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Molecular Cell Biology

Keratin 19Expression inHepatocellularCarcinomaIs Regulated by Fibroblast-Derived HGF via aMET-ERK1/2-AP1 and SP1 AxisHyungjin Rhee1, Hye-Young Kim1, Ji-Hye Choi2,3, Hyun Goo Woo2,3,Jeong Eun Yoo1, Ji Hae Nahm1, Jin-Sub Choi4, and Young Nyun Park1,5

Abstract

Keratin (KRT) 19 is a poor prognosticmarker for hepatocellularcarcinoma (HCC); however, regulatory mechanisms underlyingits expression remain unclear. We have previously reported thepresence of fibrous tumor stroma in KRT19-positive HCC,suggesting that cross-talk between cancer-associated fibroblasts(CAF) and tumor epithelial cells could regulate KRT19 expression.This was investigated in this study using an in vitro model ofparacrine interaction between HCC cell lines (HepG2, SNU423)and hepatic stellate cells (HSC), a major source of hepaticmyofibroblasts. HSCs upregulated transcription and translationof KRT19 inHCC cells via paracrine interactions. Mechanistically,hepatocyte growth factor (HGF) from HSCs activatedc-MET and the MEK–ERK1/2 pathway, which upregulated KRT19expression in HCC cells. Furthermore, AP1 (JUN/FOSL1)and SP1, downstream transcriptional activators of ERK1/2,

activated KRT19 expression in HCC cells. In clinical specimensof human HCC (n ¼ 339), HGF and KRT19 protein expressioncorrelated with CAF levels. In addition, HGF or METprotein expression was associated with FOSL1 and KRT19 expres-sion and was found to be a poor prognostic factor. Analysis ofdata from The Cancer Genome Atlas also revealed KRT19expression was closely associated with CAF and MET-mediatedsignaling activities. These results provide insights into the molec-ular background of KRT19-positive HCC that display an aggres-sive phenotype.

Significance: These findings reveal KRT19 expression inhepatocellular carcinoma is regulated by cross-talk betweencancer-associated fibroblasts and HCC cells, illuminating newtherapeutic targets for this aggressive disease. Cancer Res; 78(7);1619–31. �2018 AACR.

IntroductionKeratin 19 (KRT19) is a small (approximately 40 kDa) type I

cytokeratin, which lacks the tail domain common amongcytokeratins (1). KRT19 is a marker for hepatic stem/progenitorcells (2, 3). In the normal liver, mature hepatocytes expressKRT8 and KRT18, while cholangiocytes additionally expressKRT7 and KRT19 (4). Although most hepatocellular carcino-mas (HCC) do not express KRT19 like normal hepatocytes,10%–28% of HCCs were reported to express KRT19, and werefound to display a more invasive phenotype and poorer out-comes than those that do not, when treated using hepatic

resection or radiofrequency ablation (2, 3, 5–7). KRT19 proteinexpression or KRT19 expression–related signatures have alsoemerged as predictors of HCC recurrence and survival after livertransplantation (8, 9).

Despite the clinical significance of KRT19 expression inHCC, the regulatory mechanism underlying KRT19 expressionremains unclear. Previously, at least a subset of KRT19-positiveHCCs was considered to have originated from hepatic pro-genitor cells expressing KRT19 (10). Conversely, a recent studyusing lineage-tracing rodent HCC models demonstrated thatKRT19-positive HCC cells originated from mature hepatocytesand were not observed in the early clonal expansion of hepa-tocarcinogenesis (11, 12). Therefore, KRT19 expression wasproposed to be acquired during hepatocarcinogenesis.

Increasing evidence supports the notion that tumor microen-vironmental components including cancer-associated fibroblasts(CAF), the extracellularmatrix, angiogenic cells, and immune cellsare important factors for growth, migration/invasion, and angio-genesis in various tumors including HCC (13). CAFs, whichproduce the fibrous tumor stroma, are known to contributeto an aggressive cancer phenotype, facilitating invasiveness,proliferation, and immune suppression (14). Moreover, theywere also reported to stimulate cancer cell stemness by activatingthe WNT or NOTCH signaling pathway in colon, breast, andprostate cancers (15). We have previously reported that HCCsexpressing KRT19 exhibited fibrous tumor stroma. In contrast,that therewas little or nofibrous stroma inHCCs in the absence ofKRT19 expression, while HCCs with abundant fibrous stroma(namely, "scirrhous HCC") commonly expressed KRT19 (3, 16).

1Department of Pathology, Brain Korea 21 PLUS Project for Medical Science,Integrated Genomic Research Center for Metabolic Regulation, Yonsei Univer-sity College of Medicine, Seoul, Korea. 2Department of Physiology, Ajou Uni-versity School of Medicine, Suwon, Korea. 3Department of Biomedical Science,Graduate School, Ajou University, Suwon, Korea. 4Department of Surgery,Yonsei University College of Medicine, Seoul, Korea. 5Severance BiomedicalScience Institute, Yonsei University College of Medicine, Seoul, Korea.

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

Current address for H. Rhee: Department of Radiology, Yonsei UniversityCollege of Medicine, Seoul, Korea.

Corresponding Author: Young Nyun Park, Department of Pathology, YonseiUniversity College of Medicine, 50-1, Yonsei-ro, Seodaemun-gu, Seoul 03722,Korea. Phone: 822-2228-1768; Fax: 822-362-0860; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-0988

�2018 American Association for Cancer Research.

