Molecular Cell Biology

Canonical Wnt Signaling Remodels LipidMetabolism in Zebrafish Hepatocytes followingRas Oncogenic InsultYuxiao Yao1,2, Shaoyang Sun1, Jingjing Wang1, Fei Fei1, Zhaoru Dong3, Ai-Wu Ke3,RuoyuHe4, LeiWang1, Lili Zhang4,Min-Biao Ji4,Qiang Li5,MinYu1,Guo-MingShi3, Jia Fan3,Zhiyuan Gong6, and Xu Wang1

Abstract

There is limited understanding of theeffects of major oncogenic pathways andtheir combinatorial actions on lipid com-position and transformation during hepatictumorigenesis. Here, we report a negativecorrelation of Wnt/Myc activity with stea-tosis in human hepatocellular carcinoma(HCC) and perform in vivo functional stud-ies using three conditional transgenic zeb-rafish models. Double-transgenic zebrafishlarvae conditionally expressing humanCTNNB1mt and zebrafish tcf7l2 or murineMyc together with krasv12 in hepatocytes ledto severe hepatomegaly and significantlyattenuated accumulation of lipid dropletsand cell senescence triggered by krasv12

expression alone. UPLC-MS–based, nontar-geted lipidomic profiling and transcrip-tome analyses revealed that Wnt/Myc activ-ity promotes triacylglycerol to phospholip-id transformation and increases unsaturat-ed fatty acyl groups in phospholipids in a Ras-dependent manner. Small-scale screenings suggested that supplementation ofcertain free fatty acids (FA)or inhibitionof FAdesaturation significantly represses hepatic hyperplasia of double-transgenic larvaeand proliferation of three human HCC cells with and without sorafenib. Together, our studies reveal novel Ras-dependentfunctions of Wnt signaling in remodeling the lipid metabolism of cancerous hepatocytes in zebrafish and identify the SCDinhibitor MK8245 as a candidate drug for therapeutic intervention.

Significance: These findings identify FA desaturation as a significant downstream therapeutic target for antagonizing thecombinatorial effects of Wnt and Ras signaling pathways in hepatocellular carcinoma.

Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/19/5548/F1.large.jpg. Cancer Res; 78(19); 5548–60.�2018 AACR.

1Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education,Department of Biochemistry and Molecular Biology, School of Basic MedicalSciences, Fudan University, Shanghai, China. 2Department of Pediatric Surgery,Guangzhou Institutes of Pediatrics, Guangzhou Women and Children's MedicalCenter, Guangzhou Medical University, Guangzhou, China. 3Liver Cancer Insti-tute, Zhongshan Hospital, Key Laboratory of Carcinogenesis and Cancer Inva-sion, Ministry of Education, Fudan University, Shanghai, China. 4State KeyLaboratory of Surface Physics and Department of Physics, Collaborative Inno-vation Center of Genetics and Development, Key Laboratory of Micro & NanoPhotonic Structures, Ministry of Education, Fudan University, Shanghai, China.5Translational Medical Center for Development and Disease, Shanghai KeyLaboratory of Birth Defect, Institute of Pediatrics, Children's Hospital of FudanUniversity, Shanghai, China. 6Department of Biological Sciences, National Uni-versity of Singapore, Singapore.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for Y. Yao: Department of Pediatric Surgery, GuangzhouInstitutes of Pediatrics, Guangzhou Women and Children's Medical Center,Guangzhou Medical University, Guangzhou, China.

Corresponding Author: Xu Wang, Fudan University, Building 13#503, No. 130Dong'an Road, Xuhui District, Shanghai 201102, China. Phone: 8602-1542-37926;Fax: 8602-1640-33738; E-mail: wangxu2013@fudan.edu.cn

doi: 10.1158/0008-5472.CAN-17-3964

�2018 American Association for Cancer Research.

CancerResearch

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IntroductionHepatocellular carcinoma (HCC) is one of the most malig-

nant tumor diseases worldwide, causing over 750,000 deathsper year. Despite the rapid advances in surgical techniques andmedical discoveries, the overall survival rate and clinical out-comes for patients with HCC remain disappointing (1). Theliver is the central organ of metabolism, and the developmentof HCC is usually associated with subsequent metaboliclesions, including hepatic steatosis (2). Previous studies haverevealed the Ras and PI3K–Akt pathways, which are activated inover 50% of all HCC cases, as major drivers for hepatictumorigenesis with steatotic features (2–4), although theeffects of Ras and Akt on tumor lipid metabolism have beenreported to diverge in several contexts (5–9). In contrast, theWnt/b-catenin pathway, which is also activated in over 50% ofHCC cases, was reported to repress lipogenesis in white adiposetissue (WAT; ref. 10); however, its effects on lipid metabolismin normal and cancerous livers have not been well described.Therefore, the interactions between and roles of these primaryoncogenic pathways in hepatic lipid metabolism, along withthe dynamic remodeling of lipid composition during hepatictumorigenesis are of interest.

The components and transcriptional targets (e.g., Myc) of thecanonical Wnt signaling pathway are frequently altered and serveas major oncogenic drivers in HCC (11–14). Intensive studieshave used animal models to decipher Wnt functions in hepatictumorigenesis; the outcomes have varied in a highly context-dependentmanner. For example,mousemodels with conditionalloss of Axin1 (15) or Apc (16) in hepatocytes experienced HCC,whereas transgenic mice with activation of b-catenin aloneexperienced hepatomegaly (17, 18). However, additionalexpression of or mutation in Hras, Kras, or Met facilitatedb-catenin activation to quickly cause HCC (19, 20), indicatingthat certain oncogenic roles of canonical Wnt signaling dependon specific molecular backgrounds. Further observations sug-gest that the canonical Wnt pathway is downregulated innonalcoholic steatohepatitis (NASH) but upregulated in HCC(21), implying a complex connection between Wnt signalingand lipid metabolism.

