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Acta Biochim |
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doi: 10.1093/abbs/. |
TGF-b promotes invasion and metastasis of gastric cancer cells
by increasing fascin1 expression via ERK and JNK signal pathways
Hua Fu†, Zhongliang
Hu†, Jifang
Wen*, Kuansong Wang, and
Ying Liu
Department of Pathology,
contributed equally to this work.
*Correspondence address. Tel/Fax: +86-731-2650410;
E-mail: [email protected]
Transforming growth factor-b (TGF-b) is involved in actin cytoskeleton reorganization and tumor progression.
Fascin1, an actin-binding protein, increases cell
invasiveness and motility in various transformed cells. To determine whether
fascin1 is an important mediator of the tumor response to TGF-b, we applied the
small interfering RNA (siRNA) technique to silence
fascin
Keywords gastric carcinoma;
transforming growth factor-b1; Fascin1; RNA interference
Received: March 11, 2009 Accepted: April 15, 2009
Introduction
It is well known that transforming growth factor-b1 (TGF-b1) plays an important
role in tumor progression and actin cytoskeleton
reorganization [1–4]. On the one hand,
cancer cells over-expressing active TGF-b1 increased metastatic
ability [5]; on the other hand, targeting of TGF-b signaling prevented
metastasis in many cancers such as breast, prostate, etc. [6,7].
Moreover, clinical studies showed the positive correlation of TGF-b1 expression with
lymph node metastasis and poor prognosis in gastric carcinoma [8,9]. We have previously also reported that TGF-b1 significantly
promoted the invasion and metastasis of gastric cancer (GC) cells SGC7901 and
BGC823 [10]. However, the underlying mechanism of TGF-b1 responsible for
regulating tumor invasion and metastasis has not been fully elucidated yet.
Fascin1, a highly conserved actin-binding protein,
plays an important role in the organization of several types of actin-based structures [11]. Over-expression of fascin1 has
been previously reported in many cancers [12–15]. In oral squamous cell carcinoma, overexpression of fascin might
facilitate the tumor aggressiveness [16]. In addition, a high level of fascin in neoplastic cells is
associated with progressive high-grade or great metastatic potential tumors [13,15–21]. On the basis of
the facts that actin cytoskeleton is involved in TGF-b1-induced tumor
progression and metastasis [4] and that fascin1 expression is up-regulated by
TGF-b
Materials and Methods
Cell culture
GC cell line MKN45 was purchased from Type Culture Collection of the
Inhibitor study
The specific protein kinase inhibitors SB203580,
SP6001125, and PD98059 can inhibit the phosphorylation
of p38, JNK, and ERK, respectively, and then block the downstream pathway of mitogen-activated protein kinase
(MAPK) [24,25]. SB20358, SP6001125, and PD98059 (Sigma,
RNA interference
The targeting sequences corresponding to the shRNA
for fascin1 were selected from GenBank accession No. NM_003088. The shRNA-expression
pDNAs driven by human U6 promoter were constructed
from pGenesil-1 vector (Genesil Biotechnology,
siRNA transfection
MKN45 cells were plated into six-well plates (Costar,
Western blotting analysis
Total proteins were measured using BCA protein assay kit (Pierce,
Rockford, USA) according to the manufacturer’s protocol. Fifty micrograms of total
proteins were electrophoresed by 10% SDS–PAGE and transferred to a nitrocellulose membrane. The nitrocellulose
membrane was then incubated with blocking buffer [phosphatebuffered
saline (PBS) containing 5% non-fat milk] for 2 h at room temperature and with
primary antibodies overnight (anti-fascin1, 1:400 and anti-b–actin,
1:1000). The membrane was washed three times with PBS, and then incubated with
secondary antibody for 2 h at room temperature (1:1000). Protein was detected
using 3,3‘-diaminobenzidinetetra-hydrochloride.
b–Actin was used as an internal control.
Quantification of bands was performed as described above.
Cell invasiveness assay
The transwell chamber (
Cell chemotaxis assay
Chemotaxis assay was performed with transwell chambers
containing 8 mm pores [27]. Eight hundred microliters of RPMI
1640 containing 10 mg/ml fibronectin were placed in the lower
compartment of the transwell chamber. Tumor cells (1 ´ 105/well) pre-treated
without or with TGF-b1 (10 ng/ml) were added into the upper
compartment and incubated for 6 h, and then the migrated cells were counted as
described above [10].
