Proliferating EpCAM-Positive Ductal Cells in the ﬂamed ...Molecular and Cellular Pathobiology Proliferating EpCAM-Positive Ductal Cells in the Inﬂamed Liver Give Rise to Hepatocellular

Molecular and Cellular Pathobiology

Proliferating EpCAM-Positive Ductal Cells in theInflamed Liver Give Rise to HepatocellularCarcinomaTomonori Matsumoto1, Atsushi Takai1, Yuji Eso1, Kazuo Kinoshita2,Toshiaki Manabe3, Hiroshi Seno1, Tsutomu Chiba1,4, and Hiroyuki Marusawa1

Abstract

Hepatocellular carcinoma (HCC) originates from regeneratingliver cells with genetic alterations in chronically inflamed liver.Ductal cells and hepatocytes proliferate for liver regeneration, andproliferating ductal cells (PDC) derived from bile ductules havelong been considered putative liver stem/progenitor cells andcandidate cellular origins of HCC. The potential of PDC as tumor-originating cells, however, remains controversial in contrast toaccumulating evidence that HCC originates from hepatocytes.Here, we demonstrate that PDCs expressing the established sur-face and cancer stem cell marker EpCAM give rise to HCC ininflamed liver. EpCAM-expressing PDCs were specifically labeledin newly developed EpcamCreERT2 mice and traced in a chemi-cally induced liver injury model. Stepwise accumulation ofgenetic alterations in EpCAM-positive cells was induced by themutagenesis activity of activation-induced cytidine deaminase

using conditional transgenic mice. Lineage-tracing experimentsrevealed that labeled PDC differentiated into cholangiocytes,but not into hepatocytes, in the chemically damaged liver.Nevertheless, EpCAM-positive PDC with genetic alterationsgave rise to HCC after 8 months of chemical administration.PDC-derived HCC showed histologic characteristics of con-comitant ductule-like structures resembling human cholangio-locellular carcinoma (CLC) and exhibited serial transitionsfrom PDC-like CLC cells to hepatocyte-like HCC cells. TheWnt signaling pathway was specifically upregulated in the CLCcomponents of PDC-derived HCC. Our findings provide directexperimental evidence that EpCAM-expressing PDC could be acellular origin of HCC, suggesting the existence of stem/progenitor-derived hepatocarcinogenesis. Cancer Res; 77(22);6131–43. �2017 AACR.

IntroductionLiver cancers, including hepatocellular carcinoma (HCC) and

intrahepatic cholangiocarcinoma, generally arise in liver chron-ically inflamed due to various diseases such as hepatitis virusinfection and steatohepatitis (1). Chronic inflammation playsimportant roles in the occurrence of oncogenic genetic alterations,which leads to the transformation of normal cells into cancer cells(2). In addition, chronic inflammation induces persistent tissueinjury that elicits successive liver damage and regeneration, result-ing in the emergence of tumor cells from proliferating cells. Thus,inflammation-associated liver cancers are thought to derive fromregenerating liver cells that acquire stepwise genetic alterations inthe injured liver.

The liver has extraordinary potential to regenerate followingvarious injuries (3). Its high regenerative capacity is attributed tothe robust self-renewal capacity of hepatocytes. In chronicallyinflamed liver, the proliferation of ductal cells is histologicallyobserved as the expansion of ductule-like structures, referred to asa ductular reaction (4, 5). These proliferating ductal cells (PDC)derived from terminal bile ductules have long been thought to befacultative liver stem/progenitor cells and to contribute to liverregeneration in the damaged liver where hepatocyte proliferationis impaired (3). Although it remains controversial whether PDCscould differentiate into hepatocytes in vivo (6–11), the regener-ative mechanisms of chronically inflamed liver suggest that PDCsas well as hepatocytes are the origin cells of inflammation-asso-ciated liver cancers (12).

Although the transformation of PDCs with their high prolif-erative capacity into cancer cells through the accumulation ofgenetic alterations is a promising concept, it is still unclearwhether PDCs actually give rise to HCCs. Previous reports usinggenetically engineered reporter mice revealed that all of the HCCsexamined derived from hepatocytes (13, 14). Lineage-tracinganalyses with ductal cell–labeled mice also revealed that noneof the HCCs originated from biliary components (13–15). Incontrast, however, several models with enhanced proliferation ofPDCs develop HCCs with great frequency, suggesting that PDCsare the cellular origins of liver tumors (12, 16–18). Consistently,PDC proliferation in the nontumorous liver is associated with atumor recurrence risk after resection of HCC and combinedhepatocellular-cholangiocarcinoma (19, 20). Although these

1Department of Gastroenterology and Hepatology, Graduate School of Medi-cine, Kyoto University, Kyoto, Japan. 2Evolutionary Medicine, Shiga MedicalCenter Research Institute, Shiga, Japan. 3Division of Pathology, Shiga MedicalCenter Research Institute, Shiga, Japan. 4Kansai Electric Power Hospital, Osaka,Japan.

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

Corresponding Author: H. Marusawa, Graduate School of Medicine, KyotoUniversity, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Phone:81-75-751-4319; Fax: 81-75-751-4303; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-1800

�2017 American Association for Cancer Research.

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findings support the notion that PDCs activated in the inflamedliver give rise to HCCs, no solid evidence for PDC-derived hepa-tocarcinogenesis has been reported (12).

Epithelial cell adhesion molecule (Epcam) is the most well-established surface marker of PDCs in diverse species includinghuman, rat, and mouse (3). In both humans and rodents, expan-sion of Epcam-positive PDCs is observed uponmassive or chronicliver damage (21). Epcam-positive PDCs derived from humanandmouse liver differentiate into hepatocytes and cholangiocytesin vitro, suggesting their stem/progenitor potential (22, 23).Moreover, Epcam is reported as a cancer stem cell marker ofvarious cancers including HCC, and Epcam-positive HCC cellsexhibit highly tumorigenic potential (24). Consistently, a subsetof HCCs with ectopic overexpression of Epcam shows poorprognosis, and these "hepatic stem cell-like HCCs" are presumedto derive from biliary/progenitor cells (25). However, the cell fateof Epcam-positive PDCs and their potential as tumor-originatingcells in vivo are not well defined.

