b-catenin activation in a novel liver progenitor cell type is … · tumor and stem cell biology...

Tumor and Stem Cell Biology

b-Catenin Activation in a Novel Liver Progenitor Cell Type IsSufficient to Cause Hepatocellular Carcinoma andHepatoblastoma

Sharada Mokkapati1, Katharina Niopek1, Le Huang1,2, Kegan J. Cunniff1, E. Cristy Ruteshouser1,Mark deCaestecker5, Milton J. Finegold4, and Vicki Huff1,2,3

AbstractHepatocellular carcinoma (HCC) was thought historically to arise from hepatocytes, but gene expression

studies have suggested that it can also arise from fetal progenitor cells or their adult progenitor progeny.Here, we report the identification of a unique population of fetal liver progenitor cells in mice that can serveas a cell of origin in HCC development. In the transgenic model used, mice carry the Cited1-CreERTM-GFP BACtransgene in which a tamoxifen-inducible Cre (CreERTM) and GFP are controlled by a 190-kb 50 genomicregion of Cited1, a transcriptional coactivator protein for CBP/p300. Wnt signaling is critical for regulatingself-renewal of progenitor/stem cells and has been implicated in the etiology of cancers of rapidly self-renewing tissues, so we hypothesized that Wnt pathway activation in CreERTM-GFPþ progenitors wouldresult in HCC. In livers from the mouse model, transgene-expressing cells represented 4% of liver cells atE11.5 when other markers were expressed, characteristic of the hepatic stem/progenitor cells that give rise toadult hepatocytes, cholangiocytes, and SOX9þ periductal cells. By 26 weeks of age, more than 90% of Cited1-CreERTM-GFP;Ctnnb1ex3(fl) mice with Wnt pathway activation developed HCC and, in some cases, hepato-blastomas and lung metastases. HCC and hepatoblastomas resembled their human counterparts histolog-ically, showing activation of Wnt, Ras/Raf/MAPK, and PI3K/AKT/mTOR pathways and expressing relevantstem/progenitor cell markers. Our results show that Wnt pathway activation is sufficient for malignanttransformation of these unique liver progenitor cells, offering functional support for a fetal/adult progenitororigin of some human HCC. We believe this model may offer a valuable new tool to improve understanding ofthe cellular etiology and biology of HCC and hepatoblastomas and the development of improved therapeuticsfor these diseases. Cancer Res; 74(16); 4515–25. �2014 AACR.

IntroductionHepatocellular carcinoma (HCC) is the fifth most common

cancer worldwide with a very high mortality rate (1). His-torically, HCCs were thought to arise from hepatocytes.Interestingly, gene expression profiling of human HCCs hassuggested that a subset of HCCs can also arise from a liverprogenitor/stem cell (2). Molecular analyses of HCCs haveidentified various gene mutations and dysregulated signaling

pathways in tumors, including alterations that upregulatethe Wnt/b-catenin, Ras/Raf/MEK/ERK, PI3K/mTOR, andSonic Hedgehog pathways (3). Gene mutations that activatethe Wnt/b-catenin signaling pathway are observed in 50% ofHCCs, and the most common of these is CTNNB1 mutationsthat result in stabilization of b-catenin (4). Thus, oneapproach for generating mouse models for HCCs has beento activate the Wnt signaling pathway via Ctnnb1 mutation(5). Wnt pathway activation in adult murine hepatocytes failsto induce tumors (6–8). However, introduction of geneticalterations such as Ha-Ras or Akt mutation in adult hepa-tocytes in addition to Wnt pathway activation does result inHCC (9, 10). Published data therefore indicate that activationof the Wnt pathway alone is insufficient for HCC initiation, atleast in hepatocytes.

Because the Wnt signaling pathway plays a critical role inregulating stem/progenitor cell self-renewal and because ofthe suggestion that a fetal progenitor is the cell of origin forsome human HCCs, we hypothesized that activation of theWnt pathway in a unique population of bipotential fetal livercells that we have identified could give rise to HCC in vivowithout the introduction of additional genetic events. Aspresented below, these fetal liver cells are characterized by

Authors' Affiliations: 1Department of Genetics, University of Texas MDAnderson Cancer Center; Graduate Program in 2Genes and Developmentand 3Human Molecular Genetics, UT-Houston Graduate School of Bio-medical Sciences; 4Baylor College of Medicine and Texas Children'sHospital, Houston, Texas; and 5Department of Medicine, Vanderbilt Uni-versity, Nashville, Tennessee

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

Corresponding Author: Vicki Huff, Department of Genetics, University ofTexas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,TX 77030. Phone: 713 834-6384; Fax: 713 834-6380; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-3275

�2014 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 4515

on August 1, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 21, 2014; DOI: 10.1158/0008-5472.CAN-13-3275

http://cancerres.aacrjournals.org/

their expression of the Cited1-CreERTM-GFP BAC transgene(11) and express CD45, in addition tomarkers characteristic ofhepatic stem/progenitor cells in fetal liver. They can differ-entiate, both in vitro and in vivo, into hepatocytes and cho-langiocytes. We assessed the ability of b-catenin stabilizationto transform these cells by generating mice (Tg-b-catS) thatcarried both the BAC transgene and a Ctnnb1 conditionallystabilized allele (Ctnnb1þ/ex3(fl)) (11, 12). By 26 weeks of age,91% of Tg-b-catS mice developed HCC, demonstrating thatintroduction of a stabilizing Ctnnb1mutation into a fetal liverprogenitor can result in endogenous HCCs in adult mice.Hepatoblastomas and lung metastases were also observed inmutant mice.

Materials and MethodsMouse strains

Animal work was carried out in compliance with the Insti-tutional Animal Care and Use Committee of MD AndersonCancer Center (Houston, TX). Cited1-CreERTM-GFP is a trans-genic line carrying a BAC transgene in which expression of theCre gene (and also a GFP reporter) is driven by a 190-kbfragment 50 of the Cited1 gene, and Cre function is induciblewith tamoxifen in adose-dependentmanner (11).Ctnnb1þ/ex3(fl)

and ROSA26R-LacZ mouse strains were also used in the study(11–13). Ctnnb1þ/fl;Cited1-CreERTM-GFP embryos were gener-ated and treated with tamoxifen (0.5 mg/40 g maternal bodyweight) at E14.5, which resulted in b-catenin stabilization intransgene-expressing cells (Tg-b-catS).

RT-PCR analysisConditions for quantitative RT-PCR and primers are listed in

Supplementary Methods.

Histology and IHCTissues were paraformaldehyde-fixed, paraffin-embedded,

and assessed by hematoxylin and eosin or IHC. For IHC, tissuesections were deparaffinized and the antigens were retrievedby boiling for 20 minutes in citrate buffer (pH 5). Antibodiesused are listed in Supplementary Methods.

