Amplification of glyceronephosphate O-acyltransferase and recruitment of USP30 stabilize DRP1 to promote hepatocarcinogenesis

Hepatocellular carcinoma (HCC) 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 2,783 genes encoding metabolic enzymes, GNPAT, which encodes the enzyme glyceronephosphate O-acyltransferase, is amplified, upregulated, and highly correlated with poor clinical outcome in human patients with HCC. 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 stabilization of DRP1 play a critical role in the development of HCC.Significance: This study identifies and establishes the role of the enzyme GNPAT in liver cancer progression, which may serve as a potential therapeutic target for liver cancer. Cancer Res; 78(20); 5808-19. ©2018 AACR.


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 accounts for more than 50% of all cases (1)(2)(3).Other risk factors include food contamination with aflatoxin, alcoholic and nonalcoholic cirrhosis, and nonalcoholic fatty liver disease.Excessive alcohol consumption and smoking further increase the overall risk (1)(2)(3).Gene mutations involving TP53 and CTNNB1, numerous copy-number variations, epigenetic alterations, and dysregulation of several miRNAs 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, 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 þ/þ variant of the HFE gene (1-3).In addition, ectopic expression of 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, a middle domain (MD), and a GTPase effector domain (9).Extensive investigations have revealed that defects in DRP1 can cause neurodegenerative disorders such as Alzheimer disease, Huntington disease, 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 not only leads to mitochondrial fission but also enhances HCC growth by promoting G 1 -S phase transition via pathways that involve p53 and NF-kB (14).Enforced overexpression 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 also involved in peroxisome fission, especially under peroxisome-inducing growth conditions (12,13).
Here, we report that GNPAT is upregulated in patients with HCC and is recruited by USP30 to deubiquitinate and stabilize DRP1, thus regulating mitochondrial morphology and hepatocarcinogenesis. USP30 is a deubiquitinylase 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 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.

Clinical human HCC specimens
HCC samples were used as previously described (29).Writteninformed 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 histologic review.

Cell culture and reagents
Human embryonic kidney HEK293 cells and human retinal pigmented epithelium cells immortalized with hTERT were obtained from the American Type Culture Collection in 2009, whereas normal human hepatocytes HL-7702, human liver tumor-derived cell lines BEL7402, FHCC98, Hep3B, Huh7, HepG2, HCCLM9, and MHCC97L were obtained from China Center for Type Culture Collection and Cell Bank of Type Culture Collection of Chinese Academy Sciences 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), protea-some inhibitor MG132 (M8699), and autophagy inhibitor 3methyladenine (3-MA; M9281) were purchased from Sigma-Aldrich.Antibodies used in this study are listed in Supplementary Table S1.

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 Â 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.siRNAs and short hairpin DNAs to inhibit murine Gnpat were designed and synthesized by GENEWIZ.The latter were annealed and then inserted into pLKO.1-purovector.The DRP1-luciferase vector was generated by cloning the human DRP1 coding sequences to the luciferase gene tail of pcDNA3.1(þ)-5 0flag Luc (a kind gift from Dr. Ping Wang, Life Sciences of East China Normal University, Shanghai, China; ref. 30).To detect GNPAT and DRP1 localization in peroxisomes, the peroxisomal targeting sequence serine-lysine-leucine was inserted into the pmCherry-N1 vector (Clontech; ref. 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; ref. 31).Primers used in this study are listed in Supplementary 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).

Subcellular fractionation
Subcellular fractionation was performed as described previously (32,33).In brief, cells were trypsinized, collected in PBS, resuspended 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.

Chromatin immunoprecipitation assays and qRT-PCR
Chromatin immunoprecipitation (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 immunoprecipitation 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) followed by DNase (Thermo Scientific) treatment.Reverse transcription was performed with a cDNA Synthesis kit (Promega), and qPCR was performed using SYBR Green Master Mix (Bio-Rad) using standard protocols.All primer sequences used are shown in Supplementary Table S1.b-Actin was used as an internal control.

Other metabolic assays
Cellular nonesterified fatty acid (NEFA) levels were determined with a commercially available ELISA kit (AO42-2; Jiancheng Bio.) according to the manufacturer's instruction.Serum TG levels were measured using the Infinity Triglycerides Reagent (Thermo Scientific).

