination. To
identify DUBs that could stabilize DRP1, firefly luciferase was fused to the N
terminus of DRP1 and the fusion protein (DRP1-Luc) was used as a reporter of DRP1
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stability in the presence of each of the above 14 DUBs (30). Our results showed that
ectopic expression of USP30 and USP48, increased the luciferase activity of
DRP1-Luc (Fig. 3C and Supplementary Fig. S3D). Consistent with this, both GNPAT
and DRP1 co-purified with immuno-precipitated USP30 and USP48 (Supplementary
Fig. S3E). Further assays showed that ectopic GNPAT expression increased
USP30-mediated luciferase activity of DRP1-Luc while GNPAT inhibition produced
the opposite results. In contrast, USP48-mediated luciferase activity of DRP1-Luc
was not affected by GNPAT expression or inhibition (Fig. 3D, E). Co-IP experiments
confirmed that USP30 but not USP48 was recruited by GNPAT to promote DRP1
stability (Supplementary Fig. S3F). Also USP30 inhibition in Hep3B and HepG2 cells
resulted in a marked decrease of DRP1 at the protein level (Supplementary Fig. S3G).
To investigate whether USP30 could deubiquitinate DRP1, HA-tagged wild type
USP30 and its catalytically inactive (CI, C77S) mutant were generated. Co-IP assays
showed that wild type but not mutant USP30 deubiquitinated DRP1 (Fig. 3F). Further
studies showed that ectopic GNPAT expression increased USP30-mediated DRP1
deubiquitination (Fig. 3G and Supplementary Fig. S3H) while USP30 depletion with
ectopic GNPAT expression impaired DRP1 deubiquitination (Supplementary Fig.
S3I). To identify the type of GNPAT recruiting USP30-mediated deubiquitination of
DRP1, several vectors expressing HA-tagged mutant Ubiquitin (K6O, K11O, K27O,
K29O, K33O, K48O and K63O), which contained substitutions of arginine for all
lysine resides except the lysine at this position, respectively, were used.
Deubiquitination of DRP1 was detected only in the presence of wild type (WT) and
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K48O, consistent with the idea that GNPAT enhanced USP30-mediated K48-linked
deubiquitination of DRP1, but not other position-linked deubiquitination of DRP1
(Fig. 3H). Further experiments were performed with the mutant ubiquitin K48R,
which included a single lysine to arginine substitution at position 48. Mutant on K48
couldn’t remove the ubiqutination in contrast to WT, which classified that GNPAT
downregulated the K48 ubiquitin chain on DRP1 (Supplementary Fig. S3J). These
results indicate that GNPAT promotes USP30-mediated K48-linked deubiquitination
of DRP1.
Additional DRP1 ubiquitination sites were further confirmed with USP30
inhibition. Public proteomic database assays showed three potential ubiquitin sites of
DRP1 protein (K160/238/532) all of which were mutated to arginine to generate
single, double or triple “KR” mutants. We found that DRP1 ubiquitination was
gradually decreased in parallel with the number of KR mutations. Notably, the triple
KR mutant almost completely abolished DRP1 ubiquitination (Fig. 3I). In contrast,
USP30 inhibition increased the WT DRP1 ubiquitination, but did not affect
ubiquitination of the triple DRP1 mutant (Supplementary Fig. S3K). This suggests
that these three arginine residues are all critical sites of ubiquitination.
GNPAT and DRP1 inhibition retards HCC development
We next investigated the effects of GNPAT and DRP1 modulation on HCC
development. Because GNPAT and DRP1 are highly expressed in HepG2 and Hep3B
cells, these two cell lines were exploited for further study (Supplementary Fig. S1H).
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shRNA-mediated inhibition of either GNPAT or DRP1 significantly retarded cell
proliferation in vitro (Fig. 4A, B and Supplementary Fig. S2C, S4A) and in vivo
xenograft growth (Fig. 4C, D and Supplementary Fig. S4B).
GNPAT is critical for fatty acid synthesis and DRP1 promotes glucose
metabolism, presumably by promoting mitochondrial fission, reducing reliance on
Oxidative phosphorylation and increasing glycolysis (44, 45). Depletion of GNPAT
or DRP1 also decreased lipid uptake (Fig. 4E), lipogenesis (Fig. 4F) and
non-esterified fatty acid (NEFA) levels (Fig. 4G), indicating that both proteins
regulate lipid metabolism. Knockout of GNPAT and DRP1 using a
CRISPR/Cas9-based approach confirmed the above results (Supplementary Fig.