Rodent models are traditionally used to mimic human hepaticsteatosis and tumorigenesis. Recently, zebrafish larvae emerged asunique models for studying early-stage hepatic lesions. Theirsmall size and optical transparency allow whole-liver imaging atcellular resolution and high-throughput drug screening (22).Several zebrafish models of HCC were previously generated via(conditional) expression of (pro-)oncogenes like, krasv12, Myc,xmrk, and ctnnb1 in hepatocytes (11, 23, 24). These modelsdeveloped hepatic hyperplasia before 6 days postfertilization(dpf), and ultimately achieved stable carcinogenesis at laterstages,making themvaluable resources.Here,we report anegativecorrelation between Wnt/Myc activity and steatosis in clinicalsamples and decipher the downstream effects ofWnt signaling onlipid metabolism in zebrafish hepatocytes. We provide morpho-logic and molecular evidence that Wnt/Myc activity significantlyinhibits the aberrant steatosis, remodels the lipid composition,and enhances thehepatic hyperplasia in aRas-dependentmanner.We also performed two small-scale screenings and identified FAdesaturation as the most promising therapeutic target for repres-sing the intensive proliferative responses induced by doubleoncogenic pathway activation.

Materials and MethodsHuman HCC tissue samples

Archival specimens were obtained from209 patients withHCCwho underwent curative resection between 2006 and 2008 at theLiver Cancer Institute, Zhongshan Hospital, Fudan University(Shanghai, China). Ethical approval was obtained from Zhong-shan Hospital Research Ethics Committee, and written informedconsent was obtained from each patient. The follow-up proce-dures were described in detail in Supplementary Material and ourprevious studies (25, 26). The studies were conducted in accor-dance with Declaration of Helsinki.

Zebrafish strains and experimentationZebrafish embryos, larvae, and adult fish were raised under

standard laboratory conditions at 28.5�C. The following zebra-fish lines have been described previously: AB wild-typestrain, Tg(TCFsiam:eGFP), Tg(TCFsiam:mCherry), Tg(tp1:eGFP),Tg(fabp10a:TetON;TRE:eGFP-krasv12), Tg(fabp10a:TetON; TRE:Myc), Tg(fabp10a:dsRed), and Tg(flk1:mCherry; refs. 11, 23,27, 28). The following lines were generated via tol2-meidiatedtransgene integration: Tg(fabp10a:TetON; TRE:GAL4), Tg(TRE:ABC-P2A-tcf7l2), Tg(UAS:Kaede), Tg(TRE:mKate2), Tg(hsp70l:scd-P2A-mKate2)(F0), and scd mutant. All studies involvinganimal manipulations were approved by the Fudan UniversityShanghai Medical School Animal Care and Use Committee andfollowed the NIH guidelines for the care and use of animals.The additional information of transgenic lines, tissue prepara-tion, IHC, BrdUrd labeling, TUNEL staining, lysotracker stain-ing, senescence-associated b-galactosidase (SA-b-gal) assay,Nile red and Oil red O staining, stimulated Raman scattering(SRS) microscopy setup, triglyceride measurement, real-timeRT-PCR, lipidomic profiling, RNA sequencing (RNA-seq),FA/compounds screening, and generation of scd-mutant larvaevia CRISPR–Cas9 injection were described in detail in Supple-mentary Material.

Cell lines and experimentationHCC cell lines Li-7 (TCHu183), Huh7 (TCHu182), and

HepG2 (Scsp-510) were obtained from the Cell Bank ofType Culture Collection of Chinese Academy of Sciences,Shanghai Institute of Cell Biology, Chinese Academy ofSciences (Beijing, China) between 2015 and 2016. All celllines involved in our experiments were reauthenticated byshort tandem repeat analysis every 6 months in our laboratory.Mycoplasma testing was detected through LookOut Mycoplas-ma PCR Detection Kit (Sigma, MP0035). The cells revived fromfrozen stocks were used within 10 to 20 passages or for nolonger than 2 months. Cells were cultured in DMEM (HyCloneSH30022.01) supplemented with 10% FBS (Gibco 10099133)at 37�C with 10% CO2. The cell proliferation assay wasdescribed in detail in Supplementary Material and our previ-ous studies (28).

Quantification and statistical analysisColocalization analysis, area measurements, statistical calcula-

tions, and graphs were generated with ImageJ, Microsoft Excel,andGraphPadPrism.Data are expressed as themean�SD fromatleast three samples (N is listed in each figure legend), withstatistical differences being determined by one-way ANOVA or

Combinatorial Effects of Wnt and Ras on HCC Lipid Metabolism

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one-tailed t test. For Fig. 1B and D and Supplementary Fig. S1A,the statistical significance was evaluated by the x2 test, and thesignificance of the effect of parameters on survival was evaluatedby the log-rank (Mantel–Cox) test. Overall survival was defined asthe interval between HCC resection and death, and patients aliveat the end of follow-up were removed. Drug interactions wereanalyzed by the Chou and Talalay method using the CalcuSynsoftware program (29). The combination index (CI) was simu-lated from each level of fractional effect.

ResultsWnt/Myc activity is associatedwith less steatosis inhumanHCCtissue samples

We performed confirmatory expression analyses of activeb-catenin (ABC) dephosphorylated on Ser37 or Thr41 and theputative TCF/b-catenin transcriptional target MYC in 209 humanHCC tissue samples (26). Compared with pan–b-catenin anti-body staining, ABC antibody staining improved the visual detec-tion of the Wnt-responsive cells in tumor and paratumor tissues.

Figure 1.

Wnt/Myc activity and steatosis in human HCC tissue samples. A, Representative staining of ABC, b-catenin, MYC, and Ki67 on serial sections of human HCCtissue with adjacent paratumor tissue (row 1) and high-magnification images (row 2). Scale bar, 100 mm. B, Statistics of 194 HCC tissue samples with differentABC/MYC staining pattern.C,Representative staining of ABC, MYC, andOROon serial sections of nonsteatotic HCC tissue (row 1) and the paired steatotic paratumortissue (row 2). Scale bar, 100 mm. D, Percentages of steatosis (lipidþ) in HCC tissue samples. ABC� versus ABCþ (P ¼ 0.034); Mycþ versus Myc� (P ¼ 0.001).

