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HEPATOBILIARY MALIGNANCIES

Overexpression of Forkhead Box C1 Promotes Tumor

Metastasis and Indicates Poor Prognosis in

Hepatocellular Carcinoma

Limin Xia,1* Wenjie Huang,1* Dean Tian,2 Hongwu Zhu,1 Xingshun Qi,1 Zheng Chen,1 Yongguo Zhang,1

Hao Hu,1 Daiming Fan,1 Yongzhan Nie,1 and Kaichun Wu1

Recurrence and metastasis remain the most common causes of lethal outcomes in hepatocel-lular carcinoma (HCC) after curative resection. Thus, it is critical to discover the mecha-nisms underlying HCC metastasis. Forkhead box C1 (FoxC1), a member of the Fox familyof transcription factors, induces epithelial-mesenchymal transition (EMT) and promotes epi-thelial cell migration. However, the role of FoxC1 in the progression of HCC remainsunknown. Here, we report that FoxC1 plays a critical role in HCC metastasis. FoxC1 expres-sion was markedly higher in HCC tissues than in adjacent noncancerous tissues. HCC

patients with positive FoxC1 expression had shorter overall survival times and higher recur-rence rates than those with negative FoxC1 expression. FoxC1 expression was an independ-ent, significant risk factor for recurrence and survival after curative resection. FoxC1overexpression induced changes characteristic of EMT and an increase in HCC cell invasionand lung metastasis. However, FoxC1 knockdown inhibited these processes. FoxC1 transacti-

vated Snai1 expression by directly binding to the Snai1 promoter, thereby leading to the in-hibition of E-cadherin transcription. Knockdown of Snai1 expression significantlyattenuated FoxC1-enhanced invasion and lung metastasis. FoxC1 expression was positivelycorrelated with Snai1 expression, but inversely correlated with E-cadherin expression inhuman HCC tissues. Additionally, a complementary DNA microarray, serial deletion, site-directed mutagenesis, and a chromatin immunoprecipitation assay confirmed that neuralprecursor cell expressed, developmentally down-regulated 9 (NEDD9), which promotes the

metastasis of HCC cells, is a direct transcriptional target of FoxC1 and is involved in FoxC1-mediated HCC invasion and metastasis. Conclusions: FoxC1 may promote HCC metastasisthrough the induction of EMT and the up-regulation of NEDD9 expression. Thus, FoxC1may be a candidate prognostic biomarker and a target for new therapies. (HEPATOLOGY2013;57:610-624)

Hepatocellular carcinoma (HCC) is the thirdleading cause of cancer-related mortality,

with nearly 600,000 deaths occurring world-wide each year.1 Although resection is considered apotentially curative treatment for HCC patients, the5-year postoperative survival rate is 30%-40%.2 The

poor prognosis of patients with HCC is largely theresult of the high frequencies of tumor recurrenceand distant metastasis after curative resection.3 How-ever, the molecular mechanism underlying HCC me-tastasis remains unclear. Therefore, the identificationof novel molecular markers will provide new

Abbreviations: BLI, bioluminescent imaging; ChIP, chromatin immunoprecipitation; CREB, cAMP response element-binding protein; EGF, epidermal

growth factor; EMT, epithelial-mesenchymal transition; ERK, extracellular signal-related kinase; FoxC1, forkhead box C1; HBV, hepatitis B virus; HBx,

hepatitis B virus x; HCC, hepatocellular carcinoma; IF, immunofluorescence; IHC, immunohistochemical; miRNAs, microRNAs; mRNA, messenger RNA;NEDD9, neural precursor cell expressed, developmentally down-regulated 9; OS, overall survival; PCR, polymerase chain reaction; siRNA, short interfering

RNA; TNM, tumor-node-metastasis; VEGF, vascular endothelial growth factor.

From the1State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xian, China; and 2Department of

Gastroenterology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.Received March 23, 2012; accepted August 9, 2012.This study was supported by combined grants from the National Natural Science Foundation of China (grant nos.: 81172290, 91129723, 81090270,

81090273, 81000864, and 81120108005), the National Key and Basic Research Development Program of China (grant nos.: 2010CB529302 and

2010CB529306), the National Municipal Science and Technology Project (2009ZX09103-667 and 2009ZX09301-009-RC06), and the Chinese Postdoctoral

Science Foundation (grant nos.: 20100471776 and 201104757).*These authors contributed equally to this work.

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opportunities for the prevention of HCC recurrenceand metastasis.

Forkhead box (Fox) proteins comprise a family ofevolutionarily conserved transcriptional regulators thatplay important roles in both healthy biological proc-esses and in cancer development.4 Fox proteins aremaster regulators of epithelial-mesenchymal transition(EMT). FoxM1 induces EMT by activating the pro-tein kinase B/Snai1 pathway, which leads to metastasisin pancreatic cancer and HCC.5,6 FoxF1 and FoxQ1promote EMT and breast cancer metastasis throughthe inhibition of E-cadherin transcription.7,8 In con-trast, FoxA1 and FoxA2 antagonize EMT through thetransactivation of E-cadherin expression and mainte-nance of the epithelial phenotype. FoxA1 and FoxA2are known to inhibit the metastasis of pancreatic duc-tal adenocarcinoma and lung cancer.9,10 These studies

indicate that Fox protein-mediated EMT is involved intumor metastasis. The critical role of EMT in theinduction of invasiveness and metastasis in HCCsuggests that Fox proteins may be involved in HCCmetastasis. Importantly, FoxM1 overexpression pro-motes HCC metastasis through the up-regulation ofstathmin, lysyl oxidase, and lysyl oxidase like-2 expres-sion and indicates poor prognosis.6,11 In a previousstudy, we found that FoxM1 promoted HCC metasta-sis by transactivating matrix metalloproteinase-7,RhoC, and ROCK1 expression, and that the FoxM1expression level was an independent risk factor for

recurrence and survival in HCC patients after curativeresection.12 However, the involvement of other Foxproteins in HCC metastasis is unknown.

