Download Akt Activation, but not Extracellular Signal–Regulated Kinase

Akt Activation, but not Extracellular Signal–Regulated
Kinase Activation, Is Required for SDF-1A/CXCR4–
Mediated Migration of Epitheloid Carcinoma Cells
Sheng-Bin Peng, Victoria Peek, Yan Zhai, Donald C. Paul, Qinyuan Lou,
Xiaoling Xia, Thomas Eessalu, Wayne Kohn, and Shaoqing Tang
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana
Abstract
Emerging evidence shows that the stromal cell – derived
factor 1 (SDF-1)/CXCR4 interaction regulates multiple
cell signaling pathways and a variety of cellular
functions such as cell migration, proliferation, and
survival. There is little information linking the cellular
functions and individual signaling pathways mediated
by SDF-1 and CXCR4 in human cancer cells. In this
study, we have shown that human epitheloid carcinoma
HeLa cells express functional CXCR4 by reverse
transcription-PCR, immunofluorescent staining, and
125
I-SDF-1A ligand binding analyses. The treatment of
HeLa cells with recombinant SDF-1A results in
time-dependent Akt and extracellular signal – regulated
kinase 1/2 (ERK1/2) activations. The SDF-1A – induced
Akt and ERK1/2 activations are CXCR4 dependent as
confirmed by their total inhibition by T134, a
CXCR4-specific peptide antagonist. Cell signaling
analysis with pathway-specific inhibitors reveals that
SDF-1A – induced Akt activation is not required for
ERK1/2 activation and vice versa, indicating that
activations of Akt and ERK1/2 occur independently.
Functional analysis shows that SDF-1A induces a
CXCR4-dependent migration of HeLa cells. The
migration can be totally blocked by phosphoinositide
3-kinase inhibitors, wortmannin or LY294002, whereas
mitogen-activated protein/ERK kinase inhibitors,
PD98059 and U0126, have no significant effect on
SDF-1A – induced migration, suggesting that Akt
activation, but not ERK1/2 activation, is required for
SDF-1A – induced migration of epitheloid carcinoma
cells. (Mol Cancer Res 2005;3(4):227 – 36)
Introduction
Chemokines and their receptors play an important role in
immune and inflammatory responses by mediating the
directional migration and activation of leukocytes (1-3).
These molecules have also been implicated in hematopoiesis,
angiogenesis, and embryonic development (4-6). Much of
Received 11/19/04; revised 2/11/05; accepted 2/22/05.
The costs of publication of this article were defrayed in part by the payment of
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accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Sheng-Bin Peng, Lilly Research Laboratories, Lilly
Corporate Center, Indianapolis, IN 46285. Phone: 317-433-4549; Fax: 317-2761414. E-mail: [email protected]
Copyright D 2005 American Association for Cancer Research.
what is currently known about chemokines and their
receptors has been learned from studies of hematopoietic
cells. Stromal cell – derived factor 1 (SDF-1/CXCL12) is a
highly efficient chemotactic factor for T cells, monocytes,
pre – B-cells, dendritic cells, and hematopoietic progenitor
cells (1-6). It binds only to one receptor, CXCR4, for which
SDF-1 is the only known ligand. Like other chemokine
receptors, CXCR4 is a seven-transmembrane-domain Gprotein – coupled receptor (7-9). The chemotactic effect of
SDF-1 on hematopoietic progenitor cells has been shown to
be mediated via the CXCR4 receptor. Targeted disruption in
mice of either the SDF-1 or CXCR4 gene results in a very
similar phenotype, is lethal, and accompanied by many
severe developmental defects, including the absence of both
lymphoid and myeloid hematopoiesis in the fetal bone
marrow (10, 11). In addition, it was found that SDF-1 and
CXCR4 play a critical role in the engraftment of hematopoietic stem cells to bone marrow (12). Collectively, these
results suggest that SDF-1 and CXCR4 regulate hematopoiesis by modulation of migration and homing of hemotopoietic stem and progenitor cells.
In addition to the prominent role in regulating leukocytes
and hematopoietic progenitor cells, recent research suggests
that SDF-1 and CXCR4 also play an important role in
tumorigenesis. CXCR4 was found to be expressed or
overexpressed in a variety of cancer cell lines and tissues
including breast cancer (13), prostate cancer (14), lung cancer
(15), ovarian cancer (16), colon (17), pancreatic cancer (18),
kidney cancer (19), and brain cancer (20-22), as well as nonHodgkin’s lymphoma (23) and chronic lymphocytic leukemia
(24). The emerging evidence suggests that CXCR4 plays
important roles in multiple phases of tumor progression,
including tumor growth, invasion, and metastasis. In a
preclinical mouse model of human high-grade non-Hodgkin’s
lymphoma, CXCR4 neutralization of Namalwa cells significantly delayed tumor growth (23). In vitro data showed that
CXCR4 neutralization enhanced apoptosis of tumor cells,
decreased cell proliferation, and inhibited cell migration and
pseudopodia formation (23). In human glioblastoma and
medulloblastoma xenograft models, the CXCR4-specific
antagonist AMD3100 inhibited tumor growth by inhibiting
cell proliferation and promoting apoptosis of the tumor cells
(25). The role of CXCR4 in promoting brain tumor cell
proliferation and survival was also observed under in vitro
conditions (20-22).