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Thefibrous stromaofHCCswas reported to be generated byCAFs,recruited and transdifferentiated from peritumoral myofibro-blasts through the cross-talk with HCC cells (17). Hepatic myofi-broblasts are a heterogenous group of cells, and activated hepaticstellate cells represent one of the major sources of hepatic myofi-broblasts (18, 19). In light of these results, we hypothesized thatKRT19 expression in HCCsmight be regulated by fibrous stromalcomponents of the tumor microenvironment.

Here, we established an in vitro interaction model of HCCcells and CAFs using HCC and hepatic stellate cell lines. Wediscovered that the hepatic stellate cell-derived hepatocytegrowth factor (HGF) and the subsequent activation of theMET-MEK-ERK1/2 pathway activates KRT19 transcription viathe activator protein 1 (AP1) and specificity protein 1 (SP1)transcription factors. This regulatory axis was validated in alarge cohort (n ¼ 339) of patients with HCC, with IHC stainingand gene expression profiles from HCC cohorts from TheCancer Genome Atlas (TCGA; n ¼ 371). Our results providea better understanding of the molecular background of KRT19-positive HCCs with an aggressive biological behavior.

Materials and MethodsCell lines and reagents

A human hepatoblastoma cell line, HepG2, and two hepa-tocellular carcinoma cell lines, Hep3B and PLC/PRF/5, werepurchased from ATCC (20–22). The Huh7, SNU182, SNU423,and SNU475 cell lines were purchased from the Korean CellLine Bank (23, 24). Two hepatic stellate cell lines, hTERT-HSCand LX-2, were obtained as gifts from David Brenner (Univer-sity of California, San Diego, San Diego, CA; ref. 25) and ScottFriedman (Icahn School of Medicine at Mount Sinai, New York,NY 26), respectively. Cell lines were maintained in DMEMsupplemented with 10% FBS and penicillin/streptomycin(Invitrogen). Cell lines were authenticated using short tandemrepeat analysis, and the databases of ATCC or Korean Cell LineBank were used as reference. The primary human hepaticstellate cells were purchased from ScienCell, maintained indedicated media, and used in the experiments up to 8 passages.The cell lines in use were regularly screened to monitor myco-plasma infections per several months, using PCR-based detec-tion kit (iNtRON Biotechnology). U0126 was purchased fromCell Signaling Technology; mithramycin A was purchased fromTocris Bioscience; SCH772984, SU11274, and PHA-665752were purchased from Selleckchem; doxycycline and S3I-201were purchased from Sigma-Aldrich.

Human HCC tissue samples and clinicopathologic and IHCanalyses

We enrolled a total of 339 patients with HCC who did notundergo any kind of preoperative treatment before hepatic resec-tion. Consecutive HCC tissue samples were obtained by hepaticresection between March 2006 and February 2011 at the Sever-ance Hospital, Yonsei University Medical Center. Their clinico-pathologic characteristics are described in Supplementary TableS1. The representative paraffin-embedded sections of HCC wereused for tissue microarray (TMA) construction and IHC analysis.For TMA construction, two core biopsies of 2 mm in diameter,were taken from individual HCC donor paraffin blocks andarranged into recipient TMA blocks using a trephine apparatus(Superbiochips Laboratories).

IHC staining for KRT19, HGF, MET, and FOSL1 was per-formed using an automated staining system (Ventana MedicalSystems, Inc.). a-Smooth muscle actin (aSMA) and CD90staining was performed using an Envision kit (Dako) accordingto the manufacturer's instructions. All slides were counter-stained with hematoxylin. Detailed information on antibodiesand criteria for IHC analysis are described in SupplementaryTables S2–S4.

The clinical outcomes of the patients were retrospectivelyobtained by reviewing the electronic medical records at SeveranceHospital. The last update for the HCC patient cohort was Sep-tember 2016. The endpoints were defined as follows: disease-specific survival was defined as the time from surgery to death inpatients with HCC involving >50% of the liver, HCC with exten-sive portal vein tumor thrombosis, or HCC with extrahepaticmetastasis (27); disease-free survival was defined as the time fromsurgery to an initial diagnosis of recurrence regardless of location.This study was in accordance with the ethical guidelines of theDeclaration of Helsinki, and was approved by the InstitutionalReview Board of the Severance Hospital (4-2016-0878), and therequirement for informed consent was waived.

Gene expression profile analysisGene expression profiles of HCC cohorts were obtained from

TCGA (https://cancergenome.nih.gov, LIHC, n¼ 371), excludingthe profiles of nontumor tissues. The expression enrichment of agene signature in an individual specimen was calculated by usingthe preranked gene-set enrichment analysis (GSEA, http://software.broadinstitute.org/gsea/index.jsp).

Statistical analysisStatistical analyses were performed using the SPSS software

(version23.0, SPSS Inc.). TheMann–WhitneyU test,c2 test, Fisherexact test, or Spearman correlations were used as deemed appro-priate. Survival analyses were performed using the Kaplan–Meiermethod and the log rank test.

For detailed information on experimental procedures, pleaserefer to the Supplementary Materials and Methods and Supple-mentary Tables S5–S7.