FoxC1, which is a member of the Fox transcription

factor family, is crucial for the formation and matura-

tion of vasculature through interaction with Notch

and vascular endothelial growth factor (VEGF) path-

ways.13,14 FoxC1-knockout mice display cardiovascular

defects and die either perinatally or soon after birth.15

FoxC1 levels are dramatically decreased in adult

tissues, but FoxC1 expression during embryogenesis is

activated by the canonical Wnt and epidermal growthfactor/extracellular signal-related kinase (EGF/ERK)-

signaling pathways.16,17 We wondered whether the

deregulation of FoxC1 might be involved in tumor

progression. Interestingly, FoxC1 is reported to induce

EMT. FoxC1 induces EMT through the inhibition of

E-cadherin expression in mammary epithelial cells and

promotes their migration and invasion. Additionally,

FoxC1 overexpression is strongly correlated with poor

survival in breast cancer patients.18,19

Several recentstudies also reported that FoxC1 increases the migra-

tion and invasion of breast cancer cells, and that

FoxC1 overexpression predicts poor overall survival

(OS) in patients with breast cancer.20,21 These studies

indicate that FoxC1 might promote tumor metastasis

and malignant progression by inducing EMT.To date, no studies have reported on the clinico-

pathologic significance of FoxC1 in HCC. In thisstudy, we present the first evidence that FoxC1 pro-motes HCC invasion and metastasis by not onlyinducing EMT, but also by up-regulating NEDD9expression. FoxC1 overexpression predicts poor prog-nosis in HCC patients after curative resection. Themolecular mechanism of these effects involves thetransactivation of Snai1 and NEDD9 expression byFoxC1 through direct binding to their promoters.

Materials and MethodsPlasmid Construction. Plasmids were constructed

according to the standard procedures in our previousstudy.12 All of the primers are shown in Supporting

Table 2. The Snai1 promoter construct(1511/140)snail was generated from human

genomic DNA corresponding to the sequence from1511 to 140 (relative to the transcriptional start site)of the 5-flanking region of the human Snai1 gene. Thisconstruct was generated with the forward and reverse pri-mers incorporatingKpnI andHindIII sites at the 5- and3-ends, respectively. The polymerase chain reaction(PCR) product was cloned into the KpnI and HindIIIsites of the pGL3-Basic vector (Promega, Madison, WI).The 5-flanking deletion constructs of the FoxC1 pro-moter ([922/140]Snail, [694/140]Snail, and[354/140]Snail) were similarly generated with the(1511/140)Snail construct as a template. Other pro-moter constructs ([2056/121]NEDD9, [1762/121]NEDD9, [1324/121]NEDD9, [1007/

Address reprint requests to: Kaichun Wu, M.D., Ph.D., or Yongzhan Nie, M.D., Ph.D., State Key Laboratory of Cancer Biology and Xijing Hospital ofDigestive Diseases, Fourth Military Medical University, Xian 710032, Shaanxi Province, China. E-mail: [email protected] or [email protected]; fax:

86 29 8253 9041.

CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.

DOI 10.1002/hep.26029

Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of this article.

HEPATOLOGY, Vol. 57, No. 2, 2013 XIA ET AL. 611

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121]NEDD9, [478/121]NEDD9, and pGL3-E-cadherin) were cloned in the same manner.FoxC1-binding sites in the Snai1 and NEDD9 pro-moters were mutated with a QuikChange II site-directedmutagenesis kit (Stratagene, La Jolla, CA). MutatedSnai1 and NEDD9 promoter constructs were cloned inthe same manner. The constructs were confirmed byDNA sequencing.

Luciferase Reporter Assay. The luciferase activitywas detected with the Dual Luciferase Assay (Prom-ega), according to the manufacturers instructions.Transfected cells were lysed in culture dishes with lysisbuffer, and lysates were centrifuged at maximum speedfor 1 minute in an Eppendorf microcentrifuge. Therelative luciferase activity was determined by a Modu-lus TD20/20 Luminometer (Turner Biosystems,Sunnyvale, CA), and the transfection efficiency was

normalized to Renilla activity.A detailed description of the materials and methodsused in this study can be found in the online Support-ing Materials.

ResultsFoxC1 Is Significantly Up-regulated in HCC

Tissues, and High FoxC1 Expression Predicts PoorPrognosis in HCC Patients. To explore the role ofFoxC1 in determining clinical outcomes for HCCpatients, we assessed its expression in a tissue microar-

ray of 406 paired HCC samples. Immunohistochemi-cal (IHC) assays showed that FoxC1 was primarilylocalized in the nucleus. FoxC1 expression was foundin 257 of 406 (63.3%) primary HCC tissues, com-pared with only 98 of 406 (24.1%) adjacent nontumortissues (P < 0.01) (Fig. 1A1,A2). Up-regulation ofFoxC1 was confirmed in an additional 40 pairedHCC samples using real-time PCR. Levels of FoxC1messenger RNA (mRNA) were significantly increasedin HCC tissues, compared to adjacent nontumor tis-sues (Fig. 1A3). To investigate the role of FoxC1 inHCC metastasis, FoxC1 expression was compared

in primary and metastatic HCCs using an IHC assayin an HCC tissue microarray containing 20 pairs ofHCC specimens. Overall, 11 pairs of HCCs (55%)showed higher levels of FoxC1 expression in metastaticlesions, compared with the corresponding primarytumor samples (Fig. 1A4).

Overexpression of FoxC1 was significantly correlatedwith tumor number, tumor size, microvascular inva-sion, poor tumor differentiation, and tumor-node me-tastasis (TNM) stage (Table 1). HCC patients withpositive FoxC1 expression had shorter OS and higher

recurrence rates than those without FoxC1 expression(Fig. 1B). Coxs multivariate proportional hazardsmodel indicated that FoxC1 expression was anindependent predictor of recurrence (P 0.002) andsurvival (P 0.001) in HCC after curative resection(Table 2).