S4C-F). Collectively, these results indicate that both GNPAT and DRP1 are critical
for HCC tumor growth in vitro and in vivo and that this growth might be influenced
by lipid levels.
DRP1 is necessary for and mediates GNPAT malignant phenotypes
Because GNPAT and DRP1 inhibition diminish the malignant phenotypes of
HCC cells, we explored whether GNPAT’s oncogenic effects were DRP1-dependent.
HepG2 and Hep3B cell lines stably expressing ectopic GNPAT or DRP1 grew more
rapidly than their control counterparts both in vitro and in vivo. These effects were
reversed by inhibiting DRP1 indicating that it was the direct mediator of GNPAT’s
growth-promoting effects (Fig. 5A-F and Supplementary Fig. S5A). Further ectopic
over-expression of DRP1 but not DRP1 ∆GTP or GNPAT ∆CD was able to rescue
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the phenotypic changes in HCC cells mediated by GNPAT inhibition (Fig. 5A-F and
Supplementary Fig. S2C, S4A, S5A-C). These results indicate that DRP1 is necessary
for and mediates GNPAT malignant phenotypes.
GNPAT and DRP1 repression attenuates DEN/CCl4-induced
hepatocarcinogenesis in mice
We next examined the expression of GNAPT and DRP1 in an autologous HCC
model induced by a combination of diethylnitrosamine (DEN) and carbon
tetrachloride (CCl4). Consistent with our prior observation in human samples,
GNAPT and DRP expression were all up-regulated at various times during disease
development (Fig. 6A and Supplementary Fig. S1G, S6A).
To determine whether GNPAT and DRP1 inhibition could attenuate autologous
HCC tumor growth in the above model, concentrated lentivirus containing shGnpat
and shDrp1 expression vectors were administered to DEN/CCl4-treated mice for four
weeks (Weeks 8-11) and the tumor burden was assessed at week 28. Tumors treated
with lentivirus expressing shGnpat and shDrp1 were smaller and showed reduced
expression of GNAPT and DRP1 as well as genes involved in lipogenesis and fatty
acid uptake (Fig. 6B-F and Supplementary Fig. S6B-D). These mice also showed
lower serum levels of NEFA and TG (Fig. 6G and Supplementary Fig. S6E).
GNPAT expression is activated by a transcription complex composed of c-Myc
and KDM1A
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GNPAT mRNA levels tended to be expressed at much higher levels in HCC
tissues and cell lines than could be accounted for even by gene amplification. This
suggested an additional level of gene copy number-independent control that we
hypothesized was transcriptionally mediated. Examination of the GNPAT promoter
identified conserved c-Myc (Myc) binding E-boxes, located within CpG islands 5kb
upstream and 2kb downstream of the transcription start site (TSS) (Fig. 7A). Myc is a
master transcriptional factor whose targets contribute to growth and metabolism under
both normal and neoplastic conditions (46). As shown in Fig. 7B and 7C, Myc
inhibition resulted in down-regulation of both GNPAT mRNA and protein levels in
both HepG2 and MHCC97L HCC cells but not GNPAT-amplified Hep3B cells
(Supplementary Fig. S7A, B) thereby indicating that at least some portion of GNPAT
up-regulation is likely mediated by the excessive levels of Myc expression associated
with HCC. Indeed, a chromatin IP (ChIP) assay performed in HCC cells revealed that
Myc binds the GNPAT promoter region containing the most conserved and proximal
upstream E-boxes (Fig. 7D and Supplementary Fig. S7C) thereby supporting the
notion that GNPAT expression levels are directly regulated by Myc.
Because Myc associates with several histone demethylases to facilitate cancer
progression (47-50), we hypothesized that its interaction with one or more of these
regulates GNPAT transcription. Therefore CHIP experiments was performed to
investigate which of the seven known histone demethylases bind to the GNPAT
promoter in association with Myc. Our results showed that immuno-precipitation of
KDM1A efficiently enriched the GNPAT promoter region containing the proximal
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E-box in control HepG2 and MHCC97L cells but not in cells in which Myc levels
were depleted by the expression of a short-hairpin RNA (Fig. 7D and Supplementary
Fig. S7C, D). The direct interaction between endogenous Myc and KDM1A was also
confirmed in HepG2 and MHCC97L cells by Co-IP experiments (Supplementary Fig.