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Staining on serial sections showed that ABC, MYC, and Ki67, aproliferative marker, were all enriched in tumor tissue comparedwith paratumor tissue (Fig. 1A). Tissue microarray analysis sug-gested that the tumors positively stained by both ABC and MYCoccupied 43.8% of all 194 quantifiable samples, and the pre-sences of ABC and MYC were associated with statistical signifi-cance (P ¼ 0.004; Fig. 1B).

Interestingly, steatosis, which features an enlarged clear cyto-plasm due to the existence of lipid droplets, was frequentlyobserved in both tumor and paratumor tissues. In a few cases,the paratumor tissueswith severe steatosiswere observed adjacentto ABCþ and/or MYCþ non-steatotic tumor tissues, and the OilRedO staining was performed on the preserved frozen sections ofselected samples (Fig. 1C). Further observation of all samplessuggested a negative correlation between Wnt/MYC activity andthe accumulation of lipid droplets, as only 16.8% ABCþ and15.4% MYCþ tumor tissues were found to contain steatotic cells,whereas 29.6%ABC� and 35.9%MYC� tumor tissues were rich inlipids (Fig. 1D).

The full tabulated information about the clinical HCC samplesand their donors is listed in Supplementary Table S1. The relevantsurvival rates, tumor size, nodule size and number, and cancergrade are diagrammed in Supplementary Fig. S1A–S1D. Collec-tively, our data indicated that ABC staining coexists with MYCexpression in human HCC, and the HCC samples with high levelofWnt/Myc activitymostly contain fewer lipid droplets stained byOil Red O.

Generation of transgenic zebrafish with canonical Wntsignaling activated in hepatocytes

Clinical observations suggested a potential involvement of theWnt/b-catenin/Myc pathway in regulating hepatic lipid metabo-lism. Here, we used zebrafish as a model to identify the detailedeffects of Wnt activity on lipid accumulation in vivo. Although thecanonical Wnt pathway often has conserved roles between zebra-fish andmammals (30), it was unclear whether such conservationexists in the liver.

To achieve better characterization ofWnt-responsive cells in thezebrafish liver, we employed TCFSiam:eGFP (TCF:eGFP) andTCFSiam:mCherry (TCF:mCherry), twowidely used transcriptionalreporter lines for canonicalWnt activity (27).Weonly found a fewnonhepatocyte Wnt-responsive cells in normal livers. Those cellswere present infibrocyte-likemorphology at 6 dpf into adulthood(Fig. 2A; Supplementary Fig. S2A), and almost all Wnt-responsivecells were found between the main bile duct (2F11/2þ) andhepatocytes (fabp10a:eGFPþ) without overlapping (Supplemen-tary Fig. S2B–S2F).

To activate the canonical Wnt signaling pathway in hepato-cytes, two stable transgenic zebrafish lines (liver-driver andWnt-effector) were generated using the TetON system (Fig. 2B).On the one hand, the liver-driver line was designed to restain theexpression of effector genes only in the hepatocytes at the righttime. The liver-driver line was generated by integrating a trans-genic cascade: fabp10a:TetON; TRE:GAL4-VP16, which expressesthe inducible transactivator TetON via the liver-specific fabp10apromoter (31) and subsequently activates expression ofGAL4-VP16 and other downstream genes in the effector lines viathe TRE promoter in the presence of doxycycline. The F0 chimeraliver-driver lines were crossed to the established effector linesTRE:mKate2 or UAS:Kaede to generate F1 offspring that may carryfluorescence in the liver for screening purpose (Supplementary

Fig. S3A). Interestingly, the expression of mKate2 driven by TREpromoter displayed ubiquitous red fluorescence in the liver,whereas the expression of green Kaede driven by UAS promoterwas mosaic and heterogeneous due to the random silencingof UAS promoter in zebrafish genome (SupplementaryFig. S3B). On the other hand, the Wnt-effector line was designedto activate the canonical Wnt signaling pathway after beinginduced by the liver-driver line in hepatocytes (Fig. 2B; ref. 14).The Wnt-effector line was generated by introducing transgeniccascade: TRE:CTNNB1P33, C37, A41, P45-P2A-tcf7l2, which expressedhumanb-catenin carrying four typicalmutations reported inHCCwhole-genome sequencing and zebrafish tcf7l2 driven by TREpromoter (Fig. 2B). Crossing the liver-driver with theWnt-effectorresulted in double-transgenic offspring (Tg(Wnt)), which activat-ed the canonical Wnt signaling pathway ectopically in the liverupon doxycycline treatment; TCF:eGFP was crossed to (Tg(Wnt))for visual confirmation (Fig. 2C).

Wnt/Myc and Ras activities induce modest to excessive hepatichyperplasia

The Tg(Wnt) was investigated in parallel with a previouslygenerated zebrafish line fabp10a:TetON; TRE:Myc (Tg(Myc))that conditionally expresses murine Myc (one of the Wnt targetgenes) in the liver (11). For assessing the combinatory rolesof multiple oncogenic pathways in lipid metabolism, wealso employed a previously generated strain fabp10a:TetON;TRE:eGFP-krasv12 (Tg(Kras)) that conditionally activates Rassignaling (23), because Ras signaling is frequently altered inhuman HCC samples (Supplementary Fig. S4A and S4B), and ispartially responsible for steatosis during hepatic tumorigenesis(4). Tg(Wnt) and Tg(Myc) were further crossed to Tg(Kras) togenerate Tg(Wnt&Kras) and Tg(Myc&Kras) with double onco-genic pathways activated.