ResultsKRT19 expression in HCC is upregulated by paracrine factorsfrom hepatic stellate cells

The correlation between KRT19 expression and fibrous stro-mal components was confirmed in a cohort of patients withHCC (n¼ 339) using TMA. The expression of the KRT19 proteinwas higher in HCCs with a higher proportion of aSMA-positiveCAFs (Fig. 1A and B). We analyzed the degree of KRT19 expres-sion and the amount of aSMA-positive CAFs in a semiquanti-tative manner, and observed a significant correlation (Spearmancorrelation coefficient r ¼ 0.155, P ¼ 0.004). There was also apositive correlation between the degree of KRT19 expressionand the amount of CD90-positive CAFs (r ¼ 0.124, P ¼ 0.023;Supplementary Fig. S1A). In addition, the association betweenthe expression of KRT19 and fibrous stromal markers includingASMA, FAP, and VIM was evaluated using gene expression datafrom TCGA (LIHC, n¼ 371). KRT19 expression was significantlycorrelated with ASMA (r ¼ 0.25, P ¼ 7.56 � 10�7), FAP (r ¼0.30, P ¼ 4.06 � 10�9), and VIM expression (r ¼ 0.43, P ¼ 5.63� 10�18), respectively (Fig. 1C).

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To establish an in vitro model for evaluating the interactionsbetween epithelial tumor cells and CAFs in HCC, we tested twohepatic stellate cell lines: hTERT-HSC and LX-2. hTERT-HSCsshowed a higher expression for fibroblast activation-relatedmarkers, including FAP and aSMA (28), when compared withthat in LX-2 cells (Supplementary Fig. S1B and S1C). Interest-ingly, conditioned media (CM) from hTERT-HSCs induced an

increase in the expression of KRT19 in HepG2 cells, whereasCM from LX-2 did not (Supplementary Fig. S1D).

Next, we treated the HepG2 and SNU423 cell lines with CMfrom hTERT-HSCs for 12–72 hours. The CM from hTERT-HSCsincreased the KRT19 mRNA and protein expression levels, andthe difference was apparent at 24–72 hours (Fig. 1D). We alsoconducted indirect cocultures using permeable cell culture

Figure 1.

KRT19 expression in HCC isupregulated by paracrine factorsfrom hepatic stellate cells. A,Representative cases of HCC with orwithout the aSMA-positive CAFs.Scale bars, 100 mm. B, Correlationbetween the aSMA-positive CAFs andKRT19 protein expression. C,Correlation between the geneexpression level of KRT19 and fibrousstromal markers including ASMA, FAP,and VIM in HCC data from TCGA(n ¼ 371). D, mRNA and proteinexpression levels of KRT19 aftertreatment with hTERT-HSC CM. HCCcells (HepG2 and SNU423) wereharvested at the indicated times, andmRNA and protein expression levelswere analyzed by qRT-PCR orWestern blot analysis. E, mRNA andprotein expression levels of KRT19after coculture with hTERT-HSC. F,Morphologic changes in HepG2 andSNU423 induced by coculture withhTERT-HSC or CM from hTERT-HSC.Scale bars, 100 mm. Each qRT-PCRexperiment was independentlyconducted two times in triplicate, anda representative result is representedin the figure. The bar graph representsthe mean � SD, and statisticalsignificance is indicated (� , P < 0.05;�� , P < 0.01; �� , P < 0.001, t test).

KRT19 in HCC Is Regulated by CAF via HGF-MET-AP1 and SP1

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inserts with 1-mm pore sizes. The sharing of the culture mediabetween the HCC cell lines and hTERT-HSCs increased KRT19mRNA and protein expression in HepG2 and SNU423 cells(Fig. 1E). Similarly, CM from primary hepatic stellate cellsincreased KRT19 mRNA and protein expression (SupplementaryFig. S1B, S1E and S1F). CM from hTERT-HSC or primary hepaticstellate cells, and indirect cocultures with hTERT-HSCs resultedin similar features, leading to cell scattering and elongation(Fig. 1F; Supplementary Fig. S1G). These results suggest thatparacrine factors from hTERT-HSCs regulate KRT19 expressionin HCC cell lines.

hTERT-HSC CM regulates KRT19 expression through theMEK–ERK1/2 pathway

Considering that KRT19-positive HCCs display an aggressivebiological behavior (3), oncogenic signaling pathways includ-ing MAPK, AKT, and STAT3 were investigated to determine the

mechanism underlying the upregulation of KRT19 upon treat-ment with CM from hTERT-HSCs. The CM treatment inducedthe phosphorylation of ERK1/2 in both HepG2 and SNU423,and the phosphorylation of STAT3 in HepG2. Other signalingpathways, including p38 MAPK, JNK, and AKT, were unaffected(Fig. 2A). Treatment with the STAT3 inhibitor (S3I-201)decreased KRT19 expression in HepG2, but not SNU423, sug-gesting that the STAT3 pathway is not a common regulator ofKRT19 in both cell lines (Supplementary Fig. S2A).

When the MEK–ERK1/2 pathway was inhibited with theMEK inhibitor (U0126) or ERK1/2 inhibitor (SCH772984;ref. 29), the upregulation of KRT19 induced following treat-ment with CM from hTERT-HSCs was abolished in HepG2and SNU423 cells (Fig. 2B). KRT19 expression also increas-ed following 12-O-Tetradecanoylphorbol-13-acetate (TPA)treatment, and the combination of the MEK or ERK1/2 inhib-itor with TPA decreased KRT19 expression (Supplementary

Figure 2.

