HBx-K130M/V131I Promotes Liver Cancer in Transgenic Mice ...K130M/V131I, which overlaps both the HBx gene and basal core promoter (BCP), have been consistently shown to have the high-est

Signal Transduction and Functional Imaging

HBx-K130M/V131I Promotes Liver Cancer inTransgenic Mice via AKT/FOXO1 SignalingPathway and Arachidonic Acid MetabolismAmyP.Chiu1,2, Barbara R.Tschida3,Tung-Ting Sham1,2, Lilian H. Lo1,2, Branden S. Moriarity3,Xiao-Xiao Li1,2, Regina C. Lo4, David E. Hinton5, Dewi K. Rowlands6, Chi-On Chan1,2,Daniel K.W. Mok1,2, David A. Largaespada3, Nadia Warner7, and Vincent W. Keng1,2

Abstract

Chronic hepatitis B viral (HBV) infection remains a highunderlying cause for hepatocellular carcinoma (HCC) world-wide, while the genetic mechanisms behind this remainunclear. This study elucidated the mechanisms contributingto tumor development induced by the HBV X (HBx) geneof predominantly Asian genotype BHBV and its commonHBxvariants. To compare the potential tumorigenic effects ofK130M/V131I (Mut) and wild-type (WT) HBx on HCC, theSleepingBeauty (SB) transposon systemwasused todeliverHBxMut and WT into the livers of fumarylacetoacetate hydrolase(Fah)-deficientmice and in the context of transformation relatedprotein 53 (Trp53) deficiency. From our results, HBx Mut hada stronger tumorigenic effect than its WT variant. Also,inflammation, necrosis, and fibrosis were evident in HBxexperimental animals. Reduction of forkhead box O1(FOXO1) with increased phosphorylation of upstream ser-

ine/threonine kinase (AKT) was detected under HBx Mutoverexpression. Thus, it is proposed that HBx Mut enhancesdisease progression by reducing FOXO1 via phosphorylationof AKT. At the metabolomic level, HBx altered the expressionof genes that participated in arachidonic acid (AA) metabo-lism, as a result of inflammation via accumulation of proin-flammatory factors such as prostaglandins and leukotriene inliver. Taken together, the increased rate of HCC observed inchronic hepatitis B patients with K130M/V131I-mutated Xprotein, may be due to changes in AA metabolism and AKT/FOXO1 signaling.

Implications: Our findings suggested that HBx-K130M/V131I–mutant variant promoted HCC progression by activat-ing AKT/FOXO1 pathway and inducing stronger inflamma-tion in liver via AA metabolism.

IntroductionLiver cancer is the fourthmost common cause of cancer-related

deathsworldwide, accounting for about 700,000deaths annually.Themajor type of primary liver cancer is hepatocellular carcinoma

(HCC), which accounts for 85% to 90% of total liver cancercases (1–3). Chronic hepatitis B viral (HBV) infection has beenshown to have a strong relationship with liver cirrhosis andHCC (4). HBV infection accounts for more than 50% of all HCCcases globally (5). HBV infection is an endemic disease, in whichthe prevalence of chronic HBV infection diverges widely in dif-ferent parts of the world. About 50% of all new liver cancer casesdiagnosed were from mainland China (1–3, 6, 7). HBV is cate-gorized into 10 genotypes (A to J) and various subgenotypesaccording to its geographic distributions (3). HBV GenotypesB and C are widely distributed in East and Southeast Asia (3)and are predominantly found in theHan population inmainlandChina (8).

HBx is encoded by the X open reading frame of HBV. It plays acrucial role in hepatocarcinogenesis, functioning as a transcrip-tional activator that interacts with nuclear transcription factorsand modulates cytoplasmic signaling pathways, such as the RAS/RAF/MAP and WNT/CTNNB1 signaling pathways (9, 10). HBxcan also promote replication of HBV and regulate biologicalprocesses, such as host gene transcription, cell-cycle progression,apoptosis, and oxidative stress (3, 11, 12). HBx has also beenreported to induce inflammation, angiogenesis, immuneresponses, and multidrug resistance, accelerating HCC progres-sion (10, 13, 14).

Mutations in the HBV genome are very common in patientschronically infected with HBV and can be biomarkers for HCCdevelopment (15). Several HBx gene mutation hotspots,

1State Key Laboratory of Chinese Medicine and Molecular Pharmacology(Incubation), The Hong Kong Polytechnic University Shenzhen Research Insti-tute, Shenzhen, China. 2Department of Applied Biology and Chemical Technol-ogy, The Hong Kong Polytechnic University, Hong Kong, China. 3Center forGenome Engineering, Department of Pediatrics, Masonic Cancer Center, Uni-versity of Minnesota, Minneapolis, Minnesota. 4Department of Pathology,The University of Hong Kong, Hong Kong, China. 5Nicholas School of theEnvironment, Duke University, Durham, North Carolina. 6Laboratory AnimalServices Centre, The Chinese University of Hong Kong, Sha Tin, New Territories,Hong Kong, China. 7Victorian Infectious Diseases Reference Laboratory, ThePeter Doherty Institute for Infection and Immunity, Victoria, Australia.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Vincent W Keng, Department of Applied Biology andChemical Technology, The Hong Kong Polytechnic University, Y803, Lee ShauKee Building, The Hong Kong Polytechnic University, Hung Hom, Hong Kong.Phone: 852-3400-8728; Fax: 852-2364-9932; E-mail:[email protected]

