0022-3565/08/3243-1227–1233$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics
JPET 324:1227–1233, 2008
Vol. 324, No. 3
129643/3309278
Printed in U.S.A.
Erythropoietin Stimulates Cancer Cell Migration and Activates
RhoA Protein through a Mitogen-Activated Protein
Kinase/Extracellular Signal-Regulated Kinase-Dependent
Mechanism
Sumaya N. Hamadmad1 and Raymond J. Hohl
Departments of Pharmacology (S.N.H., R.J.H.) and Internal Medicine (R.J.H.), Carver College of Medicine, University of Iowa,
Iowa City, Iowa
Received August 2, 2007; accepted December 11, 2007
Erythropoietin (Epo) is a cytokine that regulates red blood
cell production through binding to its cell surface receptor
expressed in erythroid progenitor cells (D’Andrea et al.,
1989). Binding of Epo to its receptor induces dimerization of
two receptor subunits and subsequent activation of the associated Janus kinase (Jak)2 (Witthuhn et al., 1993). This leads
to phosphorylation of several tyrosine residues in Epo receptor (EpoR) and recruitment of SH2-containing proteins,
which result in activation of several cascades of signal transduction pathways, notably the Ras/extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase
This work was supported by the Roy J. Carver Charitable Trust as a
Research Program of Excellence and the Roland W. Holden Family Program
for Experimental Cancer Therapeutics.
1
Current affiliation: Department of Pharmacology, Yale University School
of Medicine, New Haven, CT.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.129643.
signaling pathways: the mitogen-activated protein kinase
(MAPK) pathway and the RhoA GTPase pathway. We show that
Epo activates both pathways in a Janus kinase-dependent
manner and that this activation is required for Epo effects on
cell migration. Furthermore, we use both pharmacological and
genetic inhibitors to demonstrate that the activation of RhoA
GTPase is dependent on the activity of the MAPK pathway,
providing the first evidence for interaction between these two
signaling cascades.
(MAPK) (Torti et al., 1992) pathway and the phosphatidylinositol 3/Akt kinase pathway (Damen et al., 1995). Jak2 also
phosphorylates signal transducer and activator of transcription (Stat)5 and Stat3 (Kirito et al., 1997), which then translocate to the nucleus to act as transcription factors and mediate Epo effects. In addition to Ras, Epo has been shown to
activate other members of the Ras superfamily such as Rac
(Arai et al., 2002) and Rap1a (Arai et al., 2001).
Recent findings have shown that EpoR is expressed in
many tumor types, including cancer of the breast (Acs et al.,
2001, 2002; Arcasoy et al., 2002), female reproductive tract
(Shenouda et al., 2006), lung (Acs et al., 2001), head and neck
(Arcasoy et al., 2005), and central nervous system (Yasuda et
al., 2003). Although the exact role that Epo plays in cancer is
not well understood, it is thought that EpoR expression in
cancer cells might contribute to proliferation (Acs et al., 2001;
Pajonk et al., 2004), migration (Lai et al., 2005; Lester et al.,
2005), and resistance to treatment (Belenkov et al., 2004;
ABBREVIATIONS: Epo, erythropoietin; Jak, Janus kinase; EpoR, erythropoietin receptor; Erk, extracellular signal-regulated kinase; MAPK,
mitogen-activated protein kinase; Stat, signal transducer and activator of transcription; MEK, MAPK kinase; AG490, ␣-cyano-(3,4-dihydroxy)-Nbenzylcinnamide; PD98059, 2⬘-amino-3⬘-methoxyflavone; LPA, lysophosphatidic acid; Y-27632, N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; FR180204, 5-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-amine; MEM, minimum essential medium; PAGE,
polyacrylamide gel electrophoresis; GST, glutathione S-transferase; RBD, Rho binding domain; MTT, 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; IGF, insulin-like growth factor; DN, dominant-negative form of MEK1; ROCK, Rho kinase; GEF, guanine exchange factor; MLCK,
myosin light chain kinase.
1227
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 8, 2017
ABSTRACT
Erythropoietin (Epo) receptor (EpoR) is expressed in several
cancer cell lines, and the functional consequence of this expression is under extensive study. In this study, we used a
cervical cancer cell line in which EpoR was first found to be
expressed and to correlate with the severity of the disease. We
demonstrate that Epo is a chemoattractant for these cancer
cells, enhancing their migration under serum-starved conditions. Using a Transwell migration system, we show that Epo
enhances cancer cell migration in a dose- and time-dependent
manner. The effect of Epo is dependent on the activity of two
1228
Hamadmad and Hohl
Materials and Methods
Antibodies and Reagents. RhoA, Erk, and phospho-Erk antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). The Jak inhibitor, AG490 (tyrphostin), the MEK inhibitor, PD98059, and lysophosphatidic acid (LPA) were purchased from
Sigma-Aldrich (St. Louis, MO). The RhoA kinase inhibitor, Y-27632,
and the Erk inhibitor, FR180204, were both obtained from EMD
Chemicals (San Diego, CA).
