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Prepublished online as a Blood First Edition Paper on November 14, 2002; DOI 10.1182/blood-2002-09-2813.
 Previous Article | Table of Contents | Next Article 
Blood, 1 April 2003, Vol. 101, No. 7, pp. 2762-2769
NEOPLASIA
CD40 induces human multiple myeloma cell migration via
phosphatidylinositol 3-kinase/AKT/NF- B signaling
Yu-Tzu Tai,
Klaus Podar,
Nicholas Mitsiades,
Boris Lin,
Constantine Mitsiades,
Deepak Gupta,
Masaharu Akiyama,
Laurence Catley,
Teru Hideshima,
Nikhil C. Munshi,
Steven P. Treon, and
Kenneth C. Anderson
From the the Jerome Lipper Multiple Myeloma Center,
Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA;
and Department of Medicine, Harvard Medical School, Boston, MA.
 |
Abstract |
Multiple myeloma (MM) is characterized by clonal expansion of
malignant plasma cells in the bone marrow and their egress into peripheral blood with progression to plasma cell leukemia. Our previous
study defined a functional role of CD40 activation in MM cell homing
and migration. In this study, we examine signaling events mediating
CD40-induced MM cell migration. We show that cross-linking CD40, using
either soluble CD40L (sCD40L) or anti-CD40 monoclonal antibody (mAb),
induces phosphatidylinositol 3-kinase (PI3K) activity and
activates its downstream effector AKT in MM.1S cells. CD40 activation
also activates the MAP kinase (MEK) pathway, evidenced by
phosphorylation of extracellular signal-regulated mitogen-activated
protein kinase (ERK), but not c-jun amino-terminal kinase
(JNK) or p38, in a dose- and time-dependent manner. Using pharmacologic inhibitors of PI3K and MEK, as well as
adenoviruses expressing dominant-negative and constitutively expressed
AKT, we demonstrate that PI3K and AKT activities are required for
CD40-induced MM cell migration. In contrast, inhibition of ERK/MEK
phosphorylation only partially (10%-15%) prevents migration,
suggesting only a minor role in regulation of CD40-mediated MM
migration. We further demonstrate that CD40 induces nuclear
factor (NF)- B activation as a downstream target of PI3K/AKT
signaling, and that inhibition of NF-B signaling using specific
inhibitors PS1145 and SN50 completely abrogates CD40-induced MM
migration. Finally, we demonstrate that urokinase plasminogen activator
(uPA), an NF-B target gene, is induced by CD40; and conversely, that
uPA induction via CD40 is blocked by PI3K and NF-B inhibitors. Our
data therefore indicate that CD40-induced MM cell migration is
primarily mediated via activation of PI3K/AKT/NF-B signaling, and
further suggest that novel therapies targeting this pathway may inhibit
MM cell migration associated with progressive MM.
(Blood. 2003;101:2762-2769)
© 2003 by The American Society of Hematology.
| |
Introduction |
CD40, a member of the tumor necrosis factor
receptor (TNFR) superfamily, was first identified and functionally
characterized on B lymphocytes. It is activated as a trimer after
interaction with CD40 ligand (CD40L) expressed on activated T cells.
The interaction between CD40 and CD40L plays a central role in immune
regulation, autoimmune diseases, and many human cancers, including
multiple myeloma (MM).1,2 We and others have identified
diverse biologic sequelae of CD40 activation in MM cells: up-regulation
of cell-surface proteins (eg, B7, CD18, CD11a, CD49d,
CD54)3; induction of interleukin (IL)-6
secretion3; and increased expression of adhesion
molecules, including the Ku86 and Ku70 autoantigens, on the MM cell
surface.4,5 Our studies show that triggering MM cells via
CD40 ligation induces either proliferation or growth arrest and
apoptosis of MM cells, depending upon their p53 status.6 We recently further demonstrated that CD40 induces MM cell migration and vascular endothelial growth factor (VEGF) secretion, suggesting a
functional role of CD40 activation in MM homing and
angiogenesis.7 Interestingly, CD40 ligation-triggered
VEGF secretion and angiogenesis have also been reported in endothelial
cells,8 synovial fibroblasts,9 and Kaposi
sarcoma cells.10 These studies not only define a mechanistic link between the immune response and angiogenesis, but also
suggest that CD40 activation may promote tumor progression.
