Download HGF

Hepatocyte growth factor increases the invasive potency of human prostate cancer
PC-3 cells via ERK/MAPK pathway
Abstract
Objectives:
Hepatocyte
growth
factor
(HGF)
has
been
implicated
in
epithelial-mesenchymal transition (EMT) in many cancers. However, No documented
evidence has been shown in prostate cancer. In this study, the effect of HGF on EMT,
the invasive potency and possible molecular mechanism involved were investigated in
human prostate cancer.
Methods: The study was conducted from January to July 2013 in affiliated Beijing
Anzhen Hospital of Capital Medical University. PC-3 cells were treated with HGF at
different concentrations over different times points. The EMT associated proteins and
ERK/MAPK pathway proteins were examined by western blot. RT-PCR and western
blot measured C-met expression. MTT, Wound-healing, Transwell and Soft agar
assay observed the influence of HGF on proliferation, migration, invasion,
tumorigenecity of PC-3 cells.
Results: EMT-like changes were observed in PC-3 cells after treatment of HGF, and
these changes could be blocked by knockdown of C-met by siRNA. EMT-like changes
improved the invasive potency of PC-3 cells. Further study indicated that
HGF-mediated activation of C-met played an important role in the EMT-like changes.
ERK, phospho-ERK and Zeb-1 were involved in EMT in prostate cancer.
Conclusion: HGF-mediated induction of EMT increases the invasive potency of
human prostate cancer PC-3 cells via ERK/MAPK pathway.
Key words: Hepatocyte growth factor, epithelial-mesenchymal transition, prostate
cancer, ERK/MAPK pathway, Zeb-1
Introduction
Prostate cancer (PCa) is the second most commonly diagnosed form of cancer and
the sixth leading cause of cancer-related deaths among men worldwide [1].The most
mortality in PCa results from the metastasis of cancer cells to secondary sites,
particularly bone. Some researches reported 80% of patients had bone metastasis at
autopsy and about 80% of patients dye from bone metastasis of PCa [2, 3].But the
mechanisms leading to the metastasis of PCa cells keep unknown. In recent years,
Epithelial-Mesenchymal Transition (EMT) in tumor aggressiveness is now widely
accepted to interpret this question.
Originally, the definition of EMT was reported in embryology. It was identified to
participate into embryogenesis as a crucial differentiation and morphogenetic
process. But now, EMT has been well proved in tumor progression and metastasis [4].
When EMT occurs, cancer cells loses their epithelial characteristics and acquires
mesenchymal properties simultaneously, including fibroblastoid morphology,
characteristic gene-expression changes, increased potential for motility, even though
obtains some cancer stem-cell characteristics. These changes promote cancer cells
invasion, metastasis, and resistance to chemotherapy [5-7].
Accumulating evidence has revealed that EMT could be trigged by many factors.
These factors, including transforming growth factor beta (TGFβ), epidermal growth
factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF),
platelet-derived growth factor (PDGF), insulin-like growth factor
(IGF)[8],hypoxia[8,9], microRNA[10], could induce cancer cells EMT via different
signaling pathways , for example, Wnt, Hedgehog, Notch, etc[11,12].
In this article, we investigated the relation between HGF and EMT in prostate
cancer. Other’s researches have shown the higher plasma level of HGF in prostate
cancer patients is associated with an advanced stage of malignancy and a poor
prognosis [13, 14]. Maybe the effect of HGF on occurrence of EMT in the cancer
cells is one of important reasons. However, the mechanisms by which HGF induces
EMT are not yet well understood. So our research selected human prostate cancer
PC-3 cell line as experimental object. Based on our and others’ previous studies,
among all prostate cancer cell lines, PC-3 cells are EMT negative [15-18], and C-met
positive expression [19]. We utilized HGF to induce PC-3 cells EMT to increase its
metastatic potency, and debated its possible signaling pathway.
