kruppel-like factor 4 blocks hepatocellular carcinoma ...kruppel-like factor 4 blocks...

Biology of Human Tumors

Kr€uppel-like Factor 4 Blocks HepatocellularCarcinoma Dedifferentiation and Progressionthrough Activation of Hepatocyte NuclearFactor-6Hongcheng Sun1,2, Huamei Tang3, Dacheng Xie4, Zhiliang Jia2, Zhenyu Ma5, DaoyanWei2,Lopa Mishra2, Yong Gao4, Shaojiang Zheng6, Keping Xie2, and Zhihai Peng1

Abstract

Purpose: Tumor differentiation is a behavioral index forhepatocellular carcinoma (HCC) and a prognostic factor forpatients with HCC who undergo orthotopic liver transplanta-tion (OLT). However, the molecular basis for HCC differenti-ation and prognostic value of the underlying molecules thatregulate HCC differentiation are unclear. In this study, wedefined a potential driver pathway for HCC differentiation andprognostication.

Experimental Design: The regulation and function of Kr€uppel-like factor 4 (KLF4) and hepatocyte nuclear factor-6 (HNF-6) inHCC differentiation was evaluated using human tissues, molec-ular and cell biology, and animal models, and its prognosticsignificance was determined according to its impact on patientsurvival.

Results: There was a direct relationship between the expressionlevels of KLF4 and HNF6 in HCC. Reduced KLF4 or HNF6expression correlated with high HCC grade. Poorly differentiated

HCC cells had lower expression of KLF4 or HNF6 and differen-tiation-associated markers than did well-differentiated cells. Ele-vated KLF4 of HNF6 expression induced differentiation of poorlydifferentiated hepatoma cells. Mechanistically, KLF4 trans-acti-vated HNF-6 expression. Restored HNF-6 expression upregulatedexpression of differentiation-associated markers and inhibitedHCC cell migration and invasion, whereas HNF-6 knockdowndid the opposite. Loss of KLF4 expression in primary HCCcorrelated with reduced overall survival and shortened relapse-free survival durations after OLT. Combination of KLF4 expres-sion and the Milan criteria improved prognostication for HCCafter OLT.

Conclusions: The dysregulated KLF4/HNF-6 pathway drivesdedifferentition andprogression ofHCC, andKLF4 is a biomarkerfor accurate prognostication of patients with HCC treatedby OLT when integrated with the Milan Criteria. Clin Cancer Res;22(2); 502–12. �2015 AACR.

IntroductionTumor differentiation is a well-recognized morphologic index

for assessing the biologic behavior of hepatocellular carcinoma(HCC). Histologic grade increases along with tumor burden (1),and loss of differentiation always parallels early metastasis (2).Tumor grade is widely accepted as a helpful prognostic indicatorfor HCC after surgery (2, 3) and recommended as a key deter-minant of liver transplantation for patients with HCC (4, 5).Moreover, differentiation therapy for HCC is proposed as one ofthe best strategies for treatment of this malignancy because ofinduction of cell differentiation (6). Nevertheless, the underlyingmechanisms of and molecules for regulation of HCC differenti-ation are poorly understood.

The Kr€uppel-like factors (KLF), a family of evolutionarily con-served mammalian transcription factors that share a C-terminalthree-zinc-finger DNA-binding domain, play critical roles in nor-mal development and carcinogenesis (7). Of the KLF familymembers, KLF4 is physiologically expressed in terminally differ-entiated epithelial cells and functions as an imperative regulatorof cell differentiation (8–14). However, the causal impact of KLF4expression and function on cancer differentiation has yet to be

1DepartmentofGeneralSurgery, Shanghai JiaotongUniversity Shang-hai General Hospital, Shanghai, PR China. 2Department of Gastroen-terology, Hepatology and Nutrition, The University of Texas MDAnderson Cancer Center, Houston, Texas. 3Department of Pathology,Shanghai Jiaotong University Shanghai General Hospital, Shanghai,PR China. 4Department of Oncology and Tumor Institute, ShanghaiEast Hospital, Tongji University School of Medicine, Shanghai, PRChina. 5Liver Transplantation Section, Center for Organ Transplanta-tion, Fudan University, Shanghai, China. 6Department of PathologyHainan Medical College Affiliated Hospital, Haikou, PR China.

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

Corresponding Authors: Keping Xie, Department of Gastroenterology, Hepa-tology and Nutrition, Unit 1644, The University of Texas MD Anderson CancerCenter, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-5073;Fax: 713-745-3654; E-mail: [email protected]; Shaojiang Zheng, Depart-ment of Pathology, HainanMedical CollegeAffiliated Hospital, Haikou, PR China.Phone: 86-898-66723333; E-mail: [email protected]; or Zhihai Peng,Department of General Surgery, Shanghai General Hospital, 85 Wujin Road,Shanghai, 200080, PR China. Phone: 86-21-63240090-3102; Fax: 86-21-63241377; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-0528

�2015 American Association for Cancer Research.

