ORIGINAL RESEARCH ARTICLE
published: 25 April 2012
Nodal promotes glioblastoma cell growth
Tanya De Silva, GangYe,Yao-Yun Liang, Guodong Fu, Guoxiong Xu†and Chun Peng*
Department of Biology,York University,Toronto, ON, Canada
Michael O’Connor, University of
Anna Petryk, University of Minnesota,
Osamu Shimmi, University of
S. Newfeld, Arizona State University,
Chun Peng, Department of Biology,
York University, 4700 Keele Street,
Toronto, ON, Canada M3J 1P3.
Guoxiong Xu, Jinshan Hospital, Fudan
University, Shanghai, China.
roles during embryogenesis. Recent studies in ovarian, breast, prostate, and skin cancer
cells suggest that Nodal also regulates cell proliferation, apoptosis, and invasion in cancer
cells. However, it appears to exert both tumor-suppressing and tumor-promoting effects,
depending on the cell type.To further understand the role of Nodal in tumorigenesis, we
examined the effect of Nodal in glioblastoma cell growth and spheroid formation using
U87 cell line. Treatment of U87 with recombinant Nodal significantly increased U87 cell
growth. In U87 cells stably transfected with the plasmid encoding Nodal, Smad2 phospho-
rylation was strongly induced and cell growth was significantly enhanced. Overexpression
of Nodal also resulted in tight spheroid formation. On the other hand, the cells stably trans-
fected with Nodal siRNA formed loose spheroids. Nodal is known to signal through activin
receptor-like kinase 4 (ALK4) and ALK7 and the Smad2/3 pathway. To determine which
receptor and Smad mediate the growth promoting effect of Nodal, we transfected siRNAs
targetingALK4,ALK7 , Smad2, or Smad3 into Nodal-overexpressing cells and observed that
cell growth was significantly inhibited by ALK4, ALK7 , and Smad3 siRNAs.Taken together,
these findings suggest that Nodal may have tumor-promoting effects on glioblastoma cells
and these effects are mediated by ALK4, ALK7 , and Smad3.
Keywords: Nodal,ALK4,ALK7, glioblastoma
Nodal, one of the transforming growth factor-β (TGF-β) super-
family members, was originally discovered in genetic studies as
a gene essential for the primitive streak formation and main-
tenance in mouse embryos (Conlon et al., 1994). Subsequent
studies demonstrate that Nodal plays an essential role in driving
and endoderm, specification of body axis, as well as self-renewal
and differentiation (Weng and Stemple, 2003; Shen, 2007). Sim-
ilar to other members of the TGF-β superfamily, Nodal signals
through serine/threonine receptors kinases. Two type I receptors,
activin receptor-like kinase 4 (ALK4) and ALK7, and two type
II Activin receptors (ActRIIA and ActRIIB) are known to medi-
ate Nodal signaling (Wang and Tsang, 2007). In addition, Cripto
acts as a co-receptor for Nodal. Activation of the receptors by
Nodal leads to the phosphorylation of Smad2/3 which further
bind to Smad4 and translocate into the nucleus, where a variety
of genes are transcriptionally regulated by the complexes (Schier,
Previously,we reported that Nodal is expressed in ovarian can-
cer cell lines and that overexpression of Nodal inhibited ovarian
induced ovarian cancer cell apoptosis (Xu et al., 2006; Ye et al.,
2011). Furthermore,we showed that Nodal inhibited cell prolifer-
of a growth inhibitory gene, cyclin G2 (Xu et al., 2008; Fu and
Peng, 2011). The growth inhibitory effect of Nodal has also been
observed in breast cancer cell lines (Zhong et al., 2009) and in
some prostate cancer cell lines (Vo and Khan, 2011). However,
several studies also reported that Nodal has tumorigenic effects.
For example, Nodal has been shown to promote melanoma pro-
gression (Topczewska et al., 2006). These findings suggest that
Nodal may have both tumor-suppressing and tumor-promoting
of the target organs.
