Int. J. Biol. Sci. 2012, 8
I In nt te er rn na at ti io on na al l J Jo ou ur rn na al l o of f B Bi io ol lo og gi ic ca al l S Sc ci ie en nc ce es s
2012; 8(8):1130-1141. doi: 10.7150/ijbs.4769
FOXC1 Contributes To Microvascular Invasion In Primary Hepatocellular
Carcinoma Via Regulating Epithelial-Mesenchymal Transition
Zhi-Yuan Xu 1,2*, Song-Ming Ding1,2*, Lin Zhou1,2*, Hai-Yang Xie1,2, Kang-Jie Chen1,2, Wu Zhang1,2, Chun-Yan
Xing1,2, Hai-Jun Guo1,2, and Shu-Sen Zheng1,2
1. Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health; Key Laboratory of Organ Trans-
plantation, Zhejiang Province; Hangzhou, Zhejiang, China;
2. Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University
School of Medicine; Hangzhou, Zhejiang, China.
* These authors contributed equally to this work.
Corresponding author: Shu-sen Zheng, PhD, MD, FACS. E-mail: firstname.lastname@example.org Tel: 86-571-87236570 Fax: 86-571-87236628
© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
Received: 2012.06.21; Accepted: 2012.08.28; Published: 2012.09.08
The existence of microvascular invasion (MVI) formation is one of the most important risk
factors predicting poor outcome in hepatocellular carcinoma (HCC) and its mechanism
remains largely unknown. Epithelial-Mesenchymal Transition (EMT) has been suggested to be
involved in many steps of the invasion-metastasis cascade. To elucidate the possible contri-
bution of EMT to MVI, we initially evaluated the expression of 8 EMT-related transcription
factors (TFs) in HCC patients with or without MVI and found that FOXC1 expression was
significantly higher in patients with MVI than those without MVI (P < 0.05). Knockdown of
FOXC1 expression in HCC cells resulted in a partial conversion of their EMT progresses,
mainly regulating the mesenchymal component. Ectopic expression of snail, twist or TGF-β1
could induce expression of FOXC1, but none of the expression of snail, twist, slug or TGF-β
was consistently down-regulated in response to FOXC1 silencing, suggesting FOXC1 might
operate the downstream of other EMT regulators. In addition, knockdown of FOXC1 ex-
pression led to cytoskeleton modification accompanied by decreased ability of cell prolifera-
tion, migration, and invasion. Meanwhile, some matrix metalloproteinases (MMPs) and
VEGF-A were also simultaneously down-regulated. Together, our findings demonstrate that
FOXC1 is one of candidate predictive markers of MVI, and that inhibition of FOXC1 ex-
pression can partially reverse EMT program, offering a potential molecular therapeutic target
for reducing tumor metastasis in HCC patients.
Key words: Hepatocellular carcinoma, Microvascular invasion, Epithelial-Mesenchymal Transi-
Hepatocellular carcinoma (HCC) is the fifth most
common cancer worldwide and the third leading
cause of cancer death . Currently, surgical treat-
ment, including resection and transplantation, is an
optional treatment strategy offering potential curative
treatment and better long-term outcome in patients
with HCC. Unfortunately, despite the expanded in-
dications for surgical treatment with the technical
advances, the rate of tumor recurrence and metastasis
after curative resection remains high, which still limits
patient survival . Among the factors related to tu-
mor recurrence, microvascular invasion (MVI) for-
Int. J. Biol. Sci. 2012, 8
mation has been proved to be one of the most im-
portant risk clinicopathological factors to predict the
metastatic potential of HCC . The existence of MVI
indicates that HCC has become systemic through in-
vasion of peripheral blood vessel and subsequent
spread . However, microvascular invasion to mi-
nute peripheral areas, such as the third branch of the
portal vein, is difficult to detect preoperatively with
current conventional imaging modalities. In most
cases, it can be only detected by pathological exami-
nation after surgery. No postoperative adjuvant
treatment modalities including transcatheter arterial
chemoembolization (TACE), can effectively improve
the outcomes in this group of patients. This was con-
sistent with numerous studies showing that early re-
currence within 2 years is associated with MVI after
curative liver resection and even orthotopic liver
transplantation which totally eradicate the disease
liver. Hence, it is urgent to understand the under-
lying genetic basis for MVI and then seek measure to
Increasingly, more attention is currently being
given to changes in cellular architecture and polarity
for insights into cancer behavior . The epitheli-
al-mesenchymal transition (EMT) is such process
which allows polarized epithelial cells to acquire
mesenchymal properties including fibroblastoid
morphology, characteristic gene express changes, in-
creasing potential for motility, and in the case of can-
cer, increased invasion, metastasis, resistance to
chemotherapy . The functional hallmark of carci-
noma EMT is acquisition of ability to transmigration
through extracellular matrix (ECM) as a single cell .