Conditioned media regulates KRT19 expression through the MEK–ERK1/2 pathway. A, The activation status of selected oncogenic pathways in HepG2 andSNU423 by CM from hTERT-HSC. B, KRT19 mRNA and protein levels after CM treatment combined with/without a MEK or ERK inhibitor. mRNA andprotein expression levels were analyzed by qRT-PCR or Western blot analysis. C, Upregulation of KRT19 mRNA and protein levels in HepG2 andSNU475 cells by the transient expression of a constitutively active MEK1 (S218D/S222D) or ERK1. The amounts of plasmid used in transfections (per 35-mmdish) are indicated. Each qRT-PCR experiment was independently conducted two times in triplicate, and a representative result is represented in the figure.The bar graph represents the mean � SD, and statistical significance is indicated (� , P < 0.05; �� , P < 0.01; �� , P < 0.001, t test).

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Fig. S2B). To further confirm that KRT19 expression is regu-lated by the MEK–ERK1/2 pathway, the constitutively activeform of MEK1 (S218D/S222D) and wild-type ERK1 wereoverexpressed in HepG2 and SNU475 cells (30). These celllines were chosen for the overexpression experiment, insteadof SNU423, because the transfection efficiency of SNU423 wasquite low, compared with that in other cells (SupplementaryFig. S2C). The overexpression of MEK1 (S218D/S222D) andERK1 increased KRT19 expression by 2- to 6-fold (Fig. 2C).Taken together, CM from hTERT-HSCs increased KRT19expression in HCC cell lines via the MEK–ERK1/2 pathway.

HGF in hTERT-HSC CM upregulates KRT19 via MET and theMEK–ERK1/2 pathway

To specify which paracrine factors and receptors activatethe MEK–ERK1/2 pathway in HCC cells, a phospho-receptortyrosine kinase array was performed in HepG2 and SNU423cells and three MET, ALK, and FGFR3 receptors were foundto be commonly phosphorylated (Fig. 3A). Consideringthat MET is known to drive the invasive growth of HCC viavarious signaling pathways including MEK-ERK1/2 (31), METand its well-known ligand, HGF, were further investigated.We confirmed the phosphorylation of MET upon treatmentwith CM from hTERT-HSCs in HepG2 and SNU423 cells byWestern blot analysis (Fig. 3B). The concentration of HGF,determined by ELISA, was much higher in CM from hTERT-HSCs (10.8 ng/mL), compared with that in CM from HepG2and SNU423 (<0.2 ng/mL for both; Fig. 3C). Treatment withrecombinant HGF upregulated KRT19 expression in bothHCC cell lines, and the combined treatment with MAPKinhibitors decreased the HGF-induced upregulation of KRT19expression (Fig. 3D).

In addition, CM from LX-2, where the HGF concentrationwas very low (<0.1 ng/mL), did not increase KRT19 expression(Supplementary Fig. S3A). The HGF concentration in CM fromprimary hepatic stellate cells (30.4 ng/mL) was even higherthan that in CM from hTERT-HSCs (Supplementary Fig. S3B).HCC cell lines with low MET expression, including SNU182and PLC/PRF/5, also showed low KRT19 expression (Supple-mentary Fig. S3C). The transient overexpression of MET inSNU475 cells increased phospho-MET and phospho-ERK1/2,as well as KRT19 expression at both the mRNA and proteinlevel. In PLC/PRF/5, the transient overexpression of MET alsoincreased phospho-MET and phospho-ERK1/2, and KRT19mRNA levels. (Supplementary Fig. S3D).

To determine whether HGF is indeed responsible for theKRT19 upregulation induced by CM from hTERT-HSCs, weestablished doxycycline-inducible knockdown versions ofhTERT-HSCs (hT-shHGF-1, hT-shHGF-2). Two days of doxy-cycline treatment (1 mg/mL) prior to CM generation efficientlydepleted HGF (7.2 ng/mL vs. 0.17 ng/mL in hT-shHGF-1, 8.4ng/mL vs. 0.13 ng/mL in hT-shHGF-2, Fig. 3E). Treatment ofHepG2 and SNU423 cells with CM from nondoxycycline-trea-ted hT-shHGF-1 or hT-shHGF-2 upregulated KRT19 expressionat both the transcriptional and translational levels, whereastreatment with CM from doxycycline-treated hT-shHGF-1 orhT-shHGF-2 did not (Fig. 3F). In addition, doxycycline reversedthe cell scattering induced by the CM from hT-shHGF-1 and 2(Supplementary Fig. S4A). Further, HGF neutralization withanti-HGF immunoglobulins reversed the CM-induced KRT19upregulation and cell scattering (Supplementary Fig. S4B

and S4C). When MET selective inhibitors (SU11274 andPHA-665752) were combined with CM, the upregulation ofKRT19 also decreased (Fig. 3G). Taken together, our datasuggest that HGF paracrine signaling from hepatic stellate cellsupregulates KRT19 expression via MET and the MEK-ERK1/2pathway.

The AP1 and SP1 transcription factor binding sites in theKRT19 promoter are important for KRT19 expression

To determine important cis-regulatory elements in theKRT19 promoter, we constructed luciferase reporter plasmidsof approximately 3 kb in length (�2,885 bp from þ41 bprelative to the transcription start site) and serial deletionmutants (Fig. 4A). When measuring the promoter activity ofthe constructs in HepG2 and SNU423 cells, we found it to becommonly and significantly decreased at two segments:between �2,167 and �1,967 and between �152 and �69.To confirm that these two promoter segments are truly impor-tant for KRT19 regulation, we also measured the promoteractivity of deletion constructs lacking these segments: D2167-1967 and D152-69. The constructs elicited a >70% decreasein promoter activity in the case of D2167–1967 and >25% forD152-69 (Fig. 4B).