Since TGF-β has been reported to inhibit the growth of
glioblastma cell lines (Piek et al., 1999) and ALK7 is highly
expressed in the adult brain (Ryden et al., 1996; Tsuchida et al.,
Glioblastma cells in culture have the ability to form spheroids or
adherent growth (Witusik-Perkowska et al., 2011), therefore, we
used cell growth and spheroid formation assays, along with over-
expression and gene silencing approaches, to determine the effect
and signaling pathway of Nodal in U87 cells.We demonstrate that
Nodal promotes cell growth and spheroid formation in U87 cells
through ALK4,ALK7, and Smad3.
MATERIALS AND METHODS
CELL LINES AND CELL CULTURE
U87 cell line was obtained from American Type Culture Collec-
tion (Rockville, MD, USA). The cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Hyclone, Logan, UT, USA)
supplemented with 100IU/ml penicillin and 100μg/ml strepto-
mycin (Invitrogen Canada, Inc., Burlington, ON, USA) in the
cancer cell lines (OV2008) expressing Nodal or its control vec-
tor were generated and cultured as previously reported (Ye et al.,
April 2012 | Volume 3 | Article 59 | 1
De Silva et al. Nodal enhances proliferation
GENERATION OF NODAL AND shNODAL STABLE CELL LINES
To generate Nodal-overexpressing cells, full-length Nodal cod-
ing sequence was cloned into mammalian expression vec-
tor pcDNA3.1/V5-His (Invitrogen), followed by Lipofectamine
transfection into U87 cells. Stable cells were selected using
Small hairpin sequence targeting human Nodal (shNodal; 5?-
ATCTGAAACCGCTTTTTG-3?) was cloned into the pSUPER
retroviral vector (Oligoengine, Seattle, WA, USA). To pro-
duce shNodal-expressing retroviruses, 293T cells were plated at
106cells/60-mm-diameter tissue culture dishes and transfected
retroviral packaging vector by the calcium phosphate method.
At 20h post-transfection, the medium was replaced with fresh
DMEM containing 10% FBS and cells were grown for an addi-
tional 24h. The conditioned medium containing recombinant
pore-size polysulfonic filters. The supernatants were mixed with
6μg/ml of polybrene (Sigma) and applied immediately to U87
and U87-Nodal cells. At 24h after infection, cells were selected
using 2μg/ml of puromycin (Invitrogen).
TRANSIENT TRANSFECTION OF ALK7 AND ALK4 siRNAs
Transient transfections were carried out using lipofectamine 2000
(Invitrogen). Briefly, 200nM siRNAs (siALK4, siALK7, siSmad2,
siSmad3, or negative control (NC) siRNA (GenePharma, Shang-
hai, China) and lipofectamine 2000 were incubated with OPTI
MEM1 medium (OMEM, Invitrogen) at room temperature for
20min. The mixture was then added to the cells. After 6h incu-
bation, the OMEM was replaced by normal cultured medium
supplemented with 10% FBS. Cells were allowed to recover for
24h prior to cell growth assays. The sequences of NC, siALK7
and siALK4 and confirmation of their efficiency have been
reported (Nadeem et al., 2011). Smad2 siRNA (sense GUCC-
CAUGAAAAGACUUAATT, and anti-sense UUAAGUCUUUU-
CAUGGGACUU; Pannu et al., 2007) and Smad3 siRNA (sense
CUGUGUGAGUUCGCCUUCAUU and anti-sense UGAAGGC-
GAACUCACACAGUU; Kobayashi et al., 2006) sequences were
taken from publications.
DETERMINATION OF CELL GROWTH
Cell growth was determined by manual cell counting. U87 cells
were cultured in 24 well culture plates at a cell density of
48h (10ng/ml TGF-β1, and 100ng/ml or 500ng/ml Nodal, R&D
systems, Minneapolis). Stable cells expressing Nodal or shNodal
were plated at the density of 1×105and cultured for 2days. At
was determined by trypan blue exclusion assay.