FoxC1 Promotes HCC Cell Invasion In Vitro andLung Metastasis In Vivo. FoxC1 mRNA and proteinlevels increased progressively from healthy liver cells toHCC cells with low metastatic potential and, finally,to HCC cells with high metastatic potential (Fig.1C1). To evaluate the role of FoxC1 in the migrationand invasion of HCC cells, we established two stablecell lines (denoted SMMC7721-FoxC1 andHCCLM3-shFoxC1) after infection with theLV-FoxC1 or LV-shFoxC1 lentivirus, respectively. Boththe up-regulation and knockdown of FoxC1 expression

were confirmed by western blotting analysis. Threetarget sites were selected for knockdown of FoxC1expression. Target site three was the most effective siteand was chosen for further study (Fig. 1C2). Up-regu-lation of FoxC1 significantly enhanced the migrationand invasion capacities of SMMC7721 cells (low ini-tial metastatic potential). Conversely, silencingendogenous FoxC1 expression markedly reduced cellmigration and invasion in HCCLM3 cells (high initialmetastatic potential) (Fig. 1D).

To further explore the role of FoxC1 in tumor metas-tasis in vivo, cells were transplanted into livers of nude

mice. Representative bioluminescent imaging (BLI) ofthe different groups is shown in Fig. 1E1. Histologicalanalysis (Fig. 1E5) further confirmed that the incidenceof lung metastasis in the SMMC7721-FoxC1 group

was significantly increased, compared to that in thecontrol group (60% versus 10%). In the HCCLM3-shcontrol group, all of the mice developed lung metas-tases; however, only 5 mice in the HCCLM3-shFoxC1group developed lung metastases (100% versus 50%;Fig. 1E1,E2). The number of lung metastatic nodulesin the SMMC7721-FoxC1 group was increased, com-pared to that in the SMMC7721-control group;

however, the number of lung metastatic nodules in theHCCLM3-shFoxC1 group was significantly reduced,compared to that in the HCCLM3-shcontrol group(Fig. 1E3). Furthermore, the SMMC7721-FoxC1group had a shorter OS time than the SMMC7721-control group, whereas the HCCLM3-shFoxC1 grouphad a longer OS time than the HCCLM3-shcontrolgroup (Fig. 1E4). These data suggested that FoxC1promoted HCC invasion and metastasis.

FoxC1 Induces EMT in HCC Cells. EMT plays acritical role in metastasis. Specifically, EMT induces

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tumor-associated epithelial cells to obtain mesenchymalfeatures, which results in reduced cell-cell contact andincreased motility.22 Up-regulation of FoxC1 inSMMC7721 cells resulted in the decreased expressionof epithelial markers (E-cadherin and O-catenin) andincreased expression of mesenchymal markers (vimen-tin and fibronectin), as evidenced by immunofluores-cence (IF), western blotting analysis, and real-time

PCR. After FoxC1 knockdown in HCCLM3 cells,expression of epithelial markers was significantlyincreased and expression of mesenchymal markers wasmarkedly decreased (Fig. 2A-C). These findingssuggested that FoxC1 induced EMT in HCC cells.

FoxC1 Inhibits E-Cadherin TranscriptionThrough the Transactivation of Snai1 Expression.Functional loss of E-cadherin is considered a hallmark

Fig. 1. FoxC1 is significantly up-regulated in HCC tissues and promotes HCC cell invasion and lung metastasis. (A1) IHC analysis of FoxC1

expression in healthy liver and 406 paired HCC tissues. (A2) Statistical analysis of FoxC1 expression in HCCs. (A3) Real-time PCR analysis of

FoxC1 expression in healthy liver (n 10) and 40 pairs of HCC and adjacent nontumorous tissues. (A4) Representative FoxC1 expression in pri-mary and metastatic tissues detected by IHC methods. (B) Kaplan-Meiers analysis of the correlation between FoxC1 expression and recurrence

or OS of HCC patients. (C1) Real-time PCR and western blotting analysis of FoxC1 expression in different HCC cell lines. (C2) After cells were

infected with LV-FoxC1 or LV-shFoxC1-1, -2, or -3, level of FoxC1 protein expression was detected by western blotting analysis. (D) Up-regulationof FoxC1 expression using LV-FoxC1 enhanced SMMC7721 (low metastatic potential) cell migration and invasion in vitro, whereas inhibition of

FoxC1 expression using LV-shFoxC1 decreased HCCLM3 (high metastatic potential) cell migration and invasion. (E) In vivo metastasis assay. The

above four cell lines were transplanted into livers of nude mice. (E1) Ten weeks after orthotopic implantation, BLI showed the presence of lung

metastases in the mice implanted with SMMC7721-FoxC1 cells and the absence of metastasis in the mice implanted with SMMC7721-controlcells. Mice implanted with HCCLM3-shcontrol cells showed lung metastases, whereas no metastasis was detected in mice implanted with

HCCLM3-shFoxC1 cells. Black arrows indicate metastatic lung nodules. (E2) Incidence of lung metastasis in each group of nude mice. (E3) Num-

ber of metastatic lung foci observed in each group. (E4) OS of nude mice in each group. (E5) Images showing representative hematoxylin andeosin staining of lung tissue samples from the different experimental groups. * P< 0.05.


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Table 1. Correlation Between FoxC1, Snai1, E-cadherin, and NEDD9 Expression and Clinicopathological Characteristics of

406 HCCs

Clinicopathological

Variables

Tumor FoxC1 Expression

PValue

Tumor Snai1 Expression

P Value

Tumor E-cadherin

Expression

P Value

Tumor NEDD9

Expression

PValue

Negative

(n 149)

Positive

(n 257)

Negative

(n 210)

Positive

(n 196)

Negative

(n 156)

Positive

(n 250)

Negative

(n 189)

Positive

(n 217)

Age 50.5 (9.1) 51.4 (9.3) 0.3 25 51.5 (9. 0) 50 .6 (9.4) 0.306 50.4 (9.5) 51.5 (9.1) 0.212 50.6 (8.9) 51.5 (9.5) 0.3 67