Materials and Methods
The study was conducted from January 2013 to July 2013 in affiliated Beijing
Anzhen Hospital of Capital Medical University. PC-3 cells were maintained in
DMEM medium (Gibco, USA) supplemented with 10% (V/V) fetal bovine serum
(Gibco, USA) and incubated at 37℃ with 5%CO2. Cells were treated with
recombinant human HGF (Sigma, USA) at different concentrations (20, 40, 60ng/ml,
respectively) over different time periods (12, 24, 36 hours, respectively) after
starvation for one night.
C-met siRNA or control siRNA plasmids (Santa Cruz, USA) were transfected into
PC-3 cells using Lipofectamine 2000 transfection reagent (Invitrogen, Canada)). 24
hours after transfection, stable transfectants were selected in puromycin (Life
Technologies, USA) at a concentration of 10 mg/ml. Then, the selection medium was
replaced every 3 days. After 2 weeks of selection, clones of resistant cells were
isolated. Cells were treated with recombinant human HGF as above describe.
5×103 cells/0.2 ml was stimulated in 96-wells plates with HGF (60ng/ml) for 0, 24,
48, 72 hours. Cultures were incubated with thiazolyl blue tetrazolium bromide (MTT,
5 mg/ml) for 4 hours, after which the metabolic product was dissolved in 200μl
buffered DMSO and measured at 570 nm with microplate reader.
Total proteins from cells were extracted and 30μg clarified protein lysates were
electrophoretically resolved on denaturing SDS-PAGE, and electro-transferred onto
nitrocellulose membranes. The immunoblots were incubated in 3% bovine serum
albumin, 10mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, and 0.1% Tween-20 at room
temperature and probed with primary and appropriate secondary antibodies (Santa
cruz, USA and Cell signaling, USA). Imaging was obtained by chemiluminescence
method.
Total RNA was isolated from cells using the Trizol reagent (Invitrogen, USA). The
RNA was reverse-transcribed into cDNA using oligo (dT) primers and AMV Reverse
Transcriptase (Takara, Japan).10 μL of cDNA was used for PCR in a final reaction
volume of 50 μL. The human C-met primers: sense 5′-GTTTCCCAATTTCTGACC-3′
and antisense 5′-TATATCAAAGGTGTTTAC-3′, the product length was 516 bp. The
β-actin primers: sense 5′-TGGGCATGGGTCAGAAGGAT-3′ and antisense 5′-AAGCATTTGCGGTGGACGAT-3′, the product length was 991bp. DNA amplification
conditions included an initial 5 min denaturation step at 95ºC and 30 cycles of 30s at
95ºC, 30s at 60ºC and 40s at 72ºC, followed by a final elongation of 7 min at 72ºC.
The RT-PCR samples were electrophoresed on a 1.5% agarose gel and stained with
ethidium bromide (0.5μg/mL). The gels were then photographed under ultraviolet
transillumination.
Cells were seeded in 6-well plates and grown to 60-70% confluence. Cells were
then incubated in serum free medium overnight, and were treated with HGF
(60ng/mL). Before adding HGF, 2-mm scratches were made in the confluent cell
monolayer with a 200μl tip. Cell migration into the denuded areas was assessed 12
and 24 hours after treatment by optical microscope.
8μm polycarbonate filter (Millipore, USA) were coated with 50 μg/cm2 of
reconstituted Matrigel (Sigma, USA). 5×103 cells in 300μl serum-free growth medium
were seeded into the upper chamber. Cells were incubated in normoxia condition and
allowed to migrate toward complete growth medium for 24h and 48h, non-invading
cells were removed mechanically using cotton swabs, and the cells on the lower
surface were then counted microscopically.
Cells were re-suspended in 2 ml of top agar medium (DMEM containing 0.4%
low-melting agarose and 10% FBS), and then quickly overlaid on 2 ml of bottom agar
medium (DMEM containing 0.8% low melting agarose and 10% FBS) in 6-well
culture plates. After 2-3 weeks, Colonies larger than 0.1 mm in diameter were scored
as positive. Colony-formation efficiency was counted under light microscopy.
