VEGFR-1 activation-induced MMP-9-dependent invasion in hepatocellular carcinoma

Abstract

Aim:
VEGFR-1 can promote invasion through epithelial-mesenchymal transition induction in hepatocellular carcinoma (HCC). This study aims to elucidate VEGFR-1 impact on proteolytic enzymes profile involved with invasion.

Materials & methods:
The effect on cell invasion was evaluated by invasive and migration assays with and without VEGFR-1 activation. The mechanism was investigated by real-time PCR, western blot and gelatin zymography using inhibitors for MMP-9. In total, 95 HCC patients were enrolled for its clinical value evaluation.

Results:
VEGFR-1 activation induced invasion in HCC cells with an increase in the expression and activity of MMP-9 and Snail. MMP-9 blockage effectively inhibited VEGFR-1-induced invasion. High coexpression of both in HCC predicted a worse clinical outcome.

Conclusion:
Data show a novel VEGFR-1 activation-to-MMP-9 mechanism promoting HCC invasion.

Full text

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ISSN 1479-6694
Future Oncol. (2015) 11(23), 3143–3157
part of
10.2217/fon.15.263 © 2015 Future Medicine Ltd
RESEARCH ARTICLE
VEGFR-1 activation-induced
MMP-9-dependent invasion in
hepatocellular carcinoma
Tao Li1, Yuhua Zhu1, Lihui Han2, Wanhua Ren1, Hui Liu1 & Chengyong Qin*,1
1Department o f Infectious diseases, Shan dong Provincial Hospital Affiliated to Shandong Univer sity, Jinan 250021, China
2Departm ent of Immunology, Shandong University Scho ol of Medicine, Jinan 250012, China
*Author for correspondence: qchengy@163.com
Aim: VEGFR-1 can promote invasion through epithelial–mesenchymal transition induction in
hepatocellular carcinoma (HCC). This study aims to elucidate VEGFR-1 impact on proteolytic
enzymes prole involved with invasion. Materials & methods: The eect on cell invasion
was evaluated by invasive and migration assays with and without VEGFR-1 activation. The
mechanism was investigated by real-time PCR, western blot and gelatin zymography using
inhibitors for MMP-9. In total, 95 HCC patients were enrolled for its clinical value evaluation.
Results: VEGFR-1 activation induced invasion in HCC cells with an increase in the expression
and activity of MMP-9 and Snail. MMP-9 blockage eectively inhibited VEGFR-1-induced
invasion. High coexpression of both in HCC predicted a worse clinical outcome. Conclusion:
Data show a novel VEGFR-1 activation-to-MMP-9 mechanism promoting HCC invasion.
KEYWORDS
• hepatocellular carcinoma
• invasion • MMP-9 • Snail
•V EGFR -1
Hepatocellular carcinoma (HCC) is an aggressive disease with a poor prognosis due to the tumor
invasiveness, intrahepatic spread and extrahepatic metastasis [1] . HCC metastasis is frequently associ-
ated with epithelial–mesenchymal transition (EMT) and extracellular matrix (ECM) degradation
using proteases including matrix metalloproteinases (MMPs) [2, 3]. Understanding of the molecular
mechanisms regulating the metastatic and invasive behavior of this malignant tumor is essential
for improving the treatments.
Activation of growth factor receptors is one of the most important mechanisms for survival and
invasion of human cancers. VEGFR-1 is one of three typical membrane-bound tyrosine kinase
receptors that specifically bind VEGF-B and PGF. VEGFR-1-mediated signaling promotes cancer
metastasis via three major mechanisms: promotion of angiogenesis, activation of tumor cell prolifera-
tion and induction of tumor EMT, which endows tumor cell with a more invasive phenotype [4 ,5] .
Activation of VEGFR-1 on the endothelial cells by its ligands results in the enhanced angiogenesis
and metastasis. VEGFR-1 signaling facilitates malignant angiogenesis through the enhancement of
endothelial migration and activity [6 ] . VEGFR-1 activation in breast cancer cells promotes tumor
growth via activation of MAPK and PI3K/Akt signaling [7, 8] . Moreover, activation of VEGFR-1
in cancer cells can also induce EMT, a critical mechanism for acquisition of invasive potential and
promotion of cancer cell metastasis [4,9–10] . Furthermore, it is reported that activation/induction of
proteolytic enzyme-mediated degradation of the ECM is also one of the essential steps in carcinoma
invasion [1]. Many growth factors including TGF-β, EGF and IGF-1 have the ability to enhance the
cancer cell invasiveness through activation of proteolytic enzymes [11 –13 ] . While the mechanisms
of angiogenesis and EMT are well established, the VEGFR-1 signaling pathways that regulate the
activation of proteolytic enzymes are poorly understood.
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MMPs, a family of metalloendopeptidases
that cleave the protein components of the ECM
and endothelial cell basement membrane, play
a pivotal role in tumor-associated angiogenesis
and cancer metastasis [14– 16 ] . MMP-9, a mem-
ber of MMPs family, is particularly interest-
ing, because it is correlated with the tumor
recurrence and survival in patients with HCC.
Quantitative real-time PCR studies have also
shown that the expression of MMP-9 was signifi-
cantly upregulated in the high-metastatic HCC
cells lines [1] . Moreover, animal studies have
confirmed that blocking MMP-9 can inhibit
tumor cell invasion and metastasis. Importantly,
it is hypothesized that MMP-9 enhances tumor
angiogenesis through the VEGF–VEGFR sign-
aling system [17 ] . Recent studies have shown
that VEGFR-1 activation markedly promotes
pulmonary metastasis through the induction of
MMP-9 secretion in premetastatic lung endothe-
lial cells and macrophages [18] . However, the
mechanisms of MMP-9 regulation and its func-
tion of VEGFR-1 activation in HCC remain to
be established.
In this study, we investigated one possible
mechanism of MMP-9 regulation and its role in
activation of VEGFR-1-mediated invasion. We
showed that Snail, an EMT regulator induced
by activation of VEGFR-1, was involved in the
regulation of the expression of MMP-9. We
also showed a strong correlation of high coex-
pression of VEGFR-1 and MMP-9 with the
patient tumor invasion and demonstrated that
high coexpression of VEGFR-1 and MMP-9 is
a prognostic marker for HCC.
Materials & methods
●●Reagents
Recombinant human VEGF-B167 was purchased
from R&D Systems, Inc. (MN, USA). Purified
human immunoglobulin (Sigma, MO, USA),
a nonspecific IgG, was used as a control. Both
of them were added to cultures at a final con-
centration of 50 ng/ml. The broad spectrum
MMPs inhibitor (GM6001) was purchased from
Chemicon (CA, USA). Anti-MMP-9 monoclo-
nal antibody (Ab-1) for functional blocking
was purchased from Calbiochem (CA, USA).
Monoclonal anti-VEGFR-1 and polyclonal anti-
Snail were purchased from Abcam Cambridge
(MA, USA). Polyclonal anti-MMP-9 and
anti-β-actin were purchased from Santa Cruz
Biotechnology (CA, USA). FITC-conjugated
antirabbit IgG, horseradish peroxidase
(HRP)-conjugated antirabbit IgG and HRP-
conjugated antigoat IgG were purchased from
Zhongshan Biotechnology (Beijing, China).
●●Cell cultures
All cell lines, except for those with particular
notes, were obtained from Cell bank of Chinese
Academy of Science. MHCC97H was obtained
from the Liver Cancer Institute of Zhongshan
Hospital, Fudan University, China. The human
umbilical vein endothelial cells (which are used
as positive control of VEGFR-1 expression) and
NIH3T3 were kindly provided by Professor Ling
Gao (Central Laboratory of Provincial Hospital
Affiliated to Shandong University, China). HCC
cell line and NIH3T3 was cultured as described
previously [19] . SMMC7721 and HepG2 cell lines
were routinely cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Hyclone, USA) sup-
plemented with 10% FBS (Gibco, NY, USA) and
antibiotics (100 U/ml penicillin and 100 μg/ml
streptomycin; Gibco), at 37°C in 5% CO2 and
95% air. Results from all studies were confirmed
in at least three independent experiments.
●●Patients, follow up & tissue microarrays
construction
A total of 95 HCC patients who underwent cura-
tive resection treatment at Provincial Hospital
Aff iliated to Shandong University during
October 2006 to August 2007 were analyzed.
The criteria for curative resection were described
previously [2 0] . No patients received any preop-
erative anticancer treatment. All pathological
features such as cirrhosis, tumor encapsulation,
tumor size and tumor number were defined
histologically. Tumor differentiation and stage
were grade by Edmondson grading system and
the 2002 International Union Against Cancer
TNM classification system (6th edition), respec-
tively. This study was approved by the Ethics
Committee of Provincial Hospital Affiliated
to Shandong University, and informed consent
was obtained from all patients. Patients’ clinical
characteristics were summarized in Tab le 1.
All of the patients received follow-up tests
including liver function, tumor markers and
ultrasonography with an interval of 4–7 months
after curative resection. In case of suspicious
tumor recurrence, computed tomography scan-
ning was used immediately. Treatment modali-
ties after relapse were administered depending
on the individual situation, including radi-
ofrequency ablation and selected transcatheter