Studies of CD40 signal transduction have revealed induction of
multiple mediators and pathways. Studies to date have focused on 3 mitogen-activated protein kinases (MAPKs): stress-activated protein
kinase/c-jun amino-terminal kinase (SAPK/JNK), p38, and extracellular
signal-regulated mitogen-activated protein kinase (ERK). The JNK and
p38 pathways are predominantly activated by CD40 stimulation in
multiple B-cell lines.11-14 Cross-linking CD40 rapidly
activates p38 in human tonsillar B cells, whereas inhibition of p38
activity with specific inhibitor SB203580 inhibits CD40-induced gene
expression and proliferation.14 Thus, p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes. In
contrast, CD40 induces little, if any, activation of
ERK.11,12 Other studies demonstrate that CD40 induces
activation of src-type protein tyrosine kinases
(lyn),15 phosphatidylinositol 3-kinase (PI3K),16 phospholipase C 2,16 and nuclear
factor (NF)-B/Rel.17 To date, however, the
signaling cascades mediating the biologic sequelae of CD40 activation
in human MM cells, including migration, are not delineated.
Several signaling molecules mediate cell migration, including
PI3K,18-20 protein kinase B (AKT),21-23 and
ERK.24,25 PI3K, consisting of a 110-kDa catalytic subunit
and a tightly associated regulatory subunit encoded by the
p85a gene, is a key intermediate in cellular responses
induced by a vast array of divergent agonists.26 Specifically, activation of PI3K is required for both
insulin-like growth factor-1 (IGF-1)-induced vascular smooth
muscle cell proliferation and migration27 and for
TGF- -mediated epithelial to mesenchymal cell
transition and migration.28 In both endothelial and MM cells, VEGF stimulates PI3K activation and migration.23,29 The AKT serine/threonine kinase is a core component of PI3K
signaling and mediates MM cell growth, survival, and drug
resistance.30,31 Recently, a major role for AKT in
regulating migration/invasion in endothelial cells,21
vascular pericytes,19 and a highly metastatic fibrosarcoma
cell line32 has been shown. Specifically, AKT potently
promotes fibrosarcoma cell HT1080 invasion by increasing cell motility
and matrix metalloproteinase-9 production, in a manner highly dependent
on its kinase activity and membrane-translocating ability.32 In contrast, multiple other studies have
indicated that cell motility is modulated by the magnitude and the
duration of ERK/MAPK activation,33,34 and that temporal
and quantitative regulation of MAPK mediates tumor cell
motility and invasion.
A large number of stimuli can activate NF-B transcriptional
factors. Ordinarily, NF-B is sequestered in the cytoplasm by the
inhibitory protein IB in its inactive form. Various stimuli induce phosphorylation of IB kinase (IKK) and ubiquitination of
IB, with subsequent targeting and degradation by proteasomes. NF-B then translocates into the nucleus, binds to appropriate consensus motifs, and regulates the transcription of various target genes. Induction of NF-B via AKT reveals a direct link between AKT
and IKK. AKT mediates IKK phosphorylation at threonine 23, whereas mutation of this amino acid blocks phosphorylation of AKT and
activation of NF-B by TNF in 293 cells; therefore, NF-B activation by TNF requires AKT kinase.35 Upon
platelet-derived growth factor (PDGF) stimulation, AKT
transiently associates in vivo with IKK and induces IKK
activation.36 NF-B is a target of the antiapoptotic
Ras/PI3K/AKT pathway induced by PDGF signaling.36 In
addition, the effect of AKT on NF-B induction is dependent upon
cellular PI3K activity.37 Since CD40 activates NF-B and its downstream targets, NF-B may play a pivotal role in CD40-induced MM cell migration.
The urokinase-type plasminogen activator (uPA) is a critical protease
mediating tumor invasion and metastasis. uPA is up-regulated in a
variety of solid malignancies, and its overexpression is induced by
constitutive NF-B/RelA activity.38-40 The NF-B
binding site located in the promoter of uPA gene controls its
expression.41 Since uPA mediates conversion of plasminogen
into plasmin, which degrades extracellular matrix (ECM)
proteins such as fibronectin, laminin, and collagen IV, uPA may
influence cancer progression.38,40 Proteolytic activity of
uPA is necessary for both cell invasion and cell
migration,38 suggesting its potential role in CD40-induced MM cell migration.