The SDF-1/CXCR4 interaction also plays a critical role in
cancer cell metastasis. Muller and colleagues found that
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CXCR4 was highly expressed in malignant but not normal
breast tissues, and its ligand SDF-1 was expressed in those
organs where breast cancer metastases were frequently found,
such as lymph node, lung, and bone marrow. SDF-1 stimulated
breast cancer cells to carry out the basics of invasion [i.e., cells
sent out extensions (pseudopodia), migrated in a directed
manner, and penetrated barriers imposed by extracellular matrix
(13)]. In a metastatic model of human breast cancer, CXCR4
neutralization significantly suppressed lymph node and lung
metastases of tumor cells (13). The involvement of CXCR4 in
cancer metastasis is not unique to breast cancer. In CXCR4expressing prostate cancer cells, SDF-1 was shown to promote
tumor cell transendothelial chemotaxis and adhesion to
osteoclastic cells (14). Myeloma cells express CXCR4 and
show a chemotactic response to SDF-1 associated with a rapid,
transient up-regulation of very late antigen-4, promoting tumor
cell adhesion to endothelial and marrow stromal cells (26). All
B-cell lymphomas express CXCR4 and migrate towards lymph
node stromal cells or SDF-1 (27). Chronic lymphocytic
leukemic cells also typically express functional CXCR4, and
SDF-1 promotes their adhesion to, and migration beneath, SDF1 producing marrow stromal cells (24). CXCR4/SDF-1 may
also play a role in the development of bone metastases in many
neuroblastomas (28). Collectively, the emerging data convincingly suggest that CXCR4 is directly involved in promoting
cancer metastases.
The role of SDF-1 and CXCR4 in angiogenesis is well
documented. It was shown that SDF-1 acts as a direct chemoattractant for endothelial cells in vitro, and as an angiogenic
factor in vivo (29-31). SDF-1 induces the expression of vascular
endothelial growth factor by endothelial cells. Vascular endothelial growth factor can, in turn, up-regulate CXCR4 levels on
endothelial cell surfaces. These observations indicate that SDF-1
and vascular endothelial growth factor act additively or
synergistically to amplify angiogenic processes. In addition,
SDF-1 alone can induce neovascularization in vivo and
formation of sprouting vessels in an ex vivo rat aortic ring
sprouting assay (30). Interestingly, knockout experiments
revealed that SDF-1 and CXCR4 play a role in blood vessel
development (32, 33). Human astrogliomas express elevated
CXCR4 and respond to SDF-1 by secretion of chemokines
expressed during angiogenesis and inflammation (21). In human
glioblastoma, SDF-1 and CXCR4 expression increased with
increasing tumor grade, and CXCR4 was also expressed in
neovascular endothelial cells, again indicating a role in
angiogenesis (22). In a prostate xenograft model expressing
high levels of CXCR4, the blood vessel density in the tumor was
4.5-fold higher than in the control model (34).
Despite the apparently important role of CXCR4/SDF-1 in
tumor cell growth, invasion, metastasis, and angiogenesis,
relatively little is known about the signaling pathways that
mediate these effects in cancer cells. In this study, we show that
CXCR4 is functionally expressed in epitheloid carcinoma HeLa
cells. SDF-1a treatment of HeLa cells results in the activation of
Akt and mitogen-activated protein (MAP) kinases extracellular
signal – regulated kinase (ERK) 1/2. SDF-1a also induces a
CXCR4-dependent HeLa cell migration. Analysis with pathwayspecific inhibitors reveals that Akt activation, but not ERK
activation, is required for SDF-1a – induced HeLa cell migration.
Results
CXCR4 Expression in HeLa Cells
Human epitheloid carcinoma HeLa cells are adherent cells
widely used for a variety of biological studies. We are
interested in using HeLa cells to study CXCR4/SDF-1 –
mediated functions in tumor cells. To ensure adequate
CXCR4 expression in HeLa cells, we first did a reverse
transcription-PCR (RT-PCR) analysis, and the RNA prepared
from cultured HeLa cells was analyzed using human
CXCR4, SDF-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene – specific primers. As shown in Fig. 1A,
CXCR4 was well expressed in HeLa cells, whereas SDF-1
expression is not detectable by RT-PCR in the given
conditions. However, both genes were expressed in a prostate
tumor tissue, a control sample used for this experiment.
GAPDH was used as an internal control to monitor if RNA
isolation and RT-PCR were reliable. As shown in Fig. 1A,
GAPDH was detected in both samples as expected.
To investigate the cell surface expression of CXCR4 in
HeLa cells, we did an immunofluorescent staining utilizing a
monoclonal antibody that specifically recognizes CXCR4 on
the cell surface. Confocal microscopic analysis revealed that
CXCR4-specific immunofluorescent staining was observed
when HeLa cells were incubated with CXCR4-specific
antibody (Fig. 1B), whereas no significant fluorescence staining
was observed when these cells were incubated with a control
immunoglobulin G in the same conditions (Fig. 1C), suggesting
that HeLa cells indeed express CXCR4 protein.