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VEGFR-1 activation-induced MMP-9-dependent invasion in hepatocellular carcinoma RESEaRch aRticlE
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arterial chemoembolization. The median follow-
up period was 38 months. Overall survival (OS)
and recurrence-free survival (RFS) were defined
as the interval between curative resection and
death or recurrence.
A total of 95 pathology-proven HCC tissues
and 33 peritumoral tissues (only corresponding
to HCC tissues of moderate differentiation) were
collected. After examining HE-stained slides for
the location and differentiation of cancerous tis-
sues and peritumoral tissues, tissue microarrays
(TMAs; collaborated with Shanghai Biochip
Co, Ltd, Shanghai, China) were constructed.
●●Immunouorescent microscopy
Paraformaldehyde-fixed, permeabilized
SMMC7721 cells cultured on glass slide for 48h
were incubated with primary antibodies over-
night at 4°C followed by incubation with second-
ary fluorescent antibodies. Nuclei were stained
with DAPI (Vector Laboratories, CA, USA),
and the resultant immunof luorescence was
observed under a fluorescent microscope (Leica
Microsystems, Wetzlar, Germany). Negative
controls were treated identically but with the
primary antibodies omitted.
●●Cell migration assay
Cell migration was examined utilizing a wound
healing assay as described previously [2 1] . Briefly,
SMCC7721 cells were digested and plated in
a six-well plate until 100% confluence. Three
parallel wounds with a size of approximately
400 μm were made in the cell monolayer using
a sterile pipette tip. The monolayer was washed
twice with PBS followed by incubation with
VEGF-B167 (R&D Systems, Inc., MN, USA) or
nonspecific human IgG (Sigma) in DMEM + 1%
FBS for 24 h. Relative cell migration was calcu-
lated as the percentage of the remaining cell-free
area among the initial wounded areas. Closure
of the wounded area was monitored using an
inverted microscope (Leica Microsystems,
Germany) attached with a digital camera. Cell
migration distance was determined by meas-
uring the width of the wound and calculated
using the following equation: cell migration
distance = initial half-width of the wound - the
width of the wound after migration/2. Results
are expressed as mean ± standard deviation of
three independent experiments.
●●Matrigel invasive assay
Invasion assay was performed using 24-well
Transwell chamber with a pore size of 8 μm
(Costar, NY, USA) and the inserts were coated
with Matrigel (BD, Bioscience, MA, USA). Cells
were digested and diluted to 1 × 106 cells/ml in
DMEM + 1% FBS. A total of 100 μl of cells
was then added on the upper chamber of the
insert. Consequently, DMEM + 10% with or
without VEGF-B167 was added to the upper
chamber. After 48 h of incubation, cells on the
upper surface of the filters were removed with
a cotton tip. The migrant cells were fixed with
4% paraformaldehyde and stained with hema-
toxylin. Numbers of cells migrating to the mem-
brane were counted in five randomly chosen fields
under a light microscope (×200). The average
number of migrated cell per microscopic field was
analyzed. Results are expressed as mean ± stand-
ard deviation of three independent experiments.
Table 1. Characteristics and univariate survival analysis of 95 hepatocellular carcinoma patients.
Factor n Mean RFS (95% CI) p-value Mean OS (95% CI) p-value
Age (≤50 years/>50 years) 41/54 31.6 (27.6– 35.5 )/28 .4 (25. 3–31.5) 0.236 44.2 (41.4–47.0)/38.9 (35.8–42.1) 0.087
Gender (male/female) 79/16 30.6 (28.0–33.3)/25.3 (19.3–31.3) 0. 074 40.7 (38.3–43.2)/43.1 (38.2–47.9) 0.293
Liver cirrhosis (no/yes) 15/80 29.6 (23.7–35.5)/29. 8 (27.1–32.5) 0.724 45.8 (42.6 –49.1)/40.4 (37.9–42.9) 0.459
Tumor size (≤5 cm/>5 cm) 38/57 38.6 (35.3– 41.8)/23.7 (21.3–26.1) 0.0001* 47.5 (4 5.4 –4 9.6) /37.0 (3 4. 0– 39. 9) 0.0001*
Tumor number (single/multiple) 50/45 35.5 (32.2–38.9)/23.3 (20.8–32.2) 0.0001* 47.1 (45.1–49.2)/34.8 (31.7–38.0) 0.0001*
Tumor tissue encapsulation (yes/no) 53/42 35.2 (32.2–38.2)/22.5 (19.8–25.3) 0.0001* 45.6 (43.1–48.1)/35.5 (32.3–38.6) 0.0001*
TNM stage (I–II/III–IV) 50/45 36.9 (34.0–39.7)/21.5 (19.1–23.9) 0.0001* 47.6 (45.7–49.4)/34.0 (30.9–37.0) 0.0001*
VEGFR-1 (low/high)†46/43 33.2 (29.4 –36.9)/25.1 (22.2–27.9) 0.0001* 44.5 (41.9–47.0)/37.2 (33.6–40.8) 0.023*
Snail (low/high)†43/49 34.0 (30.4–37.6)/24.7 (22.0–27.4) 0.0001* 47.4 (45.1–49.8)/35.1 (32.2–37.9) 0.0 01*
MMP-9 (low/high)†53/33 30.7 (27.3–34.1)/28.5 (24.6–32.5) 0.327 42.2 (39.0–45.4)/40.1 (36.9–43.4) 0. 274
VEGFR-1/MMP-9 high coexpression
(yes/no)
19/60 23.2 (19.7–26.6)/30.4 (27.3–33.6) 0.003* 35.6 (30.6–40.7)/42.5 (39.8–45.2) 0.02*
†Cores of six (V EGFR-1), three (Snail) and nine (MMP-9) patient s were detached from tissues microarrays duri ng immunstaining.
*p < 0.05 was considere d statistically significant .
OS: Overall survival; RFS: Recurrence-free survival.