In this study, we examined PI3K/AKT/NF-B and MAPK signaling
mediating migration in MM.1S MM cells. Using specific pharmocokinetic inhibitors and adenoviruses expressing dominant-negative AKT, as well
as constitutively expressed AKT, we show that the PI3/AKT/NF-B pathway mediates MM cell migration induced by CD40. Furthermore, we
found that uPA is a downstream target of CD40-induced PI3K/AKT/NF-B signaling in MM cells.
| |
Materials and methods |
Cells and stimulation
MM.1S cells were kindly provided by Dr Steve Rosen (Northwestern
University, Chicago, IL). The cells were cultured in RPMI1640 medium
containing 10% fetal bovine serum, 2 mM
L-glutamine, 100 U/mL penicillin, and 100 µg/mL
streptomycin. Cells were serum starved overnight and were stimulated by
CD40 cross-linking as previously reported.7 In brief,
cells (2 × 106 or 20 × 106 per sample for
total lysates or immunoprecipitations, respectively) were stimulated at
37°C with 5 µg/mL of G28.5 anti-CD40 monclonal antibody
(mAb) unless otherwise indicated. In some cases, cells were
CD40 cross-linked using 626.1 anti-CD40 mAb or 200 ng/mL of a
recombinant CD40L-FLAG-tag fusion protein (sCD40L; Alexis Biochemicals, San Diego, CA) supplemented with 1 µg/mL CD40L enhancer (Alexis Biochemicals), according to the manufacturer's protocol.
Cell migration assay
MM.1S cells were serum starved overnight and then resuspended in
70 µL of RPMI1640 medium/0.5% fetal calf serum with or
without indicated inhibitors. Cell migration was conducted in 24-well, 6.5-mm internal-diameter Transwell cluster plates (Corning Costar; Cambridge, MA). Briefly, cells (2.5 × 105/70 µL)
pretreated with or without specific inhibitors were loaded onto
polycarbonate membranes (8-µm pore size) separating 2 chambers of a
transwell. Medium/0.1% FCS (500 µL) containing agonist anti-CD40 mAb
(G28.5 or 626.1) or sCD40L (Alexis Biochemicals) was added to the lower
chamber of the Transwell cluster plates. After 6 hours, cells migrating
into the lower chamber were counted using a Coulter counter ZBII
(Beckman Coulter, Miami, FL), as well as by hemacytometer. A
mouse immunoglobulin G (IgG1) MOPC 21 was used as
a control when anti-CD40 mAb was used as a stimulant.
Reagents
PI3K inhibitors LY 294002 (LY) and wortmannin (Wort; Sigma, St
Louis, MO), as well as MEK 1/2 inhibitor PD98059 (Cell Signaling Technology, Beverly, MA), were dissolved in dimethyl sulfoxide and further diluted in RPMI1640 medium. Actinomycin D (act D) and
cycloheximide (chx) were obtained from Sigma. Antibodies (Abs) for Western blotting were obtained from the following sources: anti-pERK and anti-IB were from Santa Cruz Biotechnology (Santa Cruz, CA); antiphosphorylated JNK, anti-JNK,
anti-phosphorylated p38, anti-AKT, and anti-pAKT (detects
phosphorylation on S-473 residue) were from Cell Signaling Technology;
and anti-uPA mAb was from Oncogene Research Products (San Diego, CA).
Western Blotting and immunoprecipitation
Total cell lysates were subjected to 8% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred onto polyvinylidene fluoride membranes, as previously
reported.7
PI3K kinase activity assay
PI3K activity in antiphosphotyrosine (4G10) and anti-CD40
immunoprecipitates was performed as described.29
Radioactive lipids were separated by thin-layer chromatography, using
N-propanol: 2M HOAc (65/35, vol/vol). After drying, the plates were autoradiographed.
Analysis of AKT activity
AKT kinase assay was performed using the AKT kinase assay kit,
according to the protocol provided by the manufacturer (Cell Signaling
Technology). Briefly, 500 µg of total protein from MM cells was added
to AKT Ab-coated beads and incubated at 4°C for 3 hours, followed by
washing. Phosphorylation of GSK-3 was used as an indicator of
phosphorylated AKT, since AKT negatively regulates GSK-3/ kinase
activity via phosphorylation of GSK-3 at Ser219. After the kinase
reaction, the reaction mixture was electrophoresed on a 12% SDS-PAGE
gel and Western blotted. The blots were probed with an
antiphosphorylated GSK-3/ (Ser219) Ab.
