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GASTROENTEROLOGY 2004;127:1110–1122

LINICAL–LIVER, PANCREAS, AND BILIARY TRACT

unctional Consequences of Frizzled-7 Receptor Overexpressionn Human Hepatocellular Carcinoma

HILIPPE MERLE,*,‡ SUZANNE DE LA MONTE,* MIRAN KIM,* MARC HERRMANN,*HINJI TANAKA,§ ANNETTE VON DEM BUSSCHE,* MICHAEL C. KEW,� CHRISTIAN TREPO,‡

nd JACK R. WANDS*The Liver Research Center, Department of Medicine and Pathology, Brown Medical School, Providence, Rhode Island; ‡Inserm U271, Viruses Hépatites et Pathologies Associées, Lyon, France; §Department of Surgery II, Kyushu University, Fukuoka, Japan; and �Department ofedicine, Witwatersrand University Medical School, Johannesburg, South Africa

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ackground & Aims: The molecular pathogenesis of hu-an hepatocellular carcinoma (HCC) is understoodoorly. In some tumors, activation of the Wnt/�-cateninathway as a result of �-catenin gene mutations haseen found. However, in many other HCCs, activation ofhe Wnt/�-catenin pathway has been shown in the ab-ence of such mutations. Methods: We previously havedentified the upstream human Frizzled-7 receptorFZD7) gene of this pathway. In the present study, auantitative real-time reverse-transcription polymerasehain reaction (RT-PCR) assay for FZD7 was developednd overexpression of FZD7 was detected in 90% ofumors, most of which were related to chronic hepatitisvirus infection. FZD7 also was overexpressed in the 6CC cell lines tested and functional analysis showedhat FZD7 messenger RNA (mRNA) levels correlatedith enhanced cellular motility. Results: Transfection ofCC cells with dominant-negative mutant constructs en-oding a C-terminally truncated FZD7 protein decreasedild-type �-catenin protein accumulation and reducedell motility. More importantly, we observed �-cateninccumulation in human HCC tumors containing the wild-ype �-catenin gene in the context of high-level FZD7xpression. Conclusions: These observations suggesthat the Wnt/�-catenin signal transduction pathway isnvolved much more commonly in the molecular patho-enesis of HCC than previously recognized becauseZD7 overexpression occurred early in the disease pro-ess, stabilized wild-type �-catenin levels, and contrib-ted to enhanced tumor cell migration.

epatocellular carcinoma (HCC) is the major pri-mary malignant tumor of the liver. Although

iral causative factors have been identified, the molec-lar mechanisms that contribute to tumor progressionuring hepatocarcinogenesis remain largely un-nown.1 The previous finding of inappropriate activa-ion of the Wnt/�-catenin pathway resulting from

-catenin gene mutations has provided clues towardnderstanding this process.2 The upstream receptorsf this signal transduction pathway have been identi-ed recently. The Frizzled family is composed of 10 orore 7-transmembrane proteins that act as receptors

or Wnt proteins, which serve as the ligands.3 Thent/Frizzled signaling network influences diverse bi-

logical processes ranging from cell fate determinationo cell motility and proliferation.4

The �-catenin molecule is an important multifactorialrotein. It is involved in cell– cell adhesion by strength-ning the linkage between cadherin and �-catenin to thectin cytoskeleton.5 In the absence of Wnt/Frizzled sig-aling, �-catenin is phosphorylated by interactions withlycogen synthase kinase (GSK)-3�, and forms a com-lex with axin and the adenomatous polyposis coli pro-ein (APC); subsequently, �-catenin is targeted for deg-adation by the ubiquitin-proteasome system.6 In contrast,inding of a Wnt ligand to its Frizzled receptor will lead totabilization of intracellular �-catenin through the inhibi-ion of GSK-3� enzymatic activity. There is subsequentranslocation of the �-catenin protein into the nucleus inssociation with high mobility group domain factors such ascf/Lef. This complex is associated with transcriptionalp-regulation of growth regulatory and cell migration–elated genes.7 In Drosophila, the Wnt/Frizzled signal isransduced by the small G-protein RhoA, which in turn canead to changes involving the reorganization of the cytoskel-

Abbreviations used in this paper: APC, adenomatous polyposis colirotein; 18SrRNA, 18S ribosomal RNA; FZD7, human Frizzled-7 recep-or gene; FZD7-�T�C, human Frizzled-7 receptor gene with a C-termi-al truncation; GFP, green fluorescence protein; GSK, glycogen syn-hase kinase; RT-PCR, reverse-transcription polymerase chaineaction.

© 2004 by the American Gastroenterological Association0016-5085/04/$30.00

doi:10.1053/j.gastro.2004.07.009

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October 2004 FRIZZLED GENE OVEREXPRESSION IN HCC 1111

ton through the activation of downstream kinases such as-Jun NH2-terminal kinase. Finally, studies performed inebra fish and Xenopus showed that the Wnt/Frizzled sig-aling can increase intracellular calcium via phospholipase�, leading to downstream activation of the Ca2�-sensitivenzymes, Ca2�-calmodulin–dependent protein kinase IInd protein kinase C.8

Cytoplasmic and/or nuclear accumulation of �-catenins a frequent event in various human tumors includingolorectal, lung, breast, cervix, skin, and liver.9–17 Dur-ng hepatocarcinogenesis, it has been reported that 8%–3% of HCCs have �-catenin mutation(s), whereas 33%–9% of these tumors display aberrant �-catenin proteinccumulation in the cytoplasm and/or nucleus.18–20 Amongther candidate genes involved in stabilization of �-catenin,xin1 was found to be mutated in a small fraction (7%–%) of HCCs,21,22 whereas APC mutations were found toe exceptionally rare.23 Thus, it is estimated that in 35%–0% of HCCs, the aberrant �-catenin cellular accumulations not associated with a mutation affecting the �-catenin,xin1, or APC genes of the Wnt/�-catenin signal trans-uction cascade, and the explanation for this observation haset to be determined. Although it was reported recentlyhat the p53 gene mutation may contribute to aberrantccumulation of wild-type �-catenin,24 it is likely thatther components of this signal transduction cascade maye involved as well.