S7E). Furthermore, the use of deletion mutants showed that the region encompassing
residues of 320-439 of Myc and containing the entirety of its bHLH-ZIP dimerization
domain, is required to interact with KDM1A (Supplementary Fig. S7F, Left) while
residues 474-852 of KDM1A were required to interact with Myc (Supplementary Fig.
S7F, Right). KDM1A is the main histone demethyltransferase that demethylates
histone H3 at lysine 4 (H3K4me2), thereby mediating epigenetic activation (46). We
therefore determined whether the H3K4me2 could also be detected at the GNPAT
promoter. Indeed, immuno-precipitation of H3K4me2 efficiently enriched the
E-box-containing GNPAT promoter region in control HepG2 and MHCC97L but not
in Myc-depleted cells (Fig. 7D and Supplementary Fig. S7C). These results suggest
that Myc recruits KDM1A to bind the GNPAT promoter at a functional E-box site and
that KDM1A-mediated histone demethylation of H3K4me2 contributes to
Myc-induced GNPAT activation.
Next, the potential role of KDM1A in regulating GNPAT expression was
evaluated. ShRNA-mediated KDM1A inhibition significantly decreased GNPAT
mRNA and protein levels in control HepG2 and MHCC97L cells but not if they were
first depleted of Myc, but not in Hep3B cells (Fig. 7B, C and Supplementary Fig.
S7A-C). We then tested the effects of Myc and KDM1A in a luciferase reporter assay
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of the GNPAT promoter. Co-expression of both plasmids in HepG2 and MHCC97L
cells increased luciferase activity in reporters containing either of the wild-type E-box
sites but not in those with mutated sites. Either of the two E-boxes appeared to be
sufficient for up-regulating the activity of the reporter although their effects did not
appear to be additive (Fig. 7E and Supplementary Fig. S7G).
GNPAT partially mediates and is required for Myc oncogenic phenotypes
Previous studies have shown that Myc is activated in and essential for HCC
progression (46, 51). In HepG2 and MHCC97L cells, the ectopic expression of
GNPAT but not a dominant-negative form of the protein could partially rescue the
oncogenic phenotypes in Myc-depleted HCC cells (Fig. 7F: Left, Fig.7G, H and
Supplementary Fig. S7H: Top). This suggested that GNPAT is a relatively important
Myc target with regard to transformation and can recapitulate this complex function in
the presence of reduced or absent Myc levels as has been reported for only a limited
number of previously described Myc targets (52, 53).
Finally, we asked whether GNPAT is required for the full expression of Myc
oncogenic phenotypes. We first showed that the stable expression of Myc in HepG2
and MHCC97L cells enhanced both their the in vitro growth and in vivo
tumorigenicity. However, these phenotypes were impaired following GNPAT
inhibition (Fig. 7F: Right, Fig.7G, H and Supplementary Fig. S7H: Bottom). These
results suggest that GNPAT partially mediates and is required for the optimal
expression of Myc-mediated oncogenic phenotypes.
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Discussion
Since reprogrammed metabolism is now appreciated to be a hallmark of cancer
and a promising therapeutic target (54), the identification of key enzymes that mediate
this re-programming and contribute to cancer growth and/or survival is of paramount
importance. In the current work, an unbiased bioinformatic screening of 2783
metabolic enzymes identified GNPAT, a member of the ether lipid biosynthetic
pathway as an important facilitator of HCC development. While some of GNPAT’s
over-expression is due to gene amplification of a critical region on Chr.1q42.2, the
remainder appears to involve transcriptional activation mediated by a complex
comprised of Myc and the histone demethylase KDM1A.
Surprisingly, GNPAT was found to interact with and stabilize DRP1 located in
mitochondria and peroxisomes. Recent reports have revealed that DRP1 interacts with
the head group of phosphatidic acid and saturated acyl chains of phospholipid (55),
which further affirms our results. It is well known that DRP1 plays a key role in
mitochondrial fission (9), indicating that the interaction of GNPAT and DRP1 is
essential for mitochondrial function. Significant cross-talk between mitochondrial and
peroxisomal compartments also has been documented (8) as evidenced by the fact that
DRP1 is important for mediating fission events in both organelles. It is tempting to
speculate that GNPAT is one of the factors that mediate this activity and/or their
physical interaction.