PROTEIN EXTRACTION AND WESTERN BLOT ANALYSES
cold PBS and lysed with RIPA buffer (50mM Tris–HCl, 150mM
ing complete protease inhibitor cocktail. Protein concentrations
were determined by Bradford assay and equal amount of proteins
The membrane was blocked with TBST [10mM Tris– HCl (pH
8.0), 150mM NaCl, and 0.05% Tween 20] containing 5% non-fat
dry milk powder at room temperature for 60min. The membrane
body developed in our lab (1:1000), or phospho Smad2, Smad2/3
(Cell Signaling, 1:1000), and β-actin (Sigma, 1:5000) prepared in
with TBST and incubated for 1h with a horseradish peroxidase
conjugated secondary antibody (1:5000 dilutions). After washing
as described above, the bound antibodies were detected using an
enhanced chemiluminescence (ECL) kit (GE Healthcare,Quebec)
according to instructions from the manufacturer.
SPHEROID FORMATION ASSAYS
Hanging drop cultures were performed by placing 20μl drops
(5000cells/drop) of U87 cells onto the inner surface of lids of
100mm culture dishes. The covers were then inverted and placed
to allow the formation of spheroids. At the end of each experi-
ment, spheroids were examined and photographed using a phase
contrast microscope. Spheroid area was measured with image J.
Results are expressed as the mean±SEM. Student’s t-test was
used to determine the differences between groups. Statistical
significance was defined as P <0.05.
NODAL ENHANCED THE GROWTH OF U87 CELLS
To test the effect of Nodal on U87 cell growth, cells were treated
with recombinant Nodal or TGF-β1 for 48h. Treatment with low
dose of Nodal did not change cell number but higher dose of
Nodal significantly increased cell numbers. In contrast, TGF-β1
significantly reduced the number of U87 cells (Figure 1).
To further investigate the effect of Nodal on U87 cells, four
stable cell lines including control (U87–EV),Nodal shRNA (U87-
shNodal), Nodal-overexpressing (U87-Nodal), and Nodal plus
Nodal shRNA (U87-Nodal/shNodal) were generated. As shown
in Figure 2A, the U87-Nodal cells showed strong overexpres-
sion of Nodal. In the U87-Nodal cells co-expressing shNodal,
Nodal expression level was reduced but Nodal level was still
much higher than the control cells. In control U87 cells stably
transfected with shNodal, there was a decrease in Nodal expres-
sion levels (Figure 2A). To confirm that Nodal overexpression
leads to the activation of its signaling pathways, we measured
phosphor-Smad2 levels in these cell lines. In U87-Nodal cells,
Smad2 phosphorylation was strongly induced. Smad2 activation
was also observed in U87-Nodal/shNodal cells, although to a
lesser extent (Figure 2A). In cell growth assays, U87-Nodal cells
grew significantly faster than the control cells and this effect was
(Figure2B).We have demonstrated that Nodal reduced prolifera-
tion and induced apoptosis in an ovarian cancer cell line,OV2008
(Xu et al., 2004; Ye et al., 2011). Here, we compared the effect
of Nodal on cell growth between OV2008 and U87 cells. Over-
expression of Nodal in OV2008 decreased cell density; however,
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April 2012 | Volume 3 | Article 59 | 2
De Silva et al.Nodal enhances proliferation
FIGURE 1 | Effects of Nodal andTGF-β1 onglioblastoma cell
growth. U87 cells cultured in 24 well plates were treated with either
recombinant Nodal (100 or 500ng/ml) orTGF-β1 (10ng/ml) for 48h.
Cell numbers were determined by trypan blue exclusion.
Representative cell images and summary graph are shown. Data
represent mean±SEM (n=3).The experiment was repeated three
times with similar results. *P <0.05 vs. control,#P <0.05 vs. all
the opposite effect was observed when Nodal was overexpressed
in U87 cells (Figure 2C).
EFFECTS OF NODAL ON SPHEROID FORMATION
Hanging drop technique was used to examine the role of Nodal
in spheroid formation of U87 cells. As shown in Figure 3A,
Nodal stable cells exhibited an enhanced ability to form tight and
dense spheroid compared with control. The spheroid structure
was totally disintegrated in U87-shNodal cells, showing a loose
and uneven cell aggregate and the cell distribution area is much
larger than that of other cells (Figure 3B). Although tight struc-
NODAL ENHANCED U87 GROWTH BY ACTING THROUGH ALK4, ALK7,
Since Nodal signaling is mainly mediated by ALK4 and ALK7
and the Smad pathway, we next examined the contribution of
these receptors to the growth promoting effects of Nodal in U87
cells. We transfected siRNAs targeting either ALK4 or ALK7 into
the U87-Nodal cells and found that both of the siRNAs signif-
icantly decreased cell numbers (Figure 4A). Since Smad2 and
Smad3 are downstream mediators of ALK4/7,we also determined
their involvement in Nodal-regulated cell growth. As shown in
Figure 4B, knockdown of Smad3 significantly suppressed cell
growth. However, knockdown of Smad2 only slightly decreased
ined in several cancer cell lines and yields paradoxical findings.