Notably, the carcinoma EMT has be regarded as a
critical behavior for cancer cell to initial escape from
primary site by endowing individual cell with en-
hanced invasive potential through barrier matrix and
subsequent acquisition of intravasation, enabling
cancer cells to enter the blood vessel. Hence, we
hypothesis that larger subpopulations of cells in pri-
mary HCC with MVI, compared with that without
MVI, may have undergone an EMT for the purpose of
generating MVI. Interesting, similar hypothesis has
been previously proposed when studying a putative
role of EMT process in circulating tumor cells (CSC)
formation  and it appears today that EMT pro-
cesses contribute at least in part to tumor heterogene-
ity  and CSC formation, supporting and validating
the implication of EMT in MVI formation. In view of
above understanding, our present study aimed to
demonstrate the mechanism of MVI formation from
the perspective of EMT process.
Currently, the exact mechanism of EMT pro-
gress remained largely unexplored. However, the
critical role of some transcriptional factors (TFs) in the
activation of EMT has been well documented. These
TFs include the homeobox protein Goosecoid
(Gsc), the zinc-finger proteins Snai1 (Snail)and
Snai2 (Slug)[13, 14], the basic helix-loop-helix protein
Twist1 (Twist), the forkhead box proteins
FOXC1[16, 17] and FOXC2 and the zinc-finger,
E-box-binding proteins Zeb1 and Sip1(Zeb2)[19, 20].
Additionally, a number of molecular processes have
also been proposed to be engaged in order to initiate
EMT. However, both developmental studies in vari-
ous organisms and studies in tumor metastasis have
yielded that TFs may serve as major signaling medi-
ators in response to various inductive signals , For
example, TGF-β signaling, which is the best studied
pathway responsible for inducing EMTs, represses the
expression of E-cadherin through inducing snail, slug
and twist[20, 21]. Together, these studies have high-
lighted the major contribution of TFs during the exe-
cution of EMT program and subsequent metastatic
cascade. Undoubtedly, the expression profile analysis
and functional studies of these TFs maybe contribute
to elucidate the possible involvement of EMT in MIV
formation. In present study, we initially assessed by
comparing the expression profiles of known
EMT-related TFs between HCC tissues with and
without MVI, and found that FOXC1 was the TF
which presented prominently higher level in HCC
tissues with MVI. We then explored the functional
role of FOXC1 in regulating EMT, cell proliferation,
metastatic potential, and VEGF-A expression for fur-
ther understanding the mechanism underlying MVI
formation of HCC.
Material and method
50 HCC patients who underwent curative he-
patic resection between March 2009 and June 2011
were recruited from our institute. The study was ap-
proved by the Ethics Committee of our hospital.
Specimens were obtained immediately after surgical
resection. None of patients were treated by any pre-
operative therapy such as TACE, percutaneous etha-
nol injection. Patients with macroscopic vascular in-
vasion were excluded. There were 34 men and 16
women, ranging in age from 31 to 78 years, with a
median age of 54 years. The presence of MVI was de-
termined from histopathological reports stored in a
prospectively maintained computerized clinical da-
tabase. Tumor stage was defined according to Amer-
ican Joint Committee on Cancer/International Union
Against Cancer tumor, node, metastasis (TNM) clas-
sification system . Clinical data such as date of
Int. J. Biol. Sci. 2012, 8
sufficient to regulate the Vimentin expression. Besides
the IF network, actin cytoskeleton organization and
polymerization have a coordinately role in cell mi-
gration. We also observed that down-regulation of
FOXC1 was associated with depolymerization of
F-actin cytoskeleton and diminution of F-actin content
accompanied by morphological changes, indicating
the repressed cell mobility. Although the regulatory
mechanism of actin cytoskeleton reorganization in-
duced by FOXC1 was waiting to be elucidated, some
molecular mediators which play a central role in
modulating the actin cytoskeleton, such as Rho
GTPase and integrins  have been shown to be
closely regulated during EMT processes.
Tumor cell invasion itself is not sufficient to
produce MVI formation. Stimulation of angiogenesis
and intravasation of tumor cell are important aspects.
Nowadays, extensive documents have explored the
EMT implication in regulation of angiogenesis and
revealed that EMT can promote angiogenesis by
modulating pro-angiogenic factors such as VEGF-A.