The expression of Wnt ligands and Frizzled receptorsave been found in esophageal, colon, and skin carcino-as.25–27 However, the expression and/or functional role

f any Frizzled gene family member has not yet beenxplored during human hepatocarcinogenesis. We previ-usly identified and cloned the Frizzled-7 receptorFZD7), also called Frizzled-E3, and showed overexpres-ion in human esophageal carcinoma resulting in activa-ion of downstream APC/�-catenin signals.26 In theresent study, we have developed a quantitative real-ime reverse-transcription polymerase chain reactionRT-PCR) method to measure the expression of FZD7 inuman HCC cell lines, as well as tumors and adjacentninvolved liver. The functional association of FZD7eceptor expression with cellular motility was explorednd experiments were performed to determine its rela-ionship with �-catenin stabilization. This report showshe FZD7 gene commonly is overexpressed at the mes-enger RNA (mRNA) and protein level in most ofepatitis B virus–related HCCs. In this context, there isnhanced cell motility and migration. Most importantly,verexpression appears to be an early event during theultistep process of hepatocyte transformation and pro-

ides novel molecular targets for therapy of this seriousiver disease.

Materials and Methods

HCC Tumors and Cell Lines

HCC tumor tissues were obtained from Taiwan andouth Africa. Individuals from Taiwan had undergone surgeryor resection of tumor (n � 16). Ages ranged from 36 to 64ears. All were men with hepatitis B virus–related disease.here were 14 samples from South Africa. Ages ranged from8 to 57 years. All were men. Ten were hepatitis B virus, 1epatitis C virus, 2 related to hemochromatosis, and 1 was ofnknown cause. There was a matched, uninvolved, nontumor-us liver sample for all 30 HCCs. Cirrhosis and/or fibrosis wasresent in 85%. Huh7, Focus, HepaRG,28 Hep3B, and HepG2epatoma cell lines were grown in minimum essential Eagleedium (Mediatech, Herndon, VA), and PLC/PRF/5 was grown

n Dulbecco’s modified Eagle medium (Mediatech), supple-ented with 10% (vol/vol) fetal calf serum (Sigma, St. Louis,O), 1� minimum essential medium nonessential amino acid

olution (Sigma), and 1% (vol/vol) penicillin/streptomycinSigma).

Real-Time RT-PCR Assay

The copy number of FZD7 mRNAs was quantified innknown samples by measuring the Ct value followed byormalization to 18S ribosomal RNA (18SrRNA) after com-arison with a standard curve for both FZD7 and 18SrRNA.his ratio of FZD7/18SrRNA subsequently was normalized tocalibrator (mean value obtained from normal livers) and

xpressed as relative abundance of FZD7 mRNA. Standardsere prepared with 10-fold dilutions of the correspondingCR products cloned into the pCR 2.1 Vector (Invitrogen,ife Technologies, Rockville, MD). The specificity of theZD7 and 18SrRNA inserts was provided by sequence analy-is. Serial dilution of FZD7- and 18SrRNA-plasmids wasliquoted and stored at �20°C until use. Primers were se-ected by using the Primer3 website (http://www-enome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Prim-rs for FZD7 and 18SrRNA, respectively, (Invitrogen, Lifeechnologies) were as follows: (1) FZD7, 5=-GCCGCTTCTAC-ACAGACT-3= (forward) and 5=-TTCATACCGCAGTCTC-CC-3= (reverse) to yield a 54-bp amplicon; (2) human 18SrRNA,=-GGACACGGACAGGATTGACA-3= (forward) and 5=-AC-CACGGAATCGAGAAAGA-3= (reverse) to give a 50-bp am-licon. We conducted Nucleotide-Nucleotide Basic Local Align-ent Search Tool searches against database of Expressed Sequenceags and the nonredundant set of GenBank database sequences toonfirm the gene specificity of the nucleotide sequences chosen forhe primers to confirm the lack of DNA polymorphism.

Total RNA was extracted from liver specimens and HCCell lines by using TRIzol reagent (Invitrogen, Life Technol-gies). The quality of the RNA samples was determined bylectrophoresis through agarose gels and staining withthidium bromide; the 18S and 28S rRNA bands were visu-lized under ultraviolet light. A total amount of 250 ng ofotal RNA was treated with DNase-I, RNase-free, and reverseranscribed with random hexamers and the AMV reverse tran-

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1112 MERLE ET AL. GASTROENTEROLOGY Vol. 127, No. 4

criptase (all from Roche Diagnostics Corporation, Indianap-lis, IN). All PCR reactions were performed using an iCyclerQ Multi-Color Real Time PCR Detection System (Bio-Rad,ercules, CA) with a mix composed of 1� SYBR Green PCRaster Mix (Applied Biosystems, Foster City, CA) 500 nmol/L

ach primer, and 5 ng complementary DNA (cDNA) (equiv-lent total RNA) from unknown samples. The thermal cyclingonditions comprised an initial step at 95°C for 10 minutes,ollowed by 40 cycles at 95°C for 15 seconds, and 60°C for 1

inute. Experiments were performed in duplicate. Each PCRun included FZD7 and 18S standard curves, a nontemplateontrol, and the unknown cDNAs analyzed for FZD7 and8SrRNA copy numbers.

Cell Motility Assay

A luminescence-based assay was used to evaluate cellotility and migration. This assay assesses nonmigrated, migrat-

d-adherent, and migrated nonadherent hepatoma cells throughncoated polyvinylpyrrolidone-free polycarbonate filters, as pre-iously described,29 in chambers partitioned with 13-mm diam-ter, 8-�mol/L pore polycarbonate membranes (Osmonics, Inc.,innetonka, MN). After 72 hours of growth in 1% fetal calf

erum, cells were resuspended in serum-free medium with soy-ean trypsin inhibitor (Sigma) at 0.5 mg/mL. In the lower cham-er, collagen-I (Sigma) was diluted to 100 �g/mL in 200 �L oferum-free medium.30 After assembly, 1 � 105 of the resus-ended cells were added to the upper chamber and incubated forhours in a CO2 humidified incubator to allow cell migration to

roceed. The cells remaining on the upper surface of the mem-rane (nonmigrated) were harvested with a sterile cotton swab andlaced into a well containing adenosine triphosphate (ATP) lysisuffer (Packard Instrument Company, Meriden, CT). To harvestigrated adherent cells, the membrane (devoid of nonmigrated

ells) was placed into another well containing ATP lysis buffer.he migrated nonadherent cells were harvested by resuspending

he cells in the lower compartment of the blind chamber andlacing them into a third well containing ATP lysis buffer.TPLite substrate was added to each well and luminescent countser second were measured in a TopCount Microplate readerPackard Instrument Company). The results were analyzed usingxcel (Microsoft Corporation, Seattle, WA) to calculate percent-ges of nonmigrated, migrated-adherent, and migrated nonadher-nt cells in each assay.