Mol Cancer Res 2019;17:1582–93

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including A12T, V44I, A66T, H86Y, E109D, and K130M/V131Ican be found in HBV carriers. The dual amino acid changes atK130M/V131I, which overlaps both the HBx gene and basal corepromoter (BCP), have been consistently shown to have the high-est potential to promote HCC progression (10, 16–18). HBVpatients with chronic hepatitis, fulminant hepatitis, or HCC areoften detected with mutations at K130M/V131I sites and diag-nosed as HBeAg negative (18–20). A recent meta-analysis of 54studies examining the association between these mutations andHCC showed that X_K130M/V131I are detected in 66.5%ofHCCcases, compared with 39.8% of non-HCC cases (21). These Xprotein changes are encoded by nucleotide changes in the BCPregion A1762T/G1764A (BCP_A1762T/G1764A), and have beenshown to be associated with decreased PreC/C RNA synthesis anddecreased expression of HBeAg (20). However, the effect of theencoded protein changes on the tumorigenic potential of HBx isnot well understood. Although several studies have reported theinvolvement of HBx inHCCdevelopment, the differential tumor-igenicity of HBx mutations and the genetic mechanism(s) bywhich HBx induces and/or contributes to HCC developmentremains uncertain, and most studies have been performed usingtransformed liver cell lines such as Huh7.

The fumarylacetoacetate hydrolase (Fah)-deficient/Sleeping Beautytransposase 11 knock-in (Fah/SB11) transgenic mouse model hasbeen established and used to test the oncogenic role of candidateliver cancer genes (9, 22–26). This mouse model allows forselective repopulation of hepatocytes with the expression ofdelivered transgenes. This Fah-deficientmousemodel has a defectin hydrolyzing acetoacetate and fumarate in the tyrosine catabolicpathway. Therefore, it must be treated with nitisinone (NTBC) inthe drinking water to prevent tyrosinemia (27). Codelivery ofoncogenes and Fah cDNA in a transposon vector and removal ofNTBC after gene delivery, the constitutively expressed SB trans-posases recognize and integrate the transposon into the mousegenome in a cut-and-pastemanner (23). Eventually, Fah-deficienthepatocytes will die, while hepatocytes with stable integrationand expression of delivered transgenes can survive and repopulatethe liver (9, 23–25).

The aim of the study reported, herein, was to discover theimpact of K130M/V131I mutant and wild-type (WT) variantsof HBx-B on HCC progression and explore the genetic mech-anism(s) involved with liver tumorigenesis using the Fah/SB11transgenic mouse model.

Materials and MethodsGeneration of Fah-deficient/Rosa26-SB11 transposasetransgenic mice

All animal work was conducted under approved animalwelfare protocols of The Chinese University of Hong Kongand The Hong Kong Polytechnic University (Hong Kong, SAR).Fah-deficient mice were bred with Rosa26-SBTg/Tg to obtainexperimental animals that were deficient for Fah and transgenicfor SB transposase (Fah/SB11). All Fah-deficient mice weremaintained on 6 mg/mL 2-(2-nitro-4-trifluoromethylben-zoyl)-1,3-cyclohexanedione (NTBC, Swedish Orphan Interna-tional AB) in the drinking water until hydrodynamicallyinjected with transposon vector containing the Fah cDNA (27).The genotypes of Fah/SB11 transgenic mice were confirmed byPCR genotyping using primers specific for Fah alleles andRosa26-SB11 as described previously (9).

Plasmid constructionThe PCR products of K130M/V131I mutant andWT variants of

HBx (GOIs) were provided by N. Warner (Victorian InfectiousDiseases Reference Laboratory, The Peter Doherty Institute forInfection and Immunity, Victoria, Australia). These GOIs wereintroduced into theGateway entry clonepENTR11Dual SelectionVector (Life Technologies) to produce pENTR-GOI. The GOI inthe entry clone was introduced into the destination vector (pT2/GD-DEST-EGFP for in vivo experiments or pPB/SB-DEST-EGFP forin vitro experiments) by LR clonase reaction using Gateway LRclonase II Enzyme mix (Life Technologies) to give the pT2/GD-GOI-EGFP or pPB/SB-GOI-EGFP (Supplementary Fig. S1). Ashort hairpin RNA directed against the Trp53 gene was alsoincorporated into a transposon vector (shp53) for stable integra-tion into the mouse hepatocyte genome (refs. 9, 24, 25, 28;Supplementary Fig. S1A). All vectors used for hydrodynamic tailvein injections were prepared using EndoFree Plasmid Maxi Kit(Qiagen).

Hydrodynamic tail vein injectionTwenty micrograms of pT2/GD-HBx-B_Mut-EGFP or pT2/GD-

HBx-B_WT-EGFP was coinjected with shp53-containing plasmidinto 45-day old Fah/SB11mice hydrodynamically. After injection,NTBC water was replaced with normal drinking water. The FahcDNA in the expression vectors was coexpressed and allowed forselective repopulation of hepatocytes with stable transposonvector integration. These mice were allowed to age and sacrificedaround 150 to 200 days posthydrodynamic injection (PHI; Sup-plementary Fig. S1B). Historical controls for pT2/GD-Empty-EGFP with pT2/shp53 were used in this study.

Liver tumor analysesThe whole liver was removed from the euthanized animal,

weighed, rinsed, and placed in cold PBS. The number of livertumor nodules on rostral and caudal surfaces of all liver lobeswere counted and carefully removed using fine forceps and placedin fresh cold PBS. These isolated tumor nodules were then cut intosections forDNA, RNA, andprotein extraction using a sterile razorblade. Histology sections were taken from larger tumor nodules.Tissue samples for RNA extractionwere kept in 200 mL of RNAlaterStabilization Solution (Life Technologies) and stored at �80�C.DNA extraction was done as described in the PCR genotypingsection. RNA was extracted using TRIzol reagent (Life Technolo-gies) following the protocol provided by the manufacturer. Pro-tein was extracted using QproteomeMammalian Protein Prep Kit(Qiagen) following the protocol provided by the manufacturer.