Our present study also revealed that down-regulation
of FOXC1 was associated with repressed expression
of VEGF-A. In addition, MMP1 and MMP4 expres-
sions, which have definite role in enhancing angio-
genesis, were observed to be down-regulated by
FOXC1 knockdown. These finding indicated the pos-
sible involvement of FOXC1 in promoting angiogen-
esis. Consistence with our result, FOXC1 expression
has been reported to influence the proliferation and
differentiation of endothelial development [30, 31]. As
for intravasation of tumor cell, the participation of
EMT-related genes in the regulation of transendothe-
lial migration is nowadays becoming clear. For ex-
ample, N-cadherin has been reported to play a major
role in transendothelial migration by contributing to
heterotypic contacts between endothelial cells and
melanoma cells. β-catenin translocation from cell-cell
adhesion contact to nucleus also contribute to enhance
transendothelial cell migration. In present study,
both decreased expression of N-cadherin and redis-
tribution of β-catenin were observed. These data pro-
vided the possibility that FOXC1 might participate the
regulation of intravasation and further favored the
MVI generation. But this hypothesis requires further
analysis in mouse models.
In summary, high FOXC1 expression was corre-
lated with occurrence of MVI formation in HCC pa-
tients and suppression of endogenous FOXC1 ex-
pression resulted in a partial conversion of EMT pro-
gress in HCC cell lines, mainly influencing their
mesenchymal component. Moreover, inhibition of
FOXC1 expression in HCC cells represents a promis-
ing option to prevent HCC metastasis through recon-
struction of cellular behavior including proliferation,
migration and invasion, angiogenesis and intravasa-
Table S1. List of proteins tested by antibodies and
characteristics of the corresponding antibodies used.
Table S2. Sequence of the oligonucleotides for plasmid
construct-making and real-time PCR.
HCC: Hepatocellular carcinoma; MVI: Micro-
vascular invasion; TFs: Transcriptional factors; EMT:
This study was supported by the National S&T
Major Project (No. 2012ZX10002-017), NSFC for In-
novative Research Group (81121002) and National
Basic Research Program of China (973 Program)
(No.2009CB522403). We thank the surgeons and
nurses who kindly facilitate the recruitment and col-
lection of patient information.
The authors have declared that no competing
1. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Long-term therapy
with adefovir dipivoxil for HBeAg-negative chronic hepatitis B for up to
5 years. Gastroenterology. 2006; 131: 1743-1751.
2. Poon RT, Fan ST, Tsang FH, et al. Locoregional therapies for hepatocel-
lular carcinoma: a critical review from the surgeon's perspective. Ann
Surg. 2002; 235: 466-486.
3. Hu J, Wang Z, Fan J, et al. Genetic variations in plasma circulating DNA
of HBV-related hepatocellular carcinoma patients predict recurrence af-
ter liver transplantation. PLoS One. 2011; 6: e26003.
4. Roayaie S, Frischer JS, Emre SH, et al. Long-term results with multi-
modal adjuvant therapy and liver transplantation for the treatment of
hepatocellular carcinomas larger than 5 centimeters. Ann Surg. 2002; 235:
5. Yao FY, Ferrell L, Bass NM, et al. Liver transplantation for hepatocellular
carcinoma: comparison of the proposed UCSF criteria with the Milan
criteria and the Pittsburgh modified TNM criteria. Liver Transpl. 2002; 8:
6. Kurrey NK, Jalgaonkar SP, Joglekar AV, et al. Snail and slug mediate
radioresistance and chemoresistance by antagonizing p53-mediated
apoptosis and acquiring a stem-like phenotype in ovarian cancer cells.
Stem Cells. 2009; 27: 2059-2068.
7. Taube JH, Herschkowitz JI, Komurov K, et al. Core epitheli-
al-to-mesenchymal transition interactome gene-expression signature is
associated with claudin-low and metaplastic breast cancer subtypes.
Proc Natl Acad Sci U S A. 2010; 107: 15449-15454.
8. Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the cross-
roads of development and tumor metastasis. Dev Cell. 2008; 14: 818-829.
9. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transi-
tion. J Clin Invest. 2009; 119: 1420-1428.
10. Bonnomet A, Brysse A, Tachsidis A, et al. Epithelial-to-mesenchymal
transitions and circulating tumor cells. J Mammary Gland Biol Neo-
plasia. 2010; 15: 261-273.