We next searched for putative transcription factor binding sitesat positions �2167��1967 and �152��69 using online pro-grams (PROMO; http://alggen.lsi.upc.es, and Tfsitescan; http://www.ifti.org/cgi-bin/ifti/Tfsitescan.pl). And mutant constructsfor the AP1, MAZ, ELK, ETS, and SP1 binding sites were generatedusing site-specific mutagenesis (Fig. 4C). Of these mutant con-structs,mutants of the�2021AP1 site and�72 SP1 site revealed asignificant reduction in activity. Therefore, AP1 and SP1, asdownstream transcriptional activators of ERK1/2, were identifiedto be important for KRT19 regulation in HCC.

The binding of JUN, FOSL1, and SP1 to the KRT19 promoteris important for the regulation of KRT19 expression

The AP1 binding site sequence at the �2021 position in theKRT19 promoter (TGACTCA) is a well-known TPA-responsiveelement, to which various heterodimers of the JUN and FOSfamily may bind (32). To identify specific AP1 proteins thatregulate KRT19 expression in HCC cell lines, we performedsiRNA screening for AP1 family members. At first, we screenedthe transcriptional levels of seven JUN and FOS genes, includingJUN, JUNB, JUND, FOS, FOSB, FOSL1, and FOSL2. Amongthese seven genes, we selected JUN, JUND, FOSL1, and FOSL2,which showed a relatively higher mRNA expression than othergenes in the JUN and FOS family (Fig. 5A). To target these genes,a transient knockdown with two pooled siRNAs was carried out;the knockdown efficiency was greater than 80% (SupplementaryFig. S5A). JUN or FOSL1 knockdown generally decreased KRT19expression in HepG2 and SNU423 cells (Fig. 5B). To confirmthat these results did not represent off-target effects, each siRNAwas transfected separately. By transfecting siJUN-1, siJUN-2,siFOSL1-1, and siFOSL1-2, both JUN and FOSL1 were success-fully knocked down, at the transcriptional and translationallevel. Moreover, KRT19 mRNA levels also declined in bothHepG2 and SNU423 cells (Fig. 5C, Supplementary Fig. S5B).Following treatment with an SP1 inhibitor (mithramycin A),KRT19 expression decreased at both the transcriptional andtranslational levels (Fig. 5D). Conversely, the overexpressionof JUN, FOSL1, a heterodimeric fusion protein of JUN and





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Figure 3.

Hepatocyte growth factor upregulates KRT19 via MET and the MEK–ERK1/2 pathway. A, The phosphorylation status of receptor tyrosine kinases in HepG2 andSNU423 treated with CM from hTERT-HSC. The increased phosphorylation of three receptors (MET, ALK, and FGFR3) was commonly observed in HepG2 andSNU423 cells. B, Increased phosphorylation of MET following CM treatment was confirmed in HepG2 and SNU423 cells. C, HGF concentrations in CM fromhTERT-HSC, HepG2, and SNU423 cells determined by enzyme-linked immunosorbent assay. D, KRT19 mRNA and protein levels after HGF treatment combinedwith/without a MEK or ERK inhibitor. mRNA and protein expression levels were analyzed by qRT-PCR or Western blot analysis. E, HGF concentrations incomplete media (DMEM þ 10% FBS) and CM from the hT-shHGF-1 and hT-shHGF-2 stable cell lines with/without doxycycline treatment. F, KRT19 mRNA andprotein levels after treatment with CM from the hT-shHGF-1 and hT-shHGF-2 stable cell lines with/without doxycycline treatment. G, KRT19 mRNA and proteinlevels after CM treatment combined with/without MET inhibitors. For qRT-PCR experiments, the bar graph represents the mean � SD, and statisticalsignificance is indicated (� , P < 0.05; �� , P < 0.01; �� , P < 0.001, t test).

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FOSL1 (JUN�FOSL1) (33), and SP1 increased KRT19 transcriptlevels (Fig. 5E). In accordance with a previous report stating thatFOSL1 phosphorylation by ERK1/2 resulted in decreased pro-teasomal degradation and increased stability (34), HepG2 andSNU423 cells treated with CM from hTERT-HSCs displayedincreased phosphorylation and FOSL1 levels (Fig. 5F).

To confirm the binding of JUN, FOSL1, and SP1 to theKRT19 promoter, we performed DNA pull-downs and ChIPassays. We synthesized a 26-bp-long biotinylated double-stranded DNA, mimicking the AP1 and SP1 sites in the KRT19promoter (e.g., �2021 for the AP1 site, and �72 for the SP1site). Mutant biotinylated DNAs were also synthesized, inwhich AP1 or SP1 binding motifs were altered. Western blotanalyses of the DNA binding assay revealed the binding of JUNand FOSL1 to the �2021 AP1 site and that of SP1 binding tothe �72 SP1 site in HepG2 and SNU423 cells (Fig. 5G). ChIPassays confirmed the increased binding of JUN and FOSL1 atthe �2021 AP1 site and that of SP1 at the �72 SP1 sitefollowing CM treatment (Fig. 5H).