Histologic analysesFormalin-fixed, paraffin-embedded liver tissues were pro-

cessed, sectioned at 5-mm using a standard microtome (Leica),mounted, and heat-fixed onto glass slides at 55�Covernight usingstandard protocols. These tissue slides were then stained withhematoxylin and eosin (H&E) following standard protocols, andIHC staining as described previously (9). Dilution of primaryantibodies used was as follows: pAKT (Ser473; Cell SignalingTechnology) was diluted in ratio of 1:250, b-Catenin (D10A8) XP(Cell Signaling Technology) was diluted in ratio of 1:500, Ki67(Abcam) was diluted in ratio of 1:200, FAH (Abcam) was dilutedin ratio of 1:250, and p53 (DO-1; Santa Cruz Biotechnology) wasdiluted in ratio of 1:100). Board-certified pathologist performedthe histopathologic analyses.

Potential Pathways Contribute to HBx-K130M/V131I-Induced HCC

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Semiquantitative RT-PCRRNA from liver tissues was extracted using TRIzol reagent (Life

Technologies) following the protocol provided by the manufac-turer. The first-strand cDNA was synthesized from 250 ng mRNAusing the SuperScript First-Strand Synthesis System for RT-PCR(Life Technologies). Reactions with (RTþ) and without (RT-) thereverse transcriptase were performed for all samples. This cDNAwas used as template for subsequent PCR with various primerpairs (Supplementary Materials and Methods). PCR conditionswere similar to PCR genotyping with reduced cycles to avoidamplicon saturation. Semiquantitative analyses of unsaturatedamplicons were measured using ImageJ software. Intensity ofbands was calculated as an arbitrary value relative to actin, beta(Actb) expression level.

Quantitative RT-PCRqPCR was performed to determine the expression level of

common downstream genes involved with cancer signalingpathways. The mRNA was extracted as mentioned previouslyusing 1 mg of mRNA as cDNA synthesis template and qPCR wasperformed using the QuantStudio 7 Flex Real-Time PCR System(University Research Facility in Life Sciences of The Hong KongPolytechnic University, Hong Kong). PCR reaction mixtureconsisted of a 1:10 dilution of cDNA in the SYBR Green Icontaining qPCR Master Mix (GoTaq qPCR Master Mix, Pro-mega) with specific primers (0.2 mmol/L final concentration ofeach primer). Primer sequences listed in Supplementary Mate-rials and Methods.

Western blot analysesProtein was extracted from liver sections of injected mice

using Qproteome Mammalian Protein Prep Kit (Qiagen) follow-ing the protocol provided by the manufacturer. Protein concen-tration was detected using standard protein assay (Bio-Rad)and 30 mg of protein was loaded into the SDS-PAGE and trans-ferred onto polyvinylidene difluoride membrane. Primary anti-bodies phospho-AKT, total AKT, non-phospho. (active)CTNNB1,total CTNNB1, FOXO1, and ACTB (Cell Signaling Technology)were diluted in 5% BSA at 1:1,000 concentration. Secondaryantibodies (anti-mouse or anti-rabbit) were diluted in 5% BSAat 1:2,000 concentration. Membrane was blocked with 5% BSA,then incubated with primary antibody at 4�C overnight followedby secondary antibody incubation at room temperature for anhour. The membrane was then washed with 1� TBST for three

times in 10-minute interval. Afterwards, membrane was visual-ized using ImmobilonWesternChemiluminescentHRPSubstrate(Millipore).

Cell culture and AKT inhibitor treatment assaySNU-449 were cultured with RPMI medium (Gibco, Thermo

Fisher Scientific) supplemented with 10% heat-inactivated FBS(Gibco, Thermo Fisher Scientific) and 1% antibiotic-antimycotic(Anti-Anti; Gibco, Thermo Fisher Scientific). Immortalizedhuman hepatic cell line, HHL7, was cultured with MEM supple-mented with 10% FBS and 1% Anti-Anti. HHL7 cells (5 � 104

cells) were transfected with 1 mg of pPB/SB-GOI-EGFP and 0.5 mgof transposase vector using ViaFect transfection reagents (Pro-mega) with a plasmid to reagent ratio of 1:3 in 6-well plate(Supplementary Fig. S1C). Puromycin was used for the selectionof positively transfected cells. SNU-449 and positively transfectedHHL7 cells (4,000 cells) were treated with various concentrations(3.125, 6.25, 12.5, 25, 50, 100, and 200 mmol/L) of AKT proteinkinase inhibitor (AZD5363,MedChemExpress) for 4 days in a 96-well plate. MTS assays were performed using the CellTiter 96Aqueous One Solution Reagent (Promega) according to themanufacturer's protocol (Supplementary Fig. S1C). Absorbanceof each well was measured at 492 nm. Percentage of cell survivalwere calculated by dividing the absorbance of treated cohorts tothe untreated control.

Serum metabolomic analysesBlood from HBx-B Mut/shp53, WT/shp53, and Empty/shp53-

injected mice was extracted during necropsy, kept at 4�C over-night and serum isolated. The procedure to prepare serum forUltra Performance Liquid Chromatography-Orbitrap-Massspectroscopy (UPLC-Orbitrap-MS), the UPLC separation meth-od and mass spectrometer parameters can be found in Supple-mentary Materials (Supplementary Materials and Methods). Anequal volume of each serum sample was pooled, vortexed, andaliquoted to provide pooled quality control (QC) samples andwent through the same extraction protocols of LC-MS asdescribed. They were injected to UPLC-Orbitrap-MS intermit-tently between samples for stability check of the instrumentsthroughout the runs.