To further characterize the CXCR4 expression and function
in HeLa cells, we established a whole cell – based 125I-SDF-1a
ligand binding assay. As shown in Fig. 2, HeLa cells bind
125
I-SDF-1a in a dose-dependent manner with a K d of 0.2566
nmol/L. This 125I-SDF-1a binding can be specifically replaced
by cold SDF-1a, confirming the specificity of SDF-1a
binding. This ligand binding result further confirmed the
CXCR4 expression and function in HeLa cells. Collectively,
the results from RT-PCR, immunofluorescent staining, and
ligand binding assays suggest that HeLa cells express
endogenous CXCR4, and this endogenous CXCR4 expression
avoids the potential conformational change of the receptor that
could occur with overexpression by artificial transfection.
Therefore, we believe that HeLa cell is a highly valid tumor
cell line for CXCR4 functional studies.
SDF-1a Treatment Activates Phosphoinositide 3-Kinase/
Akt Pathway
Akt is a known downstream effector of the phosphoinositide 3-kinase (PI3K) – dependent signaling cascade. It has
recently been shown that some chemokines stimulate PI3K,
leading to the activation of protein Akt in some cell lines. The
PI3K/Akt pathway is an important mediator of chemotaxis in
many cell types (35-38). We were interested in investigating
whether SDF-1a treatment could activate PI3K/Akt pathway
in epitheloid carcinoma HeLa cells. For this purpose, we
measured the phosphorylation of Akt by Western blot using a
phospho-Akt – specific antibody. As revealed in Fig. 3A, SDF1a treatment of HeLa cells stimulated a time-dependent and
prolonged Akt phosphorylation. The phospho-Akt level
reached a maximum 5 to 10 minutes after SDF-1a treatment,
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SDF-1a/CXCR4 – Mediated Migration of Carcinoma Cells
FIGURE 1. Expression of CXCR4 in human epitheloid carcinoma HeLa cells. CXCR4 expression was analyzed by RT-PCR (A) and immunofluorescent
staining with a CXCR4 monoclonal antibody (B) or a control immunoglobulin G (C). An RNA sample prepared from a prostate cancer sample (lane 1 ) was
used as a control for RT-PCR. GAPDH was used as an internal control.
and the significantly enhanced Akt phosphorylation could still
be observed 60 minutes after SDF-1a treatment, whereas no
significant change in total Akt expression was observed over
the course of the experiment. To address whether the SDF1a – induced Akt activation was specifically via CXCR4, we
synthesized T134, a peptide antagonist that has been
characterized to block the CXCR4/SDF-1 interaction specifically (39). As revealed in Fig. 3B, the SDF-1a – activated Akt
phosphorylation was inhibited by T134 in a dose-dependent
manner. Complete inhibition was observed when 0.5 Amol/L
T134 was included in the reaction. These results suggest that
SDF-1a – induced Akt activation is specifically through its
monogamous receptor, CXCR4.
SDF-1a/CXCR4 Interaction Activates Mitogen-Activated
Protein Kinases Extracellular Signal – Regulated Kinase
1/2 and Their Downstream Effectors
To further study cell signaling mediated by the CXCR4/
SDF-1 interaction, we also investigated whether SDF-1a could
activate MAP kinases ERK1/2, p38, or c-jun-NH2-kinase.
Western blot analysis revealed that SDF-1a treatment of HeLa
cells resulted in a dramatic and rapid activation of both ERK1
and ERK2. The maximum phospho-ERK1/2 level was
observed 5 minutes after SDF-1a treatment, with phosphoERK1/2 level returning to near the basal level 20 to 40 minutes
after SDF-1a treatment. No significant change was noticed in
total ERK1/2 protein expression over the course of the
investigation (Fig. 4A). In contrast to ERK1/2, the phosphorylation of other MAP kinases, p38 or c-jun-NH2-kinase, was not
significantly affected by SDF-1a in these assay conditions (data
not shown). To further confirm the ERK pathway activation,
Western blot analysis was done for phospho-Elk-1 and phospho90rsk, the two immediate downstream effectors of ERK1/2. As
shown in Fig. 4A, both phospho-Elk-1 and phospho-90rsk
levels were increased in response to SDF-1a treatment in a timedependent manner similar to ERK1/2 activation, indicating that
ERK1/2 pathway was indeed activated. Again, T134, a peptide
antagonist specific for CXCR4, blocked SDF-1a – induced
ERK1/2 activation in a dose-dependent manner, and 0.5 Amol/L
T134 completely abolished the activation (Fig. 4B). This
observation suggests that SDF-1a – induced ERK1/2 activation
in HeLa cells was via its receptor, CXCR4.
SDF-1a/CXCR4 – Mediated Akt and ERK Activations Are
Independent from Each Other in HeLa Cells
Intracellular signaling involves a complex network of often
interacting pathways. We are interested in investigating the
relationship between SDF-1a – induced activation of Akt and
ERK, and in defining whether activation of the PI3K/Akt cascade
is required for SDF-1a – induced ERK activation and vice versa.
For this purpose, we did some pathway-specific inhibitor studies.