Cell adhesion
Each well of the 96-well plates was coated with 50 ml of Matrigel,
In vivo experiments
Thirty BALB/c nude mice aged 4–6 weeks were purchased
from Shanghai Laboratory Animal Company (
Statistical analysis
Results were presented as mean±SD.
One-way ANOVA was used for statistical analysis. In all statistical
comparisons, P< 0.05 was used to indicate a statistically significant difference.
Results
TGF-b1 induced the expression of fascin1
As shown in Fig. 1(A), GC cells treated with TGF-b1 induced the expression of fascin1 as
demonstrated by western blotting analysis. Compared with corresponding control,
the expression of fascin1 was increased 2.1 or 2.16 folds after 24 or 48 h
treatment, respectively.
Fascin1 shRNA effectively
suppressed fascin1 expression
To test the inhibitory efficiency of fascin-1 shRNAs,
fascin1 expression level was tested. Fascin1 expression level was efficiently
reduced by 64.2±2.1% with CLONE1
(pGen-1- fascin
Fascin1 siRNA attenuated the
TGF-b1-induced invasiveness
To investigate whether fascin1 is an important mediator in the TGF-b1-induced tumor
invasion, non-transfected (NT),
pGen-1-con-transfected (CON), and siFascin1-transfected (siFascin1) cells with
or without TGF-b1 (10 ng/ml) treatment were used in transwell invasion assays. The numbers of cells penetrating
through the membrane were counted. Compared with untreated NT (49±5) and CON (46±6) cells, the siFascin1 cells showed decreased invasiveness (19±5) (P< 0.05). TGF-b1 significantly promoted the invasiveness of NT (110±6) and CON (107±5) cells, but did not
have any effect on the invasiveness of siFascin1 cells (24±6) when compared with
untreated siFascin1 cells (P< 0.05) [Fig. 2(A)].
Fascin1 siRNA reduced the chemotactic ability induced by TGF-b1
To evaluate whether the suppression of fascin1 expression would alter the
TGF-b1-induced cell migration, the motility of cells to fibronectin
was assessed by counting the numbers of cells migrated through the membrane.
The siFascin1 cells (18±5) exhibited reduced motility compared with NT (40±6) and CON (41±7) cells (P< 0.05). Cells treated with 10 ng/ml of TGF-b1 showed increased chemotactic activity to fibronectin
in NT (100±5) and CON (98±6) cells, but not in siFascin1 (22±7) cells when compared
with untreated siFascin1 cells (P< 0.05) [Fig. 2(B)].
Fascin1 siRNA attenuated the
adhesiveness induced by TGF-b1
Compared with NT and CON cells, siFascin1 cells possessed lower adhesion
potential (P< 0.05). Pre-treatment with TGF-b1 promoted the adhesiveness of NT and CON
cells to Matrigel and fibronectin
(P< 0.05). However, TGF-b1 could not notably promote the
adhesiveness of siFascin1 cells when compared with untreated siFascin1 cells (P< 0.05) (Table 2).
Fascin1 siRNA inhibited in vivo tumor metastasis induced
by TGF-b1
To further investigate whether the suppression of fascin1 expression would
alter the TGF-b1-induced tumor cell migration in vivo animal model, cells were injected into the abdominal cavity of nude mice and
the numbers of tumor colonies were counted. As shown in Fig. 3, without TGF-b1 pre-treatment, the
siFascin1 cells (2.1±1.3) exhibited lower metastasis than NT (5.2± 1.5) and CON (4.9±1.2) cells (P< 0.05). TGF-b1 pretreatment could increase liver metastasis in NT (23± 1.6) and CON (21±2) cells (P< 0.05). However, TGF-b1 treatment could not promote the
metastatic potential in siFascin1 cells (2.2±1.1) when compared
with untreated siFascin1 cells (P< 0.05) (Fig. 3).