Transfection and Retroviral TransductionStudies

Because the ectodomain of Frizzled receptors functions asnatural antagonist of Frizzled-mediated signal transduction, autant cDNA with a C-terminal truncation (FZD7-�T�C) was

enerated as previously described.26 The cDNA FZD7-�T�CDNA was subcloned into a pcDNA3 mammalian expressionector (Invitrogen) and certified for protein expression by an initro translation reaction.26 The in vitro effect of stable expressionf FZD7-�T�C was examined in human hepatoma cells. ThecDNA3/FZD7-�T�C and pcDNA3/empty vectors were trans-ected by electroporation (250 V/15-ms pulse with Gene Pulser

I; BioRad, Hercules, CA). Stable transfectants were selected with00 �g/mL of Geneticin (Gibco, Carlsbad, CA) added to theulture medium. Expression of the FZD7-�T�C gene was as-essed by quantitative real-time RT-PCR and Western blot anal-sis.

Two different FZD7 truncated mutants either with dele-ions in the intracellular domain alone (FZD7-�C) or in bothhe intracellular and transmembrane domains (FZD7-�T�C),s well as a green fluorescence protein (GFP) control, wererepared by PCR using the Pfu-polymerase (Stratagene, Laolla, CA). Constructs were cloned into the lentiviralLenti6/V5 directional TOPO vector (Invitrogen) downstreamf the cytomegalovirus promoter. All constructs were verifiedy sequence analysis of both strands. Virions were produced in93FT cells, and viral stocks were frozen at �80°C. Hepatomaells were transduced at a multiplicity of infection of 5, andtable clones were selected in the presence of blasticidin4 �g/mL). Quantitative real-time RT-PCR and Western blotnalysis were used to detect gene expression. A mutant con-truct of �-catenin with biologic activity (�N/�C) was pre-ared by PCR using the Pfu-polymerase (Stratagene) as pre-iously described31 and after transfection; HCC motility wasssessed 72 hours later.

We performed transient transfection assays to assess for Tcfranscriptional activity. In brief, cells were seeded in 6-welllates at a density of 3 � 105 cells/well the day beforeransfection. Transfections were performed with either TOP-ash (Tcf Reporter Plasmid) or FOPflash (mutant Tcf bindingites) and �-galactosidase– expressing plasmid in triplicate us-ng Lipofectamine 2000 (Life Technologies). Focus, Huh7, andep3B cells were harvested 48 hours after transfection and

uciferase activity was measured by a luminometer TopCounticroplate reader (Packard Instrument Company). Transfec-

ion efficiency was normalized by measurement of �-galacto-idase activity. The experiment was performed 3 times.

Immunoprecipitation Studies

Transfected cells were washed with cold phosphate-uffered saline, and pelleted at 2000 rpm for 5 minutes andesuspended in 500 �L of solubilization buffer (136 mmol/LaCl, 2.7 mmol/L KCl, 12 mmol/L Na2HPO4, 1.8 mmol/LH2PO4, pH 7.4, 1% NP-40 with protease inhibitors). Ly-

ates were incubated for 3 hours at 4°C on a rocker platformnd then microcentrifuged at 13,000 rpm for 15 minutes at°C to pellet-out cell debris. Lysates were immunoprecipitatedn a 1.0-mL total volume with 500 �L of lysate, 480 �L ofolubilization buffer, and 20 �L of goat anti-human V5 anti-odies coupled to agarose beads (Bethyl Laboratories, Inc.,ontgomery, TX) for 12 hours at 4°C with constant shaking.he beads were washed twice with the solubilization buffer.ound proteins were eluted by boiling for 5 minutes in

odium dodecyl sulfate sample buffer. The proteins were sep-rated on 10% polyacrylamide gel and transferred to a poly-inylidene difluoride membrane (NEN Life Science Products,oston, MA).

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Protein Extraction and Western BlotAnalysis

For whole-protein extraction, hepatoma cells at 50%onfluence were homogenized in lysis buffer (30 mmol/L Tris,H 7.5, 150 mmol/L NaCl, 1% NP-40, 0.5% Na deoxy-holate, 0.1% sodium dodecyl sulfate, 10% glycerol, and 2mol/L ethylenediaminetetraacetic acid) with protease inhib-

tors (Roche Molecular Biochemicals, Indianapolis, IN) andonicated. Protein concentration was determined with theCA Protein Assay Kit (Pierce, Rockford, IL) using bovine

erum albumin as standard. Subcellular fractionations wereerformed as previously described.32

Aliquots of proteins were resolved on sodium dodecyl sul-ate–polyacrylamide gel electrophoresis and transferred ontoolyvinylidene difluoride membranes (NEN Life Science Prod-cts) by electroblotting. The membranes were blocked with% nonfat dry milk in Tris-buffered saline containing 0.1%ween 20 and then probed with a mouse monoclonal anti-�-atenin antibody diluted at 1:500 (Transduction Laboratories,an Diego, CA), or a mouse monoclonal antiproliferating cell

igure 1. Real-time PCR assayor frizzled-7 gene. �, Standardurves for (A) 18SrRNA and (B)ZD7. �, Mean values of un-nown HCC tumor samples per-ormed in duplicate. The stan-ard curves show a 5 order ofagnitude linear dynamic range.