Nuclear NF-B pull-down assay
MM.1S cells (5 × 106/time point) were incubated
with G28.5 mAb after pretreatment with PS1145 or SN50, and nuclear
extracts were prepared. Cells were pelleted and resuspended in 0.4 mL
hypotonic lysis buffer (20 mM HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.9, 10 mM
KCl, 1 mM EDTA (ethylenediaminetetraacetic acid), 0.2% Triton
X-100, and 1 mM sodium orthovanadate plus protease inhibitors) and kept
on ice for 20 minutes. After centrifugation at 14 000g for
5 minutes at 4°C, the nuclear pellet was extracted with 0.1 mL
hypertonic lysis buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA,
and 1 mM sodium orthovanadate plus protease inhibitors) on ice for a
further 20 minutes. After centrifugation at 14 000g for 5 minutes at 4°C, the supernatants were diluted to 100 mM NaCl and
incubated with 25 µL of agarose beads conjugated to a consensus
NF-B binding oligonucleotide (Santa Cruz Biotechnology) for 1 hour
at 4°C. After 3 washes, 25 µL of 2 × sample buffer was added and
boiled for 5 minutes. The result was analyzed by SDS-PAGE and Western
Blotting using an anti-p65 NF-B Ab (Santa Cruz Biotechnology).
Recombinant adenovirus
Replication-defective adenovirus vectors expressing
dominant-negative (Ad dnAKT) and constitutively active forms
of AKT (Ad myrAKT) driven by CMV promoter were kindly provided by Dr
Kenneth Walsh (St Elizabeth's Medical Center, Boston,
MA).42 The Ad dnAKT containing 3 amino acid mutations at 3 critical phosphorylation sites functions as a dominant negative for
endogenous AKT. The Ad myrAKT has an in-frame fusion of the c-src
myristoylation sequence to the N-terminus of the wild-type AKT coding
sequence, thereby targeting the fusion protein to the membrane. Ad
-gal recombinant adenoviruses43 were used as a negative
control. All viruses were produced in 293 cells and purified by 2 runs
of ultracentrifugation through CsCl gradient, as published
previously.43 To obtain transduction efficiencies of more
than 85%, multiplicities of infection of 200 were used to
infect MM.1S cells, without any significant toxicity. Cells were
typically infected with adenoviruses for 2 hours, followed by
replacement with fresh medium overnight, and then treatment with or
without test reagents (Wort, LY, PS1145, SN50) for 1 to 2 hours.
Transmigration assay was performed as described in "Cell migration assay."
uPA secretion
Supernatants from control or CD40-activated MM.1S cells
incubated in serum-free RPMI1640 were collected after 12 hours.
Supernatants from CD40-activated MM.1S cells with or without PI3K
inhibitors (Wort, 0.2 µM; LY, 30 µM) and NF-B inhibitors
(PS1145, 10 µM; SN50, 1 µM) were collected after 48 hours. The
medium was concentrated 10-fold by using a VIVASPIN concentrator
(VIVASCIENCE, Cambridge, MA). Secretion of uPA was detected by Western
blotting analysis of supernatants with anti-uPA mAb (Oncogene Research
Products, Cambridge, MA).
Statistical analysis
Statistical significance of differences observed in
CD40-activated versus control cells was determined using the Student
t test. The minimal level of significance was a
P value less than .05.