Cell Line and Culture. HeLa cells were obtained from American
Type Culture Collection (Manassas, VA). Cells were cultured in
minimum essential medium (MEM) supplemented with 10% fetal
bovine serum, 100 mM sodium pyruvate, 0.1 mM MEM nonessential
amino acids, 100 units/ml penicillin, and 100 ␮g/ml streptomycin.
Each culture was maintained at 37°C, 5% CO2 and saturating
humidity.
Cell Transfection. The expression constructs encoding dominant-negative MEK1 (K97R) was kindly provided by Stefan Strack
(University of Iowa, Iowa City, IA). One day before transfection, cells
were plated in 10-cm culture dishes so that they would reach approximately 90% confluency the next day. Lipofectamine and Plus
Reagent (both obtained from Invitrogen, Carlsbad, CA) were used to
transiently transfect the plasmids in Opti-MEM I Reduced Serum
Medium without serum. Cells were incubated at 37°C for 5 h before
the medium was changed to MEM with 10% serum. Cells were
assayed 24 to 48 h after transfection.
Cell Lysing and Immunoblotting. Cells were lysed in radioimmunoprecipitation assay lysing buffer [1% sodium deoxycholate, 1%
Triton X-100, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 50
mM Tris, 150 mM NaCl, and protease inhibitor cocktail (SigmaAldrich)]. The clarified cell lysates were resolved by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently Western
blotted with the indicated antibodies as described previously
(Hamadmad et al., 2006).
Cell Migration Studies. HeLa cell migration was studied as
described previously (Lester et al., 2005) using 6.5-mm Transwell
chambers with 8-␮m pores (Corning Costar, Corning, NY). The bottom surface of each membrane was coated with 20% fetal bovine
serum for 2 h. Approximately 104 cells were seeded in the upper
chambers in 100 ␮l of serum-free medium. Lower chambers contained 600 ␮l of serum-free medium, 10% serum, or the indicated
concentration of Epo in serum-free medium. When treatments were
added, they were added to both chambers. After the cells were
allowed to migrate, the medium in the upper chamber was sucked
out and cells on the upper side were removed with a cotton swab.
Cells on the lower side of the membrane were fixed and stained by
Diff-Quik Staining solution (Dade-Behring, Deerfield, IL). Membranes were then cut from each Transwell chamber and transferred
to microscope slides. Cells that migrated through the membrane to
the lower surface were counted by light microscopy.
Rho Activation Assay. RhoA activation was studied using the
Rho activation kit (catalog number EKS-465; Assay Designs, Ann
Arbor, MI) according to manufacturer’s recommendations. After lysing of the cells, protein concentration was measured, and approximately 700 ␮g of protein was added to each reaction. The glutathione
S-transferase (GST)-rhotekin-Rho binding domain (RBD) fusion protein was then used to bind active Rho, and the complex was pulled
down with immobilized glutathione column. Pulled-down Rho was
then detected by Western blot analysis using a specific RhoA
antibody.
Results
Epo Stimulates HeLa Cell Migration. Previous studies
have shown that Epo stimulates invasion of squamous cell
carcinoma cells (Lai et al., 2005) as well as migration of
breast cancer cells (Lester et al., 2005). To study the effect of
Epo on HeLa cell migration, the Transwell migration assay
was used. This assay measures the migration of cells across
a porous membrane in response to potential stimuli and is a
reliable and widely used method for quantifying cell migration. Cells were added to the upper chamber and allowed to
migrate for different periods of time. Epo was tested for its
ability to promote cell migration when added to the lower
chamber in serum-free medium. As shown in Fig. 1A, an
increasing concentration of Epo from 5 to 20 U/ml stimulated
HeLa cell migration in a dose-dependent manner. A 30 U/ml
concentration did not have any added effect over the 20 U/ml
concentration, which suggests that the latter value corresponds to the concentration that leads to the maximal effect
of Epo. As shown in Fig. 1B, Epo induction of migration was
also time-dependent because it increased the number of migrating cells with time within the time period that was tested
(4 –24 h). The effect of Epo was comparable with that of
serum. To rule out an effect of Epo on cell viability and/or
proliferation, we assessed cell proliferation under the conditions used in the migration studies using 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. We
found that the different concentrations of Epo did not affect
cell proliferation within the time period (up to 24 h) that were
investigated (data not shown).
Epo induction of migration was dependent on the activity
of Jak, because inhibition of this kinase by the selective
inhibitor, AG490, abolished Epo effects without affecting
that of serum-induced migration (Fig. 1C). We used 30 ␮M
AG490, which is approximately the IC50 of the inhibitor
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 8, 2017
Pajonk et al., 2004). Some studies suggested that the Erk/
MAPK pathway plays a crucial role in these processes
(Lester et al., 2005); others have pointed to the phosphatidylinositol 3-kinase pathway as a major player (Hardee et al.,
2006). Still, little is known about the role of EpoR signaling in
tumor cells.