All larvae were treated with 40 mg/mL doxycycline from 3dpf onwards and were used for phenotypic analyses at 6 dpf.Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras) expressed a eGFP-fused Krasv12 that was recruited to the cell membrane to allowcell shape observation, and the hepatocytes in Tg(Wnt&Kras)and Tg(Myc&Kras) displayed significant morphologic abnor-malities, being transformed from polygons into irregular fusi-form cells (Fig. 2D). To better examine the general effects ofdifferent oncogenic pathways on liver morphology, all sixgroups of larvae were further crossed with LiPan (fabp10a:dsRed) to facilitate observation of liver size and shape (31).The single transgenic larvae, Tg(Wnt), Tg(Myc), and Tg(Kras),all displayed marginally enlarged livers (Fig. 2E). In contrast,the double transgenic larvae displayed significant hepatomeg-aly within 3 days of induction, and both Tg(Wnt&Kras) andTg(Myc&Kras) caused much more severe and rapid hyperplasiathan their single transgenic siblings (Fig. 2E). Three-dimensional (3d) reconstruction via Amira showed that singletransgenic larvae only displayed moderate hyperplasia,whereas Tg(Wnt&Kras) and Tg(Myc&Kras) showed the lossof typical liver shape and enlarged hepatic lobes (Fig. 2E).Meanwhile, BrdUrd labeling suggested that overproliferationwas responsible for the hyperplasia in Tg(Wnt), Tg(Myc),Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras), whereas TUNELstaining revealed modestly increased apoptosis in Tg(Wnt),Tg(Myc) with and without Tg(Kras), which only affected asmall proportion of hepatocytes (Fig. 2F; SupplementaryFig. S5A).

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Wnt/Myc activity represses the extensive hepatic steatosis andsenescence triggered by Kras expression

To investigate whether ectopic Wnt/Myc activity affectedlipid distribution patterns in the liver with and withoutKras expression, a combination of three approaches (Nile redstaining, Oil Red O (ORO) staining, and SRS; Supplementary

Fig. S5B; ref. 32) was employed to assess the existence of lipiddroplets at 6 dpf. In general, the hepatocytes in control,Tg(Wnt), and Tg(Myc) larvae did not contain many neutrallipids identified by Nile red, ORO, and SRS at 6 dpf. Althoughthe ectopic Wnt and Myc activity alone statistically reduced thenumber of small lipid droplets, the low level of lipid droplets

Figure 2.

Wnt/Myc and Ras activities induce modest to excessive hepatic hyperplasia. A, Lateral views of TCFsiam:eGFP larvae at 6 dpf in bright-field (top), greenfluorescent channel (middle), and ORO staining (bottom). Scale bar, 100 mm. Magnification images of livers on the right. B, Schematic diagram of the TetON systemwith separate liver-driver and Wnt-effector constructs. The liver-specific expression of CTNNB1mt (ABC) and tcf7l2 in double-transgenic fish harboring bothcassettes is activated in the presence of doxycycline. Red amino acids indicate themutated sites in human b-catenin.C,High-magnification images of TCFsiam:eGFPand Tg(Wnt); TCFsiam:eGFP larval liver with DAPI at 6 dpf, with doxycycline induction initiated at 3 dpf. Scale bar, 40 mm. D, Representative images of opticalsections in Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras) larval liver at 6 dpf. Scale bar, 50 mm. E, Representative images of whole livers from the lateral view,screenshots of 3D reconstruction of livers from the front view in control, Tg(Wnt), and Tg(Myc), Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras) at 6 dpf. Scale bar, 50mm.F, Statistics of liver size (pixels), proliferation, apoptosis. n ¼ 12 per group for each assay; mean � SD, with statistical differences determined by one-way ANOVA.� , P < 0.05; �� , P < 0.01; ����, P < 0.0001. The liver regions are circled in A, C, and E.

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in normal hepatocytes precluded us from reaching a clearconclusion (Fig. 3A and B). As expected, Tg(Kras) alone trig-gered intensive accumulation of lipid droplets in zebrafishhepatocytes (Fig. 3A and B), providing a lipid-rich context(Fig. 1C). Significantly, the Kras-induced lipid accumulation

was greatly compromised in Tg(Wnt&Kras) and Tg(Myc&Kras),with both the number and size of lipid droplets reduced inhepatocytes (Fig. 3A and B).

As accumulation of triacylglycerol (TG) is central to replicativesenescence (33), we also performed senescence-associated(SA)-

Figure 3.

Wnt/Myc activity represses the extensive hepatic steatosis and senescence triggered by Kras expression. A, Lipid distributions in the liver of control,Tg(Wnt), and Tg(Myc), Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras) at 6 dpf. Representative images of Z projection of confocal stacks of Nile red staining(top; scale bar, 30 mm), ORO staining on hepatic cryosections (middle; scale bar, 20 mm), and SRS scans (bottom; scale bar, 20 mm). B, Statistics of lipidcontent measurements. n ¼ 12 per group for each assay; mean � SD, with statistical differences determined by one-way ANOVA. � , P < 0.05; ���� , P < 0.0001.C, SA-b-Gal staining on the whole livers (row 1; scale bar, 50 mm) and cryosections with eosin costained (row 2; scale bar, 20 mm) for senescence detectionin the liver of control, Tg(Wnt), Tg(Myc), Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras) larvae at 6 dpf. The liver regions are circled in A.

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b-Gal staining on all six groups [Control, Tg(Wnt), Tg(Myc),Tg(Kras), Tg(Wnt&Kras), Tg(Myc&Kras)], and found that Tg(Kras),but not Tg(Wnt) or Tg(Myc), induced notable cell senescence inthe liver; senescence was significantly alleviated in Tg(Wnt&Kras)and Tg(Myc&Kras) (Fig. 3C; Supplementary Fig. S5C). However,neither supplement of palmitate nor inhibition of lipogenesis viasmall molecules caused significant effects on the degree of senes-cence in Tg(Wnt&Kras) and Tg(Myc&Kras) (Supplementary Fig.S5D), indicating that the manipulation of lipid metabolism maynot have immediate impacts on senescence in these contexts. Inaddition, the reduced senescence in double transgenic livers wasaccompanied by increased level of autophagy/lipophagy, as dem-onstrated via lysosome staining, LC3 staining, and transmissionelectronmicroscope (TEM; Supplementary Fig. S5E), aswell as thealternations of mRNA level of autophagy genes (SupplementaryFig. S5F). Interestingly, both inhibition and stimulation of autop-hagy seemed to repress the hepatic hyperplasia in double trans-genic larvae (Supplementary Fig. S5G), suggesting the potentialversatile outcomes by directly targeting autophagy/lipophagy inhepatic tumorigenesis.