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well defined. In gastrointestinal cancers, KLF4 is a tumor suppres-sor, including HCC (15–19). However, whether dysregulation ofKLF4 expression casually impacts HCC differentiation and, if so,the underlying molecular mechanism must be elucidated.

The development and differentiation of the liver are cross-regulated by several liver-enriched transcription factors (LETF),including the hepatocyte nuclear factors (HNFs). HNF-6, whichbelongs to the superclass of cut domain-containing homeopro-teins, is expressed at high levels and regulates the transcription ofhepatocyte-specific genes in the adult liver (20). HNF-6 functionsas an upstream regulator of the LETF cascade that drives thedifferentiation of cells of hepatic lineage (21–23). However, theimpacts of HNF-6 expression on HCC differentiation and clinicaloutcome remain unclear.

Orthotopic liver transplantation (OLT) offers a potential curefor HCC and concomitant liver disease and is proposed forpatients with tumors limited in size and number (24). The mostsuccessful and widely accepted patient selection criteria for OLTare the Milan criteria (25, 26). However, Milan criteria are tooconservative, leading to arbitrary exclusion of some patients withtumors at advanced stages but with favorable biology, whowouldhave benefited from OLT (27). Attempts to expand the Milancriteria have been based on pathologic features, such as tumorsize and number, histologic grade, and microvascular invasion(27–29). However, the applicability of more liberal indicationsfor OLT than the current Milan criteria based exclusively onmorphological criteria in patients with HCC remains controver-sial. Given that the inherent molecules in rather than the mor-phologyofHCC lesionpredicts tumorbehavior (30),momentumtoward developing effective molecular biomarkers for improvingprognostication and selecting candidates for OLT has increased(31, 32). Currently, no pathobiologic markers demonstrating theprognostic significance of HCC after OLT are widely accepted.

In the present study, we found that loss of KLF4 expression wasclosely correlatedwith highhistologic grade inHCC cases andwasan independent negative prognostic factor for patients with HCCafter OLT. Restoration of KLF4 expression promoted differentia-

tion ofHCC cells. HNF-6, a novel transcriptional target of KLF4, isa pivotal molecular mediator of KLF4-induced differentiation ofHCC. Therefore, integration of this novel biomarker into theconventional Milan criteria may increase the accuracy of prog-nostication for HCC.

Materials and MethodsThe Supplementary Methods contains details about the mate-

rials and methods regarding immunohistochemistry and tissuemicroarray (TMA) analysis (16, 33), Western blot analysis (16),RNA extraction and reverse transcriptase PCR (RT-PCR) analysis(Supplementary Table S1), laser capture microdissection (LCM),genotyping and gene expression analyses (10, 34), immunoflu-orescent cell staining, construction of mutant KLF4 with deletionof zinc finger domain (KLF4DZFD)-expressing vectors, construc-tion of HNF-6 promoter reporter plasmids and mutagenesis,chromatin immunoprecipitation (ChIP) assay (15), cell prolifer-ation assay (15, 16), flow cytometric analysis of the cell cycle(15, 16), scratch assay (15, 16), cell invasion assay (15, 16), and amouse xenograft tumor study (16).

Patients, clinicopathologic analysis, and tissue specimensPatient information and follow-up and clinicopathologic data

extraction were described previously in detail (33). Tumors inexplanted livers were re-evaluated using the Milan criteria on thebasis of pathologic data. Matched pairs of HCC and formalin-fixed, paraffin-embedded nontumor tissue blocks for TMA andpaired fresh HCC and nontumor tissue specimens were collectedas described previously (33). The histologic grade of HCC wasclassified as well-differentiated (grade 1), moderately differenti-ated (grade 2), or poorly differentiated (grade 3) according toWorld Health Organization criteria (35). When the tumor con-sisted of more than or equal to two grades of histologic differ-entiation, themost progressive gradewas used. The studyprotocolwas approved by the Shanghai Jiaotong University InstitutionalReview Board, and informed written consent for use of the tissuespecimens was obtained from each patient or his or her guardian.

TMA construction and immunohistochemistryThe TMA construction was described in detail previously (33).

Standard immunohistochemical procedureswere performedwithhuman HCC TMA specimens using anti-KLF4 (Santa Cruz Bio-technology) and anti-HNF-6 (Sigma) antibodies, and the stainingresults were scored by twopathologists blinded to the clinical dataas described previously (15, 16) and in Supplementary Methods.Use of archived tissue specimens was approved by the ShanghaiJiaotong University andMD Anderson Cancer Center Institution-al Review Boards.