| |
Results |
CD40 activation induces transmigration of MM.1S cells
We recently demonstrated that CD40 activation increases MM cell
migration in a transwell migration assay in which anti-CD40 mAb or
soluble human CD40L (sCD40L) is applied in the lower chamber of a
transwell system.7 Since we have not studied the biologic sequelae of CD40 activation in the CD40-expressing MM.1S MM cell line,
we first assayed transmigration triggered by CD40. MM.1S (CD38+CD45 ) cells express high levels of
CD138 (syndecan-1) and are Epstein-Barr virus-independent. In
addition, IL-6 induces AKT and NF-B activation in MM.1S cells, as in
primary myeloma patient cells.30 When 250 000 MM.1S cells
were applied to the top chamber of transwells, approximately
13 250 ± 3994.3 cells migrate to the lower chamber of transwells
following 6 hours of incubation (Figure
1A-C). Thus, the baseline migration of
MM.1S cells, without any stimulants added to the lower chamber in the
transmigration assay, is approximately 4% to 7%. As shown in Figure
1A, CD40 activation, either by anti-CD40 mAb (clone G28.5 or 626.1) or
sCD40L, induces MM.1S cell migration in a dose-dependent manner within
6 hours, with peak migration at 50 µg/mL of stimulant. When anti-CD40
mAb G28.5 was added to both upper and lower chambers in the transwells,
an even more prominent dose-dependent increase in migrating MM.1S cells
was observed (Figure 1B). Similar results were also obtained when sCD40L was added to the upper and lower chambers of the transwells (data not shown). Since adhesion plays a role in cell migration, we
compared MM cell migration in a transmigration assay using filters
separating 2 chambers in the transwell, either coated with or without
fibronectin (40 µg/mL). In the presence of fibronectin, the fold
increase in migrating cells was significantly increased at 10 and 50 µg/mL of G28.5 (P = .01 and .025, respectively; Figure 1C). These results confirm our previous finding that cell migration is
induced by CD40 activation in human MM cells.7
Interestingly, CD40 activation of MM.1S cells, even at concentrations
as high as 10 µg/mL, did not significantly alter DNA synthesis
(P = .15; Figure 1D), even though the cells proliferated
in response to 50 ng/mL IL-6 (cpm, 29 609 ± 1230).
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| Figure 1.
CD40 activation induces transmigration of MM.1S MM
cells.
(A) Serum-starved MM.1S cells were plated on a polycarbonate membrane
(8-µm pore size) in the transwell cluster plate and activated using 0 to 50 µg/mL of anti-CD40 mAb (G28.5 or 626.1) or sCD40L added to the
lower chamber. After 6 hours of incubation, migrating cells in the
lower chamber were counted. G28.5 ( ); 626.1 ( ); sCD40L ( ). (B)
Serum-starved MM.1S cells were plated at the upper chamber in the
transwell cluster plate and activated using 0 to 10 µg/mL G28.5
anti-CD40 mAb added in the lower chamber () or added both in the
upper and lower chambers (). Migrating cells into the lower chamber
were collected and quantitated. (C) Membranes separating upper from
lower chambers in the transwell were coated with () or without ()
fibronectin (40 µg/mL) overnight. Results are mean ±SE of 3 independent experiments. (D) MM.1S were incubated with CD40 stimulants
(µg/mL): G28.5 (); 626.1 (); sCD40L (). After 36 hours, the
cells were pulsed with 3H-thymidine, and DNA synthesis was
measured. Results are mean ±SE of 3 independent experiments.
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CD40 activation selectively phosphorylates AKT and ERK
CD40-induced signaling in MM cells is not well characterized. We
therefore next investigated whether CD40 activation induces phosphorylation of AKT. Since MAPK JNK, p38, and ERK are the focus of
studies of CD40 signaling in many B-lymphoma lines, we also determined
whether CD40 triggering in MM.1S cells activates these kinases. CD40
activation by anti-CD40 mAb stimulates phosphorylation of AKT and ERK
in a dose- (Figure 2A) and time- (Figure
2B) dependent manner in MM.1S cells. Peak activation of AKT is maximum
at more than 2 µg/mL of anti-CD40 mAb G28.5, whereas activation of
ERK is induced by 0.02 µg/mL of G28.5 (Figure 2A). The
phosphorylation of both AKT and ERK induced by CD40 occurs within 10 minutes and persists for at least 60 minutes after CD40 activation by
G28.5 (2 µg/mL; Figure 2B). Although phosphorylation of AKT returns to baseline within 2 hours, activation of ERK is sustained (Figure 2B).
These results therefore indicate that CD40 activation selectively activates AKT and ERK/MAPK pathways in MM.1S cells.
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| Figure 2.
CD40 induces phosphorylation of AKT and ERK in a dose-
and time-dependent manner.
Serum-starved MM.1S cells were activated by CD40 by incubation with
anti-CD40 mAb (G28.5 or 626.1) at the indicated concentrations for 15 minutes (A) or with G28.5 (2 µg/mL) for 0 to 240 minutes (B). Samples
were collected and analyzed by Western blotting with
antiphosphorylation-specific Abs. Detection of total AKT on the same
blots was used to demonstrate equal loading of samples. Lysates were
also probed for pJNK and p38 using specific antiphospho Abs.