Previous studies in our lab have demonstrated that Nodal sig-
nals through ALK7 receptor to inhibit proliferation and to induce
apoptosis in ovarian cancer cell lines (Xu et al., 2004, 2006) and
reported to be only expressed in aggressive melanomas,but not in
sion (Topczewska et al., 2006). Recent studies in prostate cancer
and DU145 cells. While it had no effect on PC3 cells prolifera-
tion, it promoted migrations in these cells (Vo and Khan, 2011).
On the other hand, Nodal enhanced the proliferation of LNCaP
cells and are expressed in malignant prostate cancer cells but not
in benign tumors (Lawrence et al., 2011). In this study, we found
that treatment with recombinant Nodal or stable transfection of a
Nodal expression construct into U87 cells promoted cell growth,
April 2012 | Volume 3 | Article 59 | 3
De Silva et al. Nodal enhances proliferation
FIGURE 2 | Effect of Nodal overexpression and knockdown on
glioblastoma cell proliferation. (A) Four cell lines, U87 transfected with a
control empty vector (U87–EV), U87 transfected with Nodal shRNA
(U87-shNodal), U87 transfected with Nodal plasmid (U87-Nodal), and 87
transfected with both Nodal expression plasmid and Nodal shRNA
(U87-Nodal/shNodal) were generated. Proteins extracted from these cell
lines were analyzed by Western blotting using antibodies against Nodal,
phosphor-Smad2 (pSmad2), total Smad2 and Smad3 (Smad2/3), and β-actin
as the loading control. Overexpression and activation of Smad2 were
confirmed in the U87-Nodal cells. Numbers on the Nodal blot are
densitometry readings of the bands. (B) Cell growth assays.The stable cell
lines were cultured for 48h and cell numbers were determined by the
trypan blue exclusion method. Data represents mean±SEM (n=3).The
experiment was done three times and similar results were obtained.
*P <0.01 vs. U87–EV and U87-shNodal,#P <0.05 vs. U87–EV and
U87-Nodal. (C) Comparison of the effect of Nodal overexpression in U87
and OV2008 cells.Two pairs of cell lines, control and Nodal-overexpressing
OV2008 and control and Nodal-overexpressing U87 cells, were cultured for
2days before pictures were taken.
FIGURE 3 | Nodal enhanced spheroid formation. U87 cells stably
transfected with Nodal and/or Nodal shRNA were cultured in hanging drops
and formation of spheroids was observed at the forth day after culture. (A)
Photographs of representative spheroids from each cell lines. (B)
Quantifications of spheroid area. Data represent mean±SEM (n>35 each
group), *P <0.001 vs. the other groups.This experiment was performed
three times with similar results.
in contrast to what we observed previously in ovarian cancer cells.
These findings further suggest that Nodal is capable of exerting
both tumor-promoting and tumor-suppressing effects.
In this study, the long-term effects of Nodal gain-of-function
and loss-of-function phenotypes were examined by generating
stable cell lines overexpressing Nodal and/or Nodal siRNA in
human U87 glioblastoma cells. The overexpressing and knock-
down effects were validated by Western blots. We demonstrated
that overexpression of Nodal induced a significant increase in cell
numbers when compared to the control. Smad2 phosphorylation
was strongly activated in Nodal-overexpressing cells, confirm-
ing that the activation of Nodal signaling pathway in these cells.