The prepared serum samples were injected into a WatersACQUITY UPLC system (Waters). Mass spectrometry analysiswas achieved by Thermo ScientificOrbitrap Fusion Lumos Tribridmass spectrometer equipped with a heated electrospray

Figure 1.Tumor burdens in Fah/SB11 mice injected withWT andmutant (Mut) variants of HBx-B genotypes.A, Number of tumors found in experimental animals at about160-days PHI (HBx-BWT/shp53 (T), n¼ 10; HBx-B Mut/shp53 (T), n¼ 10; Empty/shp53 (T), n¼ 9). All mice were coinjected with shp53. Mean� S.D.; n, numberof animals; P, unpaired student t test. B, Representative image demonstrating diverse tumor burden observed in the HBx-B Mut/shp53 and HBx-BWT/shp53experimental animals. Arrows indicate tumor nodules; the orientation of the livers were optimized to show the most tumor nodules; scale bars, 0.5 cm. C, Liver towhole body weight ratio of experimental animals. Mean� S.D.; n, number of animals; P, Student t test, �P� 0.05. D, Representative IHC images for anti-Ki67staining in controls and liver tumors of Fah/SB11 animal injected with various HBx and shp53 transgenes. Left column, no primary antibody was used. Rightcolumn, anti-Ki67 primary antibody used. Top and Middle panels, wild-typemouse liver and non-injected Fah/SB11 liver from animal under the treatment of NTBCwater showing no detectable Ki67 activity, respectively. Bottom panels, Fah/SB11 animal injected with HBx-B Mut/shp53 showing strong nuclear staining of Ki67in both tumor (T) and peripheral tissue (P) area. Scale bars, 100 mm. Magnified image shown in right corner (solid-lined box) of each slide. Scale bars, 11 mm. E,Representative H&E staining images demonstrating dysplastic hepatocyte morphology of the experimental animals. (i, v) Non-injected Fah/SB11 liver undercontinuous treatment of NTBC showing normal hepatocyte morphology. (ii, vi) Fah/SB11 liver injected with empty vector control in the context of shp53. (iii)Multinucleated cells (indicated with green arrow) and pyknotic nuclei (indicated with yellow arrow) of hepatocytes were found in HBx/shp53 injected liver. (iv)More mitotic figures (indicated with red arrow) were noted in liver sections injected with HBx/shp53. Scale bars, 11 mm. (vii) Areas of lymophocytic inflammation(arrows) seen in Fah/SB11 mice with HBx gene with co-injection of shp53. (viii) Fibrosis observed in Fah/SB11 animals with HBx-B genotype co-injected withshp53. (ix) Tumor nodule (T) observed in Fah/SB11 animals HBx gene co-injected with shp53with dysplastic hepatic morphology seen in peripheral hepatocytes(P). Scale bars, 100 mm.






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ionization (H-ESI) interface (Thermo Fisher Scientific). Data wereanalyzed by Progenesis QI software 2.0 (Nonlinear Dynamics)and peak area was normalized by all compound with reference ofQC. Quality screening was done by filtering out those unstablemetabolites (coefficient of variation >30% in all pooled QCsamples). The remaining normalized data matrix were exportedto the Extended Statistical tool (EZinfo v2.0 software, UmetricsAB) for partial least squares-discriminant analysis (PLS-DA) soas to evaluate their classification performance. The parameter Q2

is used to assess the goodness of prediction for performanceof PLS-DA in a general acceptable threshold value of 0.5. Themetabolites that had significant difference between HBx-B Mut/shp53 and WT/shp53 experimental samples were identified bycomparing their m/z, mass fragmentation patterns, and chro-matographic retention times with commercially available refer-ence standards and/or online metabolite databases, such asHuman Metabolome Database (http://metlin.scripps.edu),Metlin (http://metlin.scripps.edu), and literatures.

Statistical analysesValues are given as mean � SD. Statistical significance was

assessed by two-tailed, unpaired Student t test with P values(Prism Software). P values >0.05 were considered statisticallysignificant.

ResultsDivergent effect on tumor burden bymutant andWT variants ofHBx-B

Expression vectors for pT2/GD-HBx-B_Mut-GFP and pT2/GD-HBx-B_WT-GFP were coinjected with a short hairpin RNA thattargeted Trp53 (shp53) into the tail vein of Fah/SB11 micehydrodynamically and the number of GFP-positive tumorscounted at around 160 days PHI. HBx-B WT/shp53 and HBx-BMut/shp53 variants induced an average tumor burden of 0.14 permouse and 1.25 per mouse, respectively. HBx-B Mut/shp53–injected mice (n ¼ 10; P ¼ 0.1272) displayed a trend towardhigher tumor burden than HBx-B WT/shp53 counterpart (n ¼10; Fig. 1A and B). There was a significant difference in liver towhole body weight ratio between HBx-B Mut/shp53 and HBx-BWT/shp53 groups (P¼ 0.0473) and both groups had significantlyenlarged livers relative towhole bodyweight ratio of Empty/shp53control group (P < 0.0001; Fig. 1C).

Inflammation, proliferation, hepatocytomegaly, and necrosisphenotype in HBx-injected animals

IHC andH&E-stained liver sections displayed hepaticmorpho-logic phenotypic changes as a result of HBx overexpression. Asexpected, normal nonproliferating livers were negative for the cell

proliferation marker Ki67 by IHC, while liver tumor nodulesisolated fromexperimental animals injectedwithHBx-Bgenewerestrongly Ki67-positive (Fig. 1D). Hepatocyte nuclei were enlargedwith apparent pyknotic changes, indicating the development ofhepatocellular necrosis (Fig. 1E, iii). In addition,multi-nuclei andmitotic figures were found in these sections (Fig. 1E, iii and iv).Furthermore, different degrees of inflammationwere observed onthe basis of the amount of lymphocyte aggregation in the injectedliver sections (Fig. 1E, vii). Histopathologic analyses revealedhigher inflammation events in livers injected with HBx-B Mut/shp53 than its corresponding HBx-B WT/shp53 counterpart(Fig. 2A, i). Signs of necrosis and degenerative changes were alsoobserved in liver sections of the injected animals (Fig. 2A, ii).Moreover, mild fibrosis was observed in mice injected with eitherWT or mutant HBx-B gene (Fig. 1E, viii and 2A, iii).