In the present study, to clarify whether PDCs give rise to HCCs,we established a novel mouse model in which Epcam-positivePDCs were specifically labeled and acquired genetic alterations ininflamed liver tissues. Lineage-tracing experiments demonstratedthat HCCs originate from the Epcam-positive PDCs in the injuredliver. We also showed that PDC-derived HCCs characteristicallycontain cholangiolocellular carcinoma (CLC) components, repre-senting the divergent cell lineages of tumor cells. Our findingsprovide novel evidence for the putative potential of Epcam-expressing PDCs as cell origins of liver cancers that develop byinflammation-associated tumorigenesis.

Materials and MethodsMice

To create a bacterial artificial chromosome (BAC) construct forEpcamCreERT2 transgenic mice, an ERT2-Cre-ERT2 sequencederived from pCAG-ERT2CREERT2 (Addgene plasmid #13777)was inserted immediately before the ATG start codon of theEpcam gene in a BAC clone containing Epcam-flanking genomicsequences (clone #RP23-324L16) using the Red/ET recombina-tion system (Supplementary Fig. S1). Themodified BACDNAwasinjected into the pronuclei of fertilized C57BL/6 eggs to generatefounder mice. The transgene-positive founder mice were crossedwith C57BL/6 mice to obtain germline-transmitted EpcamCreERT2

transgenic mice. Gt(ROSA)26Sortm1Sor (RosaLacZ) mice and Gt(ROSA)26Sortm14(CAG-tdTomato)Hze (RosaTom) mice were obtainedfrom The Jackson Laboratory. The activation-induced cytidinedeaminase (AID) conditional transgenic (cTg) mice (26), AlbCre/AID cTg mice, and AicdaCre transgenic mice (27, 28) weredescribed previously. Immunodeficient NOD/SCID mice andC57BL/6 wild-type mice were obtained from CLEA Japan Inc.

Animal experimentsTamoxifen (Sigma-Aldrich) in peanut oil (Sigma-Aldrich) was

intraperitoneally injected into 6- to 8-week-old mice at a dose of150 mg/kg body weight. Chow containing 0.1% (w/w) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, Sigma-Aldrich)was fed to approximately 8-week-oldmice for 1week to 8months.For analysis of tumorigenicity, minced fragments of tumor ornontumor tissues were mixed with Matrigel (Corning) and sub-cutaneously transplanted into male NOD/SCID mice. All animalexperiments were approved by Kyoto University Animal Experi-mentation Committee and performed according to its guidelines.

Histologic analysesFor histologic analyses, liver tissues fixed with 4% paraformal-

dehyde were embedded in paraffin or optimum cutting temper-ature compound (Leica Instruments). Paraffin-embedded tissueswere sectioned and stained with hematoxylin and eosin, Wata-nabe's silver impregnation staining, alcian blue (Sigma-Aldrich),and nuclear fast red (Vector Laboratories). For immunohis-tochemistry, the sections were incubated with the primary anti-bodies and then with ImmPRESS peroxidase-conjugated second-ary antibodies (Vector Laboratories), and visualized with theImmPACT DAB Substrate Kit (Vector Laboratories). Slides werecounterstained with hematoxylin (Wako). For immunofluores-cence, frozen sections were stained with the primary antibodiesand then with the fluorescence-conjugated secondary antibodies(ThermoFisher Scientific, Jackson Laboratories, or Sigma-Aldrich), and nuclei were visualized with DAPI (Vector Labora-tories). Primary antibodies included rat anti-A6 (1:200 dilution;generous gift from Valentina M. Factor), rabbit anti–acetylated-atubulin (1:300 dilution; #5335; Cell Signaling Technology),rabbit anti–b-catenin (1:100 dilution; #8480, Cell SignalingTechnology), goat anti-keratin19 (1:100dilution; sc-33111; SantaCruz Biotechnology), rabbit anti-Epcam (1:250 dilution;ab32392; Abcam), rat anti-Epcam (1:200 dilution; ab92382;Abcam), rat anti-F4/80 (1:100 dilution; 14-4801-81; Thermo-Fisher Scientific), chicken anti-GFP (1:1,000 dilution; ab13970;Abcam), goat anti-Hnf4a (1:250 dilution; sc-6556; Santa CruzBiotechnology), and rabbit anti-Ki67 (1:300dilution; #9129; CellSignaling Technology). The area ratio of Epcam-positive cholan-giolocellular pattern within a tumor was analyzed using ImageJsoftware (National Institutes of Health, Bethesda, MD). Forb-galactosidase staining, frozen sections were stained with PBScontaining 1 mg/mL X-galactosidase, 5 mmol/L potassium fer-rocyanide, 5 mmol/L potassium ferricyanide, 2 mmol/L MgCl2,0.02%Nonidet P-40, and0.01%sodiumdeoxycholate at 37�Cfor4 hours and then counterstained with eosin (Muto Kagaku) ornuclear fast red. Forwholemount b-galactosidase staining, organswere fixed with PBS containing 4% paraformaldehyde, 0.2%glutaraldehyde, 5 mmol/L EGTA, 2 mmol/L MgCl2, and 0.02%Nonidet P-40 at 4�C for 1 hour, and stained overnight at roomtemperature with X-galactosidase staining solution as describedabove.

DNA/RNA isolationDNA was isolated from the liver using the DNeasy Blood &

Tissue Kit (Qiagen). RNAwas extracted from frozen samples usingSepasol RNA-I Super G (Nacalai Tesque). For isolation of RNAfrom tumor tissues of CLC-enriched area and CLC-deficient area,paraformaldehyde-fixed frozen sections were microscopicallydissected in reference to serial sections stained for Epcam andKrt19, followed by RNA extraction with a NucleoSpin totalRNAFFPE XS kit (Macherey-Nagel).