Western blottingProteins were extracted from snap-frozen tissues andWest-

ern blotting was performed by standard protocols (14). Anti-bodies used are listed in Supplementary Methods.

Reverse-phase protein array analysisProtein extracts from normal livers and liver tumors were

prepared and subjected to reverse-phase protein array (RPPA)as previously described (15).

FACS analysisSingle-cell suspensions of fetal liver (E12.5 –E17.5) from

Cited1-CreERTM-GFP mice were prepared by homogenizationof fetal livers and sorting forGFP expression using the BDFACSAria High Speed Digital Cell Sorter. Cell suspensions fromembryos with no transgene served as negative controls. TheCited1-CreERTM-GFP transgene is known to be expressed in

fetal kidney cells (11) and kidney suspensions from Cited1-CreERTM-GFPmicewere positive controls. Antibodies used andconditions for FACS analysis are provided in SupplementaryMethods.

Cell cultureGFP-sorted cells from fetal liver were cultured in laminin-

coated dishes for 21 days in differentiating medium (16). Cellswere photomicrographed, RNA was extracted, cDNA wassynthesized, and semiquantitative PCRanalysiswas performedas previously described (17).

X-gal stainingCited1-CreERTM-GFP;ROSA26R-LacZ embryos were treated

in utero at E14.5 with tamoxifen (3 mg/40 g maternal bodyweight). Livers were dissected at 2 months of age and stainedwith X-gal as described in Supplementary Methods.

Statistical analysisStatistical significance of the results between groups was

determined by Student t test. Statistical differences were con-sidered significant if p < 0.05 (�), <0.01 (��), and <0.001 (��).All data are represented as mean � SEM.

ResultsExpression of Cited1-CreERTM-GFP transgene in fetalliver

Transgene expressionwas detectable byGFPfluorescence inE13.5 liver [Supplementary Fig. S1A (a)]. FACS analysis of livercell suspensions (E11.5 –E17.5) showed robust GFP expressionin 4% of fetal liver cells (denoted as CreERTM-GFPþ) at E11.5.This declined to 0.3%–0.6% by E14.5, and by E17.5, the GFPþ

cells were undetectable. GFPþ cells in fetal kidney served as acontrol (Table 1). In contrast to the very low frequency of GFPþ

cells in livers from the BAC transgenic mice, widespreadendogenous CITED1 protein expression has been observed infetal liver from wild-type mice at E11.5 to E14.5 (18). Thus,while the transgene includes 190 kb from the 50 region of theCited1 gene, transgene expression is much more restrictedthan that of the endogenous locus.

CreERTM-GFPþ cells are bipotent in cell cultureSemiquantitative PCR analysis of E12.5 and E13.5 CreERTM-

GFPþ cells showed expression of albumin, a1-antitrypsin,

Table 1. FACS sorting of embryonic liver andkidney samples for GFP expression

Liver (% GFPþ) Kidney (% GFPþ)

E11.5 2.5%–4.0% Not determinedE12.5 0.8%–1.0% Not determinedE13.5 0.3%–0.6% Not determinedE14.5 0.3%–0.1% 0.5%E17.5 Undetectable 0.9%

NOTE: Expressed as percentage of total liver/kidney cells.

Mokkapati et al.

Cancer Res; 74(16) August 15, 2014 Cancer Research4516




cytokeratin 18, and vinculin but not cytokeratin 19 (Supp-lementary Fig. S1A, b). Because the GFPþ cells were so rarein fetal liver, we speculated that they might be progenitor cells.To test this, we isolated CreERTM-GFPþ liver cells by FACSsorting, plated them at a low density (3 � 103/cm2) andcultured them under conditions that induce differentiation.CreERTM-GFPþ cells clonally expanded in culture and gave riseto hepatocytes and cholangiocyte-like cells (SupplementaryFig. S1B, a–c). Semiquantitative PCR analysis showed thatthese cultures expressed markers of hepatocyte lineage (albu-min, a-fetoprotein, and a1-antitrypsin), cholangiocyte mar-kers (cytokeratin 19 and vinculin) and cytokeratins 18 and 8,which were expressed by both these lineages (SupplementaryFig. S1B, d), indicating that CreERTM-GFPþ cells were indeedbipotent.

Fate mapping of CreERTM-GFPþ cells in liverWe crossed Cited1-CreERTM-GFP mice with the Rosa26R-

LacZ reporter mice (13). Following tamoxifen treatment(3 mg/40 g body weight) of embryos at E14.5, liver sampleswere collected at 2 months and assessed for the presence ofb-galþ cells. In the liver, hepatocytes (hexagonal large cells)and cholangiocytes of both small and large bile ducts (smallcuboidal cells) were positive for b-galactosidase enzymeactivity (Fig. 1A, a–c). Immunofluorescence analysis withantibody specific for b-galactosidase on liver sectionsshowed b-galþ cells in the liver (Fig. 1A, d). Co-stainingwith Sox9, a marker for adult liver progenitors (19) andb-galactosidase antibodies, identified cells of bile ducts thatwere double positive, suggesting that the transgene-expres-sing cells give rise to the ductal cells (Fig. 1A, e).

Figure 1. Characterization ofCreERTM-GFPþ cells in fetal liver.A, P60 liver sections showing X-galþ liver cells, hepatocytes, andbile ducts (arrows, a); highermagnification shows the small(arrows, b) and large bile ducts (c).Antibody staining of cryosections(P60) for b-galactosidase inhepatocytes (arrow, d) and inSOX9-positive (red nuclei) bile ductcells (e). 10 and 100 show highermagnification. Scale bar, 160 mm(a, d) and 32 mm (e). B, FACSanalysis for DLK1 and EPCAM (aand b), CD13 and CD133 (c and d)in theCreERTM-GFPþcells at E11.5to E13.5.

Liver Cancer Genetic Model

www.aacrjournals.org Cancer Res; 74(16) August 15, 2014 4517




Expression of cell surface markers characteristic ofprogenitors in the CreERTM-GFPþ cells

We characterized the fetal liver GFPþ cells at E11.5-E13.5with markers characteristic of hepatic stem/progenitors byFACS analysis. More than 95% of the CreERTM-GFPþ cellsexpressed DLK1, a fetal stem/progenitor marker that isexpressed between E10.5 and E16.5 and is undetectable inneonatal and adult livers (Fig. 1B, a; ref. 20). EpCAM expressionwas detected in 87% of GFPþ cells in E11.5 livers (Fig. 1B, a).This decreased to 49% and 35% at E12.5 and 13.5, respectively(Fig. 1B, b). Of note, all cells positive for EpCAM were alsopositive for DLK1 (Fig. 1B, a). The percentage of CD13þ andCD133þ cells in the CreERTM-GFPþ cells declined from 95% atE11.5, to 89% at E12.5, to 64% at E13.5 (Fig. 1B, c and d). Inaddition, the expression of AFP was detectable in only 3.3%,2.5% and 13.5% of CreERTM-GFPþ cells at E11.5, E12.5, andE13.5, respectively (Supplementary Fig. S2A, a and b). Table 2summarizes these findings.