Transcriptional analyses of HBx-B–injected liverTo confirm that inflammation and tumor induction were

driven by the expression of HBx genes, semiquantitative RT-PCR was performed to examine the expression of green fluo-rescent protein (Gfp), HBx, and Fah transgenes flanked by thetransposon vectors. Both normal tissues and tumors isolatedfrom HBx-B Mut/shp53 and HBx-B WT/shp53 mouse liverexpressed the Gfp, HBx, and Fah transgenes (Fig. 2B), indicatingthe successful integration and expression of transgenes into thehepatocytes of injected experimental animals. Alpha-fetoprotein(Afp), a marker for HCC diagnosis, was detected in both HBx-BMut/shp53 and HBx-WT/shp53 groups by semiquantitative RT-PCR. Afp expression level in tumor was higher than that of itscorresponding adjacent microscopically normal liver samples(P ¼ 0.0599). The mRNA expression levels of myelocytomatosisoncogene (Myc) and cyclin D1 (Ccnd1), were analyzed in HBx-B/shp53–injected animals by qPCR. HBx-B WT/shp53 inducedsignificantly higher expression of Ccnd1 than Empty/shp53,while there were no significant differences among all groupsfor Myc mRNA expression (Fig. 2C). The rescued activity of Fahin injected mice was also confirmed by IHC, indicating thesuccessful integration, transcription, and translation of genesdelivered by the transposon vector (Supplementary Fig. S2A).As expected, control WT mouse livers were strongly FAH-positive, while noninjected Fah/SB11 control animals (underNTBC treatment) were FAH-negative by IHC staining (Supple-mentary Fig. S2A). There was no detectable TRP53 in either HBxor Empty/shp53 control cohorts that were coinjected with shp53by IHC staining (Supplementary Fig. S2B).

Increased AKT andCTNNB1protein levels inHBx-injectedmiceBoth PI3K/AKT and WNT/CTNNB1signaling pathways play

vital roles in regulating cellular differentiation, proliferation,

Figure 2.Histopathologic changes and evidence of AKT signaling involvement in hepatocytes of HBx-injected animals.A, Histopathology of livers injected with HBx-BMut/shp53 and HBx-BWT/shp53. (i) Occurrence of inflammation, (ii) necrosis, and (iii) fibrosis Levels of pathological events were graded from 0 to 3 (grade 0was the least severe and grade 3 was the most severe). B, RT-PCR of liver and tumor samples from representative experimental animals. RT-negative controlsgave no detectable bands at similar PCR cycle numbers for each gene tested (data not shown). N, peripheral normal tissue; T, tumor tissue; WT, normal WT adultliver. C, qPCR onmRNA expression level ofMyc and Ccnd1 in HBx-B Mut/shp53 and HBx-BWT/shp53 experimental animals. D, RepresentativeWestern blotanalyses of proteins extracted from liver of Fah/SB11 mice injected with HBx-B Mut/shp53 (left) and HBx-BWT/shp53 (right) using Non-phospho (active)CTNNB1, CTNNB1, pAKT, AKT, FOXO1, and ACTB antibodies (top) and its semiquantitatve protein level with respect to its corresponding ACTB level (bottom).Mean� SD; P, unpaired Student t test, � P� 0.05, �� P� 0.01, �� P� 0.001. E, Survival rate of HBx-expressing cell lines under different concentrations of AKTprotein kinase inhibitor (AZD5363). Mean� SD; P, ordinary one-way ANOVA, � P� 0.05, �� P� 0.001, �� P� 0.0001. Representative RT-PCR analyses for theHBx genes in SNU-449 and transfected HHL7 cells.





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and survival in HCC. To identify underlying mechanisms ofHBx-induced HCC, activation of these two pathways wereexamined. Western blot analyses were performed on proteinextracted from experimental cohorts using nonphospho.(active) CTNNB1 (active CTNNB1), CTNNB1, phospho-AKT(ser473) (pAKT), AKT, and FOXO1 primary antibodies(Fig. 2D). The protein levels of total CTNNB1 and total AKTwere significantly higher in HBx-B Mut/shp53 than that ofEmpty/shp53, while FOXO1 protein level was significantlylower in HBx-B Mut/shp53 than that of Empty/shp53, andthere were a trend showing higher protein level of activeCTNNB1and phospho-AKT in HBx-B Mut/shp53 than thatof Empty/shp53 (Fig. 2D, left). However, only phospho-AKT protein level was significantly increased in HBx-B WT/shp53 compared to Empty/shp53 and there were no signi-ficant differences in pAKT, active and total CTNNB1, andFOXO1 levels between HBx-B WT/shp53 and Empty/shp53(Fig. 2D, right). Furthermore, IHC of liver tumor nodulesisolated from mice injected with HBx-B Mut/shp53 and HBx-BWT/shp53 groups were strongly positive for nuclear pAKTand membranous active CTNNB1 (Supplementary Fig. S2Cand S2D). The effect of suppressing AKT signaling pathwayon cell survival was performed on SNU-449, a HBV-positiveliver cancer cell line, and immortalized human hepatic cellline HHL7 transfected with HBx genotype B mutant and WTgenes. Treatment with AKT inhibitor AZD5363 could signi-ficantly reduce the survival rate of SNU-449 and HBx-transfected HHL7 cell lines compared with nontreated cells(Fig. 2E).