Quantitative PCRThe isolated RNA was reverse transcribed using a PrimeScript

RT Reagent Kit (Takara). Quantitative real-time PCR was per-formed with LightCycler 480 System II (Roche) using thefollowing primers: Total LacZ allele—F: 50-CCTCGTGATCTG-CAACTCCA-30 and R: 50-GCGAAGAGTTTGTCCTCAACC-30;Recombined LacZ allele—F: 50-ACTCTTCGCGGTCTTTCCAG-30

and R: 50-TTGGGTAACGCCAGGGTTTT-30; NonrecombinedLacZ allele—F: 50-CAGTGGGGATCGACGGTATC-30 and R:

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50-CAGACTGCCTTGGGAAAAGC-30; 18S rRNA—F: 50-TAGAGT-GTTCAAAGCAGGCCC-30 and R: 50-CCAACAAAATAGAAC-CGCGGT-30; Sox9—F: 50-AGTACCCGCATCTGCACAAC-30 andR: 50-ACGAAGGGTCTCTTCTCGCT-30; Myc—F: 50-GGATTT-CCTTTGGGCGTTGG-30 and R: 50-ATAGGGCTGTACGGAGT-CGT-30; Ccnd1—F: 50-TGCGTGCAGAAGGAGATTGT-30 and R:50-GGAAGCGGTCCAGGTAGTTC-30. The quantified values werenormalized to an internal control gene, 18S rRNA, and the resultsare presented as relative values compared with controls.

Microarray analysisCy3-labeled complementary RNA was prepared from 0.1 mg

total RNA using the Low Input Quick Amy Labeling Kit (AgilentTechnologies), and 0.6 mg of Cy3-labeled complementary RNAwas fragmented and hybridized to SurePrint G3 Mouse GeneExpression 8 � 60K v2 (Agilent Technologies) according to themanufacturer's protocol. Arrays were washed and scanned withthe Agilent SureScan G2600D Microarray Scanner (Agilent Tech-nologies) using the one-color scan setting. The scanned imageswere analyzed with Feature Extraction Software (Agilent Technol-ogies). Hierarchical clustering analysis was performed with Rsoftware. Kyoto Encyclopedia of Genes and Genomes (KEGG)pathway enrichment analysis was performed usingDAVID ver.6.8bioinformatics resources (https://david.ncifcrf.gov/). An enrich-ment score greater than 1.3 was considered significant. Gene setenrichment analysis was performed using public softwareobtained from Broad Institute. Microarray data were depositedin the GEO database (accession number GSE95099).

Statistical analysisStatistical difference in tumor incidence and rate of HCCs

overexpressing PDC marker genes was analyzed by the c2 test.

ResultsIntestinal epithelial cells, but not quiescent hepatobiliary cells,are labeled in a tamoxifen-dependent manner in EpcamCreERT2

transgenic miceFirst, we investigated Epcam expression in various mouse

organs by immunostaining (Fig. 1A). Epcam was stronglyexpressed in the epithelial cells of the jejunum, ileum, and colon,but only weakly expressed in the intrahepatic bile ducts andterminal ductules in the liver. In contrast, endogenous Epcamimmunoreactivity was absent in hepatocytes.

To track putative hepatic stem cells residing in biliary compo-nents and their progenies, we generated a transgenic mouse inwhich tamoxifen-inducible Cre recombinase was inserted beforethe start codon of the Epcam gene (Supplementary Fig. S1). Toassess the efficacies of Cre recombination in EpcamCreERT2 mice,labeling of Epcam-expressing cells with the reporter gene wasexamined in EpcamCreERT2 mice crossed with RosaLacZ reportermice (Fig. 1B). No LacZ-labeled cells were detectable withouttamoxifen injection. Tamoxifen injection induced LacZ-labeledcells in EpcamCreERT2/RosaLacZmice, but the population density ofthe labeled cells differedmacroscopically among organs: dense inthe jejunum, sparse in the ileum and colon, and undetectable inthe hepatobiliary tract (Fig. 1C). The association between endog-enous Epcam expression and labeling with the reporter genes wasfurther examined by immunostaining the intestine and livertissues of the EpcamCreERT2/RosaTom mice. Epcam-positive epi-thelial cells were specifically labeled with tdTomato in the intes-

tine by tamoxifen injection. Although the frequency of reporter-labeled cells after tamoxifen administration was low amongEpcam-immunostained cells, we confirmed that the labeling inEpcamCreERT2/Rosareporter mice was strictly limited to Epcam-pos-itive cells, andnononspecific labelingwas observed in the Epcam-negative cells (Fig. 1D). In contrast to intestinal epithelium, noreporter-labeled cells were detectable in the liver, although cellswithin the bile ductules and bile ducts exhibited endogenousEpcam expression (Fig. 1D).

Thesefindings indicate that epithelial cellswithhigh expressionof endogenous Epcam, but not quiescent hepatobiliary cells, arespecifically labeled in a tamoxifen-dependent manner in thenewly developed EpcamCreERT2 transgenic mice.

Injection of tamoxifen into EpcamCreERT2 mice specificallylabels PDCs activated in the injured liver

Epcam is transcriptionally activated byWnt/b-catenin signaling(29), andWnt/b-catenin signaling is active in PDCs (30). Thus,weexamined whether PDCs in the injured liver were labeled by theEpcamCreERT2 system. For this purpose, a DDC diet, a well-estab-lished model of liver injury with PDC activation, was fed toEpcamCreERT2/Rosareporter mice before tamoxifen treatment (3).Feeding on the DDC diet for 1 week induced the expansion ofPDCs expressing stemness markers such as Ncam and Cd44, andincreased the intensity of b-catenin and Epcam staining in thosePDCs in EpcamCreERT2/Rosareporter mice as well as wild-type mice(Fig. 2A and Supplementary Fig. S2). Interestingly, injection oftamoxifen toEpcamCreERT2/Rosareportermice after the 1-weekDDCdiet resulted in the appearance of scattered reporter-labeled cellsresiding in ductular reactions (Fig. 2B). Immunostaining revealedthat tdTomato-labeled cells at 1day after tamoxifen injectionwerepositive for Epcam and other PDCmarkers such as Krt19 and A6,but negative for a mature cholangiocyte marker (acetylated-atubulin) and amature hepatocyte marker (Hnf4a, Fig. 2C). Thesefindings suggest that immature ductal cells proliferating inresponse to the DDC diet are marked by tamoxifen treatment inthe EpcamCreERT2/Rosareporter mouse liver.