Sex Female 25 50 0.503 35 40 0.332 31 44 0.566 40 35 0.192

Male 124 207 175 156 125 206 149 182

Serum AFP 20 ng/mL 40 42 0.011 40 42 0.550 29 53 0.524 44 38 0.149

>20 ng/mL 109 215 170 154 127 197 145 179

Virus infection HBV 103 203 0.103 156 150 0.932 118 188 0.998 141 165 0.669

HCV 16 18 18 16 13 21 17 17

HBVHCV 3 7 6 4 4 6 3 7

None 27 29 30 26 21 35 28 28

Cirrrhosis Absent 41 48 0.038 53 36 0.094 34 55 0.961 43 46 0.706

Present 108 209 157 160 122 195 146 171

Child-Pugh score Class A 123 214 0.853 175 162 0.855 133 204 0.340 163 174 0.105

Class B 26 43 35 34 23 46 26 43

Tumor number Single 113 162 0.008* 152 123 0.038* 96 179 0.035* 138 137 0.034*

Multiple 36 95 58 73 60 71 51 80

Maximal tumor size 5 cm 98 136 0.012* 129 105 0.109 88 146 0.693 119 115 0.043*

>5 cm 51 121 81 91 68 104 70 102

Tumor encapsulation Absent 34 83 0.042* 45 72 0.001* 48 69 0.493 45 72 0.038*

Present 115 174 165 124 108 181 144 145

Microvascular invasion Absent 98 131 0.004* 142 87 20 ng/mL)

0.765 0.5591.048 0.095 0.834 0.6161.130 0.241

HBV infection (no versus yes) 0.667 0.4910.906 0.010 0.597 0.4400.810 0.001 0 .826 0.5951.147 0.254

Cirrhosis

(absent versus present)

0.761 0.5591.036 0.083 0.712 0.5249.969 0.031

Child-Pugh score (A versus B) 0.815 0.5961.115 0.201 0.821 0.6001.122 0.215

Tumor number

(single versus multiple)

0.401 0.3120.515

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of EMT.23 A major mechanism of E-cadherin down-regulation is its direct transcriptional repression byrepressors, including Snai1, Twist, Slug, Zeb1, andSIP1.24 We determined whether FoxC1 inhibited

E-cadherin expression by regulating the expression ofthese repressors. Real-time PCR analysis showed thatFoxC1 markedly increased Snai1 expression, but hadno significant effect on mRNA levels of Twist, Slug,

Fig. 2. FoxC1 induces EMT in HCC cells. (A) IF staining, (B) western blotting, and (C) real-time PCR show down-regulated expression of epi-thelial markers (E-cadherin and O-catenin) and up-regulated expression of mesenchymal markers (vimentin and fibronectin) in SMMC7721-

FoxC1 cells. In contrast, knockdown of FoxC1 resulted in increased expression of epithelial markers and decreased expression of mesenchymal

markers in HCCLM3 cells. *P< 0.05.


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Zeb1, or SIP1 (Fig. 3A1). Furthermore, FoxC1 up-regulated Snai1 expression and decreased E-cadherinexpression in SMMC7721 cells, whereas the inhibitionof Snai1 expression using the lentivirus, LV-shSnai1,

significantly attenuated the loss of E-cadherin expres-sion induced by FoxC1. In contrast, knockdown ofFoxC1 decreased Snai1 expression and increased E-cadherin expression in HCCLM3 cells, whereas up-


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regulation of Snai1 using the lentivirus, LV-Snai1,markedly inhibited the increase in E-cadherin expres-sion in HCCLM3-shFoxC1 cells (Fig. 3A2). Similarresults were also observed in Huh-7 cells (data notshown). Thus, Snai1 is critical for FoxC1-inducedreduction of E-cadherin expression.

To determine whether FoxC1 regulates Snai1 andE-cadherin transcription, Snai1 and E-cadherin pro-moter luciferase constructs ([1511/140]Snai1 andpGL3-E-cadherin) were cotransfected with pCMV-FoxC1. The luciferase reporter assay showed thatFoxC1 transactivated Snai1 promoter activity, butinhibited E-cadherin transcription. Furthermore, theshort interfering RNA (siRNA)-mediated knockdownof Snai1 in FoxC1-overexpressing SMMC7721 cellspartially relieved the suppression of E-cadherin pro-moter-driven luciferase activity (Fig. 3B1).

To define the roles of the cis-regulatory elements of theSnai1 promoter in response to FoxC1 regulation, reporterconstructs containing serial 5 deletions of the Snai1 pro-moter ([1511/140]Snai1, [922/140]Snai1,[694/140]Snai1, and [354/140]Snai1) werecotransfected with pCMV-FoxC1. The luciferase reporterassay showed that a deletion from nt 1511 to nt 694had no effect on FoxC1-induced Snai1 promoter activity.However, further deletion from nt 694 to nt 354significantly decreased FoxC1-induced Snai1 promoteractivity (Fig. 3B2), indicating that the sequence betweennt 694 and 354 was critical for the activation of the

Snai1 promoter by FoxC1. The third putative FoxC1-binding site was in this region. A luciferase reporter assayshowed that mutation of the third FoxC1-binding sitesignificantly reduced FoxC1-induced transactivation ofthe Snai1 promoter (Fig. 3B2). A chromatin immunopre-

cipitation (ChIP) assay confirmed the direct binding ofFoxC1 to the third FoxC1-binding site in the Snai1promoter in HCC cells (Fig. 3C). To determine whetherFoxC1 binds to the Snai1 promoter under physiologicalconditions, three healthy liver tissues (healthy control)and three HCC tissues were collected. A ChIP assayshowed that the FoxC1-binding activity to the Snai1promoter was much higher in HCC tissues than inhealthy controls (Supporting Figure 7). These resultssuggested that FoxC1 transactivated Snai1 expression,thereby leading to the inhibition of E-cadherin transcrip-tion in HCC cells.