C) The FZD7 mRNA steady-stateevels in HCC cell lines ex-ressed as relative abundancef FZD7 mRNA. The value repre-ents the mean � SD from 3eparate experiments.

uclear antigen antibody diluted at 1:1000 (Oncogene Science,ambridge, MA). A specific rabbit polyclonal antibody wasrepared against a human FZD7 peptide QNTSDGSGG-GGGPTAYPTAPYLPD, amino acids 163–187 (NCBI Pro-ein database accession no. BAA_34668), and used at 1:5000ilution. Each primary antibody was followed by incubationith a secondary horseradish-peroxidase antibody diluted:10,000 and then revealed with the chemiluminescence im-ging Western Lightning (PerkinElmer Life Sciences, Boston,A). The specificity of the antigen-antibody interaction was

stablished by absorption of FZD7 immunoreactivity withpecific and not with nonrelevant peptides, as well as a lack ofZD7 protein detection with addition of second antibodylone (data not shown). All of the blots were standardized forqual protein loading by Ponceau S red staining.

Sequencing

PCR amplification of �-catenin exon-3, which con-ains the 4 potential sites for phosphorylation by GSK-3�, waserformed on the cDNAs derived from each tumor sample

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1114 MERLE ET AL. GASTROENTEROLOGY Vol. 127, No. 4

sing a �-catenin exon-2 forward primer, and a �-cateninxon-4 reverse primer as previously described.33 After resolu-ion of the PCR products by 2% agarose gel electrophoresisnd visualization with ethidium bromide, PCR products werexcised and cloned into the pCR 2.1 Vector (Invitrogen, Lifeechnologies). Sequencing was performed in both directionssing T7 forward and M13 reverse primers.

Statistical Analysis

The dependent or independent t tests were used forontinuous data with StatView software (version 5.0; SASnstitute Inc., Cary, NC). Tests were considered significanthen P values were 0.05.

Results

Real-Time RT-PCR Assay for FZD7

We used random hexamers for the initial reverse-ranscription step. The amplification was performed withset of primers generating a small amplicon size (�50 bp)

or both FZD7 and 18SrRNA. Figure 1 shows the standardurves for FZD7 and 18SrRNA and shows the values ob-ained for unknown tumor samples. A linear relationshipetween the Ct and the log of the starting copy number washown (R2 � 0.99). The efficiency of the reaction (E),alculated by the formula, E � 101/m � 1, where m is thelope of the standard curve,34 ranged from 90% to 100% inhe assays performed at different times. The same featuresith respect to the dynamic range and efficiency of the

eactions were observed with serial dilutions of cDNAserived from the Huh7 cell line, or from human HCCumors when using 10–0.625 ng of equivalent total RNAdata not shown). Sensitivity was 10 copies per reactionor FZD7 and 100 copies for 18SrRNA.

With this assay in hand, we measured FZD7 genexpression in HepG2, Hep3B, HepaRG, PLC/PRF/5,ocus, and Huh7; values obtained on 4 normal adultiver tissues served as a control and the mean value ofhese normal livers served as a calibrator for normaliza-ion of FZD7 mRNA levels in other unknown samples.ll cell lines were found to express the FZD7 mRNAene at higher levels than in normal liver (Figure 1C).uh7, PLC/PRF/5, and Focus cells exhibited the highest

evels of FZD7 mRNA with ratios of 354, 116, and 81,espectively, compared with normal livers. In contrast,ep3B, HepaRG, and HepG2 had lower levels of ex-

ression with ratios of 15, 11, and 9, respectively.

Correlation of Steady-State FZD7 mRNALevels and HCC Motility

Based on the observation that the Wnt-Frizzledignal transduction pathway is known to be involved inell motility and migration,27 we examined the motility

f HepG2, Hep3B, Focus, and Huh7 cells in the contextf FZD7 gene expression. In these experiments, we choseot to use fetal calf serum as chemoattractant29 becauseerum growth factors may interfere with �-catenin sta-ilization independent of the Wnt-Frizzled signals.35,36

nder the experimental conditions of using soluble col-agen-I as a chemoattractant, we observed a significantorrelation between FZD7 steady-state mRNA levelsnd percent of total migrated cells (both adherent andonadherent) (Figures 2A and B). The highest FZD7RNA levels and motility rates were found in Huh7

ells.

igure 2. Measurement of HCC cell motility. (A) The percent of non-igrated and total migrated (migrated-adherent, and migrated nonad-erent) cells were evaluated by a luminescent-based assay. Thealues (percent of cells) are expressed as mean � SD from 6 sepa-ate measurements. t test, *P 0.01, **P 0.001 as comparedith values obtained with HepG2 cells. (B) Correlation between FZD7RNA steady-state levels and the percent of total migrated cells (bothigrated-adherent and migrated-nonadherent). Z test of correlation, P0.02.

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Subcellular Localization of �-Catenin

To explore the hypothesis that Wnt-Frizzled sig-al may act through the canonical �-catenin pathway toontrol cellular motility, the �-catenin status in HCCell lines was evaluated. We found that Huh7, Focus,nd Hep3B cells have a homozygous wild-type �-catenins described previously.24 HepG2 cells have a heterozy-ous deletion in exon-3 and make both wild-type andutant �-catenin proteins.37 It is known that �-cateninay be bound to either the cytosolic GSK3�/Axin/APC

omplex or membrane-linked with E-cadherin or c-Met.35

n signaling by Wnt through its Frizzled receptor, dishev-led subsequently interacts with the GSK3�/Axin/APC/�-atenin complex, causing dephosphorylation and dissocia-ion and �-catenin from the complex, followed by itsuclear translocation. Thus, nuclear vs. cytosolic localiza-

igure 3. Western blot analysis of total cellular �-catenin and Tcfeporter assay. (A) There is no correlation of proliferating cell nuclearntigen with FZD7 mRNA. (B) However, high levels of FZD7 genexpression are associated with nuclear accumulation of �-catenin;

ower FZD7 expression levels were associated with both nuclear andytoplasmic accumulation in HepG2 and Hep3B cells. (C) Measure-ent of Tcf transcriptional activity. These HCC cell lines were cotrans-

ected with either TOPflash (�) or FOPflash (□) and �-galactosidase–xpressing plasmid. Note the high level of Tcf-mediated transcriptionctivity in all 3 cell lines. There was a general correlation betweenZD7 levels and Tcf transcriptional activity.