Epcam-positive PDCs differentiate intomature cholangiocytes,but not into mature hepatocytes, in the DDC-induced liverinjury condition

To examine whether Epcam-positive PDCs produce hepato-biliary lineage progeny, mice were continuously provided theDDC diet after tamoxifen injection (Fig. 3A). Ingestion of theDDCdiet induced the progressionof ductular reactions for as longas 4 weeks (Fig. 3B, top plots). We also observed that the labeledPDCs residing in the ductular reactions proliferated and formedductal structures after 4 continuous weeks on the DDC diet (Fig.3B, bottom plots). The number of labeled PDCs graduallyincreased with DDC treatment (Fig. 3C), and the increase in thelabeled PDCs in response to liver injury was also confirmed byquantitative PCR of Cre-recombined LacZ alleles (Fig. 3D).Immunostaining revealed that a subset of tdTomato-labeled cellswas positive for a mature cholangiocyte marker, acetylated-atubulin, after 4-week DDC treatment (Fig. 3E). In addition, alllabeled cells were positive for a ductalmarker, Krt19, and negativefor a mature hepatocyte marker, Hnf4a (Fig. 3F, left plots).Similarly, the tdTomato-labeled cells were Krt19-positive butHnf4a-negative after 8 months on the DDC diet (Fig. 3F, rightplots). DDC feeding followed by 2-week discontinuation alsoresulted in a similar pattern of lineage marker signals

HCC Derived from EpCAM-Positive Ductal Cells

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(Supplementary Fig. S3A and S3B). Immunostaining for Ki67confirmed that proliferating hepatocytes were sparsely present inthe liver after DDC treatment, suggesting that the regeneratingpotential of hepatocytes was maintained independently of PDCactivation (Supplementary Fig. S3C). Taken together, our lineagetracing demonstrated that Epcam-expressing PDCs differentiatedinto mature cholangiocytes, but not into mature hepatocytes, in

EpcamCreERT2/Rosareporter mice under the DDC-based liver injurymodel conditions.

Epcam-positive PDCs with accumulation of genetic alterationsgive rise to liver tumors

Taking advantage of the characteristic features of Epcam-expres-sing PDCs labeled in EpcamCreERT2 mice, we investigated whether

Figure 1.

Characterization of EpcamCreERT2 transgenic mice. A, Immunohistochemical staining of Epcam in mouse organs. Various epithelial tissues, including the intestinesand liver, were stained for Epcam. Intrahepatic bile ducts are indicated by arrowheads, and the inset shows the high-magnification image. Scale bars,100mm.B,Experimental strategy of tamoxifen-induced Cre-mediated cell-tracking using EpcamCreERT2/Rosareportermice. Arrows show the direction of transcription,and arrowheads indicate loxP sites. C, Macroscopic images of whole mount b-galactosidase staining of EpcamCreERT2/RosaLacZ mice. The intestines andhepatobiliary tract of EpcamCreERT2/RosaLacZ mice 1 week after two injections of tamoxifen are shown. D, Representative images of the intestines and liver ofEpcamCreERT2/Rosareporter mice with and without tamoxifen injection. Whole-mount b-galactosidase–stained EpcamCreERT2/RosaLacZ mice were sectioned andstainedwith hematoxylin and eosin. The intestines and liver of EpcamCreERT2/RosaTommicewere immunostainedwith anti-Epcam (green) andDAPI (blue). The insetsshow the high-magnification image of Epcam-positive cells.

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PDCs that accumulated genetic alterations could give rise to livercancers. One molecular link between inflammation and geneticalterations is the activity of intrinsic DNA mutator enzymes suchas AID (2). Thus, we utilized AID cTg mice expressing AID inresponse to Cre recombination and introduced AID-mediatedstepwise genotoxicity in Epcam-positive PDCs.

First, we examined whether AID was induced in PDCs in theliver inflamed by DDC using AicdaCre/RosaTom mice generatedby crossing AicdaCre transgenic mice with RosaTom reportermice (31). As AicdaCre mice express Cre recombinase in

response to activation of the AID promoter, AicdaCre/RosaTom

mice reveal the history of AID expression as tdTomato expres-sion. Histologic examination detected many tdTomato-expres-sing cells in the liver of AicdaCre/RosaTom mice fed the DDCdiet, and some of them were observed in the ductular reactions.Immunofluorescence staining revealed that tdTomato-expres-sing cells included those positive for Epcam, and those positivefor Hnf4a (Supplementary Fig. S4), indicating that AID isinduced in both PDCs and hepatocytes in the course of chronicliver inflammation.

Figure 2.

PDCs activated in response to feeding on the DDC diet are labeled by tamoxifen injection in the liver of EpcamCreERT2 mice. A, Representative images of the liver ofEpcamCreERT2 mice fed a diet with or without DDC for 1 week. Hematoxylin and eosin (H&E) staining and immunostaining for the indicated molecules areshown. Scale bars, 100mm.B, Images ofb-galactosidase staining of the liver of EpcamCreERT2/RosaLacZmice.Micewere fed aDDCdiet for 1week and injectedwith andwithout tamoxifen. Counterstaining was performed with eosin. Expansion of ductular reactions is indicated by the area surrounded by the dotted line. Blue-labeledcells are indicated by arrowheads, and the inset is a high-magnification image of these cells. Brown clusters seen in the sections are porphyrin depositionscaused by the DDC diet. Scale bars, 100 mm. C, Characterization of labeled cells in the liver of EpcamCreERT2/RosaTom mice using lineage-specific markers. Epcam,Krt19, andA6were stained asmarkers of PDCs, and acetylated-a tubulin andHnf4awere stained asmarkers ofmature cholangiocytes and hepatocytes, respectively.To distinguish tdTomato-positive cells from red fluorescence emitted from the porphyrin depositions, red channel images merged with bright-field imagesare shown side by side, and tdTomato-positive cells are indicated by arrowheads. Scale bars, 100 mm.






Figure 3.