Snai1 Is Critical for FoxC1-Induced HCC Inva-sion and Metastasis. To study the possible role ofSnai1 in FoxC1-mediated invasion and metastasis,SMMC7721-FoxC1 cells were infected with LV-shSnai1lentivirus to knock down Snai1 expression. Snai1

knockdown significantly reduced FoxC1-enhanced cellmigration and invasion (Fig. 3D). To determine theeffect of Snai1 on FoxC1-mediated metastasis, two cellslines were transplanted into livers of nude mice. Ten

weeks after orthotopic implantation, BLI showed thepresence of lung metastasis in mice implanted withSMMC7721-FoxC1 plus LV-shcontrol cells, but nolung metastasis occurred in mice implanted withSMMC7721-FoxC1 plus LV-shSnai1 cells (Fig. 3E1).Histological analysis (Fig. 3E5) further confirmed that5 mice in the control group (SMMC7721-FoxC1 plusLV-shcontrol) developed lung metastasis. However,

there was only one case of lung metastasis in the Snai1-knockdown group (SMMC7721-FoxC1 plus LV-shSnai1) (Fig. 3E2). The number of metastatic lungnodules in the Snai1-knockdown group was signifi-cantly reduced, compared to the control group (Fig.

Fig. 3. Snai1 is critical for FoxC1-enhanced HCC invasion and metastasis. (A1) Effect of FoxC1 on expression of Snai1, Twist, Slug, Zeb1,and SIP1. (A2) Snai1 is critical for FoxC1-induced reduction of E-cadherin expression. Real-time PCR and western blotting were used to detect

expression of FoxC1, Snai1, and E-cadherin. Knockdown of Snai1 expression using LV-shSnai1 significantly attenuated the loss of E-cadherin

expression induced by FoxC1. In contrast, up-regulation of Snai1 using LV-Snai1 markedly inhibited the increase in E-cadherin expression in

HCCLM3-shFoxC1 cells. (B1) FoxC1 promoted Snai1 transcription, but inhibited E-cadherin transcription. Cells were pretransfected with Snai1siRNA or control siRNA. Then, Snai1 and E-cadherin promoter luciferase constructs ([1511/140]Snai1 and pGL3-E-cadherin) were cotrans-

fected with pCMV-FoxC1. A luciferase reporter assay was used to detect promoter activities. After 48 hours, cells were cotransfected with pGL3-E-cadherin and the plasmid pRL.TK as a control for transfection efficiency. (B2) Deletion analysis and selective mutagenesis at position 620/

615 base pairs from the transcription start site identified a FoxC1-responsive region in the human Snai1 promoter. Serially truncated and

mutated Snai1 promoter constructs were cotransfected with pCMV-FoxC1, and the relative luciferase activity was determined. Schematic repre-

sentations of the constructs are shown (left), and bar graphs show the relative level of luciferase activity in each sample (right). (C) ChIP assaydemonstrating the binding of FoxC1 to the Snai1 promoter. Real-time PCR was performed to detect the amount of immunoprecipitated products.

(D) Snail knockdown significantly decreased FoxC1-enhanced cell migration and invasion. After SMMC7721-FoxC1 cells were infected with the

lentivirus, LV-shSnai1, migration and invasion abilities of the cells were detected using transwell assays. (E) In vivo metastatic assay. Two cell

lines (SMMC7721-FoxC1 and SMMC7721-FoxC1 plus LV-shSnai1) were transplanted into livers of nude mice. (E1) Ten weeks after orthotopic im-plantation, BLI showed the presence of lung metastases in mice implanted with SMMC7721-FoxC1 cells and the absence of lung metastases in

the mice implanted with SMMC7721-FoxC1 LV-shSnai1 cells. Black arrow indicates the metastatic lung nodule. (E2) Incidence of lung metas-

tases observed in the different experimental groups of nude mice. (E3) Number of lung metastatic foci observed in each group. (E4) OS time ofeach group. (E5) Images showing representative hematoxylin and eosin staining of lung tissue samples from the different experimental groups.

*P< 0.05.


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3E3). Furthermore, the Snai1-knockdown group had alonger OS time than the control group (Fig. 3E4).These results indicated that Snai1 knockdown sup-pressed FoxC1-enhanced metastasis.

Both overexpression of Snai1 and down-regulation ofE-cadherin were associated with poor prognosis(Fig. 4C,D) and aggressive tumor behavior (Table 1).IHC revealed that FoxC1 expression was positively cor-

related with Snai1 expression, but inversely correlatedwith E-cadherin expression (Fig. 4A,B). Patients weresubsequently divided into four groups, according to thecombined expression level of FoxC1 and Snai1 orE-cadherin. Kaplan-Meiers analysis showed statisticallydistinct recurrence and survival patterns among the foursubgroups, among which patients with positivecoexpression of FoxC1 and Snai1 endured the highest

Fig. 4. FoxC1 expression is positively correlated with Snai1 expression, but inversely correlated with E-cadherin expression in human HCC tis-

sues. (A) IHC analysis of Snai1 and E-cadherin expression in HCC tissues. (B) The association between the expression of FoxC1 and Snai1 or E-

cadherin. (C and D) Kaplan-Meiers analysis of Snai1 or E-cadherin expression in HCC patients after curative resection. (D and E) Kaplan-Meiersanalysis of coexpression in HCC patients after curative resection. (D) Correlation of FoxC1/Snai1 coexpression with recurrence and OS. (E) Corre-

lation of FoxC1/E-cadherin coexpression with recurrence and OS. * P< 0.05.


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recurrence rates and lowest OS (Fig. 4E). Similarly,patients with the FoxC1()/E-cadherin() expressionpattern had the highest recurrence rates and lowest OS(Fig. 4F). To further investigate the roles of FoxC1,Snai1, and E-cadherin in HCC metastasis, IHC wasused to detect their expression in 20 paired primaryand metastatic HCC tissues. A representative image ofIHC staining is shown in Supporting Fig. 2A. Higherlevels of FoxC1 and Snai1 expression were observed inmetastatic HCC samples than in primary HCC sam-ples, whereas a lower level of E-cadherin expression wasobserved in metastatic tissues than in primary HCC tis-sues (Supporting Fig. 2). Taken together, both experi-mental and clinical evidence suggested that the FoxC1-mediated Snai1/E-cadherin pathway promoted HCCmetastasis and poor prognosis.