RNA-seq revealed crucial roles ofmetabolomic reprogrammingin HBx-B–injected mice

RNA-seq was performed to quantify the RNA expressionprofile of HBx-B-injected animals. RNA from HBx-B Mut/shp53tumors (n ¼ 3) and normal tissues (n ¼ 4), HBx-B WT/shp53normal tissues (n¼ 3), and Empty/shp53 normal tissues (n¼ 3)were analyzed by Illumina sequencing and expression level ofeach gene was calculated by fragments per kilobase of exon permillion reads mapped (FPKM). Raw RNA-seq data wereuploaded to Sequence Read Archive with the accession numberPRJNA529044. On the basis of the gene expression level,differentially expressed genes (DEGs) were identified betweengroups. Fold change in gene expression between two sampleswas calculated by log 2 FPKM ratio of two samples. Significantgene expression differences were filtered with log2 ratio greateror equal to 1 (for upregulation) and smaller or equal to �1 (fordownregulation).

Thenumber ofDEGsbetween thenormal tissues ofHBx-BMut/shp53 against Empty/shp53 control, HBx-B WT/shp53 againstEmpty/shp53 control, and HBx-B Mut/shp53 against HBx-B WT/shp53 were low (Supplementary Fig. S3A). Comparing RNAexpression profiles of tumors (n ¼ 3) and normal (n ¼ 4) tissuesof HBx-B Mut–injected mice, 377 DEGs were totally identified:235 geneswere upregulated and142 geneswere downregulated inthe tumor compared with its normal tissues (Supplementary Fig.S3A and S3B). Insulin growth factor 2 (Igf2), Rous sarcoma oncogene(Src), p21 (RAC1) activated kinase 6 (Pak6), as well as well-knownoncogenes, Myc and matrix metallopeptidases (Mmps), were upre-gulated in the tumors from the HBx-B Mut/shp53 cohort (Fig. 3;

Figure 3.

The schematic summary of molecular pathways induced by HBx-B genes. Asterisks (�) represent changes in metabolites detected by UPLC-MS in serum of HBx-Bexperimental animals. Up arrows represent upregulated DEGs, and down arrows represent downregulated DEGs.

Chiu et al.





Supplementary Table S1). Transcriptional mRNA expression oftargeted genes, such as Igf2, Src, Pak6, and Mmp7 were furtherconfirmed by qPCR in nonsequenced tumor (n ¼ 5) and normal(n ¼ 5) samples. Significant upregulation of these targeted geneswas detected in HBx-B Mut/shp53 injected cohort compared withtheEmpty/shp53 control (Fig. 4A).UsingKEGGpathway analyses,theDEGs show significant alterations for both canonical signaling

and metabolic pathways. Among the canonical signaling path-ways, most of the DEGs were involved in common canceroussignal transduction pathways such as Ras signaling, PI3K–Aktsignaling, ECM–receptor interaction, MAPK signaling, and Notchsignaling (Supplementary Table S2).

Apart from the canonical signaling pathways, a large numberof genes were associated with various metabolic pathways (59/

Figure 4.

Expression level validation of targeted DEGs andmetabolite profiles of AAmetabolism pathway.A, Relative mRNA expression level of Igf2, Src, Pak6, andMmp7were shown to be significantly upregulated in tumors of HBx-B Mut/shp53–injected cohort compared with empty control. Mean� SD; P, unpaired Student t test,� , P� 0.05, �� , P� 0.01, ��, P� 0.001. B, Significantly higher ion abundance of prostaglandin B2, D2 and E2, leukotriene B4, and 12-HETE in HBx experimentalcohorts compared with empty control. Mean� SD; P, unpaired Student t test, � , P� 0.05, �� , P� 0.01, �� , P� 0.001; �� , P� 0.0001.






519 genes). This suggests that metabolic changes played asignificantly important role in promoting tumor progression.AA metabolism is one of the metabolic pathways with thehighest number of DEGs involved (13 DEGs; Fig. 3; Supple-mentary Table S2). DEGs involved in AA metabolism, includ-ing prostaglandin-endoperoxide synthase 2 (Ptgs2), secretoryphospholipase A2 (Pla2g), and arachidonate 12-lipoxygenase(Alox12) were upregulated, while cytochrome p450 monoox-ygenase, such as Cyp2 and Cyp4f, genes encoding xenobioticmonooxygenase and leukotriene-B4 (LTB4) 20-monooxygen-ase were downregulated (Fig. 3; Supplementary Table S1). Theabundance of metabolites, such as prostaglandin B2, D2 andE2, LTB4, and 12-hydroxyeicosatetraenoic acid (12-HETE),from AA metabolism were shown to be significantly higher inthe HBx genotype B mutant and WT experimental cohortscompared with the Empty/shp53 control (Fig. 4B).

Serum metabolomics analyses using UPLC-Orbitrap-MSRNA-seq results indicated metabolic disorder was highly

associated with tumor development in HBx-B–injected mice.Therefore, serum samples from experimental mice were used toperform metabolomics analysis using UPLC-Orbitrap-MS todetermine metabolic changes in the context of HBx overexpres-sion. Fold changes of metabolites were compared betweenmutant and WT cohorts. A significantly higher abundance ofxanthine (67.23-fold increase), as well as its downstream meta-bolites urate (2.15-fold increase) and 5-hydroxyisourate (3.55-fold increase)were detected in themutant cohort comparedwithits WT cohort (Fig. 3; Supplementary Fig. S4B). These metabo-lites are produced during the break down of tumor cells (29),providing further evidence of involvement of HBx-B–mutantgene in liver tumorigenesis. Furthermore, metabolites 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME) involved inlinoleic acidmetabolismwere significantly reduced (P < 0.05) inmutant serum than that of WT serum. Both serum metabolites'abundance and RNA expression level of Cyp2C and Cyp2e1,genes encoding enzymes that break down linoleic acid, weredownregulated (Fig. 3; Supplementary Fig. S4B). In addition,ion abundances of metabolites, such as taurocholate (TCA),taurochenodeoxycholate (TCDCA), and taurohyodeoxycholate(THDCA)/tauroursodeoxycholate (TUDCA), in the synthesis ofbile acids (BA) from cholesterol were significantly reduced in themutant cohort compared with its WT cohort. This finding wasconsistent with the RNA-seq result that the expression level ofCyp7a1, which encodes the key enzyme that converts cholesterolto BAs, was significantly downregulated (Fig. 3; SupplementaryFig. S4B).