Animal studies
All animal studies were approved by the Animal Care Committee of Wuhan University.For xenograft experiments, 4-weekold male BALB/c nude mice were purchased from SLAC Laboratory Animal Corporation and maintained in microisolator cages.Detailed procedures were described previously (29).
For the DEN/carbon tetrachloride (CCl 4 )-induced HCC model, C57/B6 mice were intraperitoneally administrated 25 mg/kg of diethylnitrosamine (DEN; Sigma) on day 15 of life.Following this, CCl 4 (Sigma) was then injected weekly for 12 weeks (n ¼ 10 per group).Concentrated shRNA-lentiviruses 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 hematoxylin and eosin (H&E).Ki-67 staining was performed according to the supplier's protocol (mouse monoantibody, 1:200, Sc-25280; Santa Cruz Biotechnology).
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 TFAM (1:100) for 1 hour.After exhaustive removal of the primary antibody by washing, a second incubation was performed with horseradish peroxidase-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) in PB buffer.After three washes in PB buffer, cells were scratched, collected, and centrifuged.Then cells were postfixed 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).

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 dataset.GSE datasets were analyzed from NCBI GEO databases (https://www.ncbi.nlm.nih.gov), which were reported from indicated gene expression profile array.Cancer Cell Line Encyclopedia (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 t test.Data were presented as the mean AESD.

GNPAT gene gain and amplification is predictive of poor clinical outcomes in human HCC
To identify deregulated metabolic genes involved in HCC progression, an unbiased bioinformatic screening of 2,783 metabolic enzyme genes was conducted in the HCC database from 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 and B).As tumor cells almost always harbor unstable genomic DNA (40), we sought candidates from the above group whose overexpression could be explained by gene amplification.Interestingly, 36 such examples were noted as being highly correlated (R > 0.5).Nine 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; refs. 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; Supplementary Fig. S1A and S1B), 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 levels in HCCs relative to normal hepatic tissues in all tested databases (Fig. 1E; Supplementary Fig. S1C-S1E).Human HCC cell lines also contained similarly elevated levels of GNPAT transcripts than did nontransformed 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 and S1H).In patients with HCC, those with stage II/III/IV had significantly higher GNPAT expression than those with stage I (Fig. 1F).Also in patients with HCC, 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 Fig. 1G-I and Supplementary Fig. S1J-S1L, GNPAT mRNA levels in HCCs also generally correlated with the degree of GNPAT gene amplification (Log 2 SNP > 0).Interestingly, patients with HCC 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 overexpression 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 SDS-PAGE and mass spectrometry 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; Supplementary Fig. S2A).The interaction between endogenous GNPAT and DRP1 was also confirmed by 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 coexpressed 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 and D).Immunofluorescence staining further indicated that GNPAT and DRP1 were colocalized in mitochondria and peroxisomes (Fig. 2E; GNPAT is upregulated in HCC, and high expression predicts poor HCC patient outcomes.A, Summary of bioinformatic screening results in the HCCs from TCGA database.Among 2,783 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, Relative GNPAT mRNA levels in normal liver and HCC tissues from TCGA.GNPAT is upregulated in HCCs.F, Association of GNPAT expression with clinical stages in patients with HCC from TCGA dataset.G, Relative 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 patients with HCC from TCGA dataset, with GNPAT amplification versus no amplification.P values are indicated.E-H, Significance was performed 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.Ã , P < 0.05; ÃÃÃ , P < 0.001.Supplementary Fig. S2B; refs.41,42).These results were confirmed by subcellular fractionation experiments (Fig. 2F).
Mitochondrial functions are related to mitochondrial dynamics, whereas 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 Supplementary Fig. S2C and S2D, 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 DCD or DRP1 DGTP.These findings suggest that the regulation of mitochondrial morphology mediated by the GNPAT-DRP1 interaction may be 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 Figs.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, which blocked de novo protein synthesis (Fig. 3A; Supplementary Fig. S3B).To identify the pathway(s) responsible for GNPAT-mediated DRP1 degradation, NH 4 Cl, 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 NH 4 Cl or 3-MA (Fig. 3B; Supplementary Fig. S3C).These results suggest that GNPAT inhibition mediated degradation of DRP1 through the proteasome pathway.
Given that deubiquitinases (DUB) 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 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; Supplementary Fig. S3D).Consistent with this, both GNPAT and DRP1 copurified with immunoprecipitated USP30 and USP48 (Supplementary Fig. S3E).Further assays showed that ectopic GNPAT expression increased USP30-mediated luciferase activity of DRP1-Luc, whereas 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 and  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).
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).shRNA-mediated inhibition of either GNPAT or DRP1 significantly retarded cell proliferation in vitro (Fig. 4A and B; Supplementary Figs.S2C and S4A) and in vivo xenograft growth (Fig. 4C  and D; 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 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-S4F).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 diminishes 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; Supplementary Fig. S5A).Further ectopic overexpression of DRP1 but not DRP1 DGTP or GNPAT DCD was able to rescue the phenotypic changes in HCC cells mediated by GNPAT inhibition (Fig. 5A-F; Supplementary Figs.S2C, S4A, S5A-S5C).These results indicate that DRP1 is necessary for and mediates GNPAT-malignant phenotypes.