When HeLa cells were treated with PI3K inhibitors, wortmannin
or LY294002, SDF-1a – induced Akt activation was inhibited in
a dose-dependent manner, as expected. However, no significant
difference in SDF-1a – induced ERK activation was observed
with or without a PI3K inhibitor (Fig. 5A and B), suggesting that
Akt activation is not required for ERK activation. Similarly,
when HeLa cells were treated with MAP/ERK kinase (MEK)
inhibitors, PD98059 or U0126, SDF-1a – induced ERK activation was blocked in a dose-dependent manner, whereas no
significant effect of PD98059 or U0126 on SDF-1a – induced
Akt activation was observed (Fig. 5C and D). Therefore, Akt
activation is independent of ERK activation. Overall, these
results clearly show that SDF-1 – induced activations of Akt and
ERK in HeLa cells are not linear and arise from different
signaling pathways.
SDF-1a Induces CXCR4-Dependent Epitheloid Carcinoma
Cell Migration
SDF-1a and other chemokines regulate leukocyte trafficking
by mediating the adhesion of leukocytes to endothelial cells, the
initiation of transendothelial migration, and tissue invasion
(40). These are also processes used by cancer cells during
invasion and metastasis. Chemotaxis is the central function of
SDF-1 – regulated leukocyte trafficking and tumor cell metastasis. Due to the important role of the CXCR4/SDF-1 interaction
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pathways. To determine the functional role of PI3K/Akt
activation in SDF-1a – induced migration of epitheloid
carcinoma cells, HeLa cells were treated with different
concentrations of PI3K inhibitors, wortmannin or LY294002.
Cell migration in response to SDF-1a was examined by the
migration assay described under Materials and Methods. As
shown in Fig. 7A, the treatment of HeLa cells with
wortmannin inhibited SDF-1a – induced cell migration in a
dose-dependent manner, and a nearly complete inhibition
was observed with 200 nmol/L wortmannin. In addition, we
also tested the effect of LY294002, an ATP-competitive and
specific inhibitor of PI3K, because the specificity of
wortmannin at low doses was questioned previously (41).
Similar to the result with wortmannin, treatment of HeLa
cells with LY294002 also inhibited SDF-1a – induced cell
migration in a dose-dependent manner (Fig. 7B). Collectively, we have shown that PI3K inhibitors, wortmannin and
LY294002, could completely inhibit SDF-1 – induced HeLa
cell migration, suggesting that PI3K/Akt activation is
absolutely required for SDF-1a – induced migration of
epitheloid carcinoma cells.
ERK Activation Is Not Required for SDF-1a – Induced
HeLa Cell Migration
We have shown that ERK1/2 and its downstream effectors,
Elk-1 and 90rsk, were activated by SDF-1a treatment. To
evaluate the functional role of the MEK/ERK pathway in SDF1a – induced migration of epitheloid carcinoma cells, we next
examined the effects of MEK inhibitors, PD98059 and U0126,
FIGURE 2. 125I-SDF-1a ligand binding by HeLa cells. 125I-SDF-1a at
0-1.6 nmol/L was used for determination of the 125I-SDF-1a binding
affinity (A). The total binding (red line ) and the nonspecific binding in the
presence of 200-fold excess of cold SDF-1a (black line ) were determined
by filter binding assay as described under Materials and Methods. The net
125
I-SDF-1a binding (blue line ) was calculated by subtracting nonspecific
binding from the total binding. A K d of 0.2566 nmol/L was calculated by
nonlinear progression analysis. The specificity of 125I-SDF-1a binding was
further confirmed by a replacement experiment with cold SDF-1 (B). In B,
0.1 nmol/L 125I-SDF-1a and 1-64 nmol/L cold SDF-1a were used as
indicated.
in cancer metastasis, we were interested in investigating whether
SDF-1a induces HeLa cell migration. For this purpose, we
developed a cell migration assay in which HeLa cells were
evaluated for their ability to migrate through 3-Am pores of
bare filters as described under Materials and Methods. As
shown in Fig. 6A, SDF-1a induced a dose-dependent HeLa
cell migration, and the maximum migration was observed
when 89 nmol/L SDF-1a was applied in the assay conditions.
The migration was inhibited by T134 in a dose-dependent
manner by incubation of the HeLa cells with T134, indicating
that SDF-1a – induced HeLa cell migration was specifically via
its receptor CXCR4 (Fig. 6B).
Akt Activation Is Required for SDF-1a – Induced Cell
Migration
Activation of Akt and ERK1/2 after SDF-1a treatment
indicates that SDF-1/CXCR4 signaling involves multiple
FIGURE 3. SDF-1a – induced Akt activation and its inhibition by T134.
HeLa cells were treated with 12.5 nmol/L SDF-1a for 0, 1, 2, 5, 10, 20, 40,
or 60 minutes. The cell lysates were prepared and used for Western blot
analysis with phospho-Akt, total Akt, or actin antibody to investigate Akt
activation (A). HeLa cells were preincubated with 0, 0.125, 0.25, 0.5, or
1 Amol/L of T134 for 3 hours, and then treated with 12.5 nmol/L SDF-1a for
5 minutes. The cell lysates were used for Western blot analysis to
investigate inhibition of SDF-1a – induced Akt activation by T134 (B).