ion of wild-type �-catenin was explored in these HCC cellines. The �-catenin and protein levels are shown in FigureA. It was of interest that 2 HCC cell lines, namely Huh7nd Focus, with the highest FZD7 gene expression, had a

igure 4. Construction and expression of the FZD7 mutant proteins. (A)ZD7-FL, wild-type full-length FZD7 protein; FZD7-�C, FZD7 protein trun-ated in the intracellular domain; FZD7-�T�C, secreted FZD7 proteinruncated in both the transmembrane and intracellular domains; CRD,ystein-rich domain. The Wnt-ligand interacts with the CRD of FZD7 andith the epidermal growth factor repeat (EGFR) regions 1 and 2 ofRP5/6 as represented by grey boxes. (B) Expression in a heterogenousopulation of Huh7 cells stably transfected with pcDNA3/FZD7-�T�C orcDNA3/empty vector as a control. Expression was assessed by quan-itative real-time RT-PCR with either a first set of primers located in thextracellular domain, or a second set located in the intracellular domainf FZD7 receptor. Expression levels are normalized to the values ob-ained with pcDNA3. (C) Expression of FZD7-�T�C as assessed byestern blot analysis with the monoclonal anti-M2 flag tag antibody in aeterogenous population of Huh7 cells stably transfected with FZD7-T�C or pcDNA3/empty vector. (D) Expression of various forms of FZD7

n Huh7 cells. Cells were stably transfected with pLenti6/V5-GFP, clone-4GFP-C4) served as a negative control; Huh7 cells were stably transfectedith pLenti6/V5-FZD7-�C, clones 1 to 8 (�C-C1–C8); Huh7 cells tran-iently transduced with pLenti6/V5-FZD7-�C (�C-Tr). Huh7 cells stablyransduced with pLenti6/V5-FZD7-FL, clone-1 (FL-C1) served as a posi-ive control for RT-PCR assessment with sets of primers targeting eitherhe extracellular domain or the intracellular domain of FZD7 receptor.xpression levels are normalized to the values observed with pLenti6/5-GFP-C4. (E) Expression of FZD7-�C as assessed by Western blotnalysis with rabbit polyclonal anti-human FZD7 antibody with or withoutmmunoprecipitation with goat anti-human V5 antibodies coupled togarose beads. Experiments were performed in Huh7 cells transducedith pLenti6/V5-FZD7-�C and pLenti6/V5-GFP-C4 as a negative control.

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1116 MERLE ET AL. GASTROENTEROLOGY Vol. 127, No. 4

triking nuclear subcellular localization of wild-type �-cate-in (Figure 3B). Furthermore, the levels of FZD7 mRNAppear not to correlate with the cellular proliferation index,s assessed by levels of proliferating cell nuclear antigenFigure 3A).

In addition, Huh7, Focus, and Hep3B showed highasal Tcf transcriptional activity and there was a generalorrelation between the level of activity and FZD7 re-eptor expression (Figure 3C). These results also areonsistent with the levels of �-catenin found within theell and its nuclear localization. These findings suggesthat activation of the canonical Wnt/�-catenin pathways involved in the multistep process of hepatocyte trans-ormation.

Expression of a Dominant-Negative FZD7Receptor Mutants Inhibits Wild-Type �-Catenin Accumulation and Motility of HCCCells

We assessed the motility and accumulation ofild-type �-catenin in human HCC cell lines afterown-regulation of the Wnt-FZD7 signal after ectopicxpression of 2 types of dominant-negative FZD7 mu-ant receptors: (1) the FZD7-�T�C represents a secretedZD7 receptor in which both intracellular and trans-embrane domains have been truncated; this construct

as been shown to inhibit Wnt signaling in humansophageal carcinoma cell lines26; and (2) the FZD7-�Construct in which the transmembrane FZD7 receptoras been truncated in the intracellular domain only asepicted in Figure 4A. Because FZD7-�T�C receptorcts as a soluble Wnt ligand binding protein, studiesere performed on a heterogenous population of stably

ransfected cells. HCC cells were selected in the presencef geneticin and subsequently evaluated for motility andccumulation of wild-type �-catenin. The FZD7-�Cutant receptor does not act as a soluble ligand but is

inked closely to the membrane with its 7 transmem-rane domains. The FZD7-�C cDNA was cloned intohe lentiviral vector pLenti6/V5-D-TOPO to allow for aigh transduction efficiency in both dividing and non-ividing cells. Preliminary experiments with a similarlyloned GFP construct indicated that more than 80% ofuman HCC cells will abundantly express GFP from 72ours to 3 weeks posttransduction. Ectopic expression ofZD7-�C and FZD7-�T�C was assessed by Westernlot and quantitative real-time RT-PCR by using dif-erent sets of primers targeting specifically either thextracellular or the intracellular domain of FZD7, allow-ng differential assessment of the ectopic expression ofZD7-�C and FZD7-�T�C by comparison with the

ndogenous expression of the wild-type full-lengthZD7 receptor (Figure 4B–E).The secreted FZD7-�T�C ectodomain has been

hown previously to be a functional antagonist of endog-nous FZD7 signaling by suppression of the interactionf APC with �-catenin in the KYSE150 esophagealarcinoma cell line.26 We now show that ectopic expres-ion of FZD7-�T�C or FZD7-�C causes a decrease of-catenin protein levels in all HCC cell lines with aomozygous wild-type �-catenin gene, presumablyhrough down-regulation of the Wnt-FZD7 signal trans-uction cascade (Figure 5A, B). In contrast, �-cateninRNA steady-state levels were not altered by transfec-

ion of the dominant-negative mutant constructs as as-

igure 5. �-catenin protein levels in HCC cells after ectopic expres-ion of a dominant-negative FZD7-�T�C mutant receptor–expressingonstruct. (A) Levels of �-catenin protein in Huh7, Focus, Hep3B, andepG2 HCC cell lines transfected with the secreted form of theZD7-�T�C receptor. (B) Level of �-catenin protein in Huh7 cells afterransduction with the transmembrane FZD7-�C mutant receptorloned into pLenti6/V5. (C) Motility of Huh7 cells toward solubleollagen-I under conditions of transient transfection with pLenti6/V5-ZD7-�T�C, pLenti6/V5-FZD7-�C, or pLenti6/V5-GFP as a control.D) Motility measurements of representative clones under conditionsf stable integration and expression of: C1 � FZD7-�T�C, C5 �ZD7-�C, and C6 � FZD7-�C. The percent of nonmigrated and totaligrated (migrated-adherent and migrated nonadherent) cells werevaluated by a luminescent-based assay after 3 hours at 37°C. Thealues (percent of cells), expressed as mean � SD, are from 6eparate measurements. t test, *P 0.05, **P 0.01 whenompared with GFP-negative control.