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●●Western blot analysis
Cells were grown to 70–80% confluence in
DMEM + 10%FBS and were then serum-
deprived overnight in DMEM + 1% FBS.
Subsequently, cells were treated with VEGF-B167
or nonspecific IgG in DMEM + 1% FBS. Cells
in both groups were collected by scraping, and
the whole cell lysate was prepared using lysis
buffer (1 × PBS, 0.5% sodium dexycholate,
1 mM Na3VO4, 1% NP-40, 0.1% SDS, 5 mM
EDTA, 1 mM PMSF). After centrifugation,
40 μg of total protein from each group were
loaded onto 12% SDS-PAGE gels. After elec-
trophoresis, the proteins were transferred to a
nitrocellulose membrane, which was blocked,
incubated with appropriate antibodies and
developed with chemiluminescence as described
previously [19] . The fluorescence of the protein
bands was detected and quantified with LAS-
3000 system (Fuji Systems, Tokyo, Japan).
β-actin was used as a loading control.
●●Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was
performed on ABI 7500 Real-time PCR sys-
tem (Applied Biosystems, CA, USA) using the
SYBR Premix Ex Taq (TaKaRa Biotechnology
[Dalian] Co., Ltd, Dalian, China) accord-
ing to the manufacturer’s instructions.
Primer pairs were as follows: Snail: forward
5´-TAGGCCCTGGCTGCTACAAG-3´,
reverse 5´-GAGAAGGTCCGAGC ACACG-3´;
β-actin: forward 5´-TGACGTGG ACATCC
GCAAAG-3´, reverse 5´-CTGGAAGGTGGA
CAGCGAGG-3´; MMP-9: forward 5´-GTGCT
GGGCTGCTTTGCTG-3´, reverse 5´-GT
CGCCCTCAAAGGTTTGGAAT-3’. The
data were quantified with the comparative Ct
method for relative gene expression [22] . β-actin
was used as the reference gene.
●●Gelatin zymography
Cells at 70–80% confluence were treated with
VEGF-B167 or nonspecific IgG after overnight
serum deprivation in DMEM + 1% FBS. These
conditioned media were centrifuged to remove
cell debris and concentrated 15–20-fold using
Spin-x UF concentrators (Costar, NY, USA)
before being utilized as gelatinase. Volumes for
each group were adjusted to 15 μg protein as
determined by BCA. Gelatin zymography was
performed as described previously [23] . Brie fly,
samples were mixed with SDS sample buffer
without reducing agent and separated on 10%
SDS-PAGE gels containing 0.1% gelatin. Gels
were incubated at 37°C in digestion buffer
after SDS was removed by washing with 2.5%
Triton X-100-containing buffer. Gels were
stained with Coomassie Brilliant Blue R250
and the gelatinolytic activities were detected as
clear bands against a blue background.
●●Immunohistochemistry analysis
The immunohistochemistry protocols were
described previously [24 ] . Negative controls
were treated identically but with the primary
antibodies omitted. Immunoreactivity was
evaluated independently by two pathologists.
Immunohistochemical expression of VEGFR-1
and Snail were quantified by determining the
percentage of positive tumor cells at each inten-
sity scores: 1+ (absent), 2+ (weak), 3+ (moder-
ate) and 4+ (strong). The sum of these scores
gave a final score for every core: 1, 1.01~2;
2.01~3; 3.01~4, as reported elsewhere [12 ] .
Positive expression on the endothelial cells was
not counted. Staining of MMP-9 was quan-
tified by log-transformed integrated optical
d ensity as reported previously [2] .
●●Statistical analysis
For VEGFR-1, Snail and MMP-9, the cutoff
for definition of subgroups was the median
value of 2.3, 1.9 and 5.2, respectively [3]. The
normality test indicated that the raw data
has normal distribution and data were ana-
lyzed using SPSS15.0 and expressed as mean
± standard deviation. Differences between
means were compared using either a standard
two-sided independent t-test for two-group
comparisons or one-way analysis of variance
for multiple comparisons. The association was
assessed using Pearson correlation coefficient.
Survival curves were generated using Kaplan–
Meier method, and Log-rank test was used
to compare patients’ survival between sub-
groups. p < 0.05 was considered as st atistically
significant.
Results
●●VEGFR-1 expression in HCC cell lines &
surgical specimens
Western blot demonstrated that VEGFR-1
(180 kDa) was expressed in four HCC cell lines
including Hep3B, HepG2, SMMC7721 and
MHCC97H (Figure 1A). human umbilical vein
endothelial cells was used as a positive con-
trol [25]. Immunofluorescence analysis showed