DRP1 is also involved in many post-translational modifications that regulate
mitochondrial and peroxisome function (9). In support of this, our study has shown
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that GNPAT recruits USP30 to deubiquitinate and stablize DRP1. USP30 is known to
impair PARK2-mediated mitophagy and pexophagy and, along with GNPAT and
DRP1, localizes to the peroxisome (27, 28).
The genetic suppression of either GNPAT or DRP1 profoundly impaired
xenograft growth and further attenuated DEN/CCl4-induced hepatocarcinogenesis in
mice. Because previous results had shown that GNPAT regulates free cholesterol and
plasmalogen homeostasis (44). Our results confirmed that GNPAT inhibition also
decreased lipogenesis and fatty acid uptake in HCCs. Thus, it is tempting to speculate
that GNPAT’s regulation of tumor growth may involve both its regulation of
mitochondrial morphology and metabolic functions. Such functions might include
regulating the supply of critical metabolites necessary for tumor growth as well as
protection against the high levels of reactive oxygen species that are typically
generated by rapidly dividing tumors, particularly those with elevated Myc levels.
Independent of this, DRP1 has recently been reported to increase leptin sensitivity and
glucose responsiveness (45) and our results indicate that DRP1 inhibition also
attenuated lipogenesis and fatty acid uptake. While the precise mechanism for this
remains unclear, it is conceivable that this occurs via both mitochondrial and
peroxisomal pathways involved in fatty acid oxidation. Recent work in animal models
has demonstrated that both Myc-driven HCCs and -catenin-driven hepatoblastomas
re-direct fatty acid metabolism away from oxphos-linked energy-generating pathway
in favor of anabolic pathways in which fatty acids are used directly for the purpose of
new membrane synthesis to sustain rapid growth (36, 37).
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Myc plays a pivotal role in cell proliferation, tumorigenesis and metabolism and is
highly expressed in most tumors where it regulates target genes involved in these and
other processes that support tumor growth (46, 51). We found that the complex
comprised of Myc and KDM1A directly regulates GNPAT transcription in HCC cells
and is required for maximal expression of some of the Myc-driven phenotypes. The
resultant stabilization of DRP1 and the presumed increase in mitochondrial fission,
arising as a consequence of the interaction of GNPAT and the USP30 de-ubiquitinase,
could potentially provide an explanation for the significant reduction in overall
mitochondrial DNA content (and probably mitochondrial mass) that has been
observed in HCCs, hepatoblastomas and multiple other human cancers (46, 51).
Collectively, these findings suggest that targeting of GNPAT might be and effective
therapeutic strategy in HCC, particularly for the 40-70% of tumors that are believed to
be largely Myc-driven.
In summary, we propose a working model in which HCC progression is at least
partially driven by gene amplification or a complex comprised of Myc/KDM1A
which transcriptionally activates GNPAT. The interaction of GNPAT with the USP30
de-ubiquitinase stabilizes DRP1 by inhibiting its Ub-dependent degradation thereby
regulating mitochondrial morphology, lipid metabolism and liver tumorgenesis.
Several critical new points of potential therapeutic intervention have been identified
that will need to be investigated in future studies.
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Acknowledgments
We thank Profs. Bo Zhong, Xiaodong Zhang, Zan Huang and Min Wu (Wuhan
University, Wuhan, China) for the USP30 shRNAs, the deubiquitinases vectors,
Ubiquitin vectors and demethylase antibodies, respectively and Ping Wang (East
China Normal University, Shanghai, China) for pcDNA3.1(+)-5’flag Luc vector.
This study was supported by grants from the National Natural Science Foundation
of China (81772609, 81472549) to Y. Li.
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Figure Legends
Figure 1.
GNPAT is up-regulated in HCC, and high expression predicts poor HCC patient
outcomes. A, Summary of bioinformatic screening results in the HCCs from TCGA
database. Among 2783 metabolic enzyme genes, 896 genes were upregulated and
mRNA abundance from 442 of these genes being >100 RPKM by RNA-sequence
analysis in HCCs. The expression of 36 genes is tightly correlated with gene copy
number and nine genes are localized to chromosome 1q. B, Heatmap showing the
relative mRNA expression of 442 metabolic genes in HCCs from TCGA dataset. C,
List of nine metabolic genes localized in chromosome 1q from (A). R, the correlation
between mRNA expression and DNA copy number. D, MTT assays in HepG2 cells
after transfected distinct siRNAs of nine metabolic genes listed in (C). E, Releative
GNPAT mRNA levels in normal liver and HCC tissues from TCGA. GNPAT is
up-regulated in HCCs. F, Association of GNPAT expression with clinical stages in
HCC patients from TCGA dataset. G, Releative GNPAT gene copy number in normal
liver and HCC tissues from TCGA dataset. GNPAT is amplified in HCCs. H, Relative
GNPAT mRNA levels in HCC tissues with or without GNPAT amplification from
TCGA. GNPAT mRNA levels is upregulated with GNPAT amplification. I,
Correlation of GNPAT mRNA level with its DNA copy number in HCC tissues from
TCGA dataset. Each point is an individual sample. r, Spearman correlation coefficient.