Behavior of labeled PDCs under the conditions of DDCdiet-induced liver injury.A, Schedule of theDDCdiet and tamoxifen injection. After feeding on the DDCdiet for1 week, mice were injected with tamoxifen, followed by feeding on the DDC diet. The time of tamoxifen injection was designated as week 0. B, Representativeimages of hematoxylin and eosin (H&E) staining (top) and b-galactosidase (LacZ) and eosin staining (bottom). EpcamCreERT2/RosaLacZ mice were continuously fedthe DDC diet for the indicated duration before and after tamoxifen injection. LacZ-labeled cells are indicated by arrowheads, and the insets show the high-magnification images. Scale bars, 100 mm.C,Numbers of LacZ-labeled cells in the liver of EpcamCreERT2/RosaLacZmice. The numbers of b-galactosidase–stained cellsper field (magnification, �400) are shown as mean � SEM. More than three mice were analyzed in each group, and 15 fields were counted for each mouse. D,Frequencies of Cre-recombined RosaLacZ alleles quantified by real-time PCR. The amounts of total, Cre-recombined, and nonrecombined RosaLacZ alleles in the liverof EpcamCreERT2/RosaLacZ mice were quantified using real-time PCR, and the frequencies of Cre-recombined RosaLacZ alleles were calculated. The results are shownasmean� SEM (n¼ 3). E, Immunostaining for acetylated-a tubulin. Liver sections of EpcamCreERT2/RosaTommice fed DDC diet were stained with anti–acetylated-atubulin (green) and DAPI (blue). Cilia of cholangiocytes were stained with acetylated-a tubulin. Bile ducts are indicated by the dotted line, and the insetshows the high-magnification images. Asterisks indicate porphyrin deposits. Scale bars, 100 mm. F, Immunostaining for Krt19 and Hnf4a. Mice fed the DDC diet for 4weeks or 8 months were examined. To distinguish tdTomato-positive cells from the red fluorescence emitted from porphyrin depositions, red channel imagesmerged with bright-field images are shown side by side. Scale bars, 100 mm.

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We then crossed EpcamCreERT2micewithAID cTgmice that beartransgenes encoding loxP-flanked GFP gene followed by the AIDgene. In these EpcamCreERT2/AID cTg mice, tamoxifen-dependentCre recombination induced a loss of GFP expression and aberrantexpression of AID in the Epcam-expressing cells (Fig. 4A). Togenerate mice that express AID specifically in PDCs, Epcam-

CreERT2/AID cTg mice were injected with tamoxifen after 1-weekDDC diet feeding (hereafter designated as Epcam-AID mice),followed by continuous feeding on the DDC diet for 8 months.Consistent with previous studies showing that continuous feed-ing on the DDC diet enhances hepatocarcinogenesis (18, 32),sparse minute tumors were observed in a subset of control mice

Figure 4.

Development of PDC-derived HCCs inEpcam-AID mice. A, Strategy of tracingcarcinogenesis originating from PDCsusing EpcamCreERT2/AID cTg mice.Arrows in cells show the direction oftranscription, and arrowheads indicateloxP sites.B, Incidence and size of tumorsdeveloping in Epcam-AID mice and thecontrol mice. Tumor formation in eachmouse was analyzed just after 8 monthson the DDC diet. Numbers in parenthesesare the number of mice with tumorsamong the total mice examined. � , P <0.01 compared with mice without AID ortamoxifen injection by the c2 test. C,Macroscopic images of a representativeliver tumor developing in an Epcam-AIDmouse and images of the liver of thecontrol mice. Note that the enlargementand brown discoloration of the liver weresimilarly induced by the DDC dietregardless of tamoxifen injection. D,Microscopic images of a representativeGFP-negative liver tumor developing inan Epcam-AID mouse. Liver sectionswere immunostained with anti-GFP. Thetumor area is shown by a dotted line. T,Tumor. Scale bars, 100 mm. E, Images ofcoimmunofluorescence staining of GFP(green) and Hnf4a or F4/80 (red).Tumor area is shownby a dotted line, andthe insets show the high-magnificationimages. T, Tumor. Scale bars, 100 mm.






that were not injected with tamoxifen or did not carry the AIDtransgene (Fig. 4B). In contrast, most of the Epcam-AID micedeveloped larger multiple liver tumors with significantly higherfrequency than their controlmice (Fig. 4B andC). Interestingly, 21of 31 tumors (67.7%) in Epcam-AIDmice lacked GFP expressionin the majority of cells (Fig. 4D), and the GFP-negative cells werepositive for Hnf4a (Fig. 4E). Genetic deletion of GFP cassette intumor cells was confirmed by detecting the recombined GFP-AIDallele inGFP-negativeHCCs (data not shown). Although theGFP-positive cells were observed scattered throughout the tumortissues, these cells were stromal cells, most of which were positivefor a Kupffer cell marker, F4/80 (Fig. 4E). Taken together, thesefindings suggest that theGFP-negative liver tumors that developedin Epcam-AID mice originated from Epcam-expressing PDCs.

PDC-derived HCC displayed a divergent lineage ofdifferentiation with a cholangiolocellular component

Histologic examination revealed that all of the GFP-negative,PDC-derived tumors in Epcam-AID mice had an irregular trabec-ular formation composed of hepatocyte-like tumor cells positivefor Hnf4a, showing the characteristics of HCCs (Fig. 5A andSupplementary Fig. S5A). Consistently, fat deposition and/orupregulation of alpha-fetoprotein (Afp), a well-known tumormarker for human HCC, were detected in some of these tumors(Supplementary Fig. S5B). Intriguingly, 11 of 21 (52%) PDC-derivedHCCs in Epcam-AIDmice sparsely contained ductule-likestructures expressing Krt19 and Epcam (Fig. 5A and B). Thoseductule-like tumor components comprised less than 10% of eachtumor tissue and histologically resembled CLC, a subtype ofcombined hepatocellular-cholangiocarcinoma (33).