Effects of Smad2 and Smad4 siRNA
on the fascin1 expression
To address the signaling pathway by which TGF-b1 increases the
expression of fascin1, TGF-b1-induced fascin1 expression was detected
by western blotting analysis after silencing Smad2 and Smad4. As shown in Fig. 4(A,B), the expression levels of Smad2 and Smad4 were significantly suppressed (P< 0.05) by corresponding Smad siRNA,
but the reduction of Smads did not alter the fascin1
expression (PSmad2 = 0.352; PSmad4 = 0.495).
TGF-b1 regulated fascin1 expression through
JNK and ERK pathways
TGF-b1 also activates MAP kinase pathways, mainly
JNK, ERK, and p38 pathways [28,29]. To investigate
whether TGF-b1 regulates the fascin1 expression through MAPK pathway, we treated GC
cells with the specific inhibitors, SB203580, SP6001125, and PD98059, respectively.
As shown in Fig. 4(C), PD98059 and SP600125 decreased the TGF-b1-induced fascin1
expression (by 78% and 75%, respectively), but SB203580 had no effect on
fascin1 expression as DMSO did in control cells. To further demonstrate that
JNK and ERK pathways were activated by TGF-b1, we examined the
expression of p-JNK, p-ERK, and p-p
Discussion
TGF-b plays key roles in development and carcinogenesis. TGF-b1 expression is well
correlated with actin cytoskeleton formation,
increased tumor invasiveness, and cell motility [4,30,31].
A previous study showed that TGF-b participated in
invasion and metastasis by modulating the ability of cells in invading
extracellular matrix and basement membrane [32]. In agreement with our previous
data, increased invasiveness, chemotactic, and
adhesion potential in TGF-b1-treated GC cells were further confirmed
in present study.
Fascin1, a key actin-binding protein, was first
identified in the extracts of unfertilized sea urchin eggs [11,33]. Later, it has been found to be expressed in tumor
tissue [12–21]. More studies show that downregulation of fascin expression in esophageal squamous
cell carcinoma led to a decreased motility and invasiveness [16,34]. Significant correlations between lymph node metastasis
and the fascin expression level in pulmonary adenocarcinomas have also been noted [35]. Likewise, the fascin expression in gastric carcinoma is associated with
lymph node metastasis [13]. Consistent with these reports, our study further
demonstrated that inactivation of fascin1 gene by siRNA technique resulted in a decreased
motility and metastasis of GC both in vivo and in vitro, suggesting that fascin1 contributed to the invasion and metastasis of
gastric carcinoma. The up-regulation of fascin1 expression by TGF-b
expression in GC cells have not been defined, although intracellular mediators
known as Smads are indispensable for many of the
responses to TGF-b [36]. To determine whether TGF-b1-induced fascin1 is
mediated by Smads pathway, we knock down the
expression of Smad2 and Smad4 with corresponding Smad
SiRNA. Results showed that the significant
suppression of Smad did not alter the fascin1
expression, suggesting that Smad signal pathway was
not involved in TGF-b1 induction of fascin1. Accumulating evidence suggests that TGF-b signals could act
through other pathways [28,29]. Among them, MAPK has
been identified as a key signaling pathway in response to TGF-b [28,36,37].
In the present study, MAPK pathway-specific inhibitors SB203580, SP6001125, and
PD98059 were used to assess if this pathway is involved in TGF-b1-induced fascin1
expression. The results showed that PD98059 and SP600125 effectively suppressed
fascin1 expression induced by TGF-b; however, p38 inhibitor SB203580 did not.
These data implied that TGF-b1 increased fascin1 expression through JNK
and ERK signaling pathway.
It should be noted that fascin was a target of
the b–catenin pathway in the invasive progress of
colon cancer [38]. Therefore, it is possible that TGF-b1 might increase the
activity of b–catenin in the invasive GC. For this reason, we
suppose that TGF-b1 and the Wnt pathway may work together in GC.
In conclusion, this study for the first time revealed that TGF-b promotes invasion and
metastasis of gastric carcinoma cells MKN45 by mediating fascin1 expression via
JNK and ERK pathways.
Acknowledgement
We thank Prof. Weijun Cai for generous assistance in preparing the
manuscript.
Funding
This work was supported by a grant from the Research Fund for the Doctoral
Program of Higher Education of China (No. 20060533007) and partly by a grant
from the Innovation Project of Central South University (No. 2340-76209).
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