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VEGFR-1 activation-induced MMP-9-dependent invasion in hepatocellular carcinoma RESEaRch aRticlE
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that VEGFR-1 was localized in the cytomem-
brane of SMMC7721 cells (Figure 1B, upper
panel). By contrast, the negative control with-
out addition of primary anti-VEGFR-1 anti-
body did not show specific staining (Figure1B,
lower panel). Immunohistochemical staining
was performed to determine the expression of
VEGFR-1 in the HCC tumor and peritumoral
tissues of surgical specimens. VEGFR-1 was
barely detectable in the hepatocyte of peritu-
moral tissues (Figure1Ci). However, VEGFR-1
expression was distinctly observed on the cell
membrane in the parenchyma (Figure 1Cii) and
also localized at the vascular endothelial cells
and bile duct epithelial cells in the stroma of
HCC tissues (Figure1Ciii).
Figure 1. VEGFR-1 protein expression in hepatocellular carcinoma. (A) Western blot shows
expression of VEGFR-1 protein in all four cell lines. HUVECs were used as a positives control.
(B) Immunoreactivity for the VEGFR-1 was clearly observed (arrow, upper panel). The negative
control was performed similarly without the primary antibody (lower panel). Scale bar: 50 μm.
(C)Immunohistochemical staining of VEGFR-1 was localized in hepatocytes of peritumoral tissues
(i), the cell membrane in the parenchyma (ii) and the vascular endothelial cell (upper arrow) and bile
duct epithelial cells (lower arrow) in stroma of hepatocellular carcinoma tissues (iii). Scale bar:50 μm.
HUVEC: Human umbilical vein endothelial cell.
HUVECs
Hep3B
HepG2
SMMC 7721
MHCC97H
kDa
-180
-43
VEGFR-1
VEGFR-1 DAPI Merged
β-actin
DAPI VEGFR-1
iiii
ii

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100
90
80
70
60
50
40
30
20
10
0
0
0 h
12
12 h
24
24 h
Time (h)
Relative cell migration (%)
70
60
50
40
30
20
10
0
Untreated group
Untreated group
Non-specific IgG-treated group
VEGF-B167-treated group
Untreated group
IgG
IgG
VEGF-B
VEGF-B
Invaded cells/HPF (n)
**
*
*