Recent studies showed that Epo enhances cancer cell migration both in squamous cell carcinoma (Mohyeldin et al.,
2005) and breast cancer cells (Lester et al., 2005). Activation
of the Jak2, a step indispensable to all Epo effects, was
implicated in both systems. The MAPK/Erk pathway was
also shown to be required for the stimulation of migration
seen in the breast cancer cell line. Activation of the MAPK/
Erk pathway is critical for stimulation of migration by several agents that support this process (Hinton et al., 1998; Jo
et al., 2002; Brahmbhatt and Klemke, 2003). In addition,
members of the Rho family, namely RhoA, are known to play
an important role in the process of cell migration through
their effects on the remodeling of the actin cytoskeleton (Takaishi et al., 1994; Faried et al., 2006).
In this study, we sought to understand the molecular
mechanism(s) behind the effect of Epo on cancer cell migration. We used HeLa cells, a cervical cancer cell line where
EpoR was first found to be expressed and where this expression correlates with the severity of the disease (Shenouda et
al., 2006). We show that Epo acts as a chemoattractant that
induces chemotaxis of cancer cells. We also demonstrate that
this effect is dependent on activation of two signaling pathways by Epo: the MAPK/Erk and RhoA/RhoA kinase pathways. Using chemical inhibitors and dominant-negative
forms of MAPK kinase (MEK), we show that activation of the
latter pathway is dependent on activation of the first.
Epo Activates RhoA in Cancer
Migrating Cells
A 2000
1800
1600
1400
1200
1000
800
600
400
200
0
0
5
10
20
30
Serum
Epo (U/ml)
500
400
No Epo
300
+ Epo
200
100
0
4
8
12
24
Migration Time (hr)
Percentage of Migrating Cells
C 450
400
350
300
Control
+ AG 490
250
200
150
100
50
0
-
Epo
Serum
Fig. 1. Epo produces a dose- and time-dependent induction of HeLa cell
migration. A, HeLa cells were added to the upper chamber of the Transwell system as described under Materials and Methods. Epo was added to
the lower chamber at the concentrations indicated, and the cells were
allowed to migrate for 12 h. Ten percent serum was used as a control.
Cells that migrated to the other side of the membrane were counted and
are shown (mean ⫾ S.D., n ⫽ 4). B, cells were allowed to migrate for the
indicated periods of time in the presence of 20 U/ml Epo in the lower
chamber. Cell migration is expressed as a percentage of that observed in
cells without Epo at the 4-h time point (mean ⫾ S.D., n ⫽ 4). The actual
number of cells migrating in the absence of Epo at the 4-h time point is
750 ⫾ 30. C, HeLa cells were treated or not (control) with 30 ␮M AG490
and allowed to migrate for 12 h in the absence or presence of 20 U/ml Epo
or 10% serum. AG490 was present in both chambers of the Transwell
system during the migration period. Cell migration is expressed as a
percentage of that observed in control (no AG490 added) cells without Epo
(mean ⫾ S.D., n ⫽ 4). The actual number of control cells migrating in the
absence of Epo is 860 ⫾ 53.
(Eriksen et al., 2001). AG490 did not affect cell proliferation
under the conditions tested as assessed by the MTT assay
(data not shown).
Epo Activates Erk Phosphorylation in HeLa Cells. It
is well established that Epo can activate the Erk/MAPK
pathway, and several studies have implicated this pathway
in induction of cell migration in several cell types (Nguyen et
al., 1998; Lester et al., 2005; Monami et al., 2006). Therefore,
we wanted to test the effect of Epo on Erk phosphorylation in
HeLa cells. Stimulation of cells with Epo led to phosphorylation of Erk within 2 min (Fig. 2A). This effect was maxi-
mum after 10 min of Epo stimulation and seemed to decrease
after 60 min, although it stayed well above basal level even
after 90 min of stimulation. Total protein level of Erk was not
affected by Epo stimulation. As expected, serum also activated Erk phosphorylation. As shown in Fig. 2B, the activation of Erk was occurring in a Jak-dependent manner as the
Jak inhibitor, AG490, inhibited Erk phosphorylation in response to Epo. To show specificity of AG490 toward Jak, we
tested Erk activation by insulin-like growth factor (IGF)-1,
which does not require Jak activity for Erk induction. As
shown in Fig. 2B, AG490 treatment did not affect Erk activation by IGF-1, which confirms that AG490 is inhibiting Erk
in Epo-treated cells by indirectly inhibiting the upstream
Jak.
Epo Stimulation of Cell Migration Is MAPK-Dependent. To assess the role of Erk phosphorylation in Epo stimulation of cell migration, we treated the cells with the
PD98059 (Alessi et al., 1995), a selective inhibitor of MEK,
the kinase that activates both Erk-1 and Erk-2 by phosphorylation, and tested its effect on cell migration in both the
presence or absence of Epo. The inhibitor was added to both
the upper and lower chambers of the Transwell system, and
cell migration was studied as described above. Inhibition of
the MAPK pathway completely abolished the effect of Epo on
stimulation of cell migration (Fig. 3A). Inhibition of the
MAPK pathway by PD98059 was confirmed by its ability to
inhibit Epo-induced phosphorylation of Erk protein (Fig. 6).