Hepatic colony forming units (h-cfus) from fetal liver areintegrina6–positive (low), integrinb1–positive, andnegative forat least three hematopoietic stem cell markers [Ter119, CD45,and CD117 (c-kit); ref. 21]. Like h-cfus, CreERTM-GFPþ cells didnot express CD117 or Ter119 (hematopoietic stem cellmarkers),but unlike h-cfus, a significant portion (31%) did express CD45(Supplementary Fig. S2B, a, d, and e). In addition,more than 90%of CreERTM-GFPþ cells expressed integrin b1, like h-cfus, but,unlike h-cfus, were negative for integrin a6 (Supplementary Fig.S2B, a–c). About 26% of the CreERTM-GFPþ cells expressed bothintegrin b1 and CD45 (Supplementary Fig. S2B, d and e).

qPCR analysis of RNA isolated from the CreERTM-GFPþ andCreERTM-GFP� cells fromE12.5 fetal liver indicated that expres-sion of CD13, Ecadh, Lgr5, and Dlk1 was increased in CreERTM-GFPþ cells relative to CreERTM-GFP� cells (SupplementaryFig. S1C).

Cited1-CreERTM-GFP;b-catþ/fl (Tg-b-catS)micedevelopedHCCs, hepatoblastomas, and lung metastases

To test the tumorigenic potential of CreERTM-GFPþ fetalliver cells, we mosaically and somatically stabilized b-catenin(b-catS) in the CreERTM-GFPþ fetal liver cells. Cited1-CreERTM-GFP and Ctnnb1þ/ex3(fl) littermates served as controls. Exper-imental animals displayed no reduced viability but did display

hepatomegaly (Table 3). Palpatable tumors were detected asearly as 8 weeks and, by 26 weeks, 91% (20/22) of Cited1-CreERTM-GFP; Ctnnb1þ/ex3(fl) mice (hereafter denoted Tg-b-catS) developed tumors (Fig. 2A). Mice were sacrificed whenthey became moribund and tumors were collected. Within ananimal, multiple tumors of variable sizes were randomlydistributed in all liver lobes (Fig. 2B, b). Of note, apparent lungmetastases were also grossly observed in two of the 20 tumor-bearing mice that were sacrificed at 6 months of age(arrowheads, Fig. 2B, c). Genotypic analysis confirmed thepresence of the recombined Ctnnb1 allele in the tumors (Fig.2B, d). Interestingly, loss of the wild-type allele of Ctnnb1(marked by �) was noted in some tumors (e.g., T2).

Complete histologic evaluation was carried out on liversections from 12 tumor-bearing and control mice. Small fociof aberrant hepatocytes with increased basophilic stainingwas observed in most sections (Fig. 2C, b) when comparedwith normal liver sections (Fig. 2C, a). Nodular adenoma-tous lesions (Fig. 2C, c) and HCCs were most commonlypresent (Fig. 2C, d–f). HCCs were characterized by cytolog-ical atypia, occasional mitotic figures (Fig. 2C, g), andpeliosis (arrows, Fig. 2C, d) and were very similar to humanHCC.

Unexpectedly, of the 12 HCC-bearing mice, 5 (42%) also hadhistologically detectable hepatoblastomas. HBs were typicallycomposed of undifferentiated monotypic cells, characteristicof embryonic liver cells, with scanty cytoplasm (Fig. 2C, e) andnumerous mitotic figures (Fig. 2C, h). In human tumors thishistology is classified as the embryonal undifferentiated sub-type of hepatoblastomas (22). Some mice also had moderatelydifferentiated pure fetal hepatoblastomas with steatosis (Sup-plementary Table S1 and Fig. 2C, f). These observations areconsistent with the fact that we targeted a progenitorpopulation.

Histologic evaluation of a lungmetastasis revealed cells withincreased basophilic staining, steatosis, and relatively uniformnuclei (Fig. 2C, i and j). It is not clear whether the metastasesarose from HCC or hepatoblastomas. Determining this willrequire further extensive evaluation.

HCCs, hepatoblastomas, and lung metastases showedactivation of the Wnt pathway and werehyperproliferative

Western blot analysis confirmed the expression of thetruncated b-catenin (80 kDa) encoded by the exon 3–deleted

Table 2. Expression of hepatoblast markers inCreERTM-GFPþcells

E11.5 E12.5 E13.5

CD13 95 91 85CD133 90 90 90CD13/CD133 90 89 83DLK1 98 95 97EpCAM 78 49 21DLK1/EpCAM 77 49 21AFP 4 2 14

NOTE: Expressed as a percentage of CreERTM-GFPþ cells.

Table 3. Liver weight/body weight ratio inTg-b-cats mice at 1 month of age

GenotypeLiver weight/bodyweight ratio

Numberof miceanalyzed

Foldchange

Tg-b-cats 0.1550 � 0.025 8 3.03Controls 0.0511 � 0.002 9 1

NOTE: Controls versus Tg-b-cats: p < 0.0005.

Mokkapati et al.





Ctnnb1 gene and also glutamine synthetase in the Tg-b-catS

HCCs (Fig. 3A). qRT-PCR analysis also revealed a significantincrease in the mRNA expression of glutamine synthetase andother previously reported liver-specific Wnt target genes suchas glutamate receptor 1 (Glt1), ornithine amino transferase(Oat), and leukocyte cell–derived chemotaxin 2 (Lect2)(23, 24), in Tg-b-catS tumors (Fig. 3B). A statistically significantincrease in expression ofAxin2, c-Myc, andCyclin D1, canonicalWnt targets, was also observed in the Tg-b-catS tumors (Fig.3B).b-Catenin IHC demonstrated heterogeneous expression