Statistical analysisStatistical analyses were performed using the SPSS software

program (version 17.0; IBM Corporation). For continuous vari-ables, data were expressed as medians and in the interquartilerange and compared using the Kruskal–Wallis test. For categoricalvariables, data were expressed as numeral counts and percentagesand compared using the Pearson c2 test or Fisher exact test.Differences in protein expression between thematched specimenswere examined using the marginal homogeneity test. Survivalrates were calculated and survival curves were plotted using theKaplan–Meier method, and differences were compared using the

Translational Relevance

Wehave used hepatocellular carcinoma (HCC) tissue speci-mens and molecular biology and animal models to evaluatethe activation and function of the KLF4/HNF-6 pathway inhuman HCC. Our clinical and mechanistic findings indicatethat HNF-6 is a direct transcriptional target of KLF4 and thatfrequently dysregulated KLF4 expression leads to aberrantHNF-6 expression. Moreover, both KLF4 and HNF-6 upregu-lated expression of differentiation-associated markers andinhibited HCC cell differentiation, whereas knockdown ofHNF-6 and KLF4 did the opposite, suggesting a novel molec-ular basis for the critical role of loss of KLF4 in HCC devel-opment and progression.Moreover, loss of KLF4 expression inprimary HCC correlated with reduced overall survival andshortened relapse-free survival durations after orthotopic livertransplantation (OLT). Combination of KLF4 expression andthe Milan criteria improved prognostication for HCC afterOLT. Therefore, our findings may have a significant effect onthe clinical management of patients with HCC.

Regulation of HCC Differentiation by KLF4/HNF-6

www.aacrjournals.org Clin Cancer Res; 22(2) January 15, 2016 503




log-rank test. Univariate Cox regression was used to analyzedifferences in survival among patient groups. The significantfactors in the univariate analyses were included in multivariateCox proportional hazards models. The significance of the in vitrodata in the groups was determined using a two-tailed Studentt test. Statistical significance was indicated by a conventionalP < 0.05.

ResultsDecreased KLF4 expression predicted poor differentiation ofhuman HCC

We first investigated the expression of KLF4 in 99 pairs ofprimary HCC and matched adjacent nontumor tissue specimens

obtained from patients who underwent OLT using a TMA. Wefound that loss of KLF4 expression was associated with poortumor differentiation (Fig. 1A; Supplementary Fig. S1) and thatKLF4 expression in human HCC specimens was drastically lowerthan that in paired nontumor tissue specimens (Fig. 1B and C;Supplementary Fig. S2; Supplementary Table S2). Interestingly,histologic grade was the only clinicopathological parameter neg-atively related to KLF4 expression (P ¼ 0.008; Fig. 1D; Supple-mentary Table S3). These results strongly indicated that KLF4plays a role in human HCC differentiation.

In human HCC cell cultures, well-differentiated HCC cells(HepG2, Hep3B, and PLC/PRF/5) exhibited a flattened pleomor-phic morphology and enhanced intercellular aggregation (i.e.,well-differentiated phenotype), whereas poorly differentiated

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Figure 1.KLF4 and HNF-6 protein expression inhuman HCC specimens and theirassociation with tumor grade.Immunostaining of HCC and matchedadjacent nontumor tissue specimens ina TMA was conducted using a specificanti-KLF4 or anti-HNF-6 antibody. A,representative photos of KLF4 proteinexpression in grade 1, 2, and 3 HCCsamples (�200). Note that thepercentage and intensity of positivelystained tumor cells decreasedgradually as tumor grade increased. B,representative images of similarexpression patterns for KLF4 (top,reddish brown staining) and HNF-6(bottom, gray–black staining) in thetumor border (�100), a grade 2 tumor(�200), and a grade 3 tumor (�200).N, nontumor tissue; T, tumor tissue; reddotted line, border. C, Western blotanalysis of KLF4 and HNF-6 proteinexpression in grade 1 and 3 HCC (T) andnontumor tissue (N) specimens (left).The expression of KLF4 and HNF-6protein (normalized according toexpression of GAPDH) in tumors wasquantified and expressed as the ratio tothat in nontumor tissue (right). Notethat loss of KLF4 and HNF-6 proteinwas more prominent in grade 3 thangrade 1 tumors. D, left, loss of KLF4expression was correlated with poordifferentiation (P¼ 0.008; Fisher exacttest in a comparison of grade 3 tumorswith grade 1 and 2 tumors). Middle,decreased HNF-6 expression wascorrelated with poor tumordifferentiation (P ¼ 0.013; Fisher exacttest in a comparison of grade 3 tumorswith grade 1 and 2 tumors). Right, directcorrelation between the expressionlevels of KLF4 and HNF6(n ¼ 99). N/W, negative/weak; M,moderate; S, strong.