As a representative model of hepatocyte-derived HCC, we alsoexamined HCCs developed in AlbCre/AID cTg mice (Alb-derivedHCCs) inwhichHCCs originated from albumin-expressing hepa-tocytes through AID-mediated mutagenesis (34). All of the Alb-derived HCCs were histologically consistent with well-to-mod-erately differentiated HCCs, and none of those tumors containedductule-like components (Supplementary Fig. S5C). Therewas noapparent histologic difference, other than ductule-like compo-nents, between Alb-derived HCCs and PDC-derived HCCs. Tocharacterize the distinctive ductule-like components in PDC-derived HCCs, we performed alcian blue staining, which con-firmed the lack of mucin production in tumor cells, supportingthat these ductule-like tumor cells were CLC, not cholangiocel-lular carcinoma (Fig. 5C). The CLC component in PDC-derivedHCCs was immunohistochemically positive for Ncam (Fig. 5D),which tends to be extensively expressed in human CLCs (33, 35).In sharp contrast to the GFP-positivity of the biliary ductal cells inthe nontumorous region, tumor cells of the CLC componentlacked GFP expression (Fig. 5E), indicating that those CLC cellsoriginated fromEpcam-expressing PDCswithCre recombination.Silver staining, which stains reticular fibers lining the sinusoids,revealed that CLC cells and hepatocytic tumor cells abutted eachother (Fig. 5F), suggesting possible transitions between them.Immunostaining for Ki-67 revealed that both hepatocytic HCCand ductular CLC cells were positive for Ki-67, but the Ki-67labeling index was lower in CLC cells (14.7%� 3.9%) comparedwithHCCcells (21.4%�1.7%; Supplementary Fig. S5D).We alsoconfirmed that tumors in Epcam-AID mice continued growingafter discontinuing the DDC diet for 8 months (SupplementaryFig. S6A), and allograft transplantation of PDC-derived tumorsinto NOD/SCID mice led to the development of tumors histo-

logically similar to the original tumors (Supplementary Fig. S6Band S6C). Taken together, these findings demonstrated thatEpcam-positive PDCs produced HCCs, and that a subset of theseHCCs contained CLC components, suggesting they were com-posed of two types of tumor cells, i.e., hepatocyte-like HCC cellsand PDC-like CLC cells.

A subgroup of human HCCs, in which hepatocytic tumor cellsoverexpress biliary/progenitor marker genes, exhibit aggressivetumor growth and a poor prognosis (25, 36), and they arespeculated to derive from putative liver progenitor cells. Thus,we examined the incidence of overexpression of biliary/progen-itor markers in PDC-derived HCCs. Immunohistochemical stain-ing revealed that 3 of 21 (14.3%) tumors derived from PDCsrepresented ectopic overexpression of Epcam and/or Krt19 inHCC cells (Fig. 6A and B). On the other hand, 23.5% of Alb-derivedHCCs ectopically expressed Epcamand/or Krt19 (Fig. 6B),with a frequency comparable with that observed in PDC-derivedHCCs. These results suggested that upregulation of biliary/pro-genitor markers in tumor cells is not necessarily indicative of aPDC origin.

Wnt signaling pathway is characteristically activated in CLCcomponents within PDC-derived HCC

To clarify the characteristics of PDC-derivedHCCs,we analyzedthe gene expression profiles of PDC-derived HCCs with or with-out CLC components in Epcam-AID mice and Alb-derived HCCsusing comprehensive cDNA microarrays. Hierarchical clusteringanalysis identified clusters of genes specifically upregulated in thecorresponding HCC groups (Fig. 7A and Supplementary TableS1). Pathway analyses revealed that Wnt pathway–related genes,as well as extracellular matrix–related genes, were significantlyenriched in the upregulated genes in PDC-derivedHCCswithCLCcomponents (cluster A of Fig. 7A and B, and Supplementary TableS2). In contrast, numbers of genes upregulated in PDC-derivedHCCs without CLC components and Alb-derived HCCs weresimilarly categorized into pathways associated with metabolism(clusters B and C of Fig. 7A and B).

To further examine the transcriptional activation of Wnt sig-naling in PDC-derived HCCs with CLC components, we micro-scopically isolated the tumor tissues of CLC-enriched and CLC-deficient areas from identical PDC-derived HCCs, and examinedthe expression of representative Wnt signaling–downstreamgenes, including Sox9, Myc, and Ccnd1. Quantitative reversetranscription-PCR revealed high expression of Sox9, Myc, andCcnd1, especially in the area containing CLC components (Fig.7C). Moreover, the strong expression of Sox9, Ccnd1, and b-Cate-nin in CLC cells was confirmed by immunohistochemistry (Fig.7D). These results suggest that Wnt signaling is specifically upre-gulated in the CLC components of PDC-derived HCCs.

Finally, we compared our microarray data with the geneexpression profile of human CLCs (37). Gene set enrichmentanalysis revealed that the expression profile of PDC-derivedHCCswith CLC was highly similar to that of human CLCs (Fig. 7E),suggesting that the CLC components in PDC-derived HCCsrecapitulate the gene expression profile of human CLCs.

DiscussionEpcam is a transmembrane glycoprotein expressed in the

epithelial cells of various organs (21). Epcam is a marker ofpluripotent stem cells as well as tissue stem cells, and its

Matsumoto et al.





expression fades as stem/progenitor cells differentiate intomaturecell lineages (21). In the liver, Epcam is expressed in PDCs andbiliary epithelial cells, but not in mature hepatocytes. In thisstudy, we generated EpcamCreERT2 mice and investigated the roleof Epcam-positive PDCs as a source of carcinogenesis. Lineage-tracing analysis showed that PDCs activated indamaged liverwerespecifically labeled in EpcamCreERT2mice, and those labeled PDCs

differentiated into cholangiocytes, but not into hepatocytes,under the conditions of DDC-induced liver damage. Concurrent-ly, we demonstrated that PDCs activated in response to chronicliver damage gave origin to HCCs through the accumulation ofAID-mediated genetic alterations.

Hepatic stem/progenitor cells with the potential to differentiateinto both hepatocytes and cholangiocytes are thought to reside in

Figure 5.