with increased cytoplasmic and nuclear localization in HCCs,pure fetal hepatoblastomas, and lung metastasis from Tg-b-catSmice (Fig. 3C, a, b, and d). In contrast, b-catenin stainingin the embryonal-undifferentiated hepatoblastomas was strik-ingly nuclear (Fig. 3C, c). In livers from littermate control mice,Wnt signaling activity, marked by the expression of glutaminesynthetase, was restricted to a single layer of perivenularhepatocytes (Supplementary Fig. S3A, b). Glutamine synthe-tase IHC revealed both an increased and an altered expressionpattern in all Tg-b-catS HCCs, in which it was uniformlyexpressed in all the hepatocytes of the tumor and was notrestricted only to perivenular hepatocytes (Fig. 3C, e). Incontrast, glutamine synthetase expression was heterogeneousin the pure fetal hepatoblastomas and lungmetastasis (Fig. 3C,f and h). In the embryonal-undifferentiated variant of hepato-blastomas, glutamine synthetase immunoreactivity was hardlydetectable (Fig. 3C, g). In 1-month-old Tg-b-catS mice that didnot show any overt tumors, we detected increased cytoplasmic

and nuclear localization of b-catenin, aberrant spatial expres-sion of glutamine synthetase in nonperivenular areas andincreased proliferation as detected by Ki67 (SupplementaryFig. S3B, d–f) when compared with age-matched controls(Supplementary Fig. S3B, a–c). Western blotting also con-firmed these changes (Supplementary Fig. S3C). CanonicalWnt targets were also upregulated in these mice (Supplemen-tary Fig. S3D).

Tumors from Tg-b-catS mice (Fig. 3C, i–k) and the lungmetastasis (Fig. 3C, l) showed increased proliferation (Ki67staining) compared with liver sections from littermate controlmice (Supplementary Fig. S3B, c). The embryonal-undifferen-tiated variant of hepatoblastomas displayed the highest fre-quency of proliferating cells (Fig. 3C, k).

HCCs displayed activation of the MAPK and mTORpathway

To identify key cancer signaling pathways altered in theHCCs, we performed RPPA analysis and compared the proteinexpression profile of key proteins in Tg-b-catS HCCs andnormal liver of control littermates (Supplementary Fig. S4).The increased expression of mTOR and MAPKpT202 detectedby RPPA suggested that two key cancer signaling pathways,Ras/Raf/MEK/MAPK and PI3K/mTOR, were significantlyaltered in Tg-b-catS tumors. These two pathways can be activ-ated by FGFR1 (reviewed in ref. 25), and FGFR1 (pT766) wasalso identified by RPPA as being upregulated in the Tg-b-cats

tumors (Supplementary Fig. S4). Western blot analysis vali-dated the RPPA analysis data. In the Ras/Raf/MEK/MAPK

Figure 2. Development of livertumors in Tg-b-catS mice. A,Kaplan–Meier analysis of tumorincidence in mutant animals(dotted line) and littermate controls(solid line). B, gross appearance ofnormal liver (a), liver with multifocaltumors in Tg-b-catS mice (b), andlung metastases (arrowheads, c).Representative PCR from Tg-b-catS mice showing wild-type/floxed bands in tail and liver DNA ofcontrol mice and mutant tumors(d). �, loss of the wild-type allele inT2. C, histologic analysis of livertumors (b–h) and associated lungmetastasis (i and j). Hematoxylinand eosin staining of normal liver(a), altered hepatocytes (denotedby arrow, b), adenoma(demarcated by dotted line, c),HCC (d–f), hepatoblastoma (HB)embryonal (e), andhepatoblastoma pure fetal (f).Insets in "a" and "b" provide highermagnification (�3). g and h, mitoticfigures of HCC (g) and embryonal-undifferentiated hepatoblastoma(h). i and j, lung metastasis (dottedlines, i) and higher magnification (j).Scale bar, 80 mm (a, b, d, j), 500 mm(c, e, f), and 1,280 mm (i).






pathway, we confirmed significant upregulation of MEK1/2(phospho), PKCa (total and pT492), ERK1/2 (total and pT202/204), c-MYC (a downstream target of the pathway), andFOX03a (pS318), a proapoptotic gene that is inhibited byphosphorylation by ERK1/2 (Fig. 4A). In the PI3K/mTORpathway, besides upregulation of mTOR (total and pS2448)by Western analysis, we also identified upregulation of Raptorand GbL, two TORC1 complex proteins. Interestingly, PTEN,the negative upstream regulator of the mTOR pathway whoseloss results in HCC (26), was significantly downregulated in Tg-b-catS tumors (Fig. 4B). Besides these two key signaling path-ways, upregulation of stathmin, SRC, and CHK1, all of whichhave been associated with hepatocarcinogenesis (27–29), wasdetected by RPPA analysis (Supplementary Fig. S4). IHC withPKCa (pT492) and ERK1/2 (pT202) antibodies confirmed theirexpression in HCC tissue (Fig. 4C, e and f). Similarly, upregula-tion ofmTOR (pS2448) and S6 kinase (pS235/236) was detectedin theHCC lesions (Fig. 4C, g and h).While the hepatoblastomafetal variant showed upregulation of PKCa (pT492), ERK1/2(pT202), mTOR (p2448), and S6 kinase (pS235/236) (Fig. 4C,

m–p), the embryonal-undifferentiated variant showed onlyupregulation of PKCa (pT492) and only a few phospho-S6–positive cells were detected (Fig. 4C, i and l), suggesting thatalterations in signaling pathways were specific to the cell typecomprising the tumor.

Tumors express stem/progenitor markersTo test whether the HCCs in our model expressed stem/

progenitor markers characteristic of the cells from whichthey arose, we performed qRT-PCR. A statistically significantincrease in the expression of Cited1, Dlk1, CD133, and Lgr5was observed (Fig. 5A). In addition, the expression of Afp, amarker for premature hepatocytes and also a Wnt targetgene in liver, and Sox9 and Sox4, markers for adult progeni-tors in liver, were also significantly increased in tumors (Fig.5A). Increased Cited1, Lgr5, Sox9, and Afp was also confirmedat the protein level by Western blotting (Fig. 5B). By IHC,CITED1 expression was detected uniformly in all hepato-cytes of the HCCs (Fig. 5C, a), whereas DLK1 expression wasmore heterogeneous (Fig. 5C, b) and SOX9 expression was

Figure 3. Activation of Wnt pathwayin Tg-b-catS tumors. A, Westernblot analysis of Tg-b-catS tumorsand normal control livers forb-catenin and glutaminesynthetase (GS). b-Actin andPonceau-stained membrane (P)were used as loading controls.B, qPCR analysis of liver-specificb-catenin target genes (Gs), Glt1,Oat, and Lect2 in Tg-b-catS tumors(n ¼ 3) and canonical target genesAxin2, c-Myc, and cyclin D,compared with littermate normallivers (n ¼ 3). C, b-catenin IHC inHCC (a), hepatoblastoma (HB)pure fetal (b), embryonal-undifferentiated hepatoblastoma(c), and lung metastasis (d). e–h,GS staining in HCC, fetalhepatoblastoma, embryonalhepatoblastoma, and lungmetastasis. i–l, Ki67 staining inHCC, hepatoblastoma, andmetastasis. Scale bar, 80 mm.