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PI3K and AKT activity mediate MM.1S cell migration induced by
CD40
Since phosphatidylinositol (3,4,5)P3 (PIP3)
regulates cell migration and PI3K can activate AKT, we next asked
whether CD40 activation induces PI3K activity in MM.1S cells.
Specifically, we performed PI3K and AKT kinase activity assays using
cell lysates of CD40-activated versus unstimulated MM.1S cells. As
shown in Figure 3A (upper panel), PI3K
kinase activity in antiphosphotyrosine immunoprecipitates prepared from
CD40-activated cell lysates is significantly induced 5 minutes
following CD40 activation, peaks at 15 minutes, and declines
thereafter. A time-dependent increase in p85 immunoreactivity is also
observed in these antiphosphotyrosine immunoprecipitates following CD40
activation (Figure 3A, lower panel), confirming that the increased PI3K
activity in the antiphosphotyrosine immunoprecipitates is due, at least
in part, to p85/p110 PI3K. Moreover, AKT kinase activity measured by
phosphorylation of GSK-3/, was abrogated by PI3K inhibitors Wort
(0.2 µM) and LY (30 µM) (Figure 3B). These results confirm
that CD40 activation induces PI3K activity and triggers AKT activity in
MM.1S cells. Additionally, as shown in Figure 3C, activation of AKT and
ERK is blocked by PI3K inhibitors Wort (0.2 µM) or LY (30 µM) and
MEK1/2 inhibitor PD098959 (30 µM), respectively, but neither Wort
(0.2 µM) nor LY (30 µM) inhibits CD40-induced phosphorylation of
ERK. These data indicate that activation of ERK by CD40 is mediated via
MEK, without PI3K/AKT cross-talk.
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| Figure 3.
CD40 triggers AKT activities via PI3K activation.
(A) Serum-starved MM.1S cells were activated by CD40 with 2 µg/mL
G28.5 anti-CD40 mAb and collected at indicated time intervals. PI3K
kinase assay was performed using equal amounts of lysates
immunoprecipitated with antiphosphotyrosine 4G10 mAb, and
immunocomplexes were assayed for the ability to phosphorylate
PIP2. Equal amounts of PI and PIP2 produced in
each sample demonstrate a specific induction of PI3K by CD40
activation. Control (c) indicates a PI3K kinase assay performed on
protein A alone. (B) Serum-starved MM.1S cells, with or without
pretreatment with PI3K inhibitors Wortmannin (0.2 µM) and LY294002
(30 µM) or anti-AKT peptide (16 µg/mL), were incubated with 2 µg/mL G28.5 anti-CD40 mAb for 30 minutes. Cell lysates were prepared
from each sample and immunoprecipitated with an anti-AKT Ab with (lane
5) or without (lanes 1-4) competitor peptide. Kinase activity was
measured with GSK-3/ as a substrate and visualized by Western
blotting with an antiphospho GSK-3 antibody, according to
manufacturer's protocol. Western blotting with an anti-AKT Ab (lower
panel) served as a loading control. (C) Serum-starved MM.1S cells, with
or without pretreatment with indicated inhibitors, were activated by
CD40 as described in "Materials and methods." Cells were
collected 30 minutes following CD40 activation, and cell lysates were
prepared and subjected to Western blotting using anti-pAKT and
anti-pERK Abs. Total AKT and total ERK were detected using anti-AKT and
anti-ERK Abs on the same blots, demonstrating equal loading of each
sample. PD indicates PD098959 (30 µM); Wort, Wortmannin (0.2 µM);
and LY, LY294002 (30 µM).
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We next studied whether PI3K or ERK mediates CD40-induced MM cell
migration. As demonstrated in Figure 4,
Wort and LY inhibit CD40-triggered MM cell migration in a
dose-dependent manner, whereas PD98059 (30µM), even at
concentrations that inhibit ERK activation, only partially (10%-15%)
blocks MM cell migration induced by CD40. In the presence of LY (50 µM) or Wort (0.5 µM), PD98059 did not further inhibit CD40-induced
MM cell migration. These data indicate that PI3K activity, but not ERK
activity, mediates MM cell migration stimulated by CD40.
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| Figure 4.
Effects of PI3K and ERK inhibitors on CD40-induced MM.1S
migration.