PDC-derived HCCs containing CLCcomponents that developed in Epcam-AID mice. A, Hematoxylin and eosin(H&E) staining of PDC-derived HCCs.Representative images of HCCwith andwithout CLC components are shown. Inthe images of HCC without CLCcomponents, the boundary area oftumorous and nontumorous tissues isshown and the tumor area is indicatedby the dotted line. T, Tumor. Scale bars,100 mm. B, Immunohistochemicalstaining of HCC with CLC componentsusing anti-Epcam and anti-Krt19. Scalebars, 100 mm. C, Images of alcian bluestaining of CLC components. Sectionsof HCC with CLC components werestained by alcian blue combined withhematoxylin and eosin or nuclear fastred (NFR). CLC components areindicated by arrowheads, and the insetsshow the high-magnification images.Images of the colon are also shown as apositive control. Scale bars, 100 mm. D,Immunohistochemical stainingof HCC with CLC components usinganti-Ncam. Scale bars, 100 mm. E,Immunofluorescence double-stainingfor Epcam and GFP. Note that Epcam-positive cells in tumorous tissues arenegative for GFP, whereas Epcam-positive cells in nontumorous tissuesare positive for GFP. Tumor cells areshown by a dotted line. T, Tumor. Scalebars, 100 mm. F, Silver staining aroundCLC components. Reticular fibers liningsinusoids are observed as black lines.Two different representative areas areshown as low- and high-magnificationimages. Images on the right areschemes indicating adjacent portions(arrows) of CLC cells and hepatocytictumor cells. C, CLC cells; H, hepatocytictumor cells. Scale bars, 100 mm.






the connecting area between hepatocytes and cholangiocytes, andto function as a backup source for liver regeneration via generatingPDCs (3). Indeed, regeneration of biliary trees from PDCs isclearly demonstrated by three-dimensional imaging (38). On theother hand, whether hepatocytes are regenerated from PDCs inchronically damaged liver remains controversial (6–11). In thepresent study, PDCs activated in the liver damaged by DDC werespecifically marked in EpcamCreERT2/Rosareporter mice, and thoseEpcam-expressing PDCs differentiated into cholangiocytes. Incontrast, we detected noPDCs that differentiated into hepatocytesin the liver of EpcamCreERT2/Rosareporter mice. This observationwas in accordance with the findings of several previous studiesusing other ductal cell–labeled mouse models fed the DDC diet(6–10). The lack of evidence for PDC-derived hepatocytes in theDDC-mediated liver injury model might be attributable to theregeneration capacity of the remnant hepatocytes in liver dam-aged by DDC.

Taking advantage of the lack of differentiation of PDCs intohepatocytes in the DDC-induced liver injury model, we investi-gated whether Epcam-positive PDCs could directly give origin toliver cancer. AID is upregulated in both hepatocytes and cholan-giocytes in response to inflammatory stimulation (31, 39, 40) andelicits liver carcinogenesis through the induction of somaticmutations (34, 41). AID-mediated mutagenesis in epithelial cellsunder inflammatory conditions is also attributed to inflamma-tion-associated carcinogenesis in various organs (42, 43). Thus,mice with enhanced AID expression in the inflamed liver couldserve as a good model that recapitulates human inflammation-associatedhepatocarcinogenesiswith stepwise genetic alterations.By introducing AID-mediated genetic alterations in Epcam-

expressing PDCs, we found that Epcam-AID mice developedHCCs derived from Epcam-positive PDCs under a condition inwhich thePDCsdidnot differentiate intohepatocytes. Although itis difficult to determine whether those HCCs originated directlyfrom immature PDCs or via differentiated cholangiocytes, ourfindings suggest that ductal cells in the biliary components giverise to HCCs, possibly directly, but not via differentiatedhepatocytes.

In contrast to our findings, previous lineage-tracing analysesusing various mouse models produced no evidence of directmalignant transformation of PDCs into tumor cells (13–15). Itmust be noted, however, that most of those previous studiesexamined the cellular origins of HCCs using diethylnitrosamine-based carcinogenesis models. Although Mu and colleagues intro-duced oncogenic gene alterations in PDCs using Pten-floxedmice,they found no tumorigenesis in the liver tissues (14). The dis-crepancy in the incidence of PDC-derivedHCCsbetween previousmodels and our model might be due to the difference in thecarcinogenic burden placed on the PDCs. Consistent with ourresults, Epcam-positive PDCs transduced with oncogenic H-Rasand SV40LT were reported to develop liver tumors includingHCCs after transplantation into immunodeficient mice (44).Interestingly, Zhu and colleagues recently reported that micewhose tissue stem cells in various organs harbor mutations inoncogenes and/or tumor suppressor genes developed tumors incorresponding organs, including the liver (32). They also showedthat liver injury–induced ductal cell proliferation markedlyincreased the risk of hepatocarcinogenesis, although it isunknown whether the liver tumors developed directly from thePDCs or via hepatocytes that differentiated from PDCs.

Figure 6.

HCCs with overexpression ofbiliary/progenitor marker genes.A, Representative images of HCCwith ectopic overexpression ofEpcam and Krt19. Hematoxylin andeosin (H&E) staining andimmunohistochemical staining ofEpcam and Krt19 are shown. The tumorarea is indicated by a dotted line. T,Tumor. Scale bars, 100 mm. B,Summary of HCCs overexpressingbiliary/progenitor marker genes.Tumor diameter is shown as mean� SEM. Numbers in parenthesesare the rate of HCCs, withoverexpression of Epcam and/orKrt19 by immunohistochemistry.NS, not significant.

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Figure 7.

Expression analyses of PDC-derived HCCs. A, Hierarchical clustering of genes differentially expressed among HCC groups. Differentially expressed genes (>3-fold)among HCC groups were analyzed. B, KEGG pathways enriched in each gene cluster. Clusters with an enrichment score (ES) higher than 1.3 are shown. C,Expression of Sox9, Myc, and Ccnd1 genes in CLC-enriched and CLC-depleted areas from identical HCCs. Nontumorous tissues adjacent to HCCswere also analyzed.The results are expressed as relative values compared with nontumorous tissues. Values are shown as mean � SEM (n ¼ 4). NT, nontumor; hT, CLC-depletedarea; cT, CLC-enriched area.D, Immunostaining images of CLC components in PDC-derived HCCs. Representative images stained for Sox9, Ccnd1, and b-catenin areshown. Scale bars, 100 mm. E, Gene set enrichment analysis of genome-wide expressional data of PDC-derived HCCs with CLC components compared withthe other HCCs. Microarray data of PDC-derived HCCs with CLC components were compared with those of PDC-derived HCCs without CLC components andAlb-derived HCCs. Gene sets upregulated or downregulated in human CLCs versus human HCCs were defined based on differentially expressed genes(>3-fold) reported by Coulouarn and colleagues (37). FDR, false discovery rate.