Wnt/Myc activity modulates hepatic lipid composition in aRas-dependent manner

Introduction of Ras oncogenic insult provided a neutral lipid-rich environment, allowing us to morphologically observe thepotential effects of Wnt signaling on hepatic steatosis. Biochem-ical assays were performed on livers dissected from the six groupsof larvae, and the protein-normalized TG levels in each lysatewereassessed. Our results indicated no obvious change in total TGcontent of Tg(Wnt) and Tg(Myc) livers, whereas Tg(Kras) dis-played increased TG amount over twice the control level, andTg(Wnt&Kras) and Tg(Myc&Kras) significantly attenuated the TGenrichment (Fig. 4A). These results are consistent with our mor-phologic observations.

To better describe the alterations in lipid composition, weperformed UPLC-MS–based nontargeted lipidomic profilingon 6 dpf larvae, which are ideal "closed systems" with yolk asthe constant lipid input (34). Because Tg(Myc&Kras) displayeda much more pronounced phenotype than Tg(Wnt&Kras) (Fig.4A), the analysis was performed on four groups of larvae:control, Tg(Myc), Tg(Kras), and Tg(Myc&Kras) (SupplementaryFig. S6A and S6B). A total of 1,227 lipid species were identifiedin five main categories: glycerolipids (GL), glycerophospholi-pids (GP), sterol lipids (ST), sphingolipids (SP), and sacchar-olipids (SL; Supplementary Tables S2 and S3). The major lipidclasses were TG, phosphatidylethanolamine (PE), ceramideand similar sphingolipid (Cer), phosphatidylcholine (PC),diacylglycerol (DG), cholesterol (CHOL), phosphatidylserine(PS), phosphatidylglycerol (PG), phosphosphingolipids (PI-Cer), phosphatidic acid (PA), phosphatidylinositol (PI),monoacylglycerol (MG), sphingomyelin (SM), and cardiolipin(CL). The proportions of these lipid classes in the four larvalgroups are diagrammed in Fig. 4B, and the detailed statistics arelisted in Supplementary Table S4.

Most lipids maintained relatively constant proportions amongcontrol, Tg(Myc), and Tg(Kras). However, TG percentage wasreduced from 26.04% � 1.01% in the control to 20.41% �1.03% in Tg(Myc) and raised to 31.16% � 2.37% in Tg(Kras).The biggest change occurred in Tg(Myc&Kras); TG percentageextensively decreased to 9.54%� 0.66%, with the lost proportionbeing mostly occupied by PE (9.98% � 0.89%–17.66% �

0.79%), DG (6.41%–0.64% to 12.17% � 0.54%), and Cer(12.09% � 1.01%–15.88% � 0.65%) compared with Tg(Kras)(Fig. 4B). Statistically significant alteration in abundance of 286lipids species of variable importance also followed the sametrends (Supplementary Table S5). For better visualization, aheatmap was generated on the basis of the relative abundancesof individual lipids, and it revealed that almost all TG species werereduced specifically in Tg(Myc&Kras), whereas the abundances ofDG and PE displayed more complex transformations with themain tendency of increasing (Supplementary Fig. S6C). A list ofrepresentative lipid species (TG, DG, PE, Cer) was also listedas Fig. 4C. The results suggested that Myc significantly promoteda proportional transformation from TG to GP particularly in theTg(Kras) background.

Closer examinations of the fatty acyl chains' abundance in eachlipid category and class were performed (Supplementary TableS6). For better visualization, a heatmapwas also generated on thebasis of the relative abundances of saturated fatty acyl groups(SFA), monounsaturated fatty acyl groups (MUFA), polyunsatu-rated fatty acyl groups (PUFA), and fatty acyl groupswith differentlengths in Tg(Myc), Tg(Kras), and Tg(Myc&Kras) compared withcontrol (Fig. 4D). Significantly, Tg(Myc&Kras) decreased SFA andincreased PUFA in DG, PE, and total GP compared with Tg(Kras)(Fig. 4E), whereas the alternations in MUFA were not significant,probably because the increased MUFAs were further desaturatedto become PUFAs (Fig. 4E).However, the Tg(Myc) displayed noneor even opposite effects on the desaturation of FA compared withwild-type control, indicating the role of Myc in promoting desa-turation of DG, PE, and total GP was Ras-dependent (Fig. 4E).Besides, Tg(Myc&Kras) increased the proportion of shorter chainFA in TG, PE, andCer compared with Tg(Kras) (Fig. 4E). Together,the background of Ras activation was required for Myc to increaseshorter or unsaturated fatty acyl groups in the zebrafish liver.

Wnt/Myc activity broadly regulates gene expression associatedwith lipid metabolism

Lipidomic changes were examined by analyzing the expres-sion of genes involved in lipid metabolism. As an initialscreening, we performed RNA-seq on four groups of larvae:control, Tg(Myc), Tg(Kras), and Tg(Myc&Kras). The full list ofgene expression data with Gene Ontology and KEGG pathwayinformation is attached as Supplementary Table S7. KEGGpathway analyses on differentially expressed genes indicatedthat genes in lipid metabolism were significantly affected byTg(Myc) with and without the Tg(Kras) background (Supple-mentary Fig. S7A and S7B). We then manually chose 128 liver-enriched genes associated with lipid metabolism (labeled inred in Supplementary Table S7) to generate a heatmap basedon the relative alterations of individual gene expression inTg(Myc), Tg(Myc&Kras), and Tg(Kras) compared with control.The heatmap revealed that dynamic alterations occurred ingenes responsible for almost all aspects of lipid metabolism,including those involved in TG/DG/GP transformation and FAdesaturation/elongation (Supplementary Fig. S7C).

Nineteen transcripts were verified in dissected livers ofthe six larval groups via real-time RT-PCR (SupplementaryTable S8). Alternations in the TG to DG and DG to GP transfor-mations in Tg(Wnt&Kras) and Tg(Myc&Kras) were supportedby increased expression of lipase C (lipca) and choline/ethanol-amine phosphotransferase 1 (cept1a) (Fig. 4F). However, the DGto TG transformation was complex, as two diacylglycerol

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Figure 4.