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essed by real-time RT-PCR (data not shown). However,o effects of these mutant receptor constructs were ob-erved on �-catenin levels in HepG2 cells, which have aeterozygous �-catenin deletion (Figure 5A).

If FZD7 potentially is involved in cellular motility viahe canonical �-catenin pathway, it becomes importanto evaluate the impact of down-regulation of the

nt-FZD7 signal on motility of Huh7 cells.33 Wehowed that ectopic expression of the transmembrane

igure 6. Schematic representation of �-catenin constructs and cellotility assay. (A) Approximate location of functional domains: black,rotein instability; right-leaning hatch, N-terminal transactivation do-ain; left-leaning hatch, C-terminal transactivation domain; grey, ar-adillo repeats, protein-protein interaction. (B) Motility measure-ents of FZD7-�C-C6 and GFP-C4 blasticidin-selected clonal Huh7

ell populations initially transduced with pLenti6/V5-D-TOPO lentiviralectors expressing either the FZD7-�C negative-dominant mutant orFP as control, and cotransduced once again with a pLenti6/V5-D-OPO lentiviral vector expressing a biologically active �N/�C �-cate-in mutant or GFP to keep constant the total amounts of plasmidNA. The percent of nonmigrated and total migrated (migrated-adher-nt and migrated nonadherent) cells were evaluated by a luminescent-ased assay after 3 hours at 37°C. The values (percent of cells)xpressed as mean � SD were derived from 6 separate measure-ents. Note the restoration of cell motility by the �N/�C mutant-catenin in the setting of inhibition of the signal transduction of theeceptor level with stable expression of the dominant-negative FZD7C-C6 receptor mutant protein.

nchored FZD7-�C mutant receptor was very effectiven reducing the number of motile Huh7 cells underonditions of transient expression as shown in Figure 5C.n addition, this construct was more efficient in reducingotility than the secreted FZD7-�T�C mutant recep-

or. These observations were confirmed further by strik-ng inhibitory effects on cell motility by 2 independent

igure 7. (A) Quantitative real-time RT-PCR assessment of FZD7RNA levels in human HCC tumors (T) and the corresponding peritu-orous liver parenchyma (PT), derived from Taiwan and South Africa.onparametric paired test, *P 0.0001; paired t test, P � 0.0187,hen comparing levels in tumor with peritumoral areas. (B) Westernlot analysis of FZD7 receptor protein expression in HCC tumors (T)nd the corresponding peritumorous areas (PT), as well as in Huh7nd HepG2 human hepatoma cell lines. (C) Western blot analysis of-catenin protein accumulation in cytosolic (C) or nuclear (N) enriched

ractions from 2 HCC tumors and their corresponding peritumoralreas compared with 2 normal liver samples. Both peritumoral areand tumor overexpress FZD7 mRNA as shown by values listed belowhe Western blots and expressed as relative abundance of FZD7RNA. Each tumor and peritumor region had a wild-type �-cateninxon-3 as assessed by PCR and sequencing.

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1118 MERLE ET AL. GASTROENTEROLOGY Vol. 127, No. 4

table clones expressing either FZD7-�C at high levelsFZD7-�C-C5, and FZD7-�C-C6, respectively), com-ared with a FZD7-�T�C– expressing clonal cell lineFZD7-�T�C-C1) as shown in Figure 5D.

To establish a clear link between regulation of theanonical Wnt/�-catenin pathway and the FZD7-medi-ted alteration of cell motility in Huh7 cells, experi-ents were performed to restore cell motility by ectopic

xpression of a mutant �-catenin construct in Huh7 cellsarboring a low-motility phenotype caused by overex-ression of a dominant-negative FZD7 receptor mutantrotein. We generated a pLenti6/V5-D-TOPO lenti-iral vector expressing the �N/�C �-catenin mutantFigure 6A) that had been shown previously to exhibitransactivating properties on Lef/Tcf regulated targetenes in HEK cells.31 The FZD7-�C-C6 and GFP-C4lasticidin-selected clonal Huh7 cell populations wereotransduced by the �N/�C �-catenin mutant lenti-irus construct or GFP as a control to keep constanthe total amounts of plasmid DNA within Huh7 cells.n this context, we assessed the motility of cotrans-uced Huh7 cells and showed that the low-motilityhenotype induced by stable FZD7-�C expressionould be reversed by ectopic expression of the �N/�Cutant �-catenin protein (Figure 6B). These experi-ents further support a role for activation of the

anonical Wnt/�-catenin pathway during hepatic on-ogenesis in cells overexpressing FZD7 receptors.

FZD7 mRNA is Overexpressed in HumanHCC Tumors

Of importance was to extend our in vitro observa-ions into the realm of human biology and disease. Normaliver showed low FZD7 mRNA expression (Figure 7A andable 1). Among 30 paired samples tested from 2 regions of

he world, 27 (90%) displayed significant FZD7 overex-ression in comparison with corresponding peritumorousreas (nonparametric paired test, P 0.0001; paired t test,� 0.0187) (Figure 7A). These findings were confirmed at

he protein level by Western blot analysis (Figure 7B).esults were similar between tumors derived from Southfrica and Taiwan with an average increase of 12-foldetween tumor and peritumorous areas. High levels ofZD7 mRNAs as defined by a value above the cut-off level

mean of normal liver � 3 SD, � � 0.01) were observed in9 tumors (97%) and 23 peritumoral areas (77%) comparedith completely normal adult liver controls. These obser-ations led us to believe that FZD7 mRNA up-regulationay be an early event occurring in the preneoplastic peri-

umorous area of the liver.Similar to our in vitro observations in HCC cell lines, we

xplored �-catenin levels in comparison with FZD7 gene

xpression in human tumors. It was found that, among 5ifferent HCC-expressing FZD7 mRNA at 100-foldbove the mean of normal livers, 4 of the 5 HCC weressociated with a homozygous wild-type �-catenin exon-3ene. However, 1 tumor (HCC #28) had a heterozygouseletion of 1 �-catenin allele similar to that found in theepG2 cell line. Western blot analysis of cytosolic and

uclear enriched fractions showed that wild-type �-cateninrotein also accumulates in HCC and peritumorous areas inhe context of increased FZD7 mRNA expression and inhe absence of �-catenin exon-3 mutations as shown inigure 7C. The findings in human tumors, therefore, con-rm our in vitro observations in HCC cell lines.