An intriguing feature of the PDC-derived HCCs that developedin Epcam-AID mice was the concomitant component of ductule-like structures in the tumor tissue. Indeed,more than half of PDC-derived HCC tissues characteristically contained CLC compo-nents resembling bile ductules, whereas no Alb-derived HCCshad CLC components. CLC components of PDC-derived HCCswere positive for PDCmarkers, and enhancedWnt activation wasspecifically detected in the CLC components. CLC is histologicallycharacterized by the proliferation of "antler-like" small tubulesrecapitulating bile ductules and defined as a stem-cell featuresubtype of combined hepatocellular-cholangiocarcinoma (33).HCC with CLC components sometimes develops in humanchronically inflamed liver (35). Interestingly, CLC componentsin PDC-derived HCCs histologically exhibited serial transitionsfrom ductular CLC cells to hepatocytic tumor cells, similar tohuman CLCs thatmostly accompany transitions intoHCCs at thetumor boundary (45). Notably, a recent integrative genomicanalysis of human combinedhepatocellular-cholangiocarcinomareported that the CLC subtype in particular exhibits biliary epi-thelial features, suggesting its origin as a biliary committedprecursor (46). In addition, the Wnt signaling pathway plays acritical role in PDC proliferation (47) and is more prominentlyactivated in human CLCs compared with HCCs (37). Theseobservations suggest that PDC-derived HCCs possess a carcino-genic process in which PDCs withWnt activation are transformedinto CLC cells that then differentiate into hepatocytic HCCcells. The significance of Wnt signaling would be further investi-gated to elucidate the molecular mechanism of PDC-derivedcarcinogenesis.

On the other hand, accumulating evidence reveals that aberrantupregulation of biliary/progenitor markers, including EpCAM, isdetectable in 18% to 35% of human HCCs, and such HCCs withbiliary/progenitor marker expression have a significantly poorerprognosis than HCCs without biliary/progenitor marker expres-sion (25, 36). It is unclear, however, whether hepatocytic tumorcells with biliary/progenitor marker upregulation are directlyderived from putative liver progenitor cells or via differentiatedhepatocytes. In the current models, the incidence of HCCs withupregulated biliary/progenitor marker expression in Epcam-AIDmice was comparable with that in AlbCre/AID cTgmice (14.3% vs.23.5% in Fig. 6). Consistent with our findings, recent studiesshowed that differentiated hepatocytes give rise to HCCs expres-sing biliary/progenitor markers (13, 14). Thus, these findingssuggest that hepatocytic tumor cells with aberrant upregulationof biliary/progenitor markers do not always originate from ductalcells in the inflamed liver.

Opinions vary as to the cellular origin of PDCs in the liver. Anumber of previous studies suggested that ductular reactionsemerging in the damaged liver are composed of amplifying"progenitor" cells derived from "stem" cells in the bile ductules(for reviews, see ref. 3). On the other hand, hepatocytes canconvert into reprogramed progenitor cells following liver damage(48–50). The reprogramed progenitor cells that derive from

hepatocytes are biphenotypically positive for a hepatocytemarker(Hnf4a) and a subset of biliary/PDC markers (Sox9 and osteo-pontin), and their metaplasia to cholangiocytes occurs progres-sively as the expression of Epcam and Krt19 increases (48–50). Inour EpcamCreERT2 mouse model, only PDCs with high levels ofEpcam expression could be labeled due to their limited Crerecombination efficacy, and none of the labeled cells expressedHnf4a during the analyzed period. Thus, the labeled cells withinthe ductular reactions in the damaged liver of EpcamCreERT2 micewould comprise PDCs that derived from bile ductules, and notreprogramed hepatocytes.

In conclusion, our data provide concrete experimental evidencethat PDCs expressing Epcam are the cellular origin of HCC ininjured liver with PDC activation. PDC-derived HCCs showedhistologic characteristics of concomitant CLC componentsaccompanied by the activation of Wnt signaling. We suggest thatPDC-derived HCCs successively transition between hepatocyte-like HCC cells and PDC-like CLC cells within tumor tissues.Although further studies are required to determine the molecularbasis of PDC-derived carcinogenesis and the clinical significanceof PDC-derived HCCs among human HCCs, approaching thecarcinogenic mechanisms of diverse HCCs from the viewpoint oftheir cellular origins should provide novel promising treatmentstrategies for inflammation-associated hepatocarcinogenesis.

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

Authors' ContributionsConception and design: T. Matsumoto, H. MarusawaDevelopment of methodology: T. Matsumoto, H. MarusawaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Matsumoto, Y. EsoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Matsumoto, T. Manabe, H. MarusawaWriting, review, and/or revision of the manuscript: T. Matsumoto, A. Takai,T. Manabe, H. MarusawaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Kinoshita, H. Seno, H. MarusawaStudy supervision: A. Takai, T. Manabe, H. Seno, T. Chiba, H. Marusawa

AcknowledgmentsWe thankDr. T.Honjo for the generous donation of the AID cTg and AicdaCre

transgenic mice, and Drs. K. Takahashi and Y. Ueda for helpful suggestion.

Grant SupportThis work was supported by Japan Society for the Promotion of Science

(JSPS) Grants-in-Aid for Scientific Research, KAKENHI (grant number26293172), and Grant-in-Aid for JSPS Fellows (grant number 14J02212),Japan.

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

Received June 16, 2017; revised August 11, 2017; accepted September 22,2017; published OnlineFirst September 26, 2017.

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2017;77:6131-6143. Published OnlineFirst September 26, 2017.Cancer Res Tomonori Matsumoto, Atsushi Takai, Yuji Eso, et al. Give Rise to Hepatocellular CarcinomaProliferating EpCAM-Positive Ductal Cells in the Inflamed Liver

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http://cancerres.aacrjournals.org/content/77/22/6131.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/77/22/6131


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