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Figure 2. VEGFR-1 activation enhances SMMC7721 cell migration and invasion (see facing
page). (A) Wound healing assay was performed to analyze the cell migration. Bars indicated
standard deviation; *p < 0.05 versus non-specic IgG group; **p = 0.01 versus untreated group.
(B) Photographs of cell migration at the point of 12 h and 24 h post-scratch. Scale bar: 100 μm.
(C)Matrigel invasive assay was performed to analyze the cell invasion. Data shown are representative
of three independent experiments. Bars indicated standard deviation; *p < 0.0001 versus nonspecic
IgG group. Scale bar:100 μm.
VEGFR-1 activation-induced MMP-9-dependent invasion in hepatocellular carcinoma RESEaRch aRticlE
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●●VEGFR-1 activation promotes cell
migration & invasion of SMMC7721 cell line
Having determined that VEGFR-1 is present in
HCC cell lines and tissues, we determined its role
in the tumor progression. We first tested if acti-
vation of VEGFR-1 by VEGF-B167 can enhance
the migration and invasion of SMMC7721 by
wound healing and cell invasion assays. At the
point of 12 h and 24 h postscratch, the relative
cell migration was 60.3 and 12.3%, respectively,
for the untreated group, 60.5 and 8.4%, respec-
tively, for the nonspecific IgG group, but 58.6
and 1.4%, respectively, for the VEGF-B167-treated
group. There was no significant increase in the
migration among these three groups in initial
12 h. However, the motility of the VEGF-B167-
treated cells was significantly increased compared
with the nonspecific IgG-treated cells after 24 h
(Figure2A & B).
Because the activation of VEGFR-1 appeared
to promote the migration of HCC cells, we next
assessed the effect of VEGFR-1 on invasion using
Matrigel Invasive assay. Cells were treated as
described in Material and methods and invasion
was determined at 48 h. As shown in Figure2C,
invasion of cells treated with VEGF-B167 were
significantly increased compared with that of
the cell treated with nonspecific IgG. These
data suggest that the activation of VEGFR-1 by
VEGF-B167 promotes the migration and invasion
of SMMC7721 cells.
●●VEGFR-1 activation induces MMP-9
expression in HCC cell lines
MMP-9 and MMP-2 are the major proteinases
for the proteolytic degradation of the ECM com-
ponents, leading to the cancer cell invasion [2]. In
HCC metastasis, MMP-9 possesses greater bio-
logical activity than MMP-2 [20,26]. Therefore, we
wondered whether VEGFR-1 activation-induced
cell invasion was due to the elevated expression
of MMP-9. As shown in Figure 3A, the level of
MMP-9 mRNA expression in the cell treated with
VEGF-B167 for 32 h was increased by 4.3-fold and
2.6-fold in SMMC7721 and HepG2 cells, respec-
tively. The expression level was declined at 48 h
post-treatment. Gelatin zymography assay showed
that MMP-9 gelatinolytic activity in the culture
medium from VEGF-B167-treated SMMC7721
cells was significantly higher than that in the non-
specific IgG group or untreated cells (Figure 3B,
left panel). However, MMP-2 activity was barely
detectable (Figure 3B, left panel). In contrast, in
the VEGF-B167-treated HepG2 cells, the activ-
ity of both MMP-9 and MMP-2 were increased
in comparison with the controls (Figure 3B, right
panel). The predominant band was detected at
92 kDa and 72 kDa corresponding to the proac-
tive form that becomes active during zymography.
Western blot indicated that the level of MMP-9
protein, detected as two bands at 92 kDa and
82 kDa, was increased in these representative
two cell lines (Figure 3B). Immunofluorescence
detection was performed to confirm the changes
of MMP-9. As shown in Figure 3C, the immu-
noreactivtity for MMP-9 in the cytoplasm of
both SMMC7721 and HepG2 cells was signifi-
cantly elevated in the VEGF-B167-treated group.
However, there were not significantly differences
in the immunoreactivtity for MMP-2 between
two groups of HepG2 cells (data not shown).
Taken together, these data indicated that MMP-9
expression and activity were increased in HCC
cells upon activation of VEGFR-1.
●●VEGFR-1 activation promotes
MMP-9-dependent Matrigel invasion
Our data have indicated that VEGFR-1 acti-
vation enhances the invasiveness accompanied
by an increase in expression of MMP-9 in
SMMC7721 cell line. To determine the specific
contribution of MMPs, especially MMP-9 in
this cellular invasion, a broad-spectrum phar-
macologic MMP inhibitor (GM6001), and
a functional blocking anti-MMP-9 antibody
(Ab-1) were used to block the proteinase activ-
ity. VEGFR-1 activation-induced Matrigel inva-
sion was completely blocked after addition of
GM6001, indicating that metalloproteinase
activity is required for the cellular invasion
(Figure 4A). To examine if MMP-9 is involved
in the cellular invasion, functional blocking

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5.0
4.0
3.0
2.0
1.0
0.0
0 h8 h24 h28 h32 h48 h
5.0
4.0
3.0
2.0
1.0
0.0
0 h8 h24 h28 h32 h48 h
MMP-9/β-actin
MMP-9/β-actin
SMMC7721
SMMC7721
SMMC7721
HepG2
HepG2
HepG2
VEGF-B167 treated
Non-specific IgG-
treated group
VEGF-B167-treated group
Non-specific IgG-treated
group
Untreated
group
Untreated
group
IgG IgG
VEGF-B VEGF-B
MMP-9 92 kDa
82 kDa
Fold Upper band
Down band
MMP-9
92 kDa
MMP-2
72 kDa
β-actin
1
1
1.26
1.04
3.58
1.85
1
1
1.53
1.12
2.46
1.24
Non-specific IgG group VEGF-B167-treated group
1
1
1
.
26
1.0
.0
4
4
3
.5
8
1.8
1.8
5
5
1
1
1
1
.
53
1.1
2
2
.4
6
1.2
4
DAPI MMP-9
DAPI
M
MP-
9
*
*