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SDF-1a/CXCR4 – Mediated Migration of Carcinoma Cells
vation was significantly inhibited by 2 to 50 Amol/L of
PD98059. Similarly, incubation of HeLa cells with another
MEK inhibitor, U0126, over a concentration range from 0.4 to
10 Amol/L, resulted in no significant effect on SDF-1a – induced
migration (Fig. 8B), although nearly complete inhibition of
ERK activation was achieved by 0.6 to 10 Amol/L U0126
(Fig. 5B). Thus, it seems that ERK activation is not required for
SDF-1a – induced migration of epitheloid carcinoma cells,
although ERK1/2 are dramatically activated after SDF-1a
treatment.
Discussion
FIGURE 4. SDF-1a – induced activation of ERK1/2 and their downstream effectors Elk-1 and 90rsk. HeLa cells were treated with 12.5 nmol/L
SDF-1a for 0, 1, 2, 5, 10, 20, 40, or 60 minutes. The cell lysates were
prepared and used for Western blot analysis with phospho-ERK, total
ERK, phospho-Elk-1, phospho-90rsk, or actin antibody (A). HeLa cells
were preincubated with 0, 0.125, 0.25, 0.5, or 1 Amol/L of T134 for 3 hours,
and then treated with 12.5 nmol/L SDF-1a for 5 minutes. The cell lysates
were used for Western blot analysis to investigate inhibition of SDF-1a –
induced ERK1/2 activation by T134 (B).
on the migration of HeLa cells in response to SDF-1a treatment.
As shown in Fig. 8A, the incubation of HeLa cells with PD98059
over a concentration range from 2 to 50 Amol/L did not
significantly alter cell migration. As shown in Fig. 5, ERK acti-
The chemokine SDF-1 and its cognate receptor CXCR4
have recently sparked substantial interest because of their
role in tumorigenesis including tumor growth, invasion, and
metastasis (13, 23, 25). These molecules mediate multiple
signal transduction pathways and a variety of cellular
functions such as cell migration, proliferation, and survival.
However, there is little information linking the cellular
functions and individual signaling pathways mediated by
SDF-1/CXCR4 interaction within the cells, particularly
cancer cells. In this study, we have shown that SDF-1a
treatment of epitheloid carcinoma HeLa cells results in
CXCR4-dependent activation of Akt and ERK1/2. Cell
signaling analysis using pathway-specific inhibitors revealed
that Akt and ERK activations seem to be independent of
each other. Functionally, SDF-1a induces a CXCR4dependent cell migration, and Akt activation, but not ERK
activation, is required for SDF-1a – induced migration of
epitheloid carcinoma cells.
CXCR4 is a Gi-coupled receptor, and studies have shown
that SDF-1, after binding to CXCR4, causes mobilization of
calcium, decrease of cyclic AMP within the cells, and activation
of multiple signaling pathways, including PI3K, phospholipase
C-g/protein kinase C, and MAP kinases ERK1/2 (42-45).
However, almost all of these studies were done with immune
cells or stem cells. Due to the important roles of CXCR4/SDF-1
in tumorigenesis, we are interested in investigating CXCR4/
FIGURE 5. A pathway-specific inhibitor
study showing that SDF-1a – induced Akt and
ERK activations are independent of each other.
HeLa cells, 2.5 105, were preincubated for 3
hours with different concentrations of PI3K
inhibitor, LY294002 (A) or wortmannin (B), or
MEK inhibitors, PD98059 (C) or U0126 (D),
followed by treatment with or without 12.5 nmol/
L SDF-1a for 5 minutes. The preparation of cell
lysates and Western blot analysis were done as
described under Materials and Methods.
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and U0126, at concentrations totally blocking ERK activation,
have no significant effect on Akt activation. These results
suggest that SDF-1a – induced Akt and ERK activations in
HeLa cells are independent and not linear in signal
transduction. It is in accordance with other results obtained
with human glioblastoma cells where ERK activation does not
require PI3K/Akt activation (46). However, it was reported
that SDF-1 – stimulated ERK activation was inhibited by PI3K
inhibitors in T lymphocytes, ERK1/2 activation is at least
partly dependent on PI3K activation, and both biochemical
events seem to be involved in the regulation of SDF-1 –
stimulated chemotaxis (43). Therefore, it seems that the
relationship between SDF-1 – induced Akt and ERK activations is cell type dependent.
From a functional standpoint, we found that SDF-1a
treatment induced a dose-dependent HeLa cell migration, an
important function related to cancer cell metastasis. Recent
studies show that the SDF-1/CXCR4 interaction plays a
critical role in cancer cell metastasis (13, 14, 26-28).
Therefore, it is important to define the molecular mechanisms
involved in SDF-1/CXCR4 – mediated tumor cell migration.
Accumulating data have implicated that multiple signaling
mechanisms exist to regulate cell migration. Both ERK
(47, 48) and PI3K/Akt (46, 49, 50) signaling pathways have
been shown to mediate the cell migration induced by
FIGURE 6. SDF-1a/CXCR4 – mediated epitheloid carcinoma HeLa cell
migration. In A, 0-200 nmol/L concentrations of SDF-1a as indicated were
used for this experiment, and SDF-1a induced a dose-dependent HeLa
cell migration. In B, 60 nmol/L SDF-1a was used, and SDF-1a – induced
HeLa cell migration was inhibited by T134 in a dose-dependent manner.