To further confirm our findings, we transfected the cells
with a dominant-negative form of MEK1 (DN-MEK) and
tested its effect on cell migration in response to Epo. The
results of these experiments are shown in Fig. 3B. These
results were in agreement with the effect of PD98059, although there was slightly less effect with the DN-MEK,
which is contributed to lower transfection efficiency that was
achieved with transient transfection. This confirmed that the
MAPK pathway plays an essential role in Epo induction of
cell migration. As expected, DN-MEK1 inhibited Erk phosphorylation by Epo treatment (Fig. 6).
Epo Activates RhoA Protein in HeLa Cells. To further
understand the mechanism behind Epo’s activation of HeLa
cell migration, we decided to test the effects of Epo on proteins implicated in the process of cell migration, notably
members of the Rho family of small GTPases. It was previously shown that Epo induced activation of several small
GTPases, including Ras (Torti et al., 1992), Rap1 (Arai et al.,
2001), and Rac (Arai et al., 2002). In this study, we decided to
examine the effect of Epo on RhoA, which plays an important
role in the process of cytoskeletal organization required for
cell migration (Takaishi et al., 1994). Cells were stimulated
with 20 U/ml Epo for several time points before lysing. Pulldown experiments were carried out with GST-rhotekin binding domain fusion protein that binds to the GTP-bound form
of Rho that represents the active form of the small GTPase.
Then, the bound fraction was eluted and analyzed by Western blotting with Rho-specific antibodies.
Results of these experiments are shown in Fig. 4A, which
illustrates that Epo stimulation led to activation of RhoA
protein in HeLa cells within 10 min. This activation was
transient because it disappeared within 60 min of Epo stimulation. The total protein level of RhoA was not affected by
Epo stimulation during the tested time period. The activation
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 8, 2017
Percen tag e o f Mig ratin g C ells
B 600
1229
1230
Hamadmad and Hohl
A
Epo (min)
2
-
10
30
60
90
Serum
pErk
Erk
600
500
pErk
400
Total Erk
300
200
100
0
Control
2
10
30
60
90
Fig. 2. Epo induces phosphorylation of Erk in HeLa
cells. A, HeLa cells were starved of serum for 4 h
and then stimulated with 20 U/ml Epo for the indicated periods of time or with 10% serum for 10
min. Cell lysates were subjected to SDS-PAGE and
immunoblot analysis to detect phosphorylated and
total Erk. Densitometric analysis is shown for this
experiment, which is representative of at least
three separate experiments. The 0 time point (no
Epo stimulation) was used as the control to which
the other points were compared. B, cells were
treated with 30 ␮M AG490 for 12 h before stimulation with 20 U/ml Epo for 10 min (left) or IGF-1 for
5 min (right). Cell lysates were then subjected to
SDS-PAGE and immunoblot analysis to detect
phosphorylated and total Erk.
Time of Epo Stimulation (min)
B
Control
Epo
-
AG490
AG490
+
IGF
+
-
+
pErk
Erk
of RhoA was occurring in a Jak-dependent manner as the Jak
inhibitor AG490 inhibited the activation of RhoA in response
to Epo (Fig. 4B). We used LPA, which is a known activator of
RhoA protein, as a positive control for RhoA activation (Fig.
4A). We also showed that AG490 treatment did not affect
RhoA activation by LPA, which further confirms the selectivity of AG490 toward Jak (Fig. 4B).
We next sought to determine whether the RhoA pathway
plays a role in Epo induction of migration. To this end, we
treated the cells with the Rho kinase (ROCK) inhibitor,
Y-27632, and studied its effect on Epo-induced cell migration.
Inhibition of ROCK resulted in complete abrogation of the
effect of Epo on migration (Fig. 5), which suggests that the
effects of Epo are dependent on activation of the Rho/ROCK
signaling pathway.
Epo Stimulation of RhoA Activation Is Dependent on
MEK Pathway. Because Epo activates both Erk and RhoA
pathways and both are involved in inducing cell migration,
we probed the relationship between these two pathways in
mediating Epo induction of migration. To this end, cells were
treated with the MAPK pathway inhibitor, PD98059. After
incubation for 12 h, Epo stimulation of RhoA was tested as
described previously. It is interesting to note that inhibition
of MEK activation by PD98059 inhibited Epo-induced activation of RhoA (Fig. 6).
To further confirm this finding, we transfected the cells
with the dominant-negative form of MEK1 and tested RhoA
activation in response to Epo. In agreement with the MEK
inhibitor findings, the dominant-negative MEK1 also inhibited RhoA activation in response to Epo (Fig. 6). The effect on
RhoA activation can not be attributed to changes in RhoA
protein levels because the total levels of RhoA stayed constant during the time of experiment. To show that RhoA is
downstream of Erk, the selective Erk inhibitor, FR180204,
was tested. The Erk inhibitor abolished RhoA activation
without affecting Erk phosphorylation (Fig. 6). Together,
these results suggest that RhoA is acting downstream of the
MAPK pathway to activate cell migration.