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Clin Cancer Res; 22(2) January 15, 2016 Clinical Cancer Research504




HCC cells (SNU387 and SNU423) had spindle-like shapes andappeared to be scattered (Fig. 2A). Consistently, we observed adramatic loss of a panel of hepatic differentiation–associatedmarkers in poorly differentiated HCC cells or lower expressionof them than in the well-differentiated cells (Fig. 2B, left panels);and a similar expression pattern for these markers in well-differ-entiated (grade 1) and poorly differentiated (grade 3) HCC speci-mens using LCM (Supplementary Fig. S3). Western blot analysisdemonstrated a drastic reduction in the expression of KLF4 and itsdownstream transcriptional target E-cadherin (36), an epithelialand differentiation marker of HCC (37), in the poorly differen-tiated HCC cells (Fig. 2B, right and C). These findings indicatedthat KLF4 expression is critical to the maintenance of HCCdifferentiation status.

KLF4 induced differentiation of poorly differentiatedHCC cellsTo determine the causal effect of KLF4 expression on HCC

differentiation, we transfected the KLF4 expression vector pFLAG-KLF4 or control pcDNA3.1 into poorly differentiated SNU387and SNU423 cells. Increased expression of KLF4 (Fig. 3A and B)markedly elevated a-AT, a-fetoprotein (AFP), albumin, HNF-6,C/EBPa, and E-cadherin mRNA expression (Fig. 3A) and upre-

gulated HNF-6, E-cadherin, albumin, and AFP protein expressionin these cells (Fig. 3B and C). Meanwhile, enforced KLF4 expres-sion in these cells led to a dramaticmorphology shift towardwell-differentiated phenotype (Fig. 3D). In contrast, we transfectedKLF4 siRNA or the control siRNA into PLC/PRF/5 cells, whichwere well-differentiated and had high levels of KLF4 expression(Fig. 2C). KLF4 knockdown led to a concomitant decrease ina-AT,AFP, albumin, E-cadherin, HNF-6, andC/EBPamRNA expression(Fig. 3A) and downregulation of E-cadherin and HNF-6 proteinexpression in PLC/PRF/5 cells (Fig. 3B). These results clearlydemonstrated that KLF4 expression positively impacts the differ-entiation of HCC cells.

To determine whether transcriptional activity of KLF4 isrequired for the function of KLF4 in HCC differentiation, weconstructed a KLF4 vector (KLF4DZFD) lacking a zinc fingerdomain (ZFD; ref. 38; Supplementary Fig. S4A), and we foundthat KLF4DZFDprotein wasmainly distributed in the nucleus, thesame as the full-length KLF4 (Supplementary Fig. S4B). However,KLF4DZFD lost both its transcriptional activity (SupplementaryFig. S4C) and its promoting effect onHCCdifferentiation (Fig. 3).These findings strongly suggested that KLF4 promotes HCCdifferentiation via its transcriptional activity.

Figure 2.Different morphologies of andbiomarker expression in HCC cell lineswith different grades of differentiation.A, different morphologies of HCC cellline cultures derived from well-differentiated and poorly differentiatedtumors (�200). B, distinct patterns ofexpression of hepatic differentiationmarkers in HCC cell lines establishedfrom tumors with different histologicgrades as measured using RT-PCR. C,relative mRNA expression levels ofthose molecules were quantified andnormalized according to GAPDHexpression.






HNF-6–mediated KLF4-induced HCC differentiationIncreasing evidence indicates that LETFs play important roles

in inducing differentiation of hepatoma cells (22, 23). In thepresent study, we observed higher basal levels of expression ofLETFs in well-differentiated HCC cells than in poorly differen-tiated cells (Fig. 2B and C). Moreover, treatment of SNU387cells with sodium butyrate, a chemical inducer of differentia-tion, upregulated expression of KLF4, HNF-6, and C/EBPa butnot HNF-1a, HNF-4a, HNF-6b, or DBP (Supplementary Fig.S5). Western blot analysis further demonstrated increases inKLF4 and HNF-6 but not C/EBPa protein expression (Fig. 3B),indicating an important role of KLF4 and HNF-6 in the induc-tion of HCC differentiation.