Mokkapati et al.





nuclear and confined to small cells in tumors (arrows, Fig.5C, c). In the embryonal-undifferentiated variant of hepato-blastomas, CITED1 expression was weak, whereas DLK1 andSOX9 expression was robust with almost all cells expressingthese markers (Fig. 5C, d–f). In the pure fetal hepatoblas-tomas, CITED1 expression was uniform and robust (Fig. 5C,g), high DLK1 expression was restricted to a few hepatocytecells (Fig. 5C, h), and SOX9 expression was nuclear andrestricted to a very few cells as in the HCCs (arrows, Fig. 5C,i). CITED1 expression was seen in bile ducts (blackarrows, Fig. 5D, a) and hepatocytes emerging from them(red arrows, Fig. 5D, a) and in tumors (HCCs and hepato-blastomas) around bile ducts (Fig. 5D, b and c).

DiscussionWe report here the identification of a novel population of

fetal liver progenitor cells and the development of endogenousand metastatic liver tumors when b-catenin stabilization/Wntpathway activation—alone—is targeted to these cells. Thesetumors also showed upregulation of the Ras/Raf/MEK/MAPKand PI3K/mTOR pathways.

Wnt pathway activation by CTNNB1mutation is observed in20% to 40% of human HCCs and 50% to 90% of hepatoblas-tomas (30–32). However, in mice, activation of the Wnt path-way by overexpression of wild-type or stable mutants ofb-catenin in hepatocytes failed to result in tumor development(6, 7, 33). For tumors to develop in these mice, Wnt pathway

Figure 4. Activation of the MAPKand mTOR pathways in Tg-b-catS

tumors. A and B, Western blotanalysis of MAPK and mTORpathway components in HCC fromTg-b-catS HCCs compared withcontrol livers. GAPDH andPonceau-stained membraneswere used as loading controls.C, IHC analysis of phospho-PKCa(a, e, i, m), phospho-ERK1/2 (b, f, j,n), phospho-mTOR (c, g, k, o), andpS6 (d, h, l, p) in the normal liver(a–d), HCCs (e–h), embryonalhepatoblastoma (HB; i–l), and fetalhepatoblastoma (m–p) of Tg-b-catS mice. Insets show highermagnification (e–p). �, necroticareas. Scale bar, 80 mm.






activation had to be accompanied by additional alterationssuch as activation of either Ha-Ras or Akt (9, 10) or treatmentwith hepatotoxins (34). In this study, we hypothesized thatb-catenin stabilization in an early fetal progenitor would besufficient for tumor development. Therefore, using the Cited1-CreERTM-GFP mouse strain (11), we targeted Wnt pathwayactivation to CreERTM-GFPþ cells, a unique, and previouslyuncharacterized, early fetal liver progenitor.

Both in vitro differentiation studies and in vivo lineagetracing studies demonstrated that CreERTM-GFPþ cells areprogenitors of hepatocytes and cholangiocytes in the adultliver. Thus, they are functionally like fetal hepatic stem/pro-genitors, which have been characterized, in part, by expression

of cell surface markers such as DLK1, EpCAM, E-cadherin,CD13, and CD133 and which are considered to be bipotentprogenitors for both hepatocytes and cholangiocytes (35).CreERTM-GFPþ cells also express DLK1, CD13, EpCAM, CD133,andE-cadherin, each ofwhich has been reported to be amarkerfor fetal stem/progenitor cells. However, unlike previouslycharacterized hepatic colony-forming units (h-cfus) (16), theCreERTM-GFPþ cells do not express integrin a6 and a fractiondid express CD45 (a hematopoietic stem cell marker). Notably,the expression of AFP was detected in only a small percentageof cells. The expression of CD45 is unique to the CreERTM-GFPþ cells. Recent studies in humans suggest that hepaticprogenitor-like cells also express hematopoietic markers such

Figure 5. Expression of hepaticstem/progenitor markers in Tg-b-catS tumors. A, RT-PCR analysisof HCCs (n ¼ 3) and control livers(n ¼ 3) for expression of stem/progenitor markers. B, Westernblot analysis of stem/progenitormarkers in Tg-b-catSHCCs. C, IHCshowing expression of CITED1 (a,d, g), DLK1 (b, e, h), and SOX9(arrowheads, c, i, and f) in HCC(a–c) embryonal hepatoblastoma(HB; d–f) and fetal hepatoblastoma(g–i). Scale bar, 70 mm. D, IHCshowing CITED1 expression in bileduct epithelium (black arrows, a)and in hepatocytes emergingaround the bile ducts (red arrows,a). Tumors (HCC, b andhepatoblastoma, c) originatingaround the bile ducts. Insets in "a"and "b" show CITED1 expressionin bile duct epithelium. Scale bar, aand b, 70 mm; c, 160 mm.

Mokkapati et al.





as CD45 and CD109 (36). In addition, co-immunofluorescencestudies demonstrated that CreERTM-GFPþ cells also gave riseto SOX9-expressing periductal cells in adult liver, which areconsidered to be adult liver progenitors. Thus, the CreERTM-GFPþ cells are a unique cell population that represents about4% of the liver in E11.5 mice, a time point at which there is anincrease in hepatic parenchyma and hepatoblast expansion.These cells were almost undetectable by E17.5, by which timeliver development is near to completion.In animals in which b-catenin was stabilized in the

CreERTM-GFPþ cells (Tg-b-catS mice), more than 90% devel-oped HCC by 26 weeks. This is in striking contrast to theabsence of tumors when b-catenin is stabilized in adult hepa-tocytes using aCre-expressing adenovirus or using an adolaseBpromoter–driven transgene expressing amutant stable b-cate-nin protein (7). Our conditional genetic model using the Tg-b-catS mice demonstrates that the CreERTM-GFPþ liver stem/progenitor cells can be transformed by just b-catenin activa-tion. Only a small fraction of CreERTM-GFPþ cells express AFP.Future studies will be required to determine whether thetumors in the Tg-b-catS mice arise from the AFPþ and/or theAFP� fraction of the CreERTM-GFPþ cells. Of note, activation ofWnt signaling pathway in fetal hepatoblasts using an Afp-driven Cre transgene is embryonic lethal, making it impossibleto evaluate potential tumorigenesis of the large population ofAfpþ fetal liver progenitors (37).Like human HCCs, the Tg-b-catS tumors histologically