HGF and/or MET expression correlates with a higher KRT19and FOSL1 expression in human HCC tissue samples

To validate our in vitro results regarding the regulatory axis ofKRT19, the expression levels of HGF, MET, and that of their

downstream molecules, including FOSL1 and KRT19 wereevaluated by immunocytochemical analysis in our in vitromodel (Supplementary Figs. S6 and S7), IHC in TMA samplesfrom the human HCC cohort (n ¼ 339; Figs. 1A and 6A), andgene expression data from TCGA.

The immunocytochemical staining demonstrated that HGFwas weakly expressed in hTERT-HSCs, whereas HGF expressionwas stronger in HepG2 or SNU423 cells after CM treatment,suggesting an accumulation of HGF in tumor epithelial cells(Supplementary Fig. S6A). A time-course experiment after CMtreatment showed the serial accumulation ofHGF inHepG2 cells,and its colocalization with MET, suggesting that HGF in HepG2cells is derived from CM (Supplementary Fig. S6B). We alsocarried out immunocytochemical staining for ERK1/2, phos-pho-ERK1/2, FOSL1, and phospho-FOSL1; the nuclear translo-cation of these proteins was observed after CM treatment (Sup-plementary Fig. S7A–S7D).

In the IHC analysis of TMAs in the human HCC cohort, themembrane expression of MET and HGF, cytoplasmic expres-sion of KRT19, and nuclear expression of FOSL1 were scoredas described previously (3, 35, 36). When evaluating HGFexpression, its membrane distribution in epithelial tumor cellswas focused where MET was expressed, although it was alsoobserved in CAFs from the tumor stroma and the cytoplasm of

Figure 4.

AP1 and SP1 transcription factor binding sites in the KRT19 promoter are important for KRT19 expression. A, Luciferase reporter assay results for KRT19promoter constructs. An approximately 3-kb-long KRT19 promoter and its serial deletion constructs were tested in HepG2 and SNU423 cells; the fireflyluciferase results were normalized to the thymidine kinase-promoter-driven nano-Luc luciferase results. The bar graph represents the mean � SD. B,Luciferase reporter assay results for KRT19 promoter constructs, in which the �2167��1967 or �152��69 segments were deleted from the full-length KRT19promoter using site-directed mutagenesis. C, Luciferase activity of mutant reporter plasmids, in which the putative transcription factor binding siteswere altered by site-directed mutagenesis from the full-length KRT19 promoter.






Figure 5.

JUN, FOSL1, and SP1 are important regulators of KRT19 expression. A, Relative mRNA expression levels of AP1 family genes that could bind TPA-responsiveelements. mRNA expression was analyzed by qRT-PCR. B, KRT19 mRNA expression levels after transfection of two pooled siRNAs targeting JUN,JUND, FOSL1, or FOSL2. C, KRT19 mRNA expression levels after transfection of siRNAs targeting JUN or FOSL1. The knockdown efficiency was confirmedby Western blot analysis. D, KRT19 mRNA and protein levels after 24 hours of mithramycin A treatment combined with CM. mRNA and protein expressionwere analyzed by qRT-PCR and Western blot analysis, respectively. E, KRT19 mRNA levels after transient expression of JUN, FOSL1, JUN�FOSL1(heterodimeric fusion protein consisting of JUN, glycine-rich tether, and FOSL1), and SP1. F, Changes in phosphorylation and JUN and FOSL1 levels following a24-hour CM treatment of HepG2 and SNU423 cells. G, Western blot analysis of eluted DNA-binding proteins from DNA pull-down assay. The nuclearextracts of HepG2 and SNU423 were incubated with DNA fragments mimicking the �2021 AP1 or �72 SP1 binding sites of KRT19. H, Chromatinimmunoprecipitation analysis of HepG2 and SNU423 demonstrating increased JUN and FOSL1 binding at the �2021 AP1 site and increased SP1 bindingat the �72 SP1 site following CM treatment. For qRT-PCR experiments, the bar graph represents the mean � SD, and statistical significance is indicated(� , P < 0.05; ��, P < 0.01; �� , P < 0.001, t test).

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epithelial tumor cells (Supplementary Fig. S7E). The membranedistribution of HGF and MET was observed in 17% (56/339),and 31% (104/339) of HCCs, respectively. The expression ofHGF was frequently observed in HCCs with �5% of CAF pertumor area when compared with those without, whereas the

expression of MET was not significantly different by the CAFlevels in HCC (Fig. 6B). FOSL1 and KRT19 expression wasobserved in 46% (157/339), and 10% (35/339) of HCCs,respectively. The expression of KRT19 was frequently observedin HCCs that expressed HGF or FOSL1, when compared with

Figure 6.

FOSL1 and KRT19 expression and the prognosis for patients with HCC according to the HGF/MET expression status. A, Representative HCC tissue sampleswith or without HGF, MET, and FOSL1 expression. Scale bars, 100 mm. B, Association between HGF and MET expression and the percentage of aSMA-positive CAFs per tumor area. C, Association between KRT19 expression and HGF, MET, and FOSL1 expression. D, Expression of FOSL1 and KRT19compared with the combined expression status of HGF and MET. E, Differences in disease-specific survival and disease-free survival between HGF- orMET-positive patients with HCC and HGF- and MET-double negative patients with HCC.






those that did not (P < 0.001, and P ¼ 0.001, respectively;Fig. 6C). We also analyzed the correlation between the pro-portion of aSMA-positive CAFs and the degree of KRT19, HGF,MET, and FOSL1 expression in a semiquantitative manner. Asignificant positive correlation between the degree of HGFexpression and the proportion of aSMA-positive CAFs wasobserved (r ¼ 0.160, P ¼ 0.003). Furthermore, the degree ofKRT19 expression also showed a significant positive correlationwith that of HGF (r ¼ 0.288, P < 0.001), MET (r ¼ 0.158, P ¼0.004), and FOSL1 (r ¼ 0.190, P < 0.001). Conversely, theproportion of CD90-positive CAFs was not correlated with theHGF (r ¼ 0.073, P ¼ 0.182), MET (r ¼ �0.098, P ¼ 0.071), orFOSL1 expression (r ¼ �0.014, P ¼ 0.794).