To characterize the effect of HNF-6 expression on differen-tiation of HCC cells, we transfected the HNF-6 expressionvector pHNF-6-GFP or control vector pCMV6-AC-GFP intoSNU387 and SNU423 cells, which have low levels of HNF-6expression (Fig. 2B and C). Elevated expression of HNF-6markedly increased expression of hepatic differentiation mar-kers such as AFP, a-AT, albumin, and HNF-4a (Fig. 4, left) andelicited distinguishable morphology change toward well-dif-ferentiated phenotype (Fig. 4, right). In comparison, we trans-fected HNF-6 siRNA or the control siRNA into PLC/PRF/5 cells,which have high basal levels of HNF-6 expression (Fig. 2B andC). This transfection inhibited expression of the hepatic differ-entiation markers (Fig. 4A, left). Furthermore, the levels of

Figure 3.Genetically engineered expressionof KLF4 and its impact onexpression of differentiation-associated biomarkers in andmorphologyof HCCcells. A, SNU387cells were transfected with 4 mg of acontrol pcDNA3.1 vector (Ctrl), 1 mgof a pFLAG-KLF4 vector plus 3 mg ofa pcDNA3.1 vector (pFLAG-KLF4 1mg), 4 mg of a pFLAG-KLF4 vector(pFLAG-KLF4 4 mg), and 4 mg of apFLAG-KLF4DZFD vector (pFLAG-KLF4DZFD 4 mg) in 6-well plates.PLC/PRF/5 cells were transfectedwith control siRNA (siCtrl) or KLF4siRNA (siKLF4) in 6-well plates atthe indicated doses. Total RNA washarvested from the cultures 48hours after transfection andreverse-transcribed into cDNA. Theexpression of KLF4 anddifferentiation-associated markerswas measured using RT-PCR. B, left,SNU387 and PLC/PRF/5 cells weretransfected as in A; total proteinlysates were prepared 48 hoursafter transfection and subjected toWestern blot analysis. Right,Western blot analysis of KLF4, E-cadherin, HNF-6, and C/EBPaprotein expression in SNU387 cellstreated with sodium butyrate(NaBu). C, SNU387 cells weretransfected with control pcDNA3.1(Ctrl) or a pFLAG-KLF4 expressionvector (pKLF4) or pFLAG-KLF4DZFD expression vector(pKLF4 DZFD) and submitted todouble immunofluorescent stainingfor KLF4 and albumin (left) or KLF4and AFP (right) 48 hours aftertransfection. Nuclei were stainedwith DAPI (�200). D, differentmorphologies of HCC cellstransfected with pcDNA3.1 (Ctrl) orpFLAG-KLF4 (pKLF4) plasmids(�200).

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HNF-6 expression in the primary tumors were significantlylower than those in the matched nontumor tissue (P <0.001; Fig. 1B; Supplementary Fig. S6A; SupplementaryTable S4). Also, the HNF-6 expression level in HCC specimenswas negatively correlated with histological grade (P ¼0.013; Fig. 1B; Supplementary Fig. S6; Supplementary TableS5). These data indicated that HNF-6 is a critical regulator ofHCC differentiation. Moreover, restored HNF-6 expression inSNU387 and SNU423 cells markedly suppressed their inva-siveness (Fig. 4B) and migration (Fig. 4C) but not their pro-liferation or cell-cycle progression (Fig. 4D), suggesting thataltered HNF-6 expression affects certain biologic behaviors ofHCC cells.

In addition to the similar KLF4 and HNF-6 expressionpatterns (Fig. 1B), their direct correlation in human HCC

samples (Fig. 1C; Supplementary Table S6), and the significantimpact of altered KLF4 expression on HNF-6 expression in HCCcell lines (Fig. 3A and B), KLF4 knockdown in sodium buty-rate–treated SNU387 cells led to marked downregulation ofHNF-6 expression (Fig. 5A). Consistent with these in vitrofindings, increased KLF4 expression markedly upregulatedHNF-6 expression in SNU398 xenograft tumors (Fig. 5B),whereas conditional deletion of KLF4 led to marked reductionof HNF-6 expression in murine livers (Fig. 5C; SupplementaryFig. S7). Importantly, HNF-6 knockdown markedly reversedKLF4-induced upregulation of expression of differentiation-associated markers in HCC cells (Fig. 5D). These results clearlyindicated that KLF4 upregulates HNF-6 expression and thatHNF-6 is an important mediator of KLF4-induced HCCdifferentiation.

Figure 4.Influence of HNF-6 expression ondifferentiation and biologic behavior ofHCC cells. A, SNU387 cells weretranfected with a pHNF-6-GFPexpression vector (pHNF-6) or controlvector pCMV6-AC-GFP (Ctrl) andPLC/PRF/5 cells were transfected withHNF-6 siRNA (siHNF-6) or controlsiRNA (siCtrl). The cultures wereincubated for 48 hours. Total RNA washarvested, the expression of HNF-6 anddifferentiation-associated markers wasmeasured using RT-PCR (left).Different morphologies of HCC cellstransfected with pCMV6-AC-GFP (Ctrl)or pHNF-6-GFP (pHNF-6) plasmids(�200, right). B, SNU387 and SNU423cells were treated as described above,and cell invasion was assessed.Representative photos of cell invasionare shown (left), and the control cellgroups (Ctrl) were given an arbitraryinvasion percentage of 100% (right).� ,P <0.05 in a comparison of the pHNF-6–treatedgroupwith the control group.C, SNU387 and SNU423 cells weretreated as described above for ascratch assay. Shown are photos takenat the indicated time points after thecultures were wounded by scratching(left). Quantification of cell migration isshown on the right measured byImageJ 2� software (right). D, SNU387and SNU423 cells were treated asdescribed above and subjected to cellproliferation assay (left). SNU387 cellswere prepared as described in A andsubjected to cell-cycle analysis (right).