showednuclear atypia,mitoticfigures, and peliosis and robust-ly and heterogeneously expressed glutamine synthetase andnuclear b-catenin. Approximately 40% of mice that developedHCCs also developed hepatoblastomas. In the Tg-b-catS mice,b-catenin was stabilized at �E14.5, at which time hepatoblastlineage bifurcation begins. Because of this, it is possible thatb-catenin stabilization occurred in a heterogeneous popula-tion of fetal progenitors, some of which were more differen-tiated than others. This is fully consistent with the occurrenceof both HCC and hepatoblastomas in these mice. Like humanhepatoblastomas that are classified into distinct classes basedon their histology (22), we observed two distinct histologies: apure fetal moderately differentiated hepatoblastomas and anembryonal-undifferentiated hepatoblastoma. Some mutantmice showed both histologies of hepatoblastomas. Like humanhepatoblastomas, both types of murine hepatoblastomas dis-played increased nuclear b-catenin expression, homogeneous-ly in the embryonal hepatoblastomas and heterogeneously inthe fetal histology hepatoblastomas. Glutamine synthetaseexpression in the fetal moderately differentiated hepatoblas-tomas was similar to that in the HCCs, although more het-erogeneous. Glutamine synthetase expression was lower in theembryonal-undifferentiated hepatoblastomas, consistent withthe expression of glutamine synthetase in more differentiatedcells of the liver.Lung metastases were grossly observable in some Tg-b-catS

mice, an unusual occurrence inmousemodels. Nearly 46% and44% of human patients with HCC and hepatoblastomas,respectively, have metastatic disease, primarily in the lung(38, 39). As was the case for the primary Tg-b-catS tumors, weobserved robust b-catenin expression in a lung metastasis.

The observation of HCCs and sometimes, additionally,hepatoblastomas raises a question about the cell type(s) fromwhich these tumors arose. Hepatoblastomas are thought toarise frommaturation arrest of infant liver stem cells andHCCsto develop frommore differentiated cells that are neverthelessfrom the same lineage (40, 41). For human HCC, a fetal cellorigin has been suggested for a subset of tumors based on theirfetal liver gene expression profile, and the development ofHCCs following ex vivo genetic manipulation of murine hepa-toblasts is consistentwith this (42, 43). Alternatively, HCCsmayarise following dedifferentiation of adult hepatocytes and re-expression of fetal markers during the course of malignanttransformation (2). The recent observation of CITED1 expres-sion in regenerating hepatocytes following partial hepatecto-my or DDC treatment (18) suggests that fetal markers can bere-expressed in adult hepatocytes during regeneration.

Another possibility is that the HCCs arose from the fetalCreERTM-GFPþ cell–derived SOX9þ adult progenitors that weidentified in our mice. Examination of the livers of Tg-b-catS

mice revealed that small HCCs were almost invariably locatedaround bile ducts, similar to the localization of the SOX9þ

stem/progenitors, consistent with the notion that tumors mayhave arisen from adult progenitors.

Activating mutations of the Wnt signaling pathway are verycommon in both mouse and human HCC (30), and in humantumors Wnt target genes, both canonical and liver-specifictargets are upregulated (23, 24, 44, 45). We observed increasedcytoplasmic and nuclear localization of b-catenin in the liver ofyoung (1-month-old) Tg-b-catSmice and also in hepatocytes inthe Tg-b-catS HCCs. Notably, both liver and HCCs also showedan increase in both canonical (c-Myc, cyclin D1, and Axin2) andnoncanonical Wnt targets (Gs, Glt1, Oat, and Lect2). Suchinduction of canonical Wnt targets was not observed in amodel in which stabilized b-catenin was expressed in hepa-tocytes, nor did these mice develop tumors (7). These differ-ences between the two models suggest that the ability ofWnt pathway activation to result in HCCs critically dependson the activation of canonical Wnt targets.

Tg-b-catS tumors also expressed FGFR1, commonly observedin human HCCs, and displayed activation of the Ras/Raf/MAPKand the PI3K/AKT/mTOR pathways, both of which can beactivated by FGFR1 and are present in human HCCs charac-terized by aggressive behavior (46). Specifically, PKCa, whichhas been reported to contribute toHCCproliferation,migration,and invasion via activation ofMAPK (47, 48), was upregulated inTg-b-catS tumors as was pMEK1/2 and pERK1/2. Phospho-FOXO3A, a proapoptotic protein that is negatively regulatedvia phosphorylation by pERK1/2, was increased. PTEN wasdownregulated in tumors, whereas components of the TORC1complex, p-mTOR, RAPTOR, and GbL, were upregulated, con-sistent with the known role of PTEN in negatively regulating themTOR pathway. As noted previously, tumors displayed anincrease in both liver-specific and canonical b-catenin targets.One, c-MYC, is also an effector of the MEK/ERK pathway, andanother, glutamine synthetase, has been reported to enhancetumor growth by activation of themTORpathway (49, 50). Thus,the activationof theWnt pathway engineered into theCreERTM-GFPþ fetal liver progenitors may have not only initiated






tumorigenesis but may have also enhanced the effect of mTORand MEK/ERK pathway activation. We also observed upregula-tion of Stathmin, SRC, and CHK1, which have also been impli-cated in hepatocarcinogenesis (27–29).

By RPPA analysis, Tg-b-catS HCCs uniformly displayed acti-vation of the mTOR and MEK/ERK pathways and this wassubsequently verified by IHC. In contrast toHCCs, we observeddistinct patterns of protein/phospho-protein expression in thetwo histologic types of HBs. The fetal hepatoblastomas corre-sponded more closely to HCC with upregulation of PKCa,ERK1/2, mTOR, and S6 proteins, whereas the embryonal-undifferentiated hepatoblastomas showed significant upregu-lation of only PKCa. The expression of b-catenin was alsodistinct in these two histologies of hepatoblastomas, with theembryonal-undifferentiated hepatoblastomas displaying strik-ingly nuclear localization and the pure fetal hepatoblastomasresembling the HCCs. These results suggest that the specificsignaling pathways altered in the tumors are dependent uponthe cell type within the tumor.

In summary, we have identified a unique population ofprogenitor cells in fetal liver that are marked by the expressionof the Cited1-CreERTM-GFP transgene. Somatic b-catenin sta-bilization in these cells results in the frequent development ofboth HCCs and hepatoblastomas with spontaneous lungmetastases. Our model provides valuable evidence for a com-mon origin of HCC and hepatoblastomas from the Tg-b-catS

stem/progenitor cell. This model will be extremely valuable for

understanding the pathobiology of human HCCs, in particularthose that display an especially poor prognosis and are thoughtto be of stem/progenitor origin.