Wnt/Myc activity regulates hepatic lipid composition and relevant gene expression in a Ras-dependent manner. A, Protein-normalized TG level in livers of 6 dpfcontrol, Tg(Wnt), Tg(Myc), Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras) larvae (n ¼ 3/group). B, Pie charts showing the lipid composition in 6 dpf zebrafishlarvae of control, Tg(Myc), Tg(Kras), and Tg(Myc&Kras) (n ¼ 3/assay). All abbreviations are indicated in main text. C, The normalized abundance ofsignificantly altered lipid species in 6 dpf control, Tg(Wnt), Tg(Myc), Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras) larvae. D, The normalized abundance offatty acyl groups. The purple box shows significantly altered proportion of SFA and PUFA groups in DG, PE, and all GP of control, Tg(Myc), Tg(Kras), andTg(Myc&Kras). The green box shows significantly altered proportion of fatty acyl groups with different length in TG, PE, and Cer of control, Tg(Myc), Tg(Kras),and Tg(Myc&Kras) (n ¼ 3/assay). E, Statistics of the normalized abundance of fatty acyl groups. F, mRNA expression of relevant lipid metabolism genes inlivers of 6 dpf control, Tg(Wnt), Tg(Myc), Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras) larvae (n ¼ 3/assay). All the statistics are represented as mean � SD,with statistical differences determined by one-way ANOVA. � , P < 0.05; �� , P < 0.01; ���, P < 0.001; ���� , P < 0.0001. C, control; M, Myc; K, Kras; M.K, Myc&Kras.

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acyltransferases (dgat) were differentially regulated with reduceddgat1b but raised dgat2 expression (Fig. 4F; SupplementaryFig. S7D). In addition, two FA desaturases, stearoyl-CoA desatur-ase (scd) and fatty acid desaturase 2 (fads2), were upregulated byWnt/Myc activity upon Ras insult (Fig. 4F). Moreover, we foundthat the expressions of FA elongase genes, elovl7b and elovl1a, werereduced, whereas the expressions of genes in mitochondrial/peroxisome b-oxidation, acetyl-CoA acyltransferase 2 (acaa2) and3-hydroxybutyrate dehydrogenase 1(bdh1), were increased; thismay account for the absence of (very) long-chain fatty acyl groupsand the increase of short/medium-chain fatty acyl groups incertain lipid classes (Fig. 4F).

Free fatty acid supplementary assays and inhibitor screening ontransgenic larvae identify desaturation as a potential target torepress hepatic hyperplasia

Both lipidomic and transcriptional analyses suggested thatactivation of the Wnt/Myc signaling upon Ras insult causedalterations in saturation levels and lengths of fatty acyl groupsin GP, the major phospholipid. The composition of fatty acylgroups in PE and GP determines the properties and fluidities ofcell membrane and, therefore, plays a role in the high levels ofmitosis in tumorigenesis (35).

To assess whether alterations in FA composition via supple-mentation of certain free fatty acid (FFA) are potentially beneficialfor repressing tumorigenesis, we performed a liver size-basedscreening with eleven common dietary FFAs in our transgenicmodels. We first showed that the fluorescent fatty acid analoguecan be directly injected into the yolk and absorbed by larvae, witha significant amount deposited in the liver (Fig. 5A). We theninjected various FFAs into all six groups of larvae at 3 dpf, andmeasured the liver sizes at 6 dpf (Fig. 5B). The dosage wasdetermined by MTD tests to ensure zebrafish would be alivewithout severe malformation at 6 dpf, and is approximatelyequivalent to 200 or 50mg/kg (for palmitic acid and stearic acid)for drug test in mouse. In general, most FFAs did not causea significant change in liver size; however, arachidonic acid,a-linolenic acid, and g-linolenic acid caused enhanced hyperpla-sia in Tg(Wnt&Kras) and/or Tg(Myc&Kras), whereas elaidic acid(also a FADS inhibitor) and palmitic acid significantly prohibitedhepatomegaly. Additional BrdUrd labeling and TUNEL stainingrevealed that both decrease in proliferation and increase inapoptosis were responsible for these inhibitory effects (Fig. 5C).

We further assessed whether modulation of lipid/FA metabo-lism via inhibitors can repress hepatic hyperplasia in the zebrafishmodels by performing drug tests with seven prescreened inhibi-tors: two SCD inhibitors (MK8245 and A939572), two DGATinhibitors (xanthohumol and T-863), a PPARg inhibitor(BADGE), amicrosomal triglyceride transfer protein (MTP) inhib-itor lomitapide, and sorafenib as a control (Fig. 5D). Remarkably,MK8245, one of the SCD inhibitors, displayed a striking ability toinhibit hepatomegaly in both Tg(Wnt&Kras) and Tg(Myc&Kras)),without causing malformation in larvae, and it also moderatelyreduces the control liver size due to the potential toxicity (Fig. 5DandE). BrdUrd labeling andTUNEL staining suggest thatMK8245mainly repressed the proliferation of the tumor-forming hepato-cytes (Fig. 5E). Subsequently, we performed a combination assayusing sorafenib as the standard drug. We found that MK8245further repressed the overproliferation in Tg(Wnt&Kras) andTg(Myc&Kras) liver on the basis of sorafenib treatment (Fig. 5Dand E; Supplementary Fig. S8A). Finally, to confirm SCDas a valid

target at the genetic level, we generated scdmutant in zebrafish viaCRISPR/Cas9 technique ((Supplementary Fig. S8B; ref. 36), andthe liver sizes from both Tg(Wnt&Kras) and Tg(Myc&Kras) weresignificantly reduced in heterozygous scd-mutant background(Fig. 5F); we also generated a Tol2 expression vector carryingscd-P2A-mKate cascade driven by the hsp70l promoter, and coin-jected it into zebrafish zygotes with tol2 transposase mRNA(Supplementary Fig. S8C); the injected Tg(Kras) larvae were heatshocked to induce extra scd expression, and the liver hyperplasiawas not significantly promoted (Supplementary Fig. S8D).