KLF4 transactivated HNF-6 transcriptionTo determine whether KLF4 transcriptionally upregulates

HNF-6 expression, we analyzed the HNF-6 promoter sequencefor the presence of potential KLF4-binding sites containing theCACCC motif (39) and identified three putative KLF4-bindingelements (referred to as site #1, #2, and #3) in the HNF-6promoter region and accordingly constructed the deletionmutant reporters pH6Pro-843 (not containing a binding site),pH6Pro1002 (containing #1), and pH6Pro1796 (containing#1, #2, and #3; Fig. 6A; Supplementary Fig. S8). We thencotransfected the deletion mutant reporters with the KLF4 orKLFDZFD expression vector or control pcDNA3.1 as indicatedinto SNU387 and SNU423 cells; we cotransfected pGL3-basicand pFLAG-KLF4 as negative controls. Enforced KLF4 but not

KLF4DZFD expression enhanced the promoter activity ofpH6Pro1796 and pH6Pro1002 (Fig. 6B), whereas KLF4 knock-down did the opposite (Fig. 6C). Furthermore, ChIP assaysshowed that KLF4 directly interacted with the HNF-6 promoter(Supplementary Fig. S9A and B) and this binding required itsZFD (Fig. 6D). These results clearly indicated that KLF4 acti-vated the HNF-6 promoter via KLF4-binding elements.

KLF4 expression predicted good prognosis for HCC patientstreated with OLT

Univariate survival analysis demonstrated that loss of KLF4expression in primary HCC cells was significantly associated withreduced overall survival (OS) duration (P < 0.001) and reducedrecurrence-free survival (RFS) duration (P < 0.001) after liver

Figure 5.Regulation of HNF-6 expression by KLF4 and the influence of HNF-6 expression on KLF4-induced HCC differentiation. A, SNU387 cells were transfectedwith controlsiRNA (siCtrl) or KLF4 siRNA (siKLF4) as indicated for 24 hours and then submitted to treatment with or without sodium butyrate (5 mmol/L) for 48 hours.Total RNA and protein lysates were prepared for RT-PCR (left) and Western blot (right) analysis, respectively. B, immunohistochemical analysis of KLF4and HNF-6 protein expression in consecutive sections of HCC xenograft tumors established from adenoviral KLF4 (Ad-KLF4)- and adenoviral enhancedGFP (Ad-GFP)-infected SNU398 cells. C, total RNA from the livers ofAlb-Cre�;Klf4fl/fl (1# and 2#) andAlb-Creþ;Klf4fl/fl (3# and 4#)mice at the age of 20weekswereextracted and subjected to RT-PCR analysis of Klf4 and Hnf-6 expression (left) and Western blot analysis (right). The relative levels of expression werequantified and normalized according to GAPDH expression (the gene expression in the liver of mouse #3 was used as a reference; bottom). D, SNU387 and SNU423cells were cotransfected with a control pcDNA3.1 and control siRNA (pcDNA3.1 þ siCtrl), a pFLAG-KLF4 vector and control siRNA (pKLF4 þ siCtrl), or apFLAG-KLF4 vector and HNF-6 siRNA (pKLF4þ siHNF-6) as indicated. Total RNAwas extracted from the cells 48 hours after transfection and subjected to RT-PCRanalysis (left). The relative mRNA expression levels of those molecules were quantified and normalized according to GAPDH expression (right).

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transplantation (Supplementary Fig. S10A; Supplementary TableS7), and decreased HNF-6 expression in HCC was shown tocorrelate with poor OS and RFS after OLT (Supplementary Fig.S10B; Supplementary Table S7). Multivariate analysis furtherrevealed that along with well-established prognostic factors suchas macrovascular invasion and the Milan criteria, loss of KLF4expression but not HNF-6 was an independent biomarker forunfavorable clinical outcome of liver transplantation in patientswithHCC (Supplementary Table S7). To determinewhether KLF4expression improves survival prediction when combinedwith theMilan criteria, we analyzed the patients with tumors within orbeyond the Milan criteria stratified according to KLF4 expression.We could not differentiate the RFS and OS durations in the