All values in this study were expressed as the mean ± standard deviation (SD) and
representative of an average of at least three independent experiments. t-test was used
for statistical analyses. P value of less than 0.05 was considered as statistical
significance.
Results
HGF induced EMT-like change in PC-3 cells
Down-regulation of epithelial markers and up-regulation of mesenchymal markers
are the characteristic change of EMT which are associated with the scattering-like
growth of cancer cells. These changes render cancer cells ability of cell-cell
dissociation, cell spreading, and motility. In this step, Western blot showed that HGF
could up-regulated the expression of E-cadherin and down-regulated the expression
of Vimentin in time- and dose-dependent patterns. PC-3 cells acquired the stable
EMT-like changes after 60ng/ml HGF was incubated for 36 hours (Figure 1A), which
were not manifest at other gradient of time and concentration. The changes lasted 7
days after the withdrawal of HGF (Figure 1B). These results indicated that, HGF acts
as an inducer, promoting the changes of EMT markers in PC-3 cells and this
induction is reversible. Therefore, PC-3 cells treatment of HGF (60 ng/ml) for 36 h
was used in the subsequent experiments.
Figure 1 HGF induced EMT-like changes in PC-3 cells. Compared with untreated
PC-3 cells (control), HGF (60ng/ml) induced the down-regulation of E-cadherin and
up-regulation of Vimentin by time- and dose-dependent manner (A). When suspended
the 60ng/ml HGF induction for 7 days, the expression of E-cadherin restored (B).
The change of C-met in PC-3 cells after HGF induction
To investigate the role of HGF in induction of EMT-like changes in PC-3 cells, the
mRNA and proteins of C-met, HGF receptor, were examined. The RT-PCR showed
that the transcription of C-met was increased after 36 h of HGF treatment (Figure 2A).
C-met was activated by HGF-mediated phosphorylation which in turn regulate the
down-stream of target genes. Western blot indicated that both the expression of c-met
and p-c-met (phosphorylated C-met) were promoted (Figure 2B). These results
suggested that HGF induced the up-regulation of C-met at transcriptional and protein
levels.
Figure 2 HGF increased the expression of C-met. PC-3 cells were treated with
60ng/ml HGF and the expression of C-met were examined at mRNA (A, treated for 0,
12, 24, 36h) and protein level (B, treated for 36h).
HGF increased the invasive potency of PC-3 cells
We further examined the influence of HGF on the invasive potency of PC-3 cells.
MTT assay showed that the proliferation of cancer cells was increased accompanied
by doubling time decreased (Figure 3A). In addition, increased number of developed
tumors was observed in HGF-treated cells which were indicated by the Soft agar
assay (Figure 3B). HGF-treated PC-3 cells had stronger migratory capacity
compared with untreated cells which was demonstrated by wound-healing assay
(Figure 3C). Meanwhile, Transwell test displayed more invasion of PC-3 cells into the
underneath surface of the insert through collagen (Figure 3D and E). Taken together,
these results demonstrated that HGF played an important role in promoting the
potency of invasion in PC-3 cells.
Figure 3 The influence of HGF on cell proliferation, tumorigenecity, migration and
invasion. (A): MTT assay showed that HGF stimulated PC-3 cells proliferated.
(B):Soft agar assay showed the treated PC-3 cells have stronger tumorigenecity
(t=2.773, P＜0.05). (C): Wound-healing assay displayed that the treated cells
acquired the stronger migration potential. (D, E):In contrast to PC-3 cells(control),
Transwell test illustrated that HGF improved the invasion, the difference was
statistical significance (t=2.481 and 2.532, P＜0.05 at 24 and 48h, respectively).