When HCC cases were categorized according to HGF andMET protein expression, FOSL1 and KRT19 expression weremost frequently observed in the HGF and MET double positivegroup and least frequently observed in the HGF and METdouble negative group (Fig. 6D, P < 0.001 at both). Patientswith HCC with HGF or MET expression showed higher serumalpha-fetoprotein levels and more frequent microvascularinvasion, compared with patients with HCC with HGF andMET double negative expression (P < 0.05 for all; Supplemen-tary Table S8). The expression of HGF or MET was a poorprognostic factor for disease-specific survival and disease-freesurvival in our univariate analysis; however, it was not anindependent prognostic factor (Fig. 6E; Supplementary TablesS9 and S10).

The protein expression of phospho-MET was also evaluated inHCC tissues by IHC staining; however, it was not detected in allcases (Supplementary Fig. S7F). The stability of the phospho-METprotein was assessed in our in vitro model. HepG2 cells treatedwith CM from hTERT-HSCs were harvested at various time points(0 to 30 minutes) after media exchange (CM was removed andexchanged with control media). We observed that phospho-METlevels rapidly declined, whereas FOSL1 and KRT19 expressionlevels did not change (Supplementary Fig. S7G). Therefore, thenegative expression of phospho-MET in HCC tissues is linked toits instability.

To further validate the association of KRT19 expression withaSMA-positive CAF and MET signaling, we evaluated theexpression of CAF and MET-related gene signatures obtainedfrom previous studies (37, 38). After filtering out the geneswith frequent missing values greater than 50%, the enrichmentscores of the upregulated CAF signature (CAF_UP, n ¼ 21) andthe up- and down-regulated MET signatures (i.e., MET_UP, n ¼19, and MET_DOWN, n ¼ 4) were calculated from the HCCdata of TCGA (LIHC, n ¼ 371). We found that these genesignatures were closely correlated with KRT19 expressionacross HCC samples (Fig. 7A, top). The analysis of gene setsalso revealed a significant correlation between KRT19 expres-sion and the enrichment scores for the CAF_UP (r ¼ 0.32, P ¼2.5 � 10�10), MET_UP (r ¼ 0.54, P ¼ 4.46 � 10�30), andMET_DOWN (r ¼ �0.44, P ¼ 1.05 � 10�19) signatures,respectively (Fig. 7A, bottom). Moreover, the stratification ofthe patients based on the expression levels of the CAF or METsignatures also revealed significant differences in KRT19expression levels between the groups (CAF_UP, P ¼ 4.61 �10�8; MET_UP, P ¼ 6.88 � 10�18; MET_DN, P ¼ 3.13 � 10�14;permuted t test; Fig. 7B). These results strongly suggest thatKRT19 expression is closely associated with the expression ofCAF and MET-mediated signaling activities.

DiscussionHCCs that express KRT19 are known for their aggressive

biological behavior (3, 6); however, the underlying regulatorymechanism remains unclear. Interestingly, KRT19 expressionwas closely correlated with CAFs in the fibrous tumor stroma inHCCs. Therefore, to explore the regulatory mechanism under-lying KRT19 expression, we investigated the cross-talk betweentumor epithelial cells and CAFs using an in vitro model ofparacrine interaction between HCC cell lines and hepatic stel-late cell lines, and a large cohort of patients with HCC. Our invitro and human study demonstrated that KRT19 expression inHCCs was regulated by the CAF-derived HGF via the MET-ERK1/2-AP1 and SP1 axis, thus highlighting the molecularbackground of clinically aggressive KRT19-positive HCCs (Sup-plementary Fig. S8).

Previously, a subset of HCCs exhibiting a stem/progenitor-likegene expression pattern, namely the hepatoblast signature, wasreported to indicate a poor prognosis (39). In the aforemention-ed study, HCCs with hepatoblast signatures highly expressedmarkers of hepatic stem cells, including KRT19, and the AP1transcription factor was proposed as amajor driver of hepatoblastsignature expression. Our results provide a functional connectionbetweenHCCswithKRT19 expression and thosewithhepatoblastsignatures through the MET-ERK1/2-AP1 axis. Taken together,these results suggest that the MET/HGF and AP1 signaling axis isimportant for the regulation of stemness in HCCs, and KRT19represents a valuable marker of stemness and tumor-initiatingcapacity. Accordingly, CAFs are considered a major source ofHGF in addition to endothelial cells (40), which was recentlyreported to regulate the tumor-initiating capacity of HCCs via theHGF/MET pathway (36).