GNPAT and DRP1 repression attenuates DEN/CCl 4 -induced hepatocarcinogenesis in mice
We next examined the expression of GNAPT and DRP1 in an autologous HCC model induced by a combination of DEN and CCl 4 .Consistent with our prior observation in human samples, GNAPT and DRP expressions were all upregulated at various times during disease development (Fig. 6A; Supplementary Figs.S1G  and 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/CCl 4 -treated mice for 4 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; Supplementary Fig. S6B-S6D).These mice also showed lower serum levels of NEFA and TG (Fig. 6G; Supplementary Fig. S6E).

GNPAT expression is activated by a transcription complex composed of c-Myc and KDM1A
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 5 kb upstream and 2 kb 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 C, Myc inhibition resulted in downregulation of both GNPAT mRNA and protein levels in both HepG2 and MHCC97L HCC cells but not GNPAT-amplified Hep3B cells (Supplementary Fig. S7A and  S7B), thereby indicating that at least some portion of GNPAT upregulation is likely mediated by the excessive levels of Myc expression associated with HCC.Indeed, a 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; 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)(48)(49)(50), we hypothesized that its interaction with one or more of these regulates GNPAT transcription.Therefore, ChIP experiment was performed to investigate which of the seven known histone demethylases bind to the GNPAT promoter in association with Myc.Our results showed that immunoprecipitation of KDM1A efficiently enriched the GNPAT promoter region containing the proximal E-box in control HepG2 and MHCC97L cells but not in cells in which Myc levels were depleted by the expression of an shRNA (Fig. 7D; Supplementary Fig. S7C and S7D).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), whereas 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, immunoprecipitation of H3K4me2 efficiently enriched the E-box-containing GNPAT promoter region in control HepG2 and MHCC97L but not in Myc-depleted cells (Fig. 7D; 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 and C; Supplementary Fig. S7A-S7C).We then tested the effects of Myc and KDM1A in a luciferase reporter assay of the GNPAT promoter.Coexpression of both plasmids in HepG2 and MHCC97L cells increased luciferase activity in reporters containing either of the WT E-box sites but not in those with mutated sites.Either of the two E-boxes appeared to be sufficient for upregulating the activity of the reporter, although their effects did not appear to be additive (Fig. 7E; 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, G, and H; 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 in vitro growth and in vivo tumorigenicity.However, these phenotypes were impaired following GNPAT inhibition (Fig. 7F, right, G, and H; Supplementary Fig. S7H,  bottom).These results suggest that GNPAT partially mediates and is required for the optimal expression of Myc-mediated oncogenic phenotypes.

Discussion
Because 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 reprogramming and contribute to cancer growth and/or survival is of paramount importance.In the current work, an unbiased bioinformatic screening of 2,783 metabolic enzymes identified GNPAT, a member of the EL biosynthetic pathway, as an important facilitator of HCC development.Although some of GNPAT's overexpression 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 has also 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 posttranslational modifications that regulate mitochondrial and peroxisome function (9).In support of this, our study has shown that GNPAT recruits USP30 to deubiquitinate and stabilize DRP1.USP30 is known to impair PARK2-mediated mitophagy and pexophagy and, along with GNPAT and DRP1, localize to the peroxisome (27,28).
The genetic suppression of either GNPAT or DRP1 profoundly impaired xenograft growth and further attenuated DEN/CCl 4induced 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.Although 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 b-catenin-driven hepatoblastomas redirect 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).
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 deubiquitinase, 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 an effective therapeutic strategy in HCC, particularly for the 40% to 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 deubiquitinase stabilizes DRP1 by inhibiting its Ub-dependent degradation, thereby regulating mitochondrial morphology, lipid metabolism, and liver tumorigenesis.Several critical new points of potential therapeutic intervention have been identified that will need to be investigated in future studies.

Figure 1 .
Figure 1.GNPAT is upregulated in HCC, and high expression predicts poor HCC patient outcomes.A, Summary of bioinformatic screening results in the HCCs from TCGA database.Among 2,783 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, Relative GNPAT mRNA levels in normal liver and HCC tissues from TCGA.GNPAT is upregulated in HCCs.F, Association of GNPAT expression with clinical stages in patients with HCC from TCGA dataset.G, Relative 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 patients with HCC from TCGA dataset, with GNPAT amplification versus no amplification.P values are indicated.E-H, Significance was performed 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.Ã , P < 0.05; ÃÃÃ , P < 0.001.