patients with tumors meeting the Milan criteria according toKLF4 expression (P ¼ 0.509 for RFS, P ¼ 0.613 for OS;Supplementary Fig. S10C), whereas the RFS and OS rates inthe patients with tumors exceeding the Milan criteria differen-tiated according to KLF4 expression (P < 0.001 for both RFS andOS; Supplementary Fig. S10D). Nearly two thirds (22 of 34) ofthe patients with tumors beyond the Milan criteria and exhibit-ing moderate-to-high KLF4 expression had unexpectedly favor-able 5-year RFS (70.5%) and OS (91.7%) rates (SupplementaryFig. S10D). These results indicated that KLF4 is a valuablebiomarker that can identify patients with hepatocellular tumorsoutside the Milan criteria but may have favorable outcomesafter OLT.

Figure 6.Regulation and direct binding of theHNF-6 promoter by KLF4. A, schematicstructure of the HNF-6 promoterreporters and their putative KLF4-binding sites. B, HNF-6 promoterreporters were transfected in triplicatewith pFLAG-KLF4, pFLAG-KLF4DZFD,or the control vector pcDNA3.1 intoSNU387 (left) and SNU423 (right) cells.Cotransfection of pFLAG-KLF4 andpGL3-basic was used as a negativecontrol. The activity of the promoterreporters was measured 36 hours aftertransfection, and the activities in thenegative control and treated groupswere expressed as the fold activity intheir respective control groups. C,HNF-6 promoter pH6Pro-1796 wastransfected in triplicate with KLF4siRNA (siKLF4) and/or control siRNA(siCtrl) as indicated into PLC/PRF/5cells. Cotransfection of control siRNA(siCtrl) and pGL3-basic was used as anegative control. The promoter activitywas measured and expressed asdescribed in B. � , P < 0.05 and�� , P < 0.001 in a comparison of thecontrol and treated groups. D,chromatin was extracted from SNU387cells transfected with pFLAG-KLF4(KLF4 OE), pFLAG-KLF4DZFD(KLF4DZFD OE), or the control vectorpcDNA3.1 (Ctrl; left) and fromPLC/PRF/5 cells treated with KLF4siRNA (siKLF4) or control siRNA (siCtrl;right). ChIP assays were performedusing a specific anti-KLF4 antibody orIgG as a negative control andoligonucleotides flanking the HNF-6promoter regions containing putativeKLF4-binding site #1. Chromatinfragments without IgG or the antibodywere used as input controls.






DiscussionIn the present study, we defined the crucial and mechanistic

roles of KLF4 expression in HCC differentiation and its prog-nostic value for HCC in patients who undergo OLT. First,decreased expression of KLF4 along with that of differentia-tion-associated markers correlated closely with poor differen-tiation of both human HCC cells and tumors. Second, restoredKLF4 expression induced differentiation of HCC cells. Third,KLF4 expression causally correlated with HNF-6 expression inhuman HCC specimens, HCC xenograft tumors, and KLF4-deleted murine livers. KLF4 promoted HCC differentiation byinducing HNF-6 expression. Fourth, KLF4 directly activatedHNF-6 expression by binding to its promoter. Fifth, loss ofKLF4 expression in primary HCC cells can be used to identify agreatly increased risk of reduced OS and RFS duration after OLTand integration of KLF4 expression into the conventional Milancriteria can identify patients with hepatocellular tumors exceed-ing the Milan criteria who may have favorable posttransplanta-tion outcomes. Therefore, dysregulation of KLF4/HNF-6 sig-naling critically contributes to HCC dedifferentiation and pro-gression and KLF4 is a novel, useful biomarker for identifica-tion of liver transplantation candidates among patients withHCC.

HCC progression is accompanied by a stepwise process ofdedifferentiation (40, 41), whereas the driving force for andmechanistic basis of this process remain unclear. Our recentstudy has indicated a possible role for KLF4 expression in regu-lating HCC differentiation (19). This notion is consistent withprevious studies linking KLF4 to differentiation of normal epi-thelium (8–10) and neuroblastoma cells (42). Our current studyprovided the first causal evidence that expression of KLF4 drivesdifferentiation of HCC cells. However, authors previously pro-posed that KLF4 and other stemness genes—Oct4, Sox2, and c-Myc—are putative targets for human HCC therapy (43). Thishypothesis is challenged by our previous and current findings inHCC cases, which demonstrated consistent roles of KLF4 as apotent tumor suppressor and differentiation inducer for HCC(16). This discrepancy clearly warrants further investigation intothe exact role of KLF4 expression in stem cells and cancer stemcells, including those for HCC.