able 1. FZD7 mRNA Steady-State Levels Assessed byQuantitative Real-Time RT-PCR in Paired SamplesIncluding HCC Tumor Versus the CorrespondingPeritumorous Liver

Geographicarea

HCCsample,

no. Cause

FZD7 mRNAlevels

RatioT/pTT pT

bnormal liverTaiwan 1 HBV 18.5 7.3 2.5

2 HBV 46.5 6.9 6.73 HBV 11.6 5.8 2.04 HBV 53.1 7.6 7.05 HBV 60.7 9.5 6.46 HBV 16.0 3.3 4.87 HBV 31.3 14.2 2.28 HBV 17.1 18.5 0.99 HBV 9.5 13.1 0.7

10 HBV 19.6 6.5 3.011 HBV 134.2 19.6 6.812 HBV 657.1 10.2 64.413 HBV 38.2 18.5 2.114 HBV 9.5 3.6 2.615 HBV 84.0 30.9 2.716 HBV 38.2 6.9 5.5

South Africa 17 HBV 442.9 33.5 13.218 HBV 8.4 0.7 12.019 HBV 4.0 0.4 10.020 Hemochromatosis 0.7 4.4 0.221 HBV 33.5 6.2 5.422 HBV 31.3 10.2 3.123 HBV 4.7 1.1 4.324 HBV 7.3 0.4 18.325 HCV 29.1 3.3 8.826 HBV 14.9 2.2 6.827 HBV 33.1 4.7 7.028 Unknown 296.4 2.2 134.729 Hemochromatosis 14.9 8.4 1.830 HBV 20.7 1.8 11.5

ormal liverTaiwan 31 0.4

32 0.7South Africa 33 1.8

34 1.1

, tumor; pT, peritumorous; HBV, hepatitis B virus.

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Discussion

We have provided direct evidence by real-time RT-CR that FZD7 receptor gene expression commonly isp-regulated and is an early event during the developmentf HCCs. We also have developed a rabbit polyclonal anti-ody to a unique peptide of FZD7 to confirm that thiseceptor also is up-regulated at the protein level as well asy Western blot analysis. The biologic consequences of thisvent are the stabilization of wild-type �-catenin and en-anced tumor cell migration. Defining the genetic alter-tions associated with hepatocarcinogenesis is essential fornderstanding the progression of this disease. In this regard,ctivation of cellular oncogenes (i.e., c-myc)38 and growthactors (i.e., insulin-like growth factor II and transformingrowth factor type �)39–41 in association with mutations ofumor suppressor genes (i.e., P53, RB, and IGFIIR)40,42,43

ave been described in human and animal models of HCC.owever, these genetic events are present in 50% of HCC

umors and usually occur late in the multistep process ofepatocarcinogenesis and not in precancerous lesions ofysplasia, cirrhosis, and noncirrhotic chronic hepatitis.ore recently, aberrant accumulation of the potential on-

ogenic �-catenin protein, caused by mutations of the gene,as been found in both human and murine HCC tumors,ut such mutations are unusual (15% of HCCs) and of lateccurrence because they are absent in dysplasia, cirrhosis, oriver fibrosis.2,44

Additional studies have revealed that in 35%–80% ofCCs, aberrant accumulation of �-catenin is not associ-

ted with mutations of �-catenin, Axin1, or APC genesf this Wnt inducible signal transduction pathway.18–21,45

lthough p53 gene mutations could contribute, in part, toberrant accumulation of wild-type �-catenin, the fre-uency of gene mutations is relatively low. The level ofuclear and cytoplasmic �-catenin accumulation in suchumors therefore remains unexplained.24 We explored theossibility that other members of the Wnt/�-catenin signalransduction pathway could contribute to aberrant accumu-ation of wild-type �-catenin in HCC cells. During initia-ion of the Wnt signaling cascade, binding of a Wnt ligando its target, the Frizzled receptor, may lead either to thetabilization of intracellular �-catenin protein7 or activationf downstream molecules such as c-Jun NH2-terminal ki-ase, and protein kinase C.8 However, if Frizzled receptorsere overexpressed, this signal transduction pathway mighte constitutively activated. Human HCC cell lines, as wells tumors, were evaluated and the biologic consequences ofZD7 overexpression were assessed.All HCC cell lines were found to overexpress the

ZD7 gene at different levels. These observations werextended to human disease. Indeed, analysis of human

iver tumors showed frequent FZD7 gene up-regulations compared with normal liver and adjacent uninvolvedreas. The general finding of significant overexpression inreneoplastic peritumoral tissue as compared with nor-al liver suggests that up-regulation of this gene may be

n early event in hepatocarcinogenesis. Once completeransformation has occurred, higher levels of FZD7 werebserved. These results imply that activation of the

nt/�-catenin signaling owing to FZD7 receptor over-xpression alone or possibly in association with LRP-oreceptor and Wnt ligand expression or overexpression,ay be one of the major early events of the stepwise

rocess leading to hepatocyte transformation.Activation of the canonical Wnt pathway results in ac-

umulation of the �-catenin–free pool in the cytoplasm.fter forming a transcriptional transactivator complex withcF/Lef,7 there is translocation to the nucleus. Subse-uently, transactivation of genes involved in cell migrationill occur. We found that high levels of FZD7 mRNA