The growth promoting effect of Nodal was partially reversed by
co-expression of Nodal siRNA, indicating that the higher cell
number in Nodal-overexpressing cells is indeed due to Nodal
Since three dimensional tumor cell cultures like spheroid for-
et al., 2009), we examined the effect of Nodal on spheroid forma-
tion in U87 cells. Overexpression of Nodal induced the formation
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April 2012 | Volume 3 | Article 59 | 4
De Silva et al. Nodal enhances proliferation
FIGURE 4 |TheALK4/ALK7–Smad3 pathway is involved in Nodal
induced U87 cell growth. (A) U87-Nodal cells were transiently transfected
with siRNAs targeting ALK4 (siALK4) or ALK7 (siALK7), scrambled negative
control (NC), or without siRNAs (mock transfection). Cell growth was
determined using direct cell counting at 48h after transfection. Data are
mean±SEM (n=6). *P <0.05 v. smock and NC group. (B) U87-Nodal cells
were transiently transfected with siRNAs targeting Smad2 or Smad3, or
NC, cell number was counted at 48h after transfection. SiRNA targeting
Smad3, but not Smad2, significantly reduced the cell growth. *P <0.05 vs.
control. Data are mean±SEM (n=4). Results shown are representatives
of three independent experiments.
irregular structures in control cells. In Nodal-overexpressing cells,
formation. Since compact spheroid formation has been suggested
to correlate with aggressiveness of tumors (Sodek et al., 2009),
these results suggest that Nodal has tumor-promoting effects in
The tumorigenic effect by Nodal in U87 cells has also been
recently reported (Lee et al.,2010). In agreement with our results,
Lee et al. (2010) showed that overexpression of Nodal increased
MMP-2 secretion, enhanced cell invasiveness and promoted cell
proliferation in vitro, as well as increased tumor growth in vivo.
Conversely, the knockdown of Nodal expression resulted in the
opposite phenomena (Lee et al., 2010). A subsequent report indi-
and ERK1/2-HIF-1a signaling pathway is involved in this process
(Hueng et al.,2011).
Both ALK4 and ALK7 have been reported to mediate Nodal
signaling (Reissmann et al., 2001). It has been reported that
Nodal promotes the tumorigenicity and plasticity of metastatic
melanoma in part by activating ALK4 receptor associated with
crypto-1 and knockdown of Nodal stimulated tumor differentia-
tion and regression (Topczewska et al., 2006; Strizzi et al., 2009).
Using siRNAs to silence the expression of ALK4 and ALK7, we
is involved in Nodal-stimulated glioblastoma cell growth. ALK7
has been reported to inhibit proliferation (Xu et al., 2004; Zhong
2004; Xu et al., 2004, 2006; Zhang et al., 2008; Ye et al., 2011) in
various cells lines. This study shows that ALK7 also has growth
promoting effects on some cancer cells. Similarly, gene silencing
techniques revealed that Smad3 mediates the growth promoting
effect of Nodal.
Although both TGF-β and Nodal activate the Smad2/3 path-
way (Graham and Peng, 2006), they have differential effects on
U87 cell growth. Treatment with Nodal enhanced, whereas treat-
that other signaling pathways may be differentially activated by
TGF-β and Nodal, which could interact with the Smad pathway
to differentially regulate gene expression. The complexity of TGF-
β signaling in cancer progression is well documented with both
tumor-suppressing and tumor-promoting effects (Derynck et al.,
of Nodal on different cancer cells remains to be investigated, it is
possible that the role of Nodal in tumorigenesis is dependent on
cellular microenvironment,the type of cancer,and/or the stage of
This study was supported by a grant from Canadian Institutes of
Health Research (CIHR MOP-89931) to Chun Peng. Chun Peng
was a recipient of a mid-career award from CIHR and Ontario
Women’s Health Council.
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Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential con-
flict of interest.
Received: 04 October 2011; accepted: 11
April 2012; published online: 25 April
Citation: De Silva T, Ye G, Liang
Y-Y, FuG, Xu
(2012) Nodal promotes glioblastoma
cell growth. Front. Endocrin. 3:59. doi:
This article was submitted to Frontiers in
Experimental Endocrinology, a specialty
of Frontiers in Endocrinology.
Copyright © 2012 De Silva, Ye, Liang,
Fu, Xu and Peng. This is an open-access
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Commercial License, which permits non-
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