SDF-1a – regulated cell signaling in cancer cells. For this
purpose, we treated HeLa, a widely used human epitheloid
carcinoma cell line, with SDF-1a and analyzed the regulation
of multiple signal transduction components including MAP
kinases ERK1/2, p38, and c-jun-NH2-kinase, Akt, IkBa, Stat,
and GSK. Among the components analyzed, significant and
time-dependent activation of Akt, ERK1/2, and their downstream effectors, Elk-1 and 90rsk, was observed. However, no
significant regulation of p38, c-jun-NH2-kinase, NFnB, Stat, or
GSK was observed by Western blot analysis with the antibodies
used in this study (data not shown). Due to the important role of
Akt and ERK1/2 in cancer biology, this observation triggered
us to explore further the relationship between Akt and ERK1/2
activations, and their respective roles in cellular function.
Cell signaling is a very complex network, and in many
cases it is cell type dependent. Although SDF-1/CXCR4 –
mediated Akt and ERK activations were observed in multiple
cell types, including lymphocytes and stem cells, their
relationship in cell signaling was not well characterized. In
this study, we clearly show that SDF-1a – induced Akt and
ERK activations are independent of each other in HeLa cells.
PI3K inhibitors, wortmannin and LY294002, at concentrations
totally blocking SDF-1a – induced Akt activation, have no
effect on ERK activation. Similarly, MEK inhibitors, PD98059
FIGURE 7. Akt activation is required for SDF-1a/CXCR4 – mediated
HeLa cell migration. SDF-1a – induced HeLa cell migration was dramatically inhibited by PI3K inhibitors, wortmannin (A) and LY294002 (B), in a
dose-dependent manner. SDF-1a at 60 nmol/L was used in all the cell
migration reactions.
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SDF-1a/CXCR4 – Mediated Migration of Carcinoma Cells
FIGURE 8. ERK activation is not required for SDF-1a – induced HeLa
cell migration. When HeLa cells were preincubated with MEK inhibitors,
PD98059 (A) and U0126 (B), no significant inhibition of SDF-1a – induced
HeLa cell migration was observed. SDF-1a at 60 nmol/L was used in all
the cell migration reactions.
chemokines or cytokines in different cell types. However, the
molecular mechanisms of SDF-1/CXCR4 – regulated cell
migration in tumor cells are poorly understood to date. To
investigate the functional roles of PI3K/Akt and MEK/ERK
signaling cascades in SDF-1 – induced cell migration of
epitheloid carcinoma cells, we did studies with PI3K-specific
inhibitors (wortmannin and LY294002) or MEK-specific
inhibitors (PD98095 and U0126). The results showed that
PI3K inhibitors wortmannin and LY294002 totally blocked
SDF-1a – induced HeLa cell migration. In contrast, MEK
inhibitors PD98059 at 50 Amol/L and U0126 at 10 Amol/L,
both of which totally inhibited SDF-1a – induced ERK
activation, had no significant effect on SDF-1a – induced
cell migration, indicating that PI3K/Akt activation, but not
ERK activation, is required for SDF-1a – induced HeLa cell
migration. Although activation of the ERK signaling
pathway has been shown to promote cell mobility either
by regulating gene expression in carcinoma cells (47) or
directly activating myosin light chain kinase in COS-7 cells
(48), it seems that it is not the case in SDF-1a – induced
migration of epitheloid carcinoma cells. However, our results
are similar to the results observed in T lymphocytes, where
the SDF-1/CXCR4 interaction led to activation of multiple
signal transduction components, and the MAP kinase
inhibitor PD98059 had no effect on SDF-1 – induced
chemotaxis (51). Our findings are also consistent with
results obtained with hematopoietic progenitor cells and
primary marrow CD34+ cells where PI3K/Akt seemed to be
required for SDF-1 – mediated cell migration, but ERK1/2
were not (52).
However, in some cell types and under certain stimuli,
MAP kinase signaling has been shown to regulate cell
mobility (53). In T lymphocytes, SDF-1 – induced chemotaxis
is dependent on PI3K activation, and actin polymerization
requires additional biochemical inputs. SDF-1 – stimulated
ERK activation was inhibited by a PI3K inhibitor, Wortmannin. In addition, MEK inhibitor PD98059 partially attenuated
chemotaxis in response to SDF-1. Hence, it seems that
ERK1/2 activation is dependent on PI3K activation, and both
biochemical events are involved in the regulation of SDF-1
stimulated chemotaxis (43). These results are somewhat
different from those reported by another group with the same
cells (51). The differences observed by these two groups are
likely due to the nonspecificity of PI3K inhibitor, wortmannin (41). In fact, the so-called PI3K-dependent ERK
activation was only observed at high concentration (100
nmol/L) of wortmannin (43). In human neuroepithelioma
CHP100 cells expressing functional CXCR4, inhibition of
either the ERK or the PI3K pathways blocked the SDF-1 –
induced chemotaxis, suggesting that both Akt and ERK
activations were involved in SDF-1 – regulated cell migration
(54). In human embryonic kidney 293/hCXCR4 transfected
cells, h-arrestin 2 amplified CXCR4-mediated activation
of both p38 MAP kinase and ERK, and the suppression of
h-arrestin 2 expression blocked the activation of the two
kinases (55). Interestingly, inhibition of p38 activity (but not
ERK activity) by selective inhibitors or by expression of a
dominant-negative mutant of p38 MAP kinase effectively
blocked the chemotactic effect (55). Therefore, it seems that the
molecular mechanisms involved in CXCR4-mediated cell
migration are also cell type dependent.