MK8245 suppresses proliferation of human HCC cellsTo evaluate its therapeutic potential in mammalian cells,

MK8245 was tested for antiproliferative activity against threehuman HCC cell lines: Li-7, Huh7, and HepG2. The IC50

of MK8245 for Li-7, Huh7, and HepG2 after 48-hour incuba-tion was 8.86 � 1.27 mmol/L, 6.21 � 0.83 mmol/L, and 12.69 �1.08 mmol/L, respectively; the IC50 of sorafenib for Li-7, Huh7,and HepG2 was 9.91 � 1.34 mmol/L, 6.32 � 0.79 mmol/L, and8.39 � 1.17 mmol/L, respectively (Fig. 6A), suggesting thatMK8245 displays comparable antiproliferation activity inhuman HCC cells.

In addition, the MK8245 was evaluated in combination withsorafenib. The two compounds were applied to cell culturesalone or in combination at concentrations close to their IC50,followed by a cell viability assay. The results showed thatMK8245 further decreased the viability of all three cell lineson the basis of sorafenib treatment (Fig. 6B), suggesting thatprognosis of human patients may benefit from such combina-tional treatment. Finally, because the MUFA oleate is the majorproduct of SCD activity, we performed a rescue experiment bydirectly replenishing oleic acid in the culture medium. Thissignificantly neutralized the effects of MK8245, and all threecell lines partially restored their proliferative responses (Fig.6B). To further evaluate the effect of combining treatments withMK8245 and sorafenib, the three cell lines were exposed tovarying concentrations of MK8245 and sorafenib at fixed ratiosof 9:10 in Li-7, 1:1 in Huh7, and 3:2 in HepG2. Cell viabilitywas then assessed and the Fraction effect (Fa)–CI curves foreach drug were generated (Fig. 6C). The averaged CIs forMK8245 and sorafenib at concentrations close to their IC50

were 1.159 for Li-7 (slight antagonistic effect), 0.782 for Huh7(moderate synergistic effect), and 1.009 for HepG2 (nearlyadditive effect), none of which displayed significant synergisticor antagonistic effect (CI < 0.7).

In summary, our studies suggest that the canonicalWnt signaling pathway has profound influences on the lipidmetabolism of hepatocytes in a highly context-dependentmanner. Investigations using zebrafish indicated that Ras activ-ity, a typical oncogenic and adipogenic event in hepatic tumor-igenesis, could serve as a perturbed background necessary forobserving the Wnt signaling remodeling of lipid composition.Lipidomic and transcriptional analyses revealed that Wnt/Mycactivity significantly promotes a proportional transformationfrom TG to GP, particularly in the TG-rich background inducedby ectopic Ras activity. Small-scale screenings in zebrafishmodels identified a desaturase inhibitor, MK8245, as an effec-tive reagent to block the hepatic hyperplasia. Taken together,these data reveal the effects of multiple oncogenic pathways onlipid composition and FA desaturation in hyperplasic zebrafishliver, and possibly in human HCC.

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DiscussionCanonical Wnt signaling in the liver is partially conservedbetween zebrafish and mammals

The canonical Wnt signaling pathway is highly conservedthroughout vertebrates, and our results revealed both the simi-larities and differences of Wnt signaling in zebrafish and mam-

malian livers. On the one hand, most hepatocytes in both mam-mal and zebrafish do not display canonical Wnt activity, andb-catenin is ubiquitously distributed at the cell surface throughoutthe entire liver (37, 38). Consistent with the aberrant Wnt activityinmammalianHCC,we show that ectopic expressionofb-cateninand Tcf7l2 in zebrafish livers also induces hyperplasia with or

Figure 5.

FFA supplementation and inhibitor screening on transgenic zebrafish larvae. A, An FFA analogue was injected into the yolk sac at 3 dpf (i) andabsorbed by the liver at 6 dpf (ii). Scale bar, 100 mm. B, Liver sizes (pixels) of control, Tg(Wnt), Tg(Myc), Tg(Kras), Tg(Wnt&Kras), and Tg(Myc&Kras)(n ¼ 6/group) at 6 dpf, with injection of different FFAs at 3 dpf. C, Statistics of BrdUrd labeling and TUNEL staining in the livers (n ¼ 12/group) withelaidic acid and palmitic acid injected. D, Liver sizes (n ¼ 6/group) at 6 dpf, with treatment of different inhibitors from 3 dpf. sor, sorafenib. E, Statisticsof BrdUrd labeling and TUNEL staining in the livers (n ¼ 12/group) after MK8245 and MK8245 þ sorafenib incubation. F, Statistics of liver sizes in control,Tg(Wnt&Kras), and Tg(Myc&Kras) larvae with scdþ/þ or scdþ/� as the genetic background (n ¼ 12/sample). All column data are represented as mean � SD.Statistical differences were determined by one-way ANOVA or unpaired t test. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001; ���� , P < 0.0001.

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without other oncogenic insult (Fig. 2E). On the other hand, theWnt-responsive cells are mostly observed in a subset of perive-nous hepatocytes in the mammalian liver (39), but only a fewnonhepatocyte Wnt-responsive cells surrounding the main bileduct were detected. Although those cells were stained by an OV6antibody, with a small subset being GFAPþ (Supplementary Fig.S2B and S2F), they are unlikely to be equivalent of mammalianoval cells, due to the significantly different morphology andnonoverlapping with Notch reporter (tp1:eGFPþ; SupplementaryFig. S2C; ref. 40). The nature of those cells needs further inves-tigation in both normal and regenerating livers.

Remodeling of hepatic lipid metabolism by Wnt signaling iscontext dependent

The negative correlation of Wnt/Myc activity and steatosis inclinical samples is simulated by combining Tg(Wnt) or Tg(Myc)with Tg(Kras) in zebrafish (Figs. 1C and 3A). In contrast to WAT,where both the canonical Wnt pathway and Myc activity display

clear roles of antiadipogenesis (10, 41), normal hepatic tissuedoes not harbor much accumulated neutral lipids in the firstplace, and the effects ofWnt andMycon lipid composition are notbiologically significant on their own (Fig. 3A). However, intro-duction of krasv12 expression creates a lipid-rich environment andenhances the Wnt function in remodeling lipid metabolism,including TG to GP transformation with less saturation andshorter lengths in FA chains. Interestingly, it has been commonlyrecognized that steatosis in NASH is an important risk factor forHCC. However, we do not routinely observe neutral lipid accu-mulation inmost typicalHCC tissues, suggesting that the steatosismay just be a transient state of certain tumor-causing hepatocytes.In the steatotic hepatocytes, spontaneous mutations in theWnt/Myc pathway may benefit from the TG-rich cellular envi-ronment caused by aberrant Ras signaling or activation of otheroncogenic signaling pathways, and be more functional in induc-ing tumorigenesis. Similarly, the antisenescence effect ofWnt/Mycsignaling is only observable upon Ras oncogenic insult, because

Figure 6.