A transmigration assay was performed as described in "Materials and
methods." Serum-starved MM.1S cells, with and without
pretreatment with inhibitors for 1 hour, were seeded in the upper
chamber in the transwell cluster plate. At 6 hours after CD40
activation by G28.5 anti-CD40 mAb (5 µg/mL) in lower chamber,
migrating cells in the lower chamber were collected and
counted. Data are the means ± SD of triplicate determinations.
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In order to define the functional role of AKT activity mediating
CD40-induced MM cell migration, MM.1S cells were first transduced with
adenovirus vectors expressing either dominant-negative AKT mutant (Ad
dnAKT) or Ad myrAKT. The expression of dnAKT and
myrAKT was confirmed by Western blotting using green fluorescent
protein (GFP) and hemagglutinin (HA) Abs, respectively (Figure
5A). Phosphorylation of AKT in
adenovirus Ad myrAKT MM.1S transfectants is observed even without CD40
stimulation (Figure 5B), confirming constitutive activation by Ad
myrAKT. The activation of AKT was further enhanced in Ad
myrAKT-transduced MM.1S cells at 15 minutes after CD40 stimulation (clearly seen in the films with shorter exposure). In contrast, CD40
stimulation did not induce AKT phosphorylation in MM.1S cells transduced with dominant-negative Ad dnAKT, compared with cells transduced with control Ad -gal viruses (Figure 5B, center). The
temporal sequelae of AKT activation by CD40 were similar in Ad
gal-transduced cells (Figure 5B, right), as in nontransduced MM.1S
cells (Figure 2).
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| Figure 5.
AKT mediates CD40-induced MM cell migration in a
PI3K-dependent manner.
(A) MM.1S cells were infected with Ad dnAKT or Ad myrAKT for 2 hours,
and fresh medium was then added. Cell lysates from infected cells, with
or without CD40 stimulation, were probed using an anti-HA mAb for Ad
myrAKT and an anti-GFP Ab for Ad dnAKT. (B) MM.1S cell were transduced
with indicated adenoviruses overnight and stimulated with G28.5 mAb for
indicated time intervals. Total cell lysates were prepared and
phosphorylation of AKT was detected. (C) Infected MM.1S cells were
pretreated with or without LY294002 (30 µM) for 1 hour, and then
seeded in the upper chamber in the transwell cluster plate.
Transmigration assays were performed as described in "Materials
and methods."
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We next performed transmigration assays using MM.1S cells transduced
with these adenoviruses, in the presence or absence of PI3K inhibitors.
Adenovirus infection of MM.1S cells was performed overnight, followed
by serum starvation before addition of cells to the upper chamber of
transwells. As shown in Figure 5C, CD40 activation induced migration in
MM.1S cells transduced with control Ad -gal, whereas CD40-induced MM
migration was blocked in Ad dnAKT MM.1S transfectants. Pretreatment
with LY (30 µM) for 1 hour inhibited CD40-induced migration of
control Ad -gal-transduced cells. Interestingly, transduction of Ad
myrAKT stimulated baseline migration of MM.1S cells, compared with
MM.1S cells transduced with control Ad -gal, suggesting that
constitutively active AKT, per se, can initiate cell migration. CD40
activation further enhanced (2-fold) migration of Ad myrAKT-transduced
MM.1S cells; moreover, migration triggered by CD40 activation was not
inhibited by LY (30 µM) in Ad myrAKT-transduced cells (Figure 5C).
To rule out the possibility that cell death related to Ad dnAKT or LY
treatment contributed to the inhibitory effects on cell migration, we
assayed cell viability of Ad dnAKT- or LY-treated cells using trypan
blue exclusion. All cells were viable at 24 hours of incubation (data not shown); therefore, the observed inhibition of CD40-induced migration by Ad dnAKT or LY294002 was not due to cell death. These data
indicate that AKT activity mediates MM cell migration induced by CD40
activation and further confirm that AKT is activated by CD40 through PI3K.