Although ERK activation is not required for SDF-1a –
induced migration of HeLa cells as revealed by this study,
we believe that it may play an important role in promoting
tumor cell proliferation and survival. Indeed, many studies
with cultured tumor cells or cancer xenografts imply that
CXCR4/SDF-1 interaction stimulates tumor cell growth and
promotes cell survival (20, 23, 25). The functional roles of
SDF-1 – inducd ERK1/2 and Akt activations in cell proliferation and survival of HeLa cells are currently under
investigation in our laboratory.
Materials and Methods
Reagents
Anti-ERK1/2, anti – phospho-ERK1/2, anti-Akt, anti –
phospho-Akt, anti – phospho-Elk-1, and anti – phospho-90rsk
polyclonal antibodies for Western blot analysis were purchased from Cell Signaling (Beverly, MA). Anti – h-actin
monoclonal antibody was from Sigma (St. Louis, MO). AntihCXCR4 monoclonal antibody for immunofluorescent staining was from R&D Systems (Minneapolis, MN). Recombinant
human SDF-1a was purchased from PeproTech EC Ltd.
(London, United Kingdom). PI3K inhibitors, wortmannin and
Mol Cancer Res 2005;3(4). April 2005
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233
234 Peng et al.
LY294002, and MEK inhibitors, PD98059 and U0126, were
from Calbiochem (San Diego, CA). T134, a CXCR4-specific
peptide antagonist, was synthesized based on the published
structure (35).
for 1 hour in a dark humid chamber. Finally, the cells were
counterstained with propidium iodide. After careful washing
with 1 PBS/1% BSA, the cells were visualized for cell
surface expression of CXCR4 under a confocal microscope.
Cell Culture
Human epitheloid carcinoma HeLa Cells were purchased
from the American Type Culture Collection (Rockville, MD),
and grown in RPMI 1640 supplemented with 10% (v/v) fetal
bovine serum (FBS). Adherent cultures were passaged twice
each week at subconfluence after trypsinization. Cultures were
maintained in an incubator at 37jC in an atmosphere of 5%
CO2 and 95% air. The cells used for all experiments do not
exceed 15 passages.
Ligand Binding Assay
Ligand binding assay was done in a 96-well U-bottomed
microplate. One-hundred-microliter reactions containing
100,000 cells, 50 mmol/L HEPES (pH 7.5), 1 mmol/L CaC12,
5 mmol/L MgCl2, 0.5% BSA, and different concentrations of
125
I-SDF-1a and cold SDF-1a were incubated for 2 hours at
4jC. The reaction mixtures were then transferred to a 96-well
MultiScreen-FB filter plate, which was preequilibrated with
0.3% polyethyleneimine and 0.2% BSA for 30 minutes. The
plate was then washed thrice by filtration with 300 AL of washing
buffer containing 50 mmol/L HEPES (pH 7.5), 1 mmol/L CaC12,
5 mmol/L MgCl2, 0.5 mol/L NaCl, and 0.5% BSA with a vacuum
filtration apparatus from Millipore. After washing, the filter plate
was adapted to a MultiScreen Adaptor plate (Perkin-Elmer), 100
AL of Microscint 20 were added to each well, and the
radioactivity was determined on a Microplate Scintillation
Counter from Packard (Meriden, CT).
Reverse Transcription-PCR
Total cellular RNA was prepared using Absolutely RNA RTPCR Miniprep Kit from Invitrogen (Carlsbad, CA). Briefly,
cells were grown in flasks to 80% confluence, and 3 106
trypsinized cells were lysed with lysis buffer and digested with
RNase-free DNase following the instruction of the manufacturer. For RT-PCR, cDNA was synthesized from DNase-treated
RNA (200 ng) using Moloney murine leukemia virus reverse
transcriptase. The gene-specific primers used for human
CXCR4, SDF-1, and GAPDH amplification were designed as
follows: CXCR4 , 5V-CTTCTACCCCAATGACTTGTGG-3V
(sense) and 5V-AATGTAGTAAGGCAGCCAACAG-3V (antisense); SDF-1, 5V-ATGAACGCCAAGGTCGTGGTC-3V(sense)
and 5V-CTCACATCTTGAACCTCTTGTT-3V (antisense); and
GAPDH, 5V-ATGTCGAAGCGCGACATCGTC-3 (sense) and
5V-CACGACCAGTTGTCCATTCCT-3V (antisense). For PCR
reactions, 25 AL of sample contained synthesized cDNA, 1 unit
of AmpliTaq DNA polymerase, PCR buffer, deoxynucleotide
triphosphates, and 4 Amol/L of each primer. The following PCR
reaction conditions were used in a DNA Engine PTC-200
(MJ Research): 94jC for 5 minutes, 30 cycles at 94jC for
30 seconds, 55jC for 30 seconds, and 72jC for 30 seconds. The
PCR products were electrophoresed through 2.0% agarose gel
and visualized by ethidium bromide.