DiscussionUsing the Fah/SB11 mouse model, K130M/V131I mutant

variant of HBx-B induced a higher tumor burden than itsWT counterpart and significantly increased the liver weight ofthe experimental animals. Hepatocytes of HBx-B Mut/shp53-and WT/shp53-injected mice were larger than those of Empty/shp53 control and Fah/SB11 noninjected mice, indicating hepa-tomegaly. In another experimental cohort, in which Fah/SB11mice were injected with HBx-B Mut and WT alone, the numberof tumors induced were similar to the experimental cohortsthat were coinjected with shp53 (data not shown). On the

basis of this evidence, inactivation of Trp53 did not contributeto tumorigenesis at time of necropsy (between 160 and200 PHI).

In addition, severe inflammation, mild necrosis, and fibrosiswere observed in livers injected with HBx-B Mut/shp53 indicatingthe development of chronic hepatitis. From the RNA-seq analyses,the AA metabolism pathway associated with proinflammatoryprocess was activated. From the metabolite profiles of the exper-imental cohorts, significantly higher abundance of the proinflam-matory factors, such as prostaglandin B2, D2, E2, LTB4 and theprocarcinogenic factor, 12-HETE, from AA metabolism weredetected in the HBx-mutant and WT experimental cohorts com-pared with the Empty/shp53 control (Fig. 4B). These resultssuggest that HBx-B could induce liver inflammation through theactivation of the AAmetabolism pathway, producingmore proin-flammatory factors and attract the accumulation of leukocytesinto the liver tissues.

Both IHC and Western blot analyses confirmed expression ofeither HBx-B Mut or WT could activate phosphorylation of AKTand overexpress CTNNB1 in our injected mice. Moreover, theprotein level of FOXO1 was significantly reduced in HBx-B Mut/shp53 group. FOXO1 is a transcriptional factor that mediates theexpression of downstream target genes involved in cell prolifer-ation, apoptosis, cell-cycle arrest, and metabolism (30, 31).FOXO1 is also a downstream target of AKT, which can be phos-phorylated by pAKT and translocated to the cytoplasm, andsubsequently degraded via ubiquitination (30). Therefore, wepropose that HBx-B–mutant variant increased cell proliferationand promoted cell-cycle progression by activating AKT phosphor-ylation, which in turn, phosphorylated and translocated FOXO1to cytoplasm for ubiquitination, resulting in reduced FOXO1abundance in cells. In transcriptional analysis, both HBx-BMut/shp53 and WT/shp53 induced overexpression of Afp, Myc,and Ccnd1.

RNA-seq was performed to quantify the RNA expression pro-files between tumor and its peripheral normal tissue of HBx-BMut-injectedmice. The sequencing results highlighted the impor-tance of signal transduction pathways, such as PI3K–Akt signalingpathway, andmetabolic pathways, includingAAmetabolism. ThemRNA expression levels of Igf2, Src, Pak6, and Mmps were signif-icantly increased in tumor of HBx-B Mut/shp53–injected animalscomparing with that of normal tissues. Igf2 encodes for a mito-genic peptide, namely insulin-like growth factor 2 (IGF2). OtherIGF components such as IGF2-mRNA binding protein (IGF2BP)and IGF-binding proteins (IGFBPs) are required to initiate thesignaling cascade of IGF2 (32). Ingenuity Pathway Analysisupstream regulator analyses showed that expression of Igf2 wasregulated by CTNNB1 and PI3K/AKT (Supplementary Table S3).In addition, Igfbp1, which was also upregulated in tumors of HBx-B Mut/shp53 cohort, was regulated by FOXO1 (SupplementaryTable S3). These results are consistent with our translational andtranscriptional analyses and further confirmed the activity of AKT/FOXO1 signaling pathway on regulating cell growth, prolifera-tion, and survival via IGF2. Upregulation of IGF2was significantlyassociatedwith risk of hepatoblastomadevelopment (32).On thebasis of a previous study on gene expression profiling of patientswith HCC acquired from various risk factors, IGF2 was one ofthe differentially expressed genes found exclusively in HBV-induced HCC cases (33). Activation of AKT and upregulation ofSrc in the HBx-B Mut cohort triggered downstream-cascadedevents resulting in upregulation and activation of mDia1, which

Chiu et al.





was responsible for actin regulation and associated with cellmotility. A recent study published that high levels of Srccould promote cell proliferation and invasion (34). In addition,protein tyrosine kinase 2 (PTK2)/Src complex could also activateMAPK, WNT, and PI3K–AKT signaling pathways responsible forcell proliferation and cell survival by overexpressing oncogenessuch asMyc andMmps, which were also upregulated in our DEGslist. Furthermore, Src is also an upstream target of PAK, whichis encoded by Pak6 and is an effector of RAC proteins. Activationof Rac cascade triggers actin polymerization and induces mem-brane protrusion and cell spreading, resulting in mesenchymalcell morphology (35). PAK also possesses oncogenic roles thatcan regulate cell proliferation, cell survival, adhesion, andmigration (36).