J, Survival of human HCC patients from TCGA dataset with GNPAT amplification
versus no amplification. P values are indicated. (E-H) Significance was performed
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using Wilcoxon signed rank test. The horizontal lines in the box plots represent the
median, the boxes represent the interquartile range, and the whiskers represent the
minimal and maximal values. ***P < 0.001.
Figure 2.
GNPAT interacts with DRP1 to regulate mitochondrial morphology. A, Cellular
extracts from HEK293 cells expressing Flag-GNPAT and control vector were
immuno-purified with anti-FLAG affinity columns. The eluates were resolved by
SDS-PAGE and Coomassie blue staining. The protein bands were retrieved and
analyzed by mass spectrometry. DRP1 was identified as GNPAT interacting protein. B,
Endogenous interaction between GNPAT and DRP1. Whole cell lysates from HepG2
(left) and Hep3B (right) cells were prepared and endogenous IPs were performed with
antibodies against the indicated proteins. Immunocomplexes were then
immuno-blotted using antibodies against GNPAT and DRP1. C-D, Schematic diagram
showed the structure of DRP1 (left) and GNPAT (right) and truncation mutants used.
HA-tagged DRP1 wild type or truncation mutants were coexpressed with
Flag-GNPAT in HEK293. Extracts were immunoprecipitated with HA antibody and
examined by western blotting (C). Flag-tagged GNPAT wild type or truncation
mutants were coexpressed with HA-DRP1 in HEK293. Co-IP with Flag antibody and
examined by indicated antibodies (D). E, HepG2 and Hep3B cells were fixed and
immuno-stained with anti-GNPAT and anti-TFAM before confocal microsopy. Scale
bar, 10 μm. F, GNPAT interacts with DRP1 in mitochondria. Mitochondria were
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separated and immuno-precipitation assays were performed with the indicated
antibodies. G, Top- Middle: Representative confocal images of HepG2 stably
transfected GNPAT shRNAs and stained with TOM20 antibody. Elongated,
intermediate and fragmented mitochondria were counted. Scale bars, 10 μm. Bottom:
Representative transmission electron microscopy (TEM) images of HepG2 stably
transfected GNPAT shRNAs. Scale bars represent 500 nm. Mitochondria were
indicated and counted. Data were presented as mean ± SD. *P < 0.05, **P < 0.01,
***P < 0.001.
Figure 3.
GNPAT recruits USP30 to deubiquitinate and stabilize DRP1. A, GNPAT affects
DRP1 protein turnover. HEK293 cells were transfected with indicated vectors. After
cells were treated with CHX (50 μg/ml) for the indicated time, expression of DRP1
and GNPAT was analyzed by western blotting (left); the intensity of DRP1 expression
for each time point was quantified by densitometry with β-actin as a normalizer
(right). B, Immunoblotting analysis of DRP1 protein expression in HEK293 cells
transfected with shGNPAT after treatment with NH4Cl (25 mM), MG132 (100 µM) or
3-MA (500 ng/ml) for 6 h. C, Screening for the deubiquitinating enzymes of DRP1.
The indicated DUBs overexpressed in HEK293 for 24h and used for luciferase assay.
Data were presented as mean ± SD. D-E, Luciferase assay for co-expressing the
indicated vectors in HEK293. (A-E) Data were presented as mean ± SD. *P < 0.05,
**P < 0.01, ***P < 0.001. F, USP30 deubiquitinates DRP1. Flag-DRP1 and
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Myc-Ubiquitin (Ub) were co-expressed with WT or catalytic inactive (CI, C77S)
mutant of USP30 in HEK293 cells. IP and IB analysis were performed with the
indicated antibodies. G, GNPAT increases USP30-mediated DRP1 deubiquitination.