Immunofluorescence Microscopy
Ten thousand HeLa cells in 800 AL medium (RPMI 1640 +
10% FBS) were seeded in a four-chambered cover glass (LabTek II Chamber Slides System, Nalge Nunc International,
Rochester, NY) and grown overnight at 37jC to reach about
80% confluence. The cells were fixed in 1% formaldehyde
prepared in 1 PBS for 10 minutes at room temperature,
washed thrice for 10 minutes each with 1 PBS/1% bovine
serum albumin (BSA), and then permeabilized with 1 PBS/
1% BSA/0.025% NP40 for 15 minutes at room temperature.
After permeabilization, the cells were washed twice with 1
PBS/1% BSA, and blocked for 30 minutes at room
temperature with protein blocker buffer (Dako, Carpinteria,
CA), and then incubated with anti-CXCR4 monoclonal
antibody in 1:200 dilution or a control immunoglobulin G in
1:100 dilution for 1 hour at room temperature. After incubation
with primary antibody, the cells were washed and incubated
with goat anti-mouse immunoglobulin G conjugated with
Alexa 488 (Molecular Probes, Eugene, OR) in 1:500 dilution
SDF-1a Treatment and Preparation of HeLa Cell Lysates
HeLa cells, 2 105, were seeded and grown in 1 mL of
RPMI 1640 with 10% FBS in six-well plates for overnight, then
starved for 3 hours by growing them in RPMI 1640 without
FBS. After starvation, the cells were treated with human SDF1a at 37jC for various periods as indicated in figure legends,
and then washed with 1 mL of ice-cold PBS. Cells were then
immediately lysed with 100 AL of lysis buffer consisting of
25 mmol/L Tris (pH 7.5), 2.5 mmol/L Na2H2P2O7, 150 mmol/L
NaCl, 2 mmol/L TAMP, 15 mmol/L p-nitropenyl phosphate,
5 mmol/L benzamidine, 60 mmol/L h Gly PO4, 1 mmol/L
Na-vanadate, 10 mmol/L Na-fluoride, 1 mmol/L DTT,
15 mmol/L EDTA, 5 mmol/L EGTA, 1 Amol/L okadaic acid,
1 Amol/L microsystin, 1% Triton X-100, and 1 protease
inhibitor cocktail from Roche (Indianapolis, IN). Total cell
lysates were clarified by centrifugation at 10,000 rpm for
10 minutes using an Eppendorf mini-centrifuge. Protein
concentrations were determined with Bio-Rad (Hercules,
CA) protein assay agents using BSA as a standard.
Western Blot Analysis
A total of 10 Ag of control or SDF-1a – treated cell lysate
was separated on a 4% to 20% SDS-polyacrylamide gel and
transferred electrophoretically to a nitrocellulose membrane
(Invitrogen), and the membrane was blocked in blocking
solution (5% nonfat dry milk/Tris-buffered solution/0.01%
Tween 20) and then incubated overnight with a primary
antibody in the appropriate dilution. The unbound primary
antibody was removed by washing the membrane with Trisbuffered solution/0.01% Tween 20, followed by incubation
with horseradish peroxidase – conjugated anti-rabbit or antimouse secondary antibody diluted 1:5,000 in 3% nonfat dry
milk/Tris-buffered solution/0.01% Tween 20. Protein was then
visualized using enhanced chemiluminescence solution from
Amersham (Piscataway, NY) and X-ray film.
Mol Cancer Res 2005;3(4). April 2005
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SDF-1a/CXCR4 – Mediated Migration of Carcinoma Cells
Cell Migration Assay
HeLa cells were grown in RPMI 1640 + 10% FBS to 80%
confluence on the day of experiment. The cells were harvested
and washed once with migration assay buffer [HBSS consisting
of 20 mmol/L HEPES (pH 7.5) and 0.1% BSA]. After washing,
the cells were pretreated with an inhibitor at certain
concentration for 15 minutes at 37jC with 5% CO2. Then,
1 105 cells in 250 AL of migration assay buffer were added to
each of FALCON HTS FluoroBlock insert with pore size of
3.0 Am (BD Bioscience, Palo Alto, CA). To each bottom well,
750 AL of 60 nmol/L SDF-1 or a mixture of 60 nmol/L SDF-1
and an inhibitor in certain concentration prepared in migration
assay buffer were added. After assembly of inserts and bottom
wells, the migration plate was incubated for 22 hours at 37jC
with 5% CO2. Following the incubation, the top inserts were
transferred into a second Falcon non-TC-treated 24-well plate
containing 2 Ag/well Calcein AM (Molecular Probes) prepared
in 0.5 mL of HBSS. The plate was then incubated for
90 minutes at 37jC, and the total cell migration was obtained
by measuring the fluorescence in the CytoFluor 400 microplate
spectrofluorometer using excitation/emission wavelength of
485/530 nm.
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Akt Activation, but not Extracellular Signal−Regulated
Kinase Activation, Is Required for SDF-1 α/CXCR4−Mediated
Migration of Epitheloid Carcinoma Cells
Sheng-Bin Peng, Victoria Peek, Yan Zhai, et al.
Mol Cancer Res 2005;3:227-236.
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