NEDD9 Is a Direct Transcriptional Target of

FoxC1. To further elucidate how FoxC1 promotesinvasion and metastasis in HCC cells, we conducted adetailed comparison of gene expression in HCCLM3-shFoxC1 cells and HCCLM3-shcontrol cells, empha-sizing genes involved in metastasis. FoxC1 down-regu-lation substantially reduced the expression of a numberof metastasis-related genes, including NEDD9, BOC,CNTN1, AOC3, VCAN, CCKAR, MAP4K1, CD24,CNTN2, CD34, and SMO (Supporting Table 1).Changes in expression in these downstream targets

were further validated by real-time PCR in two differ-ent cell lines (Supporting Fig. 1).

Of particular interest was NEDD9, which was down-regulated 8.7-fold in response to FoxC1 knockdown(Supporting Table 1). NEDD9 is a scaffolding proteinthat coordinates with the FAK- and Src-signaling cas-cades, which are relevant to integrin-dependent migra-tion and invasion.25 NEDD9 promotes tumor metastasisand is associated with poor prognosis in melanoma,breast cancer, and colon cancer.25,26 Considering the crit-ical role of NEDD9 in metastasis, we wanted to deter-mine whether NEDD9 was involved in FoxC1-mediatedHCC metastasis. Real-time PCR and western blottinganalyses showed that FoxC1 up-regulated NEDD9

expression in SMMC7721 cells, whereas the knockdownof FoxC1 expression decreased NEDD9 expression inHCCLM3 cells (Fig. 5A). To determine whether FoxC1regulates NEDD9 transcription, a NEDD9 promoter lu-ciferase construct, (2056/121) NEDD9, was cotrans-fected with pCMV-FoxC1. A luciferase reporter assayshowed that FoxC1 transactivated NEDD9 promoter ac-tivity (Fig. 5B1). Sequence analysis revealed four putativeFoxC1-binding sites in the NEDD9 promoter. Serial de-letion and site-directed mutagenesis showed that thethird and fourth FoxC1-binding sites were critical for

FoxC1-induced NEDD9 transactivation (Fig. 5B2). AChIP assay further confirmed that FoxC1 binds directlyto the NEDD9 promoter in HCC cells (Fig. 5B3).Furthermore, binding activity of FoxC1 to the NEDD9promoter was much higher in HCC tissues than inhealthy liver tissues (Supporting Fig. 9). These resultssuggested that NEDD9 was a direct transcriptionaltarget of FoxC1.

NEDD9 Overexpression Promotes HCC metastasisand Is Positively Correlated With Poor Prognosis inHuman HCC Patients. Western blotting analysisshowed that NEDD9 expression was much higher inhighly metastatic HCC cells than in weakly metastaticHCC cells (Fig. 5C). To determine whether NEDD9regulates the invasive capacity of HCC cells,SMMC7721 cells were infected with the lentivirus,LV-NEDD9. Up-regulation of NEDD9 expression was

confirmed by western blotting analysis, and the resultingstable cell line was named SMMC7721-NEDD9.NEDD9 overexpression significantly increased the inva-sion ability of SMMC7721 cells (Fig. 5D). BLI showedthe presence of lung metastases in mice implanted withSMMC7721-NEDD9 cells, but no lung metastasesoccurred in mice implanted with SMMC7721-controlcells (Fig. 5E1). Histological analysis (Fig. 5E5)confirmed that 7 mice in the SMMC7721-NEDD9group developed lung metastases. However, only 1mouse in the SMMC7721-control group developed lungmetastasis (Fig. 5E2). The number of metastatic lung

nodules in the SMMC7721-NEDD9 group was signifi-cantly increased, compared to that in the SMMC7721-control group (Fig. 5E3). Furthermore, theSMMC7721-NEDD9 group had a shorter OS timethan the control group (Fig. 5E4). These results sug-gested that NEDD9 overexpression promoted HCCinvasion and metastasis. Additionally, NEDD9 knock-down markedly decreased the invasion and metastasis ofHCCLM3 cells (data not shown).

IHC results showed that NEDD9 was significantlyup-regulated in HCC tissues, compared to adjacentnontumor tissues, and that NEDD9 was mainly local-

ized in the cytoplasm (Fig. 6D1). NEDD9 overexpres-sion was significantly correlated with poor tumordifferentiation and more-advanced TNM stage(Table 1). HCC patients with positive NEDD9 expres-sion had shorter OS and higher recurrence rates thanthose with negative expression of NEDD9 (Fig. 6E1).These results suggested that NEDD9 promoted HCCmetastasis and correlated with poor prognosis.

NEDD9 Knockdown Significantly AttenuatesFoxC1-Enhanced Invasion and Metastasis. The lenti-virus, LV-shNEDD9, was used to knock down NEDD9


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expression in SMMC7721-FoxC1 cells (Fig. 6A).NEDD9 knockdown decreased FoxC1-enhanced cellinvasion (Fig. 6B).In vivometastatic assays confirmed that5 mice developed lung metastases in the control group(SMMC7721-FoxC1 plus LV-shcontrol). However, there

were only two cases of lung metastasis in the NEDD9-knockdown group (SMMC7721-FoxC1 plus LV-shNEDD9) (Fig. 6C1,C2,C5). The number of metastaticlung nodules was significantly reduced in the NEDD9-knockdown group, compared to the control group


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(Fig. 6C3). Moreover, the NEDD9-knockdown grouphad a longer OS time than the control group (Fig. 6C4).

IHC assays showed that FoxC1 expression was posi-tively correlated with NEDD9 expression in humanHCC tissues (Fig. 6D1,D2). Kaplan-Meiers analysisshowed statistically distinct recurrence and survivalpatterns among the four subgroups, among whichpatients with positive coexpression of FoxC1 andNEDD9 had the highest recurrence and lowest OS(Fig. 6E2). Furthermore, NEDD9 expression washigher in metastatic tissues than in primary HCCtissues (Supporting Fig. 2). These results suggestedthat FoxC1 promoted HCC metastasis by up-regulat-ing NEDD9 expression.