MK8245 suppresses proliferation of human HCC cells. A, Dose–response curves and statistics of the IC50 values of MK8245 and sorafenib in human HCC linesdetermined by the MTT assay (n ¼ 5/assays). B, Treatment of MK8245 and sorafenib displayed antiproliferation effect on HCC cells; exogenous oleic acidattenuated the antiproliferation effect of MK8245 (n ¼ 5/assays). Data, mean � SD. Statistical differences were determined by one-way ANOVA. ���� , P < 0.0001.C, Drug combinational studies and synergy quantification of MK8245 and sorafenib in Li-7, Huh7, and HepG2 cell lines. Compounds were used in combinationin a fixed dose ratio for 48 hours, and cell viability was assessed with MTT assay. The IC50 values and fractional effect versus combination index (Fa–CI)curves were calculated and generated in CalcuSyn software.

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normal liver does not contain many senescence cells. However,the hepatic senescence was not affected by manipulating lipidmetabolism in double transgenic larvae, suggesting the repressionof senescence may not be linked to lipid remodeling at all in ourzebrafish models.

How to target the canonical Wnt signaling pathway for HCCtreatment?

Cumulative evidence supports the importance of Wnt activityin HCC diagnosis and prognosis (42). Many efforts have beenmade to develop therapeutic approaches targeting the Wnt path-way, and several relevant inhibitors or peptide drugs are in clinicaltrials. However, because theWnt pathway is necessary for homeo-stasis in normal tissues, most of these therapies display commonadverse effects including fatigue, alopecia, diarrhea, and nausea(42). In our study, we propose FA desaturation as a potentialmetabolic weakness featured by hepatocytes carrying aberrantWnt activity and show that inhibition of FAdesaturation via eithera drug or genetic approach represses zebrafish hepatic hyperplasiain Tg(Wnt&Kras) and Tg(Myc&Kras) (Fig. 5D; Supplementary Fig.S8B). Notably, neither the scd mutant nor the inhibitor-treatedlarvae display obvious developmental defects, suggesting that FAdesaturation may be a safer target to achieve a balance betweenantitumor functions and adverse side effects.

Zebrafish drug tests repurpose MK8245 as a candidate drug fortreating hepatic tumor

The most prominent candidate drug identified in the small-scale zebrafish screening is the SCD inhibitor MK8245. SCDcatalyzes the formation of MUFA (16:1 and 18:1) from SFA andis highly conserved between humans and lower organisms,including zebrafish and fruit flies (43). Recent studies suggestthat SCD is a transcriptional target of the canonical Wnt signalingpathway, and inhibition of SCD activity induces ER stress andapoptosis in several human HCC cells (44, 45).

Interestingly, the antiproliferation effect of SCD inhibition canbe circumvented by lipid scavenging in hypoxic or Ras-driventumor cells (5). The SCD inhibitor MK8245 is a liver-targetedmedicine for the treatment of diabetes and dyslipidemia currentlyin stage II clinical trials (46). In our studies using both zebrafishmodels andhumanHCCcells,MK8245displays antiproliferationeffects. AlthoughMK8245 also affects the development of normalhepatocytes, it merits further investigation for its possible effectson hepatic tumorigenesis in mammals.

Altogether, our studies not only improve our understanding ofthe complex regulatory outputs of primary oncogenic pathways

but also demonstrate zebrafish larva as a powerful and convenientin vivoplatform for target identification anddrug screening. Futurework will fully assess the biochemical functions of SCD andMK8245 using mammalian systems, and may lead to futuretherapies for treating subsets of HCC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Yao, S. Sun, F. Fei, J. Fan, X. WangDevelopment ofmethodology:Y. Yao, S. Sun, J.Wang, F. Fei, Z.Dong, L. Zhang,M.-B. Ji, X. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Yao, S. Sun, J. Wang, R. He, L. Wang, L. Zhang,M.-B. Ji, J. Fan, Z. Gong, X. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Yao, S. Sun, J. Wang, Q. Li, X. WangWriting, review, and/or revision of the manuscript: Y. Yao, F. Fei, Z. Dong,L. Wang, L. Zhang, Q. Li, X. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Sun, J. Wang, Z. Dong, A.-W. Ke, L. Zhang,M. Yu, G.-M. Shi, X. WangStudy supervision: M. Yu, X. Wang

AcknowledgmentsWe thank Drs. Qunying Lei (Fudan University) and Lijian Hui (Chinese

Academy of Sciences) for insightful discussion. This work was supportedby the National Natural Science Foundation of China (81402582,81602548, 81671725), Natural Science Foundation of Shanghai(14YF1400600 and 18ZR1404500), Foundation for Returned Oversea Chi-nese Scholars 48-12, Natural Science Foundation of Guangdong Province(2018A30310053), and National Key R&D Program of China(2016YFC0102100/A0301000). X. Wang received support from NationalNatural Science Foundation of China (81402582), Natural Science Foun-dation of Shanghai (14YF1400600 and 18ZR1404500), and Foundation forReturned Oversea Chinese Scholars 48-12. Y. Yao received support fromNatural Science Foundation of Guangdong Province (2018A30310053).Z. Dong received support from National Natural Science Foundation ofChina (81602548). M. Ji received support from National Natural ScienceFoundation of China (81671725) and National Key R&D Program of China(2016YFC0102100/A0301000).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 22, 2017; revised April 26, 2018; accepted July 24, 2018;published first July 31, 2018.

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Published OnlineFirst July 31, 2018; DOI: 10.1158/0008-5472.CAN-17-3964