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

Authors' ContributionsConception and design: S. Mokkapati, V. HuffAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Mokkapati, K. Niopek, L. Huang, K.J. Cunniff,E.C. Ruteshouser, M.J. FinegoldAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Mokkapati, V. HuffWriting, review, and or revision of themanuscript: S.Mokkapati, K. Niopek,K.J. Cunniff, E.C. Ruteshouser, V. HuffAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Niopek, L. Huang, K.J. CunniffStudy supervision: V. HuffOther (provided a previously unpublished mouse line used forthese studies and reviewed the manuscript prior to submission):M. deCaestecker

Grant SupportThis study was supported from NIH grants CA34936, DK069599, NCI CCSG

grant CA16672, CPRIT RP100329, and CPRIT RP110324. S. Mokkapati is arecipient of the Dodie P. Hawn Fellowship in Genetics at MD Anderson CancerCenter.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 15, 2013; revised April 16, 2014; accepted May 1, 2014;published OnlineFirst May 21, 2014.

References1. El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011;365:

1118–27.2. Thorgeirsson SS, Lee JS, Heo J, Libbrecht L, Chu IS, Kaposi-Novak P,

et al. A novel prognostic subtype of human hepatocellular carcinomaderived from hepatic progenitor cells. Nat Med 2006;12:410–6.

3. Whittaker S, Marais R, Zhu AX. The role of signaling pathways in thedevelopment and treatment of hepatocellular carcinoma. Oncogene2010;29:4989–5005.

4. Guichard C, Imbeaud S, Amaddeo G, Ben Maad I, Letouze E, PelletierL, et al. Landscape of somatic mutation in hepatocellular carcinoma.J Hepatol 2012;56:S8-S.

5. Monga SPS. Role of Wnt/beta-catenin signaling in liver metabolismand cancer. Int J Biochem Cell Biol 2011;43:1021–9.

6. Harada N, Miyoshi H, Murai N, Oshima H, Tamai Y, Oshima M, et al.Lack of tumorigenesis in the mouse liver after adenovirus-mediatedexpression of a dominant stable mutant of beta-catenin. Cancer Res2002;62:1971–7.

7. Cadoret A, Ovejero C, Saadi-Kheddouci S, Souil E, Fabre M, Romag-nolo B, et al. Hepatomegaly in transgenic mice expressing an onco-genic form of beta-catenin. Cancer Res 2001;61:3245–9.

8. Nejak-Bowen KN, Thompson MD, Singh S, Bowen WC, Dar MJ,Khillan J, et al. Accelerated liver regeneration and hepatocarcinogen-esis in mice overexpressing serine-45 mutant beta-catenin. Hepatol-ogy 2010;51:1603–13.

9. Harada N, Oshima H, Katoh M, Tamai Y, Oshima M, Taketo MM.Hepatocarcinogenesis in mice with beta-catenin and Ha-ras genemutations. Cancer Res 2004;64:48–54.

10. Stauffer JK, ScarzelloAJ, Andersen JB,DeKluyver RL,BackTC,WeissJM, et al. Coactivation of AKT and beta-catenin inmice rapidly inducesformation of lipogenic liver tumors. Cancer Res 2011;71:2718–27.

11. Boyle S, Misfeldt A, Chandler KJ, Deal KK, Southard-Smith EM,Mortlock DP, et al. Fate mapping using Cited1-CreER(T2) micedemonstrates that the cap mesenchyme contains self-renewing pro-

genitor cells and gives rise exclusively to nephronic epithelia. Dev Biol2008;313:234–45.

12. Harada N, Tamai Y, Ishikawa T, Sauer B, Takaku K, Oshima M, et al.Intestinal polyposis in mice with a dominant stable mutation of thebeta-catenin gene. EMBO J 1999;18:5931–42.

13. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporterstrain. Nat Genet 1999;21:70–1.

14. Bader BL, Smyth N, Nedbal S, Miosge N, Baranowsky A, Mokkapati S,et al. Compound genetic ablation of nidogen 1 and 2 causes basementmembrane defects and perinatal lethality in mice. Mol Cell Biol2005;25:6846–56.

15. Tibes R, Qiu YH, Hennessy B, Andreeff M, Miiis GB, Kornblau SM.Reverse phase protein array: validation of a novel proteomic technol-ogy andutility for analysis of primary leukemia specimensandhemato-poietic stem cells. Mol Cancer Ther 2006;5:2512–21.

16. Suzuki A, Taniguchi H, Zheng YW, Takada Y, Fukunaga K, Seino K,et al. Clonal colony formation of hepatic stem/progenitor cellsenhanced by embryonic fibroblast conditioning medium. TransplantProc 2000;32:2328–30.

17. Suzuki A, Zheng YW, Kondo R, Kusakabe M, Takada Y, Fukao K,et al. Flow-cytometric separation and enrichment of hepatic pro-genitor cells in the developing mouse liver. Hepatology 2000;32:1230–9.

18. Murphy AJ, de Caestecker C, Pierce J, Boyle SC, Ayers GD, Zhao Z,et al. CITED1 expression in liver development and hepatoblastoma.Neoplasia (New York, N Y) 2012;14:1153–63.

19. Furuyama K, Kawaguchi Y, Akiyama H, Horiguchi M, Kodama S,Kuhara T, et al. Continuous cell supply from a Sox9-expressingprogenitor zone in adult liver, exocrine pancreas and intestine. NatGenet 2011;43:34–41.

20. TanimizuN, NishikawaM,SaitoH, Tsujimura T,MiyajimaA. Isolation ofhepatoblasts based on the expression of Dlk/Pref-1. J Cell Sci 2003;116:1775–86.

Mokkapati et al.





21. Suzuki A, Zheng YW, Kaneko S, Onodera M, Fukao K, Nakauchi H,et al. Clonal identification and characterization of self-renewing plu-ripotent stem cells in the developing liver. J Cell Biol 2002;156:173–84.

22. Zimmermann A. The emerging family of hepatoblastoma tumours:from ontogenesis to oncogenesis. Eur J Cancer 2005;41:1503–14.

23. Cadoret A, Ovejero C, Terris B, Souil E, Levy L, Lamers WH, et al. Newtargets of beta-catenin signaling in the liver are involved in the gluta-mine metabolism. Oncogene 2002;21:8293–301.

24. Ovejero C, Cavard C, Perianin A, Hakvoort T, Vermeulen J, Godard C,et al. Identification of the leukocyte cell-derived chemotaxin 2 as adirect target gene of beta-catenin in the liver. Hepatology 2004;40:167–76.

25. Turner N, Grose R. Fibroblast growth factor signalling: from develop-ment to cancer. Nat Rev Cancer 2010;10:116–29.

26. Horie Y, Suzuki A, Kataoka E, Sasaki T, Hamada K, Sasaki J, et al.Hepatocyte-specific Pten deficiency results in steatohepatitis andhepatocellular carcinomas. J Clin Invest 2004;113:1774–83.