Functionally, KLF4 can act as a protein-binding partner ortranscription factor to activate or deactivate target genes and/orpathways (8, 44). Given the important roles of LETFs ininduction of HCC differentiation (22, 23, 45), we determinedthe regulatory effect of KLF4 on LETFs in HCC cells andidentified HNF-6 as the novel, specific downstream target ofKLF4. Mechanistically, KLF4 transcriptionally activates HNF-6,which plays a pivotal role in mediation of KLF4-induced HCCdifferentiation. Interestingly, the role of HNF-6 in HCC cellcycle remains unclear (46, 47), whereas we observed no impactof HNF-6 on cell-cycle progression or proliferation in HCCcells. The discrepancy in these findings may be attributed to thedistinct biologic backgrounds of the cell lines studied amongothers. However, our data did convincingly demonstrate aninhibitory role for HNF-6 expression in migration and invasionof HCC cells, suggesting that HNF-6 is a tumor suppressor. Ourclinical finding that loss of HNF-6 expression correlated withpoor histologic grade of HCC and unfavorable outcome afterOLT further substantiated. Therefore, our study not onlyexpands our knowledge of HNF-6 in the context of cancer but

also provides an experimental rationale for designing novel,effective differentiation therapy for cancer, including HCC.

Liver transplantation offers an important opportunity tostudy the potential impact of molecular signaling on theclinical course of HCC. Accumulating evidence suggests thatpatients with hepatocellular tumors beyond the Milan criteriamay not necessarily suffer posttransplantation tumor recur-rence (27, 48). Therefore, expansion of the criteria for trans-plantation is warranted so that many more patients can benefitfrom this procedure (27, 48–50). In the present study, wedemonstrated that the KLF4 expression status can be used tostratify patients with tumors outside the Milan criteria intomore homogenous groups with distinct RFS and OS durationsafter OLT. Indeed, patients with tumors beyond the Milancriteria and having moderate-to-high KLF4 expression hadexcellent 5-year RFS and OS. KLF4 thus represents a promisingnew tool to predict outcomes of HCC after OLT in patients withtumors beyond the Milan criteria, which may be profoundlybeneficial in guiding clinical decisions regarding surveillanceprotocols and therapeutic strategies after OLT. However, ourstudy was retrospective and confined to patients with tumorsmeeting the Milan criteria on the basis of preoperative imaging.In addition, we developed the biomarker and analyzed thepathologic data on the patients' explanted livers. Therefore, inthe pretransplantation setting, evaluation of the significance ofthe KLF4 expression status of tumor biopsy specimens obtainedfrom patients with tumors beyond the Milan criteria at the timeof qualification for OLT is critically important. The presentstudy suggests that accurate expansion of the Milan criteria ispossible via integration of KLF4 expression.

In summary, loss of KLF4 expression was an independentnegative prognostic factor for patients with HCC who underwentOLT and that integration of this novel biomarker of KLF4 expres-sion into the Milan criteria improved the accuracy of prognosti-cation, suggesting that routine evaluation of KLF4 expression inexplanted livers is a novel pathologic biomarker for HCC prog-nosis after OLT and helps select patients with HCC with tumorsoutside the Milan criteria who could benefit from OLT. Thedemonstrated role of the KLF4/HNF-6 signaling in HCC dedif-ferentiation and progression could also be explored for noveltherapeutic modalities to reverse HCC dedifferentiation and thuscontrol its progression.

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

Authors' ContributionsConception and design: H. Sun, L. Mishra, K. XieDevelopment of methodology: H. Sun, D. Xie, Z. Jia, K. XieAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Sun, D. Xie, Z. Jia, Z. Ma, D. Wei, S. Zheng,K. Xie, Z. PengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):H. Sun, D. Xie, Z. Jia, D. Wei, Y. Gao, S. Zheng, K. XieWriting, review, and/or revision of the manuscript: H. Sun, K. XieAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Tang, L. Mishra, S. Zheng, K. Xie, Z. PengStudy supervision: L. Mishra, K. Xie, Z. Peng

AcknowledgmentsThe authors thank Don Norwood for editorial comments.


Sun et al.




Grant SupportThe work was supported, in part, by grants R01-CA129956 and

R01-CA148954 from the NIH (K. Xie), grant 81101623 from the NationalNatural Science Foundation of China, grant #20114Y126 from theShanghai Municipal Bureau of Health (H. Sun), and grant 81460461(to S. Zheng).

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

Received March 7, 2015; revised June 5, 2015; accepted August 22, 2015;published OnlineFirst September 2, 2015.

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Sun et al.




2016;22:502-512. Published OnlineFirst September 2, 2015.Clin Cancer Res Hongcheng Sun, Huamei Tang, Dacheng Xie, et al. Nuclear Factor-6

HepatocyteDedifferentiation and Progression through Activation of Krüppel-like Factor 4 Blocks Hepatocellular Carcinoma

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