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Figure 3. VEGFR-1 activation upregulates the expression and activity of MMP-9 in hepatocellular
carcinoma cell lines (see facing page). (A) Relative expression of MMP-9 and β-actin was determined
by quantitative real-time PCR, normalized to non-specic IgG group, and arbitrarily set at 1.0. Bars
indicate standard deviation; *p<0.001 for both versus the zero time point, and the similar results
were obtained in three independent experiments. (B) The expression and activity of MMP-9 in
SMMC7721 and HepG2 cells was analyzed by western blot and gelatin zymography. Data shown are
representative of three independent experiments. (C) Immunouorescence detection of MMP-9 in
SMMC7721 (upper panel) and HepG2 cells (lower panel). Scale bar:50 μm.
VEGFR-1 activation-induced MMP-9-dependent invasion in hepatocellular carcinoma RESEaRch aRticlE
future science group www.futuremedicine.com
antibody against MMP-9 was used and the
results showed that abrogation of MMP-9 activ-
ity significantly reduced cell invasion by approxi-
mately 84% when compared with SMMC7721
cells treated with VEGF-B167 alone (Figure 4B).
These results support a key role for MMP-9 in
VEGFR-1 activation-induced invasion.
●●VEGFR-1 activation induces Snail
expression in HCC cell lines
Recent reports have shown that Snail promotes
HCC invasion by inducing EMT and/or upregu-
lating MMPs expression [2 ,3] . Moreover, Snail is
activated downstream of several reporters and bio-
active compounds including VEGFRs [1 2 , 27–29] .
Accompanied with activation of VEGFR-1, Snail
expression is significantly elevated in pancreas
cancer cells [4] . Thus, we examined the specific
role of Snail in VEGFR-1 mediated MMP-9
expression in HCC cells. The results showed
that mRNA level of Snail in SMMC7721 and
HepG2 were increased by 8.5-fold and 2.2-fold
after 28 h and 24 h of VEGF-B167 treatment,
respectively (Figure 5A). Western blot analysis also
indicated the Snail protein level was increased
after VEGF-B167 treatment (Figure 5B).
●●High expression of both VEGFR-1 & MMP-9
correlated with tumor metastasis
The results that activation of VEGFR-1 promotes
MMP-9 expression of in HCC cell lines suggest
that VEGFR-1 and MMP-9 might be involved
in the tumor invasion and metastasis. To test this
hypothesis, we investigated the clinical signifi-
cance of VEGFR-1 in HCC and its relationship
to MMP-9 and Snail expression by immunohis-
tochemistry ( Tabl e 1). The representative exam-
ples of VEGFR-1, MMP-9 and Snail staining
in HCC tissues and corresponding peritumoral
tissues were shown in Figure 6A. As expected,
VEGFR-1 was localized on the cell membrane;
Snail was localized in the nucleus, whereas the
MMP-9 was present in both cytoplasmic and
extracellular compartment. High expression of
VEGFR-1, Snail and MMP-9 was confirmed in
48.3, 53.3 and 45.3%, respectively, of all the cases.
High expression of VEGFR-1 was also associated
with a worse prognosis of HCC cases (p < 0.0001
for RFS and p = 0.023 for OS, respectively), and
there was also a trend toward a worse outcome
in patients with high expression of MMP-9
(p = 0.327 for RFS and p = 0.274 for OS, respec-
tively; Ta ble 1). HCC tissues with high expression
of VEGFR-1 also showed increased MMP-9 and
Snail expression with a Pearson correlation coef-
ficient of r = 0.232 (p = 0.036) and r = 0.418,
respectively (p < 0.001) (Figure 6B). To demon-
strate the prognostic significance of expression
pattern of VEGFR-1 and/or MMP-9 in HCC,
we divided the patients in four groups, I: high
expression of both VEGFR-1 and MMP-9, II:
low VEGFR-1 but high MMP-9 expression, III:
high VEGFR-1 but low MMP-9 expression, IV:
low expression of both VEGFR-1 and MMP-9,
and performed a Kaplan–Meier survival analy-
sis. The results showed that patients with high
expression of both VEGFR-1 and MMP-9 had
the worst prognosis when compared with other
groups (p = 0.008 for RFS and p = 0.128 for OS,
respectively) (Figure6C). Therefore, we divided
patients into groups I with high expression of both
VEGFR-1 and MMP-9 and group II without high
expression of both VEGFR-1 and MMP-9. Our
results showed that patients with high expression
of both VEGFR-1 and MMP-9 had a significantly
worse RFS and OS than the patients without high
expression of both VEGFR-1 and MMP-9 (p =
0.003 for RFS and p = 0.02 for OS, respectively)
(Figure 6C). Collectively, these results strongly sup-
port that induction of MMP-9 by VEGFR-1 acti-
vation contributes to HCC invasion and metasta-
sis, and it can be speculated a new model for caner
invasion (Figure 6D).
Discussion
In comparison with the well-established mecha-
nisms of EMT and angiogenesis, the impact of
VEGFR-1 mediated signaling on the expression
profile of proteolytic enzymes involved in inva-
siveness is not well understood. In this study, we