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 8, 2017
Percentage of Control
700
Epo Activates RhoA in Cancer
Percentage of Migrating Cells
A 450
400
350
300
250
Control
200
+ PD98059
150
100
50
0
-
Epo
Serum
400
350
300
250
Control
200
DN MEK1
150
100
50
0
-
Epo
Serum
Fig. 3. Epo activation of cell migration is MAPK-dependent. A, HeLa cells
were added to the upper chamber of the Transwell system as described
under Materials and Methods. A total of 20 U/ml Epo or 10% serum was
added to the lower chamber, and cells were allowed to migrate for 12 h.
When PD98059 was used, it was added to both chambers of the Transwell
system at a 30 ␮M concentration. Cells that migrated to the other side of
the membrane were counted, and cell migration was expressed as a
percentage of that observed in control (no inhibitor added) cells without
Epo (mean ⫾ S.D., n ⫽ 4). The actual number of control cells migrating
in the absence of Epo is 820 ⫾ 40. B, HeLa cells were transfected with
dominant-negative MEK1 as described under Materials and Methods.
Migration was studied as described in A. The actual number of control
cells migrating in the absence of Epo is 690 ⫾ 45.
Discussion
Erythropoietin receptor has been shown to be expressed in
many cancer cells, and the functional consequences of this
expression have been extensively studied recently. One of the
very first cancers where EpoR expression was demonstrated
is cancer of the female reproductive tract, most notably cervical cancer (Acs et al., 2001, 2003). EpoR was found both in
cervical cancer cell lines and in specimens from patients with
cervical cancer (Acs et al., 2003; Shenouda et al., 2006). The
level of EpoR expression was shown to correlate with the
severity of the cancer and the metastatic potential of the
disease; however, the functional consequence of this expression was not elaborated. Moreover, these cells were found to
express Epo mRNA and protein, although no studies have
definitely proven their ability to secrete Epo in the surrounding media. More recently, several studies have suggested a
role for Epo in enhancing cancer cell migration and invasion
induced by serum. These effects were demonstrated in breast
cancer cell lines (Lester et al., 2005) and squamous cell carcinoma of the head and neck (Lai et al., 2005). Thus, we
hypothesized that Epo induces migration of cervical cancer
cells and decided to investigate the pathway(s) that mediates
this effect.
Our findings demonstrate that Epo induces migration of
HeLa cervical cancer cells in serum-free medium by acting as
a chemoattractant for these cells. We also show that this
effect is dependent on the MAPK and the RhoA pathways
that are both activated by Epo stimulation. Furthermore, the
activation of RhoA is shown to be dependent on the MAPK
pathway, and this was confirmed using pharmacological and
genetic inhibitors.
Epo activation of downstream signaling pathways is dependent on the activity of the Epo receptor-associated kinase,
Jak. This requirement for Jak activity is a hallmark for Epo
acting through its receptor. We showed that the effects of Epo
on HeLa cell migration and Erk and Rho activation are
dependent on Jak activity using the Jak-selective inhibitor
AG490. The activation of Erk by Epo is a well characterized
process that starts with recruitment of Son of sevenless, the
guanine exchange factor (GEF) for Ras, to the phosphorylated tyrosine sites on EpoR that are generated by the activity of Jak. Activated Ras then triggers the signal transduction cascade known as the Raf/MEK/MAPK that culminates
in Erk activation (Hamadmad and Hohl, 2007). However,
this is the first study to show that Epo activates RhoA, and
the exact mechanism for this activation has yet to be elucidated. It is possible that a mechanism similar to the one that
leads to Ras activation exists for RhoA in these cells.
Several agents that induce migration of cancer cells were
found to activate the MAPK pathway. For example, urokinase-type plasminogen activator (Nguyen et al., 1998), epidermal growth factor, platelet-derived growth factor (Graf et
A
Epo (min)
2
-
10
30
60
LPA
90
GTP-Rho
Total Rho
B
Epo
Control
-
AG490
AG490
+
+
LPA GTP-Rho
+
Total Rho
pErk
Erk
Fig. 4. Epo activates RhoA in a Jak-dependent manner. A, HeLa cells
were starved for 4 h of serum and then stimulated with 20 U/ml Epo for
the indicated periods of time or with 30 ␮M LPA for 5 min. Cell lysates
were subjected to pull-down analysis with the GST-rhotekin-RBD fusion
protein and then to Western blot analysis with RhoA antibody. Total cell
lysates were also Western blotted to determine total RhoA. B, cells were
treated with 30 ␮M AG490 for 12 h before stimulation with 20 U/ml Epo
for 10 min (left) or 30 ␮M LPA for 5 min (right). Cell lysates were then
subjected to pull-down analysis with the GST-rhotekin-RBD fusion protein and then to Western blot analysis with RhoA antibody. Total cell
lysates were also Western blotted to determine total RhoA protein level.
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 8, 2017
Percentage of Migrating Cells
B 450
1231
1232
Hamadmad and Hohl
500
450
Percentage of Migrating Cells
400
350
300
Control
250
+ Y27632
200
150
100
50
0
-
Epo
Serum
al., 1997), and proepithelin (Monami et al., 2006) were all
found to induce cell migration through activation of the
MAPK pathway. The exact mechanism through which MAPK
affects migration is not very well defined; however, most
studies suggest that Erk acts through activation of the myosin light chain kinase (MLCK), which induces serine phosphorylation of the myosin regulatory light chain and thereby
promotes contractility of the actomyosin cytoskeleton
(Nguyen et al., 1999). This effect is thought to facilitate
retraction of the cell tail. In addition to MLCK, Erk is
thought to target other cytoskeletal-associated components,
such as caldesmon, microtubules, and Scar/WAVE, which are
likely to be involved in the process of migration (Westphal et
al., 2000).