In fact, CAFs represent a heterogeneous group with respect totheir origin, biological properties, and differentiation markers(41). CAFs are usually considered to support the extracellularmatrix and promote cancer progression via paracrine signaling(14).However, in certain circumstances, CAFs could play a tumor-restraining role, including growth arrest, and promoting antitu-mor immune responses (41, 42). In HCCs, CAFs have beenreported to promoteHCCprogression, via the secretion of variousparacrine factors including the HGF, SDF-1, IL6, TGFb, EGF, andFGF families (43). In this study, the proportion of aSMA-positiveCAFs was associated with HGF and KRT19 immunoreactivity anda poor outcome, suggesting the tumor-promoting role of aSMA-positive CAFs. Conversely, the proportion of CD90-positive CAFswas positively correlatedwith the degree of KRT19 expression, butnot with HGF, MET, or FOSL1 expression. The heterogeneity ofCAFs in HCCs remains unclear, and the specific markers fortumor-promoting and tumor-restraining CAFs remain to beuncovered.

MET, the HGF receptor, is a receptor tyrosine kinase that canactivate downstream pathways including MAPK, PI3K, andRAC1-CDC42, and promote the invasive growth of cells(31). HCCs with high MET expression are also known to beassociated with a poorer prognosis than those without (35,44). MET is an emerging therapeutic target of HCC, andcurrently, several agents targeting the HGF/MET signaling axis,including tivantinib, cabozantinib, and INC280 are undergo-ing clinical trials (45). For an optimal therapeutic applicationof MET inhibitors, a selective enrollment of patients with MET-addicted HCC is crucial. Unfortunately, no practically effective

Rhee et al.





marker is known for assessing the activation status of the HGF/MET axis (31). Considering the short half-life (less than 10minutes) of phospho-MET, as noted in our in vitro experiment,we doubt that a reliable assessment of the phosphorylationstatus of MET could be performed in human HCC tissuesamples, especially in formalin-fixed paraffin-embedded tis-sues by IHC staining. Although IHC staining may not reliablyreveal its cellular source, because paracrine factors can bind tothe extracellular matrix (46), the positive immunoexpressionof HGF was associated with a proportion of aSMA-positiveCAFs, and the degree of MET or HGF expression was also

correlated with those of their downstream targets includingKRT19 and FOSL1. Further analysis of gene expression datafrom TCGA also revealed that KRT19 expression was closelyassociated with CAF and MET-mediated signaling activities.Taken together, KRT19 may serve as an indirect marker for theactivation of HGF/MET signaling and may represent a poten-tial marker for therapeutic applications involving the inhibi-tion of HGF/MET signaling.

In our study, AP1 (JUN/FOSL1 complex) and SP1 transcrip-tion factors were identified as crucial positive regulators ofKRT19 expression in HCC. Accordingly, SP1 was reported as

Figure 7.

External validation of the associationof KRT19 expression with CAF andMET signatures. A, Expressionheatmap of CAF_UP (n¼ 21), MET_UP(n ¼ 19), and MET_DOWN genesignatures (n ¼ 4) according to theKRT19 gene expression in the HCCdata of TCGA (n ¼ 371). B,Comparison of KRT19 expressionaccording to the expression levels ofthe CAF or MET signatures. The high-and low-groups of HCC are stratifiedby the mean value of the enrichmentscores for each signature,respectively.






a transcriptional activator of KRT19 in pancreatic cancer celllines (47). To our knowledge, the AP1-binding site located 2-kbp upstream of the transcription start site has not beendescribed previously, because previous studies have used rel-atively short (less than 2 kbp) mouse KRT19 promoter con-structs to search for transcriptional activators. A previouslyreported SP1-binding site in the mouse KRT19 promoter washomologous with the SP1-binding site that we found in thehuman KRT19 promoter (Supplementary Fig. S9). Interesting-ly, the AP1-binding site is very well conserved in most mam-mals (Supplementary Fig. S9), suggesting the functional impor-tance of the AP1-driven regulation of KRT19.

In conclusion, the expression of KRT19 in HCC is regulatedby CAF via the HGF-MET-ERK1/2-AP1 and SP1 axis. The HGFand/or MET protein expression was well correlated with FOSL1and KRT19 expression in human HCC tissues. Our resultsprovide insights into the molecular background of KRT19-positive HCC with an aggressive biological behavior. Theassessment of these markers could indicate the activation ofMET signaling and predict poor outcomes for HCCs.

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

Authors' ContributionsConception and design: H. Rhee, H.-Y. Kim, J.S. Choi, Y.N. ParkDevelopment of methodology: H. Rhee, H.-Y. Kim, Y.N. ParkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Rhee, H.-Y. Kim, J.E. Yoo, J.H. Nahm, J.S. Choi,Y.N. ParkAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Rhee, H.-Y. Kim, J.-H. Choi, H.G. Woo, Y.N. ParkWriting, review, and/or revision of the manuscript: H. Rhee, H.-Y. Kim,H.G. Woo, Y.N. ParkAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.H. Nahm, Y.N. ParkStudy supervision: Y.N. Park

AcknowledgmentsThis research was supported by grants from the National Research

Foundation of Korea (NRF) funded by the Korean government (MSIP;grant number: NRF-2011-0030086, NRF-2016M3A9D5A01952416, NRF-2017R1A2B4005871, and NRF-2017M3A9B6061512).

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 April 8, 2017; revised August 12, 2017; accepted January 18, 2018;published OnlineFirst January 23, 2018.

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2018;78:1619-1631. Published OnlineFirst January 23, 2018.Cancer Res Hyungjin Rhee, Hye-Young Kim, Ji-Hye Choi, et al. Fibroblast-Derived HGF via a MET-ERK1/2-AP1 and SP1 AxisKeratin 19 Expression in Hepatocellular Carcinoma Is Regulated by

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