The role of ERK/MAPK signaling pathway in EMT induced by HGF
To investigate the molecular mechanism involved in EMT induced by HGF, The
change of ERK/MAPK after HGF induction in PC-3 cells was determined. Western
blot result indicated the increased expression of ERK and phosphor-ERK after HGF
treatment. Moreover, Zeb-1, a direct suppressor of E-cadherin, was elevated by HGF,
demonstrating the ERK/MAPK signaling pathway was involved in the induction of
EMT by HGF (Figure 4).
Figure 4 ERK/MAPK pathway is involved in induction of EMT by HGF. PC-3 cells
were treated with HGF (60 ng/ml) for 36h and expression of ERK, phospho-ERK and
Zeb-1 were examined by western blot.
C-met siRNA inhibited EMT-like changes induced by HGF
We observed the influence of knockdown of C-met by siRNA on EMT-like changes
induced by HGF. The results showed that showed that transfection of C-met siRNA
inhibited the expression of C-met in PC-3 cells, compared with PC-3 cellsb and
control-siRNA cells (Figure 5A). Under the HGF induction, PC-3 cells and
control-siRNA cells showed down-regulation of E-cadherin and up-regulation of
Vimentin in contrast to C-met siRNA cells, this demonstrated the role of C-met in
EMT-like changes induced by HGF (Figure 5B). ERK/MAPK pathway had mimic
changes. Through the action of HGF, expression of ERK, p-ERK and Zeb-1
up-regulated in PC-3 cells and control-siRNA cells but C-met siRNA cells (Figure 5C).
All data suggested that HGF induced PC-3 cells EMT-like changes in C-met
dependent manner.
Figure 5 The role of C-met in EMT induced by HGF. (A): Western blot showed the
expression of C-met in PC-3 cells (a), control siRNA cells (b), but reduction in C-met
siRNA cells (c). (B): Knockdown of C-met inhibited EMT-like changes induced by
HGF (60ng/ml) in C-met siRNA cells (c) compared with PC-3 cells (a) and control
siRNA cells (b). (C): Under induction of HGF, Up-regulation of ERK, p-ERK and
Zeb-1 in PC-3 cells (a) and control siRNA cells (b) but C-met siRNA cells (c).
Discussion
HGF binds to C-met, the receptor of C-met, and activates C-met through
auto-phosphorylation of C-met, which in turn triggers the transcription of
downstream target genes. In normal conditions, HGF/ C-met pathway regulates tissue
and organ regeneration, and plays an important role in modulation of cell
morphology and induction of both angiogenesis and lymphangiogenesis [20].
Accumulating evidences show that HGF stimulates proliferation, migration and
invasion in a variety of cancers, including colon, stomach, lungs, bladder and
prostate cancers [14]. In prostate cancer, the level of HGF is elevated in the serum of
patients with prostate cancer and is associated with metastatic disease independent of
PSA levels and age, and patients have a decreased overall survival rate [21, 22].In
addition, Duhon and his colleagues indicated that exposure of DU145 prostate tumor
cells to HGF stimulates the PI3K and MAPK pathways, leading to increased
scattering, motility, and invasion, which was prevented by addition of EGCG [23].
Although HGF accelerates the progress of prostate cancer, but the mechanisms
involved are not clear. The relationship between HGF and EMT has been shown in
different cancer models. However, no such research in prostate cancer has been
reported. A study has been shown that EMT is induced by HGF in DU145 cells [24],
but it should be noted that DU145 cells have been confirmed to be EMT-positive cells
on the basis of our and others’ research [15, 25, 26]. Therefore, we investigated the
effect of HGF on induction of EMT in PC-3 cells.
Properties typical for EMT comprise down-regulation of epithelial markers like
E-cadherin, and up-regulation of mesenchymal markers like Vimentin, N-cadherin
and α-smooth muscle actin (SMA) [27, 28]. Down-regulation of E-cadherin is often
regarded as a key step triggering EMT [29]. Because intercellular adhesions are
critical for the maintenance of epithelial phenotype, E-cadherin is essential for
adherent junctions, whereas its down-regulation would result in the loss of cell
polarity and abnormal differentiation, and finally facilitates EMT [30, 6].