FoxC1 Increases Ubiquitination and Degradationof O-catenin. O-catenin has been implicated in pro-moting HCC progression in several studies.27,28 In this

study, we found that FoxC1 overexpression decreasedexpression of O-catenin. To determine whether FoxC1regulated O-catenin transcription, a O-catenin promoterluciferase construct (pGL3-CTNNB1) was cotransfected

with pCMV-FoxC1. The luciferase reporter assayshowed that FoxC1 had no effect on O-catenin tran-scription (Supporting Fig. 6A). These data suggest thatFoxC1 did not regulate the O-catenin promoter inHCC cells. Recent studies reported that the expressionlevel of O-catenin could be regulated by multiple micro-RNAs (miRNAs).29,30 We speculate that FoxC1 maydecrease O-catenin expression through regulating

miRNA expression. Expression levels of O-catenin werealso measured in 406 HCC tissues. Increased O-cateninaccumulation was detected in 220 of 406 (54.2%) HCCtissues, compared to adjacent nontumor tissues. NuclearO-catenin staining was detected in 41 cases (41 of 220;18.6%), with the remaining cases showing staining inthe cytoplasm (Supporting Fig. 6B). These results wereconsistent with those of previous studies.27,31 However,these data were inconsistent with our findings in HCC

cell lines. These differences may be attributed to the exis-tence of other mechanisms that regulate O-cateninexpression (e.g., the Wnt pathway). In the absence of

Wnt signaling, O-catenin is bound to E-cadherin atadherens junctions. N-terminally phosphorylated O-cat-enin is targeted for ubiquitination and subsequentproteasomal degradation.32 Deregulation of E-cadherinby FoxC1 may decrease the level of O-catenin in themembrane and increase ubiquitination of O-catenin. Todetermine whether FoxC1 increased O-catenin degrada-tion, SMMC7721-control and SMMC7721-FoxC1 cells

were treated with MG-132. MG-132 treatment signifi-cantly reversed inhibition of O-catenin expression byFoxC1 (Supporting Fig. 6C). These data indicate thatFoxC1 increased ubiquitination and degradation ofO-catenin.

Hepatitis B Virus x Up-regulates FoxC1 Expres-

sion Through the ERK/cAMP Response Element-Binding Protein Signaling Pathway. Previous studiesreported that EGF/MAPK and canonical Wnt-signalingpathways up-regulated FoxC1 expression,16,17 whereasthe mechanism by which FoxC1 is reactivated in HCCremains unknown. Chronic hepatitis B virus (HBV)infection is a major risk factor for the development ofHCC in Asia.3 In our clinical samples, among the 306HBV-infected HCC tissues, 203 of 306 (66.3%) hadpositive FoxC1 expression (Table 1). Therefore, wedetermined whether HBV could induce FoxC1 expres-sion in hepatocytes. In this study, we found that hepati-

tis B virus x (HBx) significantly up-regulated FoxC1expression and transactivated its promoter activity,

whereas the other viral proteins had no effect on FoxC1expression, indicating that HBx is a critical regulator ofFoxC1 expression during HBV infection (SupportingFig. 3A-C). Gene-promoter analysis of the FoxC1 pro-moter revealed the presence of many consensus cis-ele-ments, including cAMP response element-binding pro-tein (CREB), nuclear factor kappa beta, c-Ets, and

Fig. 5. NEDD9, which promotes HCC metastasis, is a direct transcriptional target of FoxC1. (A) FoxC1 up-regulates NEDD9 expression.SMMC7721 and HCCLM3 cells were infected with LV-FoxC1 or LV-shFoxC1, and mRNA and protein levels of NEDD9 in the infected cells weredetected using real-time PCR and western blotting techniques. (B1) FoxC1 transactivates NEDD9 promoter activity. A NEDD9 promoter luciferase

construct, (2056/121)NEDD9, was cotransfected with pCMV-FoxC1, and promoter activity was detected using a luciferase reporter assay.

(B2) Deletion and selective mutation analysis identified two FoxC1-responsive regions in the NEDD9 promoter. Serially truncated and mutatedNEDD9 promoter constructs were cotransfected with pCMV-FoxC1, and relative luciferase activity was measured. (B3) ChIP assay demonstrated

the interaction of FoxC1 with two of the potential FoxC1-binding sites in the NEDD9 promoter. Real-time PCR was performed to detect the

amounts of immunoprecipitated products. (C) Western blotting analysis of NEDD9 expression in different HCC cell lines. (D) Up-regulation ofNEDD9 expression with LV-NEDD9 enhanced the invasive capacity of SMMC7721 cells (low metastatic potential) in vitro. Up-regulation of

NEDD9 expression by LV-NEDD9 was confirmed by western blotting analysis. (E) In vivo metastasis assay. (E1) Ten weeks after orthotopic implan-

tation, BLI showed the presence of lung metastases in mice implanted with SMMC7721-NEDD9 cells and the absence of metastases in mice

implanted with SMMC7721-control cells. (E2) Incidence of lung metastasis in each group of nude mice. (E3) Number of metastatic lung foci ineach group. (E4) OS in each group. (E5) Images showing representative hematoxylin and eosin staining of lung tissue samples from the different

experimental groups. *P< 0.05.


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CCAAT enhancer-binding protein binding sites (Sup-porting Fig. 4). Serial deletion and mutation assays ofthe FoxC1 promoter revealed that the CREB-bindingsite in the FoxC1 promoter was critical for HBx-induced FoxC1 overexpression (Supporting Fig. 3D). A

ChIP assay further confirmed that CREB bounddirectly to the FoxC1 promoter in response to HBxprotein (Supporting Fig. 3E). HBx is a multifunctionalprotein that activates many cellular signal-transductionpathways, such as ERK1/2, Janus kinase, and p38MAPKs.33 An ERK1/2 inhibitor markedly decreasedHBx-induced FoxC1 expression and abolished thebinding of CREB to the FoxC1 promoter (SupportingFig. 3E,F). Furthermore, knockdown of FoxC1 mark-edly decreased HBx-enhanced cell invasion (SupportingFig. 5). These studies suggested that one of the mecha-

nisms by which FoxC1 is reactivated in HCC isthrough the HBx/ERK/CREB-signaling pathway.