In this study, we demonstrate that, through activation of
the RhoA protein, another mechanism can contribute to the
MAPK pathway role in mediating cancer cell migration. Although the exact mechanism for this activation is still not
well elucidated, we predict that the kinase activity of Erk
Control
Epo
-
+
Vector
Control
PD98059 FR1802
+
+
-
+
DN MEK1
+
GTP-Rho
Total Rho
pErk
Erk
Fig. 6. Activation of RhoA is MAPK-dependent. HeLa cells were treated
with 30 ␮M PD98059 or 30 ␮M FR180204 for 12 h (lanes 3 and 4; left) or
transfected with control vector or dominant-negative MEK1 (right), as
described under Materials and Methods, before stimulation with 20 U/ml
Epo for 10 min. Cell lysates were either subjected to pull-down analysis
with the GST-rhotekin-RBD fusion protein and then to Western blot
analysis with RhoA antibody for active RhoA or directly subjected to
Western blot with the indicated antibodies to determine total RhoA,
phosphorylated Erk, and total Erk.
plays a role in this process. Erk is known to have more than
50 substrates (Kolch, 2000). It is possible that one of the
substrates that Erk phosphorylates plays a role in RhoA
activation. For example, many GEFs that activate small Gproteins like RhoA are regulated by phosphorylation (Tybulewicz, 2005). Phosphorylation is known to activate Vav, a
GEF for RhoA, leading to RhoA activation (Tybulewicz,
2005). Alternatively, GTPase activator proteins, which enhance the intrinsic GTPase activity of the small G-proteins,
thus inactivating them, are known to be governed by phosphorylation. Phosphorylation has been shown to inhibit GTPase activator protein function in some instances, leading to
enhanced activity of the G-protein (Bernards and Settleman,
2005). In addition, the function of RhoA itself has been shown
to be regulated by phosphorylation at several tyrosine and
serine sites (Loirand et al., 2006). One or more of the aforementioned proteins could be a member in the growing list of
Erk substrates.
It is well established that RhoA and other members of the
Rho family function primarily in processes that require remodeling of the actin cytoskeleton. In stationary cells, RhoA
promotes the formation of actin stress fibers and clustering of
proteins in focal adhesions (Kaibuchi et al., 1999). However,
recent studies have implicated this protein in the process of
cancer cell migration and invasion (Takaishi et al., 1994;
Faried et al., 2006). Several hypotheses were suggested to
explain this role; however, the exact mechanism is not completely understood. It is believed that the Rho kinase, by
inactivating myosin light chain phosphatase, complements
the function of MLCK in promoting myosin regulatory light
chain phosphorylation and actomyosin contractility (Amano
et al., 1996). Our finding that RhoA activation is dependent
on Erk pathway activation demonstrates how the process of
myosin light chain phosphorylation is orchestrated to generate the coordinated movement of the cell during the process
of migration. This is further revealed by the timing of activation of the two pathways; whereas Erk phosphorylation
was induced more promptly and sustained for longer time
(Fig. 2A), RhoA signaling was activated later and only transiently (Fig. 4). Rho kinase also activates a downstream
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 8, 2017
Fig. 5. Epo activation of migration requires ROCK activity. HeLa cells were added to the upper chamber of the Transwell system as described under
Materials and Methods. A total of 20 U/ml Epo or 10% serum was added to the lower chamber, and cells were allowed to migrate for 12 h. When
Y-27632 was used, it was added to both chambers of the Transwell system at a 30 ␮M concentration. Cells that migrated to the other side of the
membrane were counted, and cell migration was expressed as a percentage of that observed in control (no inhibitor added) cells without Epo (mean ⫾
S.D., n ⫽ 4). The actual number of control cells migrating in the absence of Epo is 770 ⫾ 55.
Epo Activates RhoA in Cancer
References
Acs G, Acs P, Beckwith SM, Pitts RL, Clements E, Wong K, and Verma A (2001)
Erythropoietin and erythropoietin receptor expression in human cancer. Cancer
Res 61:3561–3565.
Acs G, Zhang PJ, McGrath CM, Acs P, McBroom J, Mohyeldin A, Liu S, Lu H, and
Verma A (2003) Hypoxia-inducible erythropoietin signaling in squamous dysplasia
and squamous cell carcinoma of the uterine cervix and its potential role in cervical
carcinogenesis and tumor progression. Am J Pathol 162:1789 –1806.
Acs G, Zhang PJ, Rebbeck TR, Acs P, and Verma A (2002) Immunohistochemical
expression of erythropoietin and erythropoietin receptor in breast carcinoma.
Cancer 95:969 –981.
Alessi DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR (1995) PD 098059 is a
specific inhibitor of the activation of mitogen-activated protein kinase kinase in
vitro and in vivo. J Biol Chem 270:27489 –27494.
Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, and
Kaibuchi K (1996) Phosphorylation and activation of myosin by Rho-associated
kinase (Rho-kinase). J Biol Chem 271:20246 –20249.
Arai A, Kanda E, and Miura O (2002) Rac is activated by erythropoietin or interleukin-3 and is involved in activation of the Erk signaling pathway. Oncogene
21:2641–2651.
Arai A, Nosaka Y, Kanda E, Yamamoto K, Miyasaka N, and Miura O (2001) Rap1 is
activated by erythropoietin or interleukin-3 and is involved in regulation of beta1
integrin-mediated hematopoietic cell adhesion. J Biol Chem 276:10453–10462.
Arcasoy MO, Amin K, Chou SC, Haroon ZA, Varia M, and Raleigh JA (2005)
Erythropoietin and erythropoietin receptor expression in head and neck cancer:
relationship to tumor hypoxia. Clin Cancer Res 11:20 –27.
Arcasoy MO, Amin K, Karayal AF, Chou SC, Raleigh JA, Varia MA, and Haroon ZA
(2002) Functional significance of erythropoietin receptor expression in breast
cancer. Lab Invest 82:911–918.
Belenkov AI, Shenouda G, Rizhevskaya E, Cournoyer D, Belzile JP, Souhami L,
Devic S, and Chow TY (2004) Erythropoietin induces cancer cell resistance to
ionizing radiation and to cisplatin. Mol Cancer Ther 3:1525–1532.
Bernards A and Settleman J (2005) GAPs in growth factor signalling. Growth
Factors 23:143–149.
Brahmbhatt AA and Klemke RL (2003) ERK and RhoA differentially regulate
pseudopodia growth and retraction during chemotaxis. J Biol Chem 278:13016 –
13025.
Damen JE, Cutler RL, Jiao H, Yi T, and Krystal G (1995) Phosphorylation of tyrosine
503 in the erythropoietin receptor (EpR) is essential for binding the P85 subunit of
phosphatidylinositol (PI) 3-kinase and for EpR-associated PI 3-kinase activity.
J Biol Chem 270:23402–23408.
D’Andrea AD, Fasman GD, and Lodish HF (1989) Erythropoietin receptor and
interleukin-2 receptor ␤ chain: a new receptor family. Cell 58:1023–1024.
Eriksen KW, Kaltoft K, Mikkelsen G, Nielsen M, Zhang Q, Geisler C, Nissen MH,
Ropke C, Wasik MA, and Odum N (2001) Constitutive STAT3-activation in Sezary
syndrome: tyrphostin AG490 inhibits STAT3-activation, interleukin-2 receptor
expression and growth of leukemic Sezary cells. Leukemia 15:787–793.
Faried A, Faried LS, Kimura H, Nakajima M, Sohda M, Miyazaki T, Kato H, Usman
N, and Kuwano H (2006) RhoA and RhoC proteins promote both cell proliferation
and cell invasion of human oesophageal squamous cell carcinoma cell lines in vitro
and in vivo. Eur J Cancer 42:1455–1465.
Graf K, Xi XP, Yang D, Fleck E, Hsueh WA, and Law RE (1997) Mitogen-activated
protein kinase activation is involved in platelet-derived growth factor-directed
migration by vascular smooth muscle cells. Hypertension 29:334 –339.
Hamadmad SN, Henry MK, and Hohl RJ (2006) Erythropoietin receptor signal
transduction requires protein geranylgeranylation. J Pharmacol Exp Ther 316:
403– 409.
Hamadmad SN and Hohl RJ (2007) Lovastatin suppresses erythropoietin receptor
surface expression through dual inhibition of glycosylation and geranylgeranylation. Biochem Pharmacol 74:590 – 600.
Hardee ME, Rabbani ZN, Arcasoy MO, Kirkpatrick JP, Vujaskovic Z, Dewhirst MW,
and Blackwell KL (2006) Erythropoietin inhibits apoptosis in breast cancer cells
via an Akt-dependent pathway without modulating in vivo chemosensitivity. Mol
Cancer Ther 5:356 –361.
Hinton DR, He S, Graf K, Yang D, Hsueh WA, Ryan SJ, and Law RE (1998)
Mitogen-activated protein kinase activation mediates PDGF-directed migration of
RPE cells. Exp Cell Res 239:11–15.
Jo M, Thomas KS, Somlyo AV, Somlyo AP, and Gonias SL (2002) Cooperativity
between the Ras-ERK and Rho-Rho kinase pathways in urokinase-type plasminogen activator-stimulated cell migration. J Biol Chem 277:12479 –12485.
Kaibuchi K, Kuroda S, and Amano M (1999) Regulation of the cytoskeleton and cell
adhesion by the Rho family GTPases in mammalian cells. Annu Rev Biochem
68:459 – 486.
Kirito K, Uchida M, Yamada M, Miura Y, and Komatsu N (1997) A distinct function
of STAT proteins in erythropoietin signal transduction. J Biol Chem 272:16507–
16513.
Kolch W (2000) Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK
pathway by protein interactions. Biochem J 351:289 –305.