27. Gan L, Guo K, Li Y, Kang X, Sun L, Shu H, et al. Up-regulatedexpression of stathminmaybe associatedwith hepatocarcinogenesis.Oncol Rep 2010;23:1037–43.

28. Hong J, Hu K, Yuan Y, Sang Y, Bu Q, Chen G, et al. CHK1 targetsspleen tyrosine kinase (L) for proteolysis in hepatocellular carcinoma.J Clin Invest 2012;122:2165–75.

29. Masaki T, Okada M, Shiratori Y, Rengifo W, Matsumoto K, Maeda S,et al. pp60c-src activation in hepatocellular carcinoma of humans andLEC rats. Hepatology (Baltimore, Md) 1998;27:1257–64.

30. de La Coste A, Romagnolo B, Billuart P, Renard CA, Buendia MA,Soubrane O, et al. Somatic mutations of the beta-catenin gene arefrequent in mouse and human hepatocellular carcinomas. Proc NatlAcad Sci U S A 1998;95:8847–51.

31. Koch A, Denkhaus D, Albrecht S, Leuschner I, von Schweinitz D,Pietsch T. Childhood hepatoblastomas frequently carry a mutateddegradation targeting box of the beta-catenin gene. Cancer Rese1999;59:269–73.

32. Wong CM, Fan ST, Ng IO. beta-Catenin mutation and overexpressionin hepatocellular carcinoma: clinicopathologic and prognostic signif-icance. Cancer 2001;92:136–45.

33. Tan X, Apte U, Micsenyi A, Kotsagrelos E, Luo JH, Ranganathan S,et al. Epidermal growth factor receptor: a novel target of theWnt/beta-catenin pathway in liver. Gastroenterology 2005;129:285–302.

34. YangW, Yan HX, Chen L, Liu Q, He YQ, Yu LX, et al. Wnt/beta-cateninsignaling contributes to activation of normal and tumorigenic liverprogenitor cells. Cancer Res 2008;68:4287–95.

35. Cardinale V, Wang Y, Carpino G, Mendel G, Alpini G, Gaudio E, et al.The biliary tree–a reservoir of multipotent stem cells. Nat Rev Gastro-enterol Hepatol 2012;9:231–40.

36. Li J, Xin J, Zhang L, Wu J, Jiang L, Zhou Q, et al. Human hepaticprogenitor cells express hematopoietic cell markersCD45 andCD109.Int J Med Sci 2014;11:65–79.

37. Decaens T, Godard C, de Reynies A, Rickman DS, Tronche F, CoutyJP, et al. Stabilization of beta-catenin affects mouse embryonic livergrowth and hepatoblast fate. Hepatology 2008;47:247–58.

38. Yang T, Lu J-H, Lin C, Shi S, Chen T-H, Zhao R-H, et al. Concomitantlung metastasis in patients with advanced hepatocellular carcinoma.World J Gastroenterol 2012;18:2533–9.

39. Uchiyama M, Iwafuchi M, Naito M, Yagi M, Iinuma Y, Kanada S,et al. A study of therapy for pediatric hepatoblastoma: preventionand treatment of pulmonary metastasis. Eur J Pediatr Surg 1999;9:142–5.

40. Fiegel HC, Gluer S, Roth B, Rischewski J, von Schweinitz D, Ure B,et al. Stem-like cells in human hepatoblastoma. J Histochem Cyto-chem 2004;52:1495–501.

41. Sell S, Leffert HL. Liver cancer stem cells. J Clin Oncol 2008;26:2800–5.

42. Lee JS, Heo J, Libbrecht L, Chu IS, Kaposi-Novak P, Calvisi DF, et al. Anovel prognostic subtype of human hepatocellular carcinoma derivedfrom hepatic progenitor cells. Nat Med 2006;12:410–6.

43. Zender L, Spector MS, Xue W, Flemming P, Cordon-Cardo C, SilkeJ, et al. Identification and validation of oncogenes in liver cancerusing an integrative oncogenomic approach. Cell 2006;125:1253–67.

44. Nishida N, Fukuda Y, Komeda T, Kita R, Sando T, Furukawa M,et al. Amplification and overexpression of the cyclin D1 gene inaggressive human hepatocellular carcinoma. Cancer Res 1994;54:3107–10.

45. Yuen MF, Wu PC, Lai VC, Lau JY, Lai CL. Expression of c-Myc, c-Fos,and c-jun in hepatocellular carcinoma. Cancer 2001;91:106–12.

46. Thorgeirsson SS, Calvisi DF, Ladu S, Gorden A, Farina M, Conner EA,et al. Ubiquitous activation of Ras and Jak/Stat pathways in humanHCC. Gastroenterology 2006;130:1117–28.

47. Wu TT, Hsieh YH, Wu CC, Hsieh YS, Huang CY, Liu JY. Overexpres-sion of protein kinase C alpha mRNA in human hepatocellular carci-noma: a potential marker of disease prognosis. Clin Chim Acta2007;382:54–8.

48. WuTT,HsiehYH,Hsieh YS, Liu JY. Reduction of PKCalpha decreasescell proliferation, migration, and invasion of human malignant hepa-tocellular carcinoma. J Cell Biochem 2008;103:9–20.

49. Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B,et al. Bidirectional transport of amino acids regulates mTOR andautophagy. Cell 2009;136:521–34.

50. Haussinger D, Schliess F. Glutamine metabolism and signaling in theliver. Front Biosci 2007;12:371–91.






2014;74:4515-4525. Published OnlineFirst May 21, 2014.Cancer Res Sharada Mokkapati, Katharina Niopek, Le Huang, et al. Sufficient to Cause Hepatocellular Carcinoma and Hepatoblastoma-Catenin Activation in a Novel Liver Progenitor Cell Type Isβ

Updated version

10.1158/0008-5472.CAN-13-3275doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2014/06/03/0008-5472.CAN-13-3275.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/74/16/4515.full#ref-list-1

This article cites 50 articles, 13 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/74/16/4515.full#related-urls

This article has been cited by 5 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/74/16/4515To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-13-3275

http://cancerres.aacrjournals.org/content/suppl/2014/06/03/0008-5472.CAN-13-3275.DC1

http://cancerres.aacrjournals.org/content/74/16/4515.full#ref-list-1

http://cancerres.aacrjournals.org/content/74/16/4515.full#related-urls

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

mailto:[email protected]

http://cancerres.aacrjournals.org/content/74/16/4515


b-catenin activation in a novel liver progenitor cell type is … · tumor and stem cell biology...

Documents