In our research, the treatment of PC-3 cells with HGF resulted in EMT-like changes
that were demonstrated by down-regulation of E-cadherin and up-regulation of
Vimentin, which mean that HGF induced EMT phenotype in PC-3 cells and this
induction effect presented time- and concentration-dependent manner. The further
studies showed that the stimulation of HGF enabled cancer cells possess increased
potency of proliferation, migration, invasion and tumorigenecity. But we found that
the changes were reversible after withdrawal of HGF for 7 days, similar to EMT
induced by TGF-β1 [31]. These results suggested that the growth factors are required
to maintain the EMT phenotype. It is well known that many growth factors, including
FGF, IGF, TGF-β, HGF, secreted from the stromal cells [32]. Under the continue
stimulation of these growth factors, the cancer cells acquire stable EMT phenotype.
Therefore our results also highlight that the bidirectional interactions and
co-evolution of tumor-stroma in cancer progression.
Then, the effect of HGF on its receptor, c-met, was determined at the transcription
and protein levels. Over-expression of C-met has been found in most human cancers
[33, 34].Our study demonstrated that the expression of C-met was promoted due to
the HGF-dependent transcriptional up-regulation. This result is consistent with
Baccoccio’s study in prostate cancer [35]. It should be noted that there was marked
elevation of phospho-c-met after HGF, which identified the effect of HGF on the
activation of C-met in prostate cancer. When expression of C-met was blocked by
siRNA, C-met siRNA cells didn’t display EMT-like changes under induction of HGF in
the present study. These results demonstrated that HGF induced EMT development of
PC-3 cells by C-met-dependent manner.
Many of the oncogenic effects of HGF/ C-met are mediated by a complex
downstream signaling network, the most prominent of which are the MAPK and
PI3k/Akt pathways [36].In the present study, ERK was phosphorylated by HGF, and
PC-3 cells express high basal level of phospho-ERK and ERK. But these changes
could be blocked by knockdown of C-met by siRNA. The data suggested that the
functional expression of ERK play an important role in EMT induced by HGF in PC-3
cells. Similar result was seen in the process of EMT induced by HGF in
hepatocellular cancer [37].
Our study showed that HGF promoted the up-regulation of zinc finger E-box
binding homeobox-1(Zeb-1) in PC-3 cells. Like other several zinc finger transcription
factors, including Snail and Slug, which have been described as E-cadherin
repressors, Zeb-1 has also been linked to E-cadherin repression, thereby enhancing
the ability of the cancer cells to migrate to distal sites [38]. It has been shown that
HGF combined with early growth response factor-1(Egr-1) through MAPK pathway,
which binds to Snail1 promoter, leading to its rapid induction and execution of EMT
[39]. Another research showed that Zeb gene is activation occurs upon the activation
of Snail [40]. In the colorectal cancer, SW480 colorectal cancer cells grow in a
mesenchyme-like phenotype, characterized by loosely attached cells and lacking
membranous E-cadherin. Silence of Zeb-1 by siRNA changed the cell phenotype
resembling mesenchymal-epithelial transition (MET) [41]. Our results support these
researches and show the role of Zeb-1 in EMT in prostate cancer.
In conclusion, our results show that HGF directly promotes EMT and carcinogenic
properties in prostate cancer via ERK pathways. Specific molecular targeting of the
pathway may have clinical therapeutic benefits in prostate cancer.
AUTHOR CONTRIBUTIONS
Yi-Li Han designed of the study, participating in whole experiment and draft the
manuscript. Yong Luo, Yong-Xing Wang and Ya-Tong Chen participated in the design
of the study and some experiments. Ming-Chuan Li performed the statistical analysis.
Yong-Guang Jiang conceived of the study, coordinated the experiments performed by
the members of the research team and helped to draft the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests
ACKNOWLEDGEMENTS
This work was supported by National Natural Science Foundation of China (NO.
30700968 and 81341066).
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