Int. J. Biol. Sci. 2012, 8
11. Klymkowsky MW, Savagner P. Epithelial-mesenchymal transition: a
cancer researcher's conceptual friend and foe. Am J Pathol. 2009; 174:
12. Hartwell KA, Muir B, Reinhardt F, et al. The Spemann organizer gene,
Goosecoid, promotes tumor metastasis. Proc Natl Acad Sci U S A. 2006;
13. Batlle E, Sancho E, Franci C, et al. The transcription factor snail is a
repressor of E-cadherin gene expression in epithelial tumour cells. Nat
Cell Biol. 2000; 2: 84-89.
14. Hajra KM, Chen DY, Fearon ER. The SLUG zinc-finger protein represses
E-cadherin in breast cancer. Cancer Res. 2002; 62: 1613-1618.
15. Yang J, Mani SA, Donaher JL, et al. Twist, a master regulator of mor-
phogenesis, plays an essential role in tumor metastasis. Cell. 2004; 117:
16. Bloushtain-Qimron N, Yao J, Snyder EL, et al. Cell type-specific DNA
methylation patterns in the human breast. Proc Natl Acad Sci U S A.
2008; 105: 14076-14081.
17. Ray PS, Wang J, Qu Y, et al. FOXC1 is a potential prognostic biomarker
with functional significance in basal-like breast cancer. Cancer Res. 2010;
18. Mani SA, Yang J, Brooks M, et al. Mesenchyme Forkhead 1 (FOXC2)
plays a key role in metastasis and is associated with aggressive basal-like
breast cancers. Proc Natl Acad Sci U S A. 2007; 104: 10069-10074.
19. Eger A, Aigner K, Sonderegger S, et al. DeltaEF1 is a transcriptional
repressor of E-cadherin and regulates epithelial plasticity in breast can-
cer cells. Oncogene. 2005; 24: 2375-2385.
20. Comijn J, Berx G, Vermassen P, et al. The two-handed E box binding zinc
finger protein SIP1 downregulates E-cadherin and induces invasion. Mol
Cell. 2001; 7: 1267-1278.
21. Li H, Wang H, Wang F, et al. Snail involves in the transforming growth
factor beta1-mediated epithelial-mesenchymal transition of retinal pig-
ment epithelial cells. PLoS One. 2011; 6: e23322.
22. Lu W, Dong J, Huang Z, et al. Comparison of four current staging sys-
tems for Chinese patients with hepatocellular carcinoma undergoing
curative resection: Okuda, CLIP, TNM and CUPI. J Gastroenterol
Hepatol. 2008; 23: 1874-1878.
23. Cheng J, Xie HY, Xu X, et al. NDRG1 as a biomarker for metastasis,
recurrence and of poor prognosis in hepatocellular carcinoma. Cancer
Lett. 2011; 310: 35-45.
24. Hannenhalli S, Kaestner KH. The evolution of Fox genes and their role in
development and disease. Nat Rev Genet. 2009; 10: 233-240.
25. Zarbalis K, Siegenthaler JA, Choe Y, et al. Cortical dysplasia and skull
defects in mice with a Foxc1 allele reveal the role of meningeal differen-
tiation in regulating cortical development. Proc Natl Acad Sci U S A.
2007; 104: 14002-14007.
26. Aldinger KA, Lehmann OJ, Hudgins L, et al. FOXC1 is required for
normal cerebellar development and is a major contributor to chromo-
some 6p25.3 Dandy-Walker malformation. Nat Genet. 2009; 41:
27. Wang J, Ray PS, Sim MS, et al. FOXC1 regulates the functions of human
basal-like breast cancer cells by activating NF-kappaB signaling. Onco-
28. Sizemore ST, Keri RA. The Forkhead Box Transcription Factor FOXC1
Promotes Breast Cancer Invasion by Inducing Matrix Metalloprotease 7
(MMP7) Expression. J Biol Chem. 2012; 287: 24631-24640.
29. Yilmaz M, Christofori G. EMT, the cytoskeleton, and cancer cell invasion.
Cancer Metastasis Rev. 2009; 28: 15-33.
30. Sommer P, Napier HR, Hogan BL, et al. Identification of Tgf beta1i4 as a
downstream target of Foxc1. Dev Growth Differ. 2006; 48: 297-308.
31. Hayashi H, Kume T. Forkhead transcription factors regulate expression
of the chemokine receptor CXCR4 in endothelial cells and
CXCL12-induced cell migration. Biochem Biophys Res Commun. 2008;
32. Qi J, Chen N, Wang J, et al. Transendothelial migration of melanoma
cells involves N-cadherin-mediated adhesion and activation of the be-
ta-catenin signaling pathway. Mol Biol Cell. 2005; 16: 4386-4397.