Discussion

Recurrence and metastasis remain the most commonlethal outcomes after curative resection in HCC.3 Thus,it is critical to investigate the mechanisms underlyingHCC metastasis. In this study, we demonstrated thatFoxC1 was frequently up-regulated in human HCC tis-sues, relative to adjacent noncancerous tissues. FoxC1overexpression was correlated with increased tumor size,loss of tumor encapsulation, microvascular invasion,malignant differentiation, and more-advanced TNMstage. Additionally, HCC patients with positive FoxC1expression had worse prognoses than did patients who

Fig. 6. Knockdown of NEDD9 significantly attenuates FoxC1-enhanced invasion and metastasis. (A) After cells were infected with LV-shNEDD9,

protein expression level of FoxC1 and NEDD9 was detected by western blotting analysis. (B) NEDD9 knockdown decreased FoxC1-enhanced cell

invasion. After SMMC7721-FoxC1 cells were infected with the lentivirus, LV-shNEDD9, invasion capacity of the cells was measured in transwellassays. (C) In vivo metastatic assay. (C1) BLI showed the presence of lung metastases in mice implanted with SMMC7721-FoxC1 plus LV-shcon-

trol cells and the absence of lung metastases in mice implanted with SMMC7721-FoxC1 plus LV-shNEDD9 cells. (C2) Incidence of lung metasta-

sis in the different groups of nude mice. (C3) Number of metastatic lung foci observed in each group. (C4) OS in each group. (C5) Imagesshowing representative hematoxylin and eosin staining of lung tissue samples from the different experimental groups. (D1) IHC analysis of

NEDD9 expression in HCC tissues. (D2) Correlation between FoxC1 and NEDD9 levels. (E1) Kaplan-Meiers analysis of the correlation between

NEDD9 expression and recurrence or OS of HCC patients. (E2) Kaplan-Meiers analysis of the correlation of FoxC1/NEDD9 coexpression with re-currence and OS. *P< 0.05.


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were negative for FoxC1 expression. Furthermore, multi-variate analysis revealed that FoxC1 expression level wasan independent, significant risk factor for recurrence andsurvival after curative resection. These clinical datastrongly suggested that FoxC1 contributes to the malig-

nant progression of HCC and may be a useful prognos-tic biomarker.Several pieces of evidence in this study support a

close association between FoxC1 expression and HCCmetastasis. First, FoxC1 protein and mRNA levels

were correlated with the metastatic potential of theHCC cell lines examined. Second, FoxC1 expression

was markedly higher in metastatic lesions, comparedwith their corresponding primary tumor samples.Third, up-regulation of FoxC1 significantly promotedthe invasion and lung metastasis of HCC cells,

whereas the knockdown of FoxC1 decreased the inva-

sion and metastasis of HCC cells.EMT plays an important role in HCC invasiveness

and metastasis.34,35 The EMT transition triggered dur-ing tumor progression is controlled by several tran-scription factors, including Twist, Snai1, Slug, Goose-coid, ZEB1, and SIP1.24 In this study, we found thatthe overexpression of FoxC1 had a significant effect onEMT, as indicated by the increased expression of mes-enchymal markers (fibronectin and vimentin) anddecreased expression of epithelial markers (E-cadherinand O-catenin). In contrast, knockdown of FoxC1decreased the expression of mesenchymal markers and

increased the expression of epithelial markers. EMT isa key event in tumor invasion and metastasis; epithelialcells lose their epithelial adherence and cell-cell con-tacts and undergo remarkable cytoskeletal remodelingto facilitate cell motility and invasion.36 Thus, HCCcells overexpressing FoxC1 most likely become moreinvasive by undergoing EMT.

Disruption of the E-cadherin-mediated adhesion sys-

tem is a major event in the transition from a noninvasive

tumor to invasive malignant carcinoma and is a key bio-

marker for EMT.23 E-cadherin is directly repressed by

Snai1, which, in turn, induces mesenchymal phenotypeacquisition in epithelial tumor cells.37,38 FoxC1 increases

cell migration and invasion in mammary epithelial cells

by inhibiting E-cadherin expression.18 However, the mo-

lecular mechanism by which FoxC1 inhibits E-cadherin

expression remains unknown. This study was the first to

demonstrate that FoxC1 transactivates Snai1 expression

by directly binding to its promoter, thus leading to the

inhibition of E-cadherin transcription by its repressor,

Snai1. Inhibition of Snai1 expression significantly sup-

pressed FoxC1-enhanced invasion and lung metastasis.

In addition, in a cohort of 406 human HCC tissues, we

found that FoxC1 expression was positively correlated

with Snai1 expression, but inversely correlated with

E-cadherin expression. More important, patients exhibit-

ing FoxC1()/Snai1() coexpression had the highest re-

currence rates and lowest OS among the four subgroups,whereas patients exhibiting FoxC1()/E-cadherin()

expression had shorter OS times and higher recurrence

rates. Thus, both experimental and clinical evidence indi-

cate that the FoxC1/Snai1/E-cadherin pathway may play

an important role in promoting HCC metastasis and

producing a poor clinical outcome.In addition, we conducted a detailed analysis of

gene expression in FoxC1-knockdown cells using acomplementary DNA array. We found that knock-down of FoxC1 reduced the expression of a number ofmetastasis-related genes. Among these genes, NEDD9

was the most down-regulated upon FoxC1 knock-down. Using serial deletion, site-directed mutagenesis,and ChIP, we showed that NEDD9 is a direct tran-scriptional target of FoxC1. Inhibition of NEDD9expression markedly decreased FoxC1-mediated HCCmetastasis. Furthermore, FoxC1 expression was posi-tively correlated with NEDD9 expression, and thecoexpression of these genes was associated with poorprognosis in human HCC patients. Thus, FoxC1 pro-moted HCC metastasis by up-regulating NEDD9expression.

In conclusion, this study demonstrates that the over-expression of FoxC1 in HCC is a strong indicator ofmore-aggressive tumors and poor clinical outcome.FoxC1 promotes HCC metastasis through not onlythe induction of EMT, but also up-regulation of theadhesive molecule, NEDD9. Thus, FoxC1 may be acandidate biomarker for HCC prognosis and a targetfor new therapies.

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