DEGs involved in AA metabolism were affected significantly;Ptgs2, Pla2g, and Alox12 were upregulated, while Cyp2, Cyp4f,genes encoding xenobiotic monooxygenase, and LTB4 20-monooxygenase were downregulated. As mentioned previous-ly, AA metabolism is highly associated with inflammationevents in liver (37, 38). Thus, we hypothesized that HBx Mutcould induce severe inflammation in liver via AA metabolismpathway by upregulating expression of Ptgs2, which encodes for

prostaglandin-endoperoxide synthase that converts the unsta-ble product prostaglandin G2 (PGG2) to prostaglandin H2

(PGH2) that would be further converted to other prostaglan-dins (PGs), including PGE2, PGD2, and PGB2, resulting inoverproduction of proinflammatory factors. Studies haveshown that these PGs can promote tumor growth and arehighly abundant in colon, lung, breast, and head and neckcancers (39–43). In addition, accumulated PGs could activateRAS/ERK and glycogen synthase kinase 3 beta (GSK3B)/CTNNB1 signaling pathways and hence induce cell prolifera-tion (39). On the other hand, the gene encoding LTB4 20-monooxygenase was downregulated. LTB4 20-monooxygenaseis an enzyme that converts LTB4 to 20-OH-LTB4, the down-regulation of gene encoding LTB4 20-monooxygenase causedthe accumulation of LTB4 in cells. LTB4 is also known to be aproinflammatory factor that attracts leukocytes to the injuredtissue (44). Moreover, LTB4 could also activate MEK/ERK andPI3K/AKT pathways through the interaction with the Gprotein–coupled receptors BLT2 in human pancreatic cancercells (45, 46). Furthermore, ALOX12 has been described as apotential procarcinogenic enzyme (39). The higher abundanceof 12-HETE, product of ALOX12, was detected in

Figure 5.

The schematic image of HBx/AKT/FOXO1 signaling pathway and AAmetabolism–induced inflammation. HBx-B Mut-promoted tumor progression viaphosphorylation of AKT in PI3K/AKT signaling pathway and reduced FOXO1 production. HBx-B Mut gene induced inflammation via AAmetabolism byupregulating Ptges and downregulating gene coded for LTB4 20-monooxygenase, resulting in accumulation of proinflammatory factors, such as prostaglandins(PGs) and LTB4 around infected cells (47).






HBx-B–injected experimental cohorts compared with Empty/shp53 control, suggesting that HBx gene might also promotetumor progression by upregulation of Alox12.

All results shown herein confirm that HBx-B K130M/V131I–mutant variant displayed a stronger tumorigenic effect than itsWTcounterpart. The X_K130M/V131I amino acid changes areencoded by nucleotide changes at BCP_A1762T/G1764A, whichalso result in a decrease in PC/C mRNA and subsequent HBeAgexpression. These changes have been strongly associated with thedevelopment of HCC in many clinical studies; however, whetherthe increasedHCC is associatedwith the decrease inHBeAg levels,or mutation of the X protein remains unknown. To our knowl-edge, ours is the first study to show a direct effect of these Xmutations on HBx-associated tumorigenesis. We suggest thatHBx-B mutant variant promoted tumor progression via phos-phorylation of AKT in the PI3K/AKT signaling pathway andreduced FOXO1 production, resulting in upregulation of Igf2.Moreover, it significantly altered the metabolism of AA, whichcontributed to inflammation of HBx-B–injected animals. HBxMut induced inflammation via AA metabolism by accumulationof proinflammatory factors such as the different types of PGs,LTB4, and 12-HETE to the infected tissues (Fig. 5; ref. 47). Takentogether, these results provide a potential mechanism, wherebyHBV encoding X_K130M/V131I (BCP_A1762T/G1764A) maycontribute to the high rate of HCC observed clinically in patientcohorts containing these mutations.

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

Authors' ContributionsConception and design: A.P. Chiu, D.A. Largaespada, N. Warner, V.W. KengDevelopment of methodology: A.P. Chiu, D.K. Rowlands, D.K.-W. Mok

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.P. Chiu, B.R. Tschida, T.-T. Sham, L.H. Lo,D.K.-W. MokAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.P. Chiu, T.-T. Sham, L.H. Lo, X.-X, Li, R.C. Lo,D.E. Hinton, C.-O. Chan, D.K.-W. MokWriting, review, and/or revision of the manuscript: A.P. Chiu, B.R. Tschida,T.-T. Sham, B.S. Moriarity, D.E. Hinton, D.K. Rowlands, D.K.-W. Mok,D.A. Largaespada, N. Warner, V.W. KengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.P. Chiu, T.-T. ShamStudy supervision: D.A. Largaespada, V.W. KengOther (generated reagents): B.S. MoriarityOther (performed histopathological analysis of liver sections): D.E. HintonOther (performed the serum metabolomics analyses): C.-O. Chan

AcknowledgmentsThis project was supported by The Shenzhen Science and Technology

Innovation Commission (JCYJ20170413154748190, to V.W. Keng;JCYJ20160229173844278, to D.K.-W. Mok; and JCYJ20160330171116798,to C.-O. Chan), Health Medical Research Fund (11122171), Food andHealth Bureau, and the Hong Kong SAR Government, NSFC/RGC JointResearch Scheme (N-PolyU503/16; to V.W. Keng) and the NIH IMVTP grantno. T32 AI083196-04 (to B.R. Tschida), General Research Fund of theResearch Grant Council of the Hong Kong Special Administrative Region(Project no. 15302718), Hong Kong Chinese Materia Medica StandardsProject (to D.K.-W. Mok), the Large Equipment Funds and UniversityResearch Facility in Chemical and Environmental Analysis and Life Sciencesof the Hong Kong Polytechnic University.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedOctober 20, 2018; revised February 18, 2019; accepted April 8, 2019;published first April 11, 2019.

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2019;17:1582-1593. Published OnlineFirst April 11, 2019.Mol Cancer Res Amy P. Chiu, Barbara R. Tschida, Tung-Ting Sham, et al. AKT/FOXO1 Signaling Pathway and Arachidonic Acid MetabolismHBx-K130M/V131I Promotes Liver Cancer in Transgenic Mice via

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HBx-K130M/V131I Promotes Liver Cancer in Transgenic Mice ...K130M/V131I, which overlaps both the HBx gene and basal core promoter (BCP), have been consistently shown to have the high-est