Figure 2 .
Figure 2. GNPAT interacts with DRP1 to regulate mitochondrial morphology.A, Cellular extracts from HEK293 cells expressing Flag-GNPAT and control vector were immunopurified 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 immunoprecipitations were performed with antibodies against the indicated proteins.Immunocomplexes were then immunoblotted using antibodies against GNPAT and DRP1.C and D, Schematic diagram showed the structure of DRP1 (left) and GNPAT (right) and truncation mutants used.HA-tagged DRP1 WT or truncation mutants were coexpressed with Flag-GNPAT in HEK293.C, Extracts were immunoprecipitated with HA antibody and examined by Western blotting.Flag-tagged GNPAT WT or truncation mutants were coexpressed with HA-DRP1 in HEK293.D, Co-IP with Flag antibody and examined by indicated antibodies.E, HepG2 and Hep3B cells were fixed and immunostained with anti-GNPAT and anti-TFAM before confocal microscopy.Scale bar, 10 mm.F, GNPAT interacts with DRP1 in mitochondria.Mitochondria were separated, and immunoprecipitation 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 mm.Bottom, representative transmission electron microscopy images of HepG2 stably transfected GNPAT shRNAs.Scale bars, 500 nm.Mitochondria are indicated and were counted.Data are presented as mean AE SD.Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001.

Figure 3 .
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 cycloheximide (CHX; 50 mg/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 b-actin as a normalizer (right).B, Immunoblotting analysis of DRP1 protein expression in HEK293 cells transfected with shGNPAT after treatment with NH 4 Cl (25 mmol/L), MG132 (100 mmol/L), or 3-MA (500 ng/mL) for 6 hours.C, Screening for the deubiquitinating enzymes of DRP1.The indicated DUBs overexpressed in HEK293 for 24 hours and used for luciferase assay.Data are presented as mean AE SD.D-E, Luciferase assay for coexpressing the indicated vectors in HEK293.A-E, Data are presented as mean AE SD.Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001.F, USP30 deubiquitinates DRP1.Flag-DRP1 and Myc-ubiquitin (Ub) were coexpressed with WT or catalytic inactive (C77S) mutant of USP30 in HEK293 cells.Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies.G, GNPAT increases USP30-mediated DRP1 deubiquitination.HEK293 cells were transfected with Flag-DRP1, Myc-ubiquitin, HA-USP30, or HA-GNPAT.Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies.H, GNPAT promotes K48-linked deubiquitination of DRP1.HEK293 cells were cotransfected with Flag-DRP1, Myc-GNPAT, and HA-Ub or its mutants as indicated.H and I, Immunoprecipitation with Flag antibody and immunoblot with the indicated antibodies were done.I, Identification of DRP1 ubiquitination sites.ShUSP30, Myc-ubiquitin, and WT DRP1 or mutants of DRP1 were transfected into HEK293.

Figure 4 .
Figure 4. GNPAT and DRP1 depletion retards tumor growth.A, Western blot analysis of stably expressing GNPAT or DRP1 shRNAs and shRNAþsh-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 and D, Xenograft growth in nude mice.Cells with stable knockdown of GNPAT or DRP1 were injected subcutaneously into nude mice (n ¼ 5).Tumor volumes were measured every 3 to 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 [ 3 H] acetate into neutral lipids.G, NEFA levels in tumors derived from C. Data are presented as mean AE SD.Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001.

Figure 5 .
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 ¼ 5).D, Representative images of the indicated tumor sections stained with H&E or immunostained with Ki-67 antibody.Scale bars, 100 mm.E, Tumor weights for each group.F, NEFA levels in tumors derived from C. Data are presented as mean AE SD.Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001.

Figure 6 .
Figure 6.DRP1 and GNPAT repression attenuates hepatocarcinogenesis in DEN/CCl 4 -induced mouse HCC model.A, The schematic overview of DEN/CCl 4 -induced HCC mice model.DEN (25 mg/kg) was injected 15 days after birth (n ¼ 10).From 4 to 15 weeks, mice were intraperitoneally injected with CCl 4 (0.5 mL/kg) weekly.During this period, lentiviruses containing GNPAT shRNA and DRP1 shRNA were given once per week for four times via tail-vein injection.After 28 weeks, livers were extracted.B, Liver images extracted from the indicated mice.C and D, Tumor volume and tumor nodules of each liver.E, Representative images of indicated liver sections stained with H&E or immunostained with GNPAT, DRP1, or Ki-67 antibody.Scale bars, H&E, 200 mm; immunostaining, 100 mm.F, Western blot analysis of the indicated protein expression of liver tissues from A. G, Serum NEFA levels in mice from A. Data are presented as mean AE SD.Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001.

Figure 7 .
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 5 kb and downstream 2 kb of the 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 are presented as mean AE SD.Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001.