Inhibition of NF-B signaling inhibits MM.1S migration induced by
CD40 activation
We next determined whether CD40-induced NF-B activation in
MM.1S cells mediates CD40-induced MM cell migration. As shown in Figure
6A, IB is rapidly degraded upon
CD40 activation of MM.1S cells via G28.5 (2 µg/mL; Figure 6A);
degradation of IB was also seen uponCD40 activation of MM.1S
cells with sCD40L (2 µg/mL; data not shown). Degradation of IB
peaked at 20 minutes after CD40 stimulation and returned to baseline
within 1 hour (Figure 6A). PI3K inhibitor LY (0-30 µM), in a
dose-dependent manner, blocked the degradation of IB (Figure 6B),
with complete inhibition of degradation by LY (30 µM). These results
confirm that CD40 activates NF-B in MM.1S cells and suggest that
CD40-induced NF-B activation is mediated via PI3K/ AKT signaling.
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| Figure 6.
CD40 induces NF-B activation in a PI3K-dependent
manner.
(A) Serum-starved MM.1S cells were stimulated with 2 µg/mL G28.5
anti-CD40 mAb, and total cell lysates were made at the indicated time
points. Western blotting was performed using an anti-IB Ab. The
same blot was stripped and probed for tubulin to confirm equal loading.
(B) Serum-starved MM.1S cells pretreated with PI3K inhibitor LY (0-30 µM) were stimulated with CD40, and Western blotting using an
anti-IB Ab was performed. Again immunoblotting with
anti--tubulin served as a control loading.
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To directly define the role of CD40-induced AKT activation in
downstream NF-B signaling, we used MM.1S Ad dnAKT transfectants to
block activation of AKT and MM.1S Ad myrAKT transfectants to constitutively activate AKT. MM.1S cells transduced with either virus
were incubated with G28.5 anti-CD40 mAb (2 µg/mL), and Western blotting using an anti-IB Ab was performed on whole cell lysates at indicated time points. As seen in Figure 6A, CD40 induced IB degradation in MM.1S cells at 10 to 30 minutes. The expression of
kinase-dead AKT by Ad dnAKT blocks CD40-induced IB degradation (Figure 7A), whereas the expression of
constitutively active AKT by Ad myrAKT accelerates IB degradation
(Figure 7B). These results indicate that NF-B is a downstream target
of AKT activation induced by CD40.
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| Figure 7.
Effects of Ad dnAKT and Ad myrAKT on CD40-induced
IB degradation.
Adenoviruses expressing kinase-dead AKT (Ad dnAKT) (A) and
constitutively active AKT (Ad myrAKT) (B) were transduced into MM.1S
cells, followed by serum starvation for 2 hours. Serum-starved
adenovirus-transduced cells were incubated with 2 µg/mL G28.5
anti-CD40 mAb, and collected at the indicated time points. Cell lysates
were subjected to Western blotting using anti-IB, and with
anti--tubulin mAb as a loading control.
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We next examined CD40-induced nuclear translocation of NF-B in
MM.1S cells using nuclear NF-B pull-down assays. Nuclear extracts
prepared from control and CD40-treated MM.1S cells were incubated with
agarose beads conjugated to consensus NF-B oligomers, and nuclear
NF-B was assayed by Western blotting using anti-p65 Ab. As shown in
Figure 8A, CD40 activation induced
increased nuclear NF-B, which was inhibited in a time-dependent
manner by pretreatment with either PS1145 (10 µM) or SN50 peptide (1 µM) (Figure 8A). The novel IKK inhibitor PS114544 blocks
activation of IB, thereby interfering with NF-B translocation,
whereas cell-permeable SN50 peptide inhibits nuclear translocation of
NF-B. In order to assess the role of the NF-B signaling in
CD40-mediated MM cell migration, we used these 2 inhibitors in
transmigration assays. As shown in Figure 8B, pretreatment (1 hour)
with PS1145 (1-25 µM) or SN50 (0.5-5 µM) markedly reduces
CD40-induced cell migration, with complete inhibition by PS1145 (2.5 µM) and SN50 (0.5 µM), conditions that do not cause cell
death.44,45 Moreover, NF-B inhibitors PS1145 and SN50
did not block CD40-induced migration of MM.1S cells expressing
constitutively active AKT (Figure 8C). These results confirm that
CD40-induced AKT-dependent NF-B activation mediates CD40-triggered
MM cell migration.
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| Figure 8.
Inhibition of NF-B signaling blocks CD40-mediated
MM.1S migration, which is dependent on transcription and new protein
synthesis.
(A) Serum-starved MM.1S cells were pretreated with either 10 µM
PS1145 for 90 minutes or 1 µM SN50 peptides for 30 minutes, and then
incubated with 2 µg/mL G28.5 mAb for the indicated