Lai SY, Childs EE, Xi S, Coppelli FM, Gooding WE, Wells A, Ferris RL, and Grandis
JR (2005) Erythropoietin-mediated activation of JAK-STAT signaling contributes
to cellular invasion in head and neck squamous cell carcinoma. Oncogene 24:4442–
4449.
Lester RD, Jo M, Campana WM, and Gonias SL (2005) Erythropoietin promotes
MCF-7 breast cancer cell migration by an ERK/mitogen-activated protein kinasedependent pathway and is primarily responsible for the increase in migration
observed in hypoxia. J Biol Chem 280:39273–39277.
Loirand G, Guilluy C, and Pacaud P (2006) Regulation of Rho proteins by phosphorylation in the cardiovascular system. Trends Cardiovasc Med 16:199 –204.
Mohyeldin A, Lu H, Dalgard C, Lai SY, Cohen N, Acs G, and Verma A (2005)
Erythropoietin signaling promotes invasiveness of human head and neck squamous cell carcinoma. Neoplasia 7:537–543.
Monami G, Gonzalez EM, Hellman M, Gomella LG, Baffa R, Iozzo RV, and Morrione
A (2006) Proepithelin promotes migration and invasion of 5637 bladder cancer
cells through the activation of ERK1/2 and the formation of a paxillin/FAK/ERK
complex. Cancer Res 66:7103–7110.
Nguyen DH, Catling AD, Webb DJ, Sankovic M, Walker LA, Somlyo AV, Weber MJ,
and Gonias SL (1999) Myosin light chain kinase functions downstream of Ras/ERK
to promote migration of urokinase-type plasminogen activator-stimulated cells in
an integrin-selective manner. J Cell Biol 146:149 –164.
Nguyen DH, Hussaini IM, and Gonias SL (1998) Binding of urokinase-type plasminogen activator to its receptor in MCF-7 cells activates extracellular signalregulated kinase 1 and 2 which is required for increased cellular motility. J Biol
Chem 273:8502– 8507.
Pajonk F, Weil A, Sommer A, Suwinski R, and Henke M (2004) The erythropoietinreceptor pathway modulates survival of cancer cells. Oncogene 23:8987– 8991.
Shenouda G, Mehio A, Souhami L, Duclos M, Portelance L, Belenkov A, and Chow T
(2006) Erythropoietin receptor expression in biopsy specimens from patients with
uterine cervix squamous cell carcinoma. Int J Gynecol Cancer 16:752–756.
Takaishi K, Sasaki T, Kato M, Yamochi W, Kuroda S, Nakamura T, Takeichi M, and
Takai Y (1994) Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility. Oncogene 9:273–279.
Torti M, Marti KB, Altschuler D, Yamamoto K, and Lapetina EG (1992) Erythropoietin induces p21ras activation and p120GAP tyrosine phosphorylation in human erythroleukemia cells. J Biol Chem 267:8293– 8298.
Tybulewicz VL (2005) Vav-family proteins in T-cell signalling. Curr Opin Immunol
17:267–274.
Westphal RS, Soderling SH, Alto NM, Langeberg LK, and Scott JD (2000) Scar/
WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated
multi-kinase scaffold. EMBO J 19:4589 – 4600.
Witthuhn BA, Quelle FW, Silvennoinen O, Yi T, Tang B, Miura O, and Ihle JN (1993)
JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated
and activated following stimulation with erythropoietin. Cell 74:227–236.
Yasuda Y, Fujita Y, Matsuo T, Koinuma S, Hara S, Tazaki A, Onozaki M, Hashimoto
M, Musha T, Ogawa K, et al. (2003) Erythropoietin regulates tumour growth of
human malignancies. Carcinogenesis 24:1021–1029.
Address correspondence to: Dr. Raymond J. Hohl, Department of Internal
Medicine, SE 313 GH, University of Iowa, Iowa City, IA 52242. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on July 8, 2017
pathway that culminates in the inactivation of cofilin, an
actin-depolymerizing agent.
Although the finding that RhoA activation is dependent on
Erk is novel, several studies have suggested that the two
pathways can cross-talk and/or cooperate. In one study by Jo
et al. (2002), it was found that the stimulation of cell migration induced by active MEK1 was totally blocked by either a
dominant-negative RhoA or the ROCK inhibitor, Y-27632.
On the other hand, inhibition of the MAPK pathway by
PD98059 did not affect the migration of cells that was induced by active RhoA. These results suggest that the RhoA
pathway is acting downstream of the Erk pathway in inducing cell migration and are in agreement with our findings.
Our results further extend the role of EpoR in cancer cells
and warrant more studies that consider the benefits and
risks of Epo supplements to cancer patients, especially cancers that are modulated by Epo. These results also suggest
that targeting the Epo pathway, or more specifically the
MAPK pathway, can be a mechanism for inhibition of cancer
cell migration in vivo, which is an essential step for the
process of cancer metastasis. Both pharmacological inhibitors and antisense techniques might prove efficient for this in
the future.
1233
Téléchargement

Erythropoietin Stimulates Cancer Cell Migration and Activates