HEK293 cells were transfected with Flag-DRP1, Myc-ubiquitin (Ub), HA-USP30 or
HA-GNPAT. IP and IB analysis were performed with the indicated antibodies. H,
GNPAT promotes K48-linked deubiquitination of DRP1. HEK293 cells were
co-transfected with Flag-DRP1, Myc-GNPAT and HA-Ub or its mutants as indicated.
H-I, IP with Flag antibody and IB with the indicated antibodies were done. I,
Identification of DRP1 ubiquitination sites. ShUSP30, Myc-Ub and WT DRP1 or
mutants of DRP1 were transfected into HEK293.
Figure 4.
GNPAT and DRP1 depletion retards tumor growth. A, Western blot analysis of stably
expressing GNPAT or DRP1 shRNAs and shRNA+sh-resistant constructs of GNPAT
(top) and DRP1 (bottom) with indicated antibodies in HepG2 and Hep3B cells. B,
MTT assays showed relative absorbance at each time point with indicated cells. C-D,
Xenograft growth in nude mice. Cells with stable knockdown of GNPAT or DRP1
were injected subcutaneously into nude mice (n = 5). Tumor volumes were measured
every 3-4 days. Tumor growth curve after injecting the indicated cells (C) and
photographs of tumors from indicated cells (D). E, Quantification of neutral lipid
staining. Each of the indicated cell types was stained with BODIPY-493/503 and
assessed by flow cytometry. F, Incorporation of [3H] acetate into neutral lipids. G,
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NEFA levels in tumors derived from C. Data were presented as mean ± SD. *P < 0.05,
**P < 0.01, ***P < 0.001.
Figure 5.
DRP1 mediates and is required for GNPAT’s oncogenic phenotypes. A, Western blot
analysis of stably expressing indicated vectors in HepG2 and Hep3B cells. B, MTT
assay showed relative absorbance of cells from (A). C, Tumor growth in nude mice.
The indicated cells were injected subcutaneously into nude mice (n = 5). D,
Representative images of the indicated tumor sections stained with H&E or
immuno-stained with Ki-67 antibody. Scale bars, 100μm. E, Tumor weights for each
group. F, NEFA levels in tumors derived from C. Data were presented as mean ± SD.
*P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6.
DRP1 and GNPAT repression attenuates hepatocarcinogenesis in DEN/CCl4-induced
mouse HCC model. A, The schematic overview of DEN/CCl4 -induced HCC mice
model. DEN (25 mg/ml) was injected 15 days after birth (n=10). From 4 -15 weeks,
mice were intraperitoneal injected with CCl4 (0.5 ml/kg) weekly. During this period,
lentivirus containing GNPAT shRNA and DRP1 shRNA were given once per week for
4 times via tail-vein injection. After 28 weeks, livers were extracted. B, Liver images
extracted from the indicated mice. C-D, Tumor volume and tumor nodules of each
liver. E, Representative images of indicated liver sections stained with H&E or
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immuno-stained with GNPAT, DRP1 or Ki-67 antibody. Scale bars, H&E: 200μm;
immunostaining:100 μm. F, Western blot analysis of the indicated protein expression
of liver tissues from (A). G, Serum NEFA levels in mice from A. Data were presented
as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 7.
GNPAT expression in HCC cells without GNPAT amplification is transcriptionally
regulated by the complex composed of Myc and KDM1A. A, The schematic
representation of GNPAT promoter upstream 5kb and downstream 2kb of the
transcription start site (TSS). E-boxes and CpG islands were labeled. B, Myc and
GNPAT protein levels were analyzed by western blotting in HepG2 and MHCC97L
stably transfected with shMyc. C, GNPAT mRNA levels were determined by
qRT-PCR with Myc and KDM1A depletion cells. D, CHIP-qPCR analysis with the
indicated antibodies in HCC HepG2 cells stably transfected with shMyc. E, Dual
luciferase assays in HepG2 cells. F, Western blot analysis in HepG2 and MHCC97L
stably expressing indicated vectors with indicated antibodies. G, MTT assays showed
relative absorbance of indicated cells. H, Xenografts growth in nude mice. Data were
presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
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Published OnlineFirst August 24, 2018.Cancer Res Li Gu, Yahui Zhu, Xi Lin, et al. hepatocarcinogenesisrecruitment of USP30 stabilize DRP1 to promote Amplification of glyceronephosphate O-acyltransferase and
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