xpression were associated almost exclusively with nuclearnd/or cytoplasmic accumulation of �-catenin in HCC linesnd human tumors with the wild-type gene. However, as anxception, we observed up-regulation of FZD7 gene expres-ion in the context of a heterozygous �-catenin exon-3 geneeletion in one HCC tumor (HCC #28) and cell lineHepG2). It is possible that an internal deletion of the-catenin gene abolishes phosphorylation sites (localizedithin exon-3) and abrogates the canonical Wnt-Frizzled

ignaling via the mutated �-catenin allele.46 In this setting,ZD7 may signal through either in the canonical �-cateninathway via the wild-type �-catenin allele, or may act in aoncanonical �-catenin–independent pathway involvinghe activation of protein kinase C.8 More importantly, weave shown that down-regulation of Wnt-FZD7 signalingy ectopic expression of different dominant-negative mu-ants of FZD7 receptors (i.e., the transmembraneZD7-�C or the secreted FZD7-�T�C) is associated witharkedly reduced accumulation of �-catenin protein in the

ytoplasm and nucleus of HCC cell lines that display aomozygous wild-type �-catenin gene. However, our ob-ervations on the inefficiency of dominant-negative mutantZD7 receptors to reduce accumulation of the wild-type-catenin protein in the environment of a heterozygously-catenin–mutated gene, as we have shown in HepG2 cells,mphasizes the potential of cooperation between noncanoni-al and �-catenin–independent pathways for mediating thent-FZD7 signaling, however, further studies are required.The Wnt/Frizzled signaling network influences di-

erse biological processes.4 The �-catenin protein be-ongs to a family of structural proteins that includesatenins and cadherins. By forming a membrane-associ-ted complex, these proteins mediate adhesion and are

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1120 MERLE ET AL. GASTROENTEROLOGY Vol. 127, No. 4

ssential for the processes of cellular motility and migra-ion.33,47 Therefore, we investigated the role of FZD7ene expression in HCC cell motility. An ATP lumines-ence-based assay was used to quantify directional cellotility values in chemotaxis chambers and we observed

hat the magnitude of steady-state FZD7 mRNA levelsas correlated strongly with enhanced motility of HCC

ells. To further characterize the role of Wnt-FZD7 sig-al on cell motility, experiments showed that interferingith the Wnt-FZD7 interaction by ectopic expression ofominant-negative mutant FZD7 receptors led toarked reduction of HCC cell migration and occurred in

he context of a homozygous wild-type �-catenin gene.hese results support previous findings on the inhibitoryffect of natural secreted Frizzled-related proteins on theotility of human glioma cells.48 Of interest was the

bservation that the transmembrane FZD7-�C mutanteceptor was more effective than the secreted FZD7-�T�Coluble receptor with respect to inhibiting Huh7 cell mo-ility and migration. It is likely that Huh7 cells can secreteet unidentified Wnt ligand for binding to the FZD7eceptor. The secreted FZD7-�T�C mutant receptor maye less efficient in binding, or could be overwhelmed by theigh number of secreted Wnt ligand molecules. In contrast,he transmembrane FZD7-�C mutant receptor may beaturated and efficient because it has the capability to bindo the LRP5 co-receptor to allow for optimal transmission ofhe Wnt signal. Finally, our experiments suggest that theanonical �-catenin pathway may be involved in the FZD7-ediated regulation of Huh7 cell motility. Indeed, we have

bserved that ectopic expression of a �-catenin mutant wasble to restore high levels of motility in Huh7 cells wherehe Wnt/�-catenin–mediated signal transduction cascadeas been inhibited at the receptor level by stable expressionf a dominant-negative FZD7-�C construct.

In summary, deregulation of the Wnt-APC-�-cateninathway is found in a number of human (colorectal, lung,reast, cervix, melanoma, and HCC) tumors.9–17 Mosteports have established that this deregulation may beaused by �-catenin gene mutation.2 Surprisingly, ourtudy shows that over 90% of human HCCs have up-egulation of the FZD7 receptor gene and this phenom-non was associated functionally with stabilization ofild-type �-catenin and enhanced HCC cell motility.herefore, enhanced FZD7 gene expression is the mostommon and one of the earliest genetic abnormalitiesbserved thus far in HCC and probably is responsible forhe �-catenin accumulation in human HCC tumorsithout �-catenin, axin, or APC mutations. Molecularechanisms that may lead to up-regulation of the FZD7

ene are unknown. There are various considerations thatnclude (1) paracrine or autocrine induction by Wnt

igands, (2) gene amplification, and (3) demethylation ofZD7 gene promoter sequences. Further investigationsre required to test these possibilities.

Although we have emphasized the canonical Wnt/�-atenin signaling pathway in this report, Wnt/Frizzledignals may activate at least 2 other intracellular signal-ng pathways, including the planar cell polarity pathwayhat signals through the small GTPase Rho, and anotherignaling cascade that activates isoforms of protein kinase.27,49 In the future, we will need to focus on under-

tanding these pathways as well in the context of aomozygous wild-type �-catenin gene or in tumors with-catenin mutations and deletions.

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Received January 12, 2004. Accepted July 1, 2004.Address requests for reprints to: Jack R. Wands, M.D., The Liveresearch Center, 55 Claverick Street, 4th Floor, Providence, Rhode

sland 02903. e-mail: jack_wands_md@brown.edu; fax: (401) 444-194.Supported in part by a fellowship from Association Pour la Re-

herche sur le Cancer (France), and National Institutes of HealthBethesda, MD) grants CA-35711, AA-02666, NCRR COBRE-15578-2, and Massachusetts Institute of Technology, National Scienceoundation, Biotechnology Process Engineering Center.

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Functional Consequences of Frizzled-7 Receptor Overexpression in Human Hepatocellular CarcinomaMaterials and MethodsHCC Tumors and Cell LinesReal-Time RT-PCR AssayCell Motility AssayTransfection and Retroviral Transduction StudiesImmunoprecipitation StudiesProtein Extraction and Western Blot AnalysisSequencingStatistical Analysis

ResultsReal-Time RT-PCR Assay for FZD7Correlation of Steady-State FZD7 mRNA Levels and HCC MotilitySubcellular Localization of -CateninExpression of a Dominant-Negative FZD7 Receptor Mutants Inhibits Wild-Type -Catenin Accumulation and Motility of HCC CellsFZD7 mRNA is Overexpressed in Human HCC Tumors

DiscussionReferences