Future Oncol. (2015) 11(23)
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RESEaRch aRticlE Li, Zhu, Han, Ren, Liu & Qin
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confirmed that VEGFR-1 activation in HCC
cell lines can regulate the expression of MMP-
9, which might be a novel mechanism for HCC
invasion.
HCC usually expresses high level of VEGF/
VEGFR compared with the peritumoral tis-
sue [16 ,3 0 –3 1] . Among VEGFRs, VEGFR-
1, expressed in cancer cells, is particularly
interesting due to its direct tumor activation
via an autocrine stimulatory pathway [4,7,3 2].
Previously studies have shown that overexpres-
sion of VEGF-B (a sole ligand for VEGFR-1)
in cancerous tissues is associated with tumor
invasion and poor prognosis in HCC. In addi-
tion, isoform VEGF-B167 appears to be the clini-
cally dominant isoform of VEGF-B [33] . Using
VEGF-B167 as the ligand to activate VEGFR-1,
we found that VEGFR-1 activation can sig-
nificantly promote cancer cell migratory and
invasive ability in vitro. These results provided
experimental evidence for the clinical signifi-
cance of VEGF-B in HCC prognosis. In addi-
tion, these results are also supported by the in
vivo results showing that high level of VEGFR-1
was associated with poor prognosis of HCC
patients, which was consistent with the finding
in renal cell cancer [34 ] . However, the clinical
value of VEGFR-1 warrants further evaluation
in larger clinical trials.
Recently, a monoclonal VEGFR-1 blocking
antibody IMC-18F1 was used to block the func-
tion of VEGFR-1 to confirm that the changes in
invasiveness were mediated through VEGFR-1,
but not the aberrant expression of another VEGF
tyrosine kinase receptor or due to the activation
of neuropilin-1 in several studies [31,35] . However,
VEGFR-B167 is thought to be the sole ligand for
VEGFR-1. There are no significant effects of the
nonspecific IgG on the changes of invasiveness.
These two aspects confirmed that the elevation in
migration and invasion of HCC cells was caused
by VEGFR-1 activation rather than unspecific
stimulation.
MMP-9, which is expressed in many types of
human carcinomas including HCC, has been
closely associated with tumor invasion and poor
prognosis [20,36 –37] . Recent studies reported that
MMP-9 was significantly upregulated by many
growth factors, leading to the invasion a vari-
ety of solid cancers in vitro [11– 13] . In present
study, VEGFR-1 activation-to-MMP-9 signal-
ing pathway was investigated and confirmed as
a novel mechanism of HCC invasion in vitro
and in vivo. Additional, these findings were
reinforced by the results of immunohistochem-
istry on TMA showing that VEGFR-1 is posi-
tively correlated with the MMP-9 expression
in cancerous tissues. Moreover, the results that
Figure 4. VEGFR-1 activation promotes MMP-9-dependet Matrigen invasion by SMMC7721 cells. (A) Matrigel invasion by SMMC7721
cells was examined in the absence or presence of a broad-spectrum MMP inhibitor GM6001 (12.5 μmol/l), bars indicate standard
deviation; *p < 0.005, versus nonspecic IgG group. (B) Matrigel invasion by SMMC7721 cells was examined in the presence or absence
of function-blocking MMP-9 antibody Ab-1 (15 μg/ml). Bars indicated standard deviation; **p < 0.05 versus VEGF-B167-treated group. The
similar results were obtained in three independent experiments.
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
IgG VEGF-B IgG VEGF-B
+GM6001
+Ab-1
+Ab-1
+GM6001
IgG VEGF-B IgG VEGF-B
Invaded cells/HPF (n)
# of invaded cells/HPF
***

3153
VEGFR-1 activation-induced MMP-9-dependent invasion in hepatocellular carcinoma RESEaRch aRticlE
future science group www.futuremedicine.com
patients with high coexpression of VEGFR-1
and MMP-9 exhibited the worst clinical out-
come also suggest that overexpression of both
VEGFR-1 and MMP-9 may play a synergistic
role in the progression of HCC and can serve
as a prognostic marker for HCC.
Snail, one of the key regulators of EMT, is
involved in the process of VEGFR-mediated
cancer cell invasion [4 ,29] . Correlative stud-
ies have shown Snail also can induce MMP-9
expression at the level of mRNA and protein
in vitro [1 2 ,23,38] . In this study, we found that
a distinct increase of Snail expression was
detected in the cells with activation of VEGFR-
1. Additionally, a strong positive correlation of
them was also observed in HCC tissue. These
results indicated that Snail may participate in the
regulation of VEGFR-1 activation-to-MMP-9
signaling pathway. VEGFR-1 activation led to
the enhanced expression of EMT regulators
including Snail, Slug and Twist in pancreatic
cancer cells. Among them, Twist appears to be
regulated by VEGFR-1 activation [4] . However,
the disadvantage of the present study was that
only one regulator was detected. Further stud-
ies are required to analyze the other regulators
expression in responding to the activation of
VEGFR-1 and elucidate the exact role in this
pa t hway.
Conclusion
In conclusion, we provide evidence for a new,
stepwise signaling pathway from activation of
VEGFR-1 to MMP-9 and demonstrate the criti-
cal role of MMP-9 in VEGFR-1-mediated HCC
cell invasion. Based on the findings of this study,
VEGFR-1 is present and functional in HCC
cells and its activation significantly enhances the
Figure 5. VEGFR-1 activation promotes Snail expression in hepatocellular carcinoma cell
lines. (A)Relative expression of Snail and β-actin was determined by quantitative real-time PCR,
normalized to nonspecic IgG group, and arbitrarily set at 1.0. Bars indicated standard deviation;
*p < 0.001 for both versus the zero time point, and the similar results were obtained in three
independent experiments. (B) The expression of Snail was detected by western blot. Data shown are
representative of three independent experiments.
10.0
9.0
8.0
7. 0
6.0
5.0
4.0
3.0
2.0
1. 0
0.0
01 24824 28 32 0124
824 28 32
2.5
2.2
2.0
1. 8
1. 5
1. 2
1. 0
0.8
0.5
0.2
0.0
SMMC7721
SMMC7721
HepG2
HepG2
Snail/β-actin
Snail/β-actin
Untreated
group IgG VEGF-B
β-actin
β-actin
Snail
Snail
VEGF-B167 treated
Non-specific IgG-
treated group
VEGF-B167 treated
Non-specific IgG-
treated group
**
Time (h) Time (h)

Future Oncol. (2015) 11(23)
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RESEaRch aRticlE Li, Zhu, Han, Ren, Liu & Qin
future science group
4.0
3.5
2.5
1. 5
1. 0
2.0
3.0
4.0
3.5
2.5
1. 5
1. 0
2.0
3.0
1. 01.5 2.52.0 3.0 3.5 3.5 4.0 4.5 5.0 6.05.5
MMP-9(log)value
VEGFR-1
VEGFR-1
Snail
VEGF-B
VEGFR-1 activation
MMP-9
Cancer invasion
Months after surgery
Months after surgery Months after surgery
Months after surgery
1. 0
0.8
0.6
0.4
0.2
0.0
1. 0
0.8
0.6
0.4
0.2
0.0
1. 0
0.8
0.6
0.4
0.2
0.0
1. 0
0.8
0.6
0.4
0.2
0.0
10 20 30 40 50 60 10 20 30 40 50 60
10 20 30 40 50 60
10 20 30 40 50 60
Recurrence-free survival (%)
Overall survival (%)
Recurrence-free survival (%)Overall survival (%)
I (n = 19)
II (n = 16)
III (n = 21)
IIV (n = 23)
I (n = 19)
II (n = 16)
III (n = 21)
IIV (n = 23)
Non-high coexpression
VEGFR-1/MM-9 (n = 60)
VEGFR-1
Case83 Case88
Hepatocellular carcinoma tissuesPeritumoral tissues
Case83
MMP-9
Snail
Non-high coexpression
VEGFR-1/MMP-9 (n = 60)
High coexpression
VEGFR-1/MM-9 (n = 19)
High coexpression
VEGFR-1/MMP-9 (n = 19)
+
+
++ ++
+
++
+
+
+
++
+
+
++
+
+
++
+
+
+
++
++
+
+++
++
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

3155
Figure 6. High coexpression of VEGFR-1/MMP-9 in hepatocellular carcinoma patients indicates
the worst outcome and a proposed model of VEGFR-1 activation promoting invasion via
induction of MMP-9 (see facing page). (A) Immunohistochemistry staining of VEGFR-1, MMP-9
and Snail in two representative patients with high coexpression of three proteins (Case83) and low
for three proteins (Case88). The black arrows indicated the expression of VEGFR-1; the blue arrows
indicated the expression of MMP-9; the red arrows indicated the expression of Snail. Scale bars,
50μm. (B) Comparison of the recurrence-free survival (upper left panel) and overal survival (lower
left panel) of patients categorized by VEGFR-1/MMP-9 immunohistochemistry result. Dierence of
recurrence-free survival (upper right panel) and overall survival (low right panel) in hepatocellular
carcinoma patients with or without high coexpression VEGFR-1/MMP-9. (C) Pearson correlation
test was performed to analyze the relationship between the VEGFR-1 and Snail (upper panel) and
between VEGFR-1 and MMP-9 (lower panel). (D) A proposed model of VEGFR-1 activation induced
invasion via MMP-9.
VEGFR-1 activation-induced MMP-9-dependent invasion in hepatocellular carcinoma RESEaRch aRticlE
future science group www.futuremedicine.com
invasiveness of cell lines through its regulation of
MMP-9. This study provided a novel pathway
associated with the progression of HCC induced
by VEGFR-1 activation.
Future perspective
It is significant to further confirm the role of
Snail in the novel pathway associated with
VEGFR-1 activation in the future. It is necessary
to verify whether VEGFR-1 could be a target for
the treatment of HCC invasion and metastasis.
Author contributions
T Li and C Qin conceived and designed the study, con-
ducted most of experiments and drafted the manuscript.
Y Zhu and L Han conducted some of the experiments,
supervised the data collection and analysis, interpreted
data and assisted in writing the manuscript. W Ren and
H Liu c onducted some of the experiments and interpreted
the data.
Financial & competing interests disclosure
This study was supported by National Nature Science
Foundation of China (numbers 81472685), Shandong
Outstanding Youth Science Fund (BS2013YY037). The
authors have no other relevant af filiations or financial
involvement with any organization or entity with a finan-
cial interest in or financial conflict with the subject matter
or materials discussed in the manuscript apart from those
disclosed.
No writing assistance was utilized in the production of
this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate
institutional review board approval or have followed the
principles outlined in the Declaration of Helsinki for all
human or animal experimental investigations. In addi-
tion, for investigations involving human subjects,
informed consent has been obtained from the participants
involved.
EXEcUtiVE SUMMaRY
● Epithelial–mesenchymal transition and extracellular matrix degradation are critical for the initiation and progression of
tumor invasion.
● VEGFR-1 activation can induce EMT by decreasing the expression of E-cadherin in hepatocellular carcinoma (HCC).
However, the impact of VEGFR-1 activation on the expression prole of proteolytic enzymes involved with HCC
invasion is unknown.
● In this study, we found that activation of VEGFR-1 in HCC cell lines can signicantly increase the expression and
activity of MMP-9, and then promoted the cell invasion. Functional blockage of MMP-9 inhibited tumor cell invasion,
suggesting that VEGFR-1-induced cell invasion is dependent on the function of MMP-9.
● Snail, an epithelial–mesenchymal transition regulator induced by VEGFR-1, was increased by VEGFR-1 activation,
indicating that Snail is involved in the regulation of the MMP-9 expression and cell invasion.
● High coexpression of VEGFR-1 and MMP-9 in HCC, which is associated with the patient tumor invasion and metastasis,
is predictive of a worse clinical outcome.
● These data showed a novel VEGFR-1 activation-to-MMP-9 signaling pathway that promotes HCC invasion.

Future Oncol. (2015) 11(23)
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RESEaRch aRticlE Li, Zhu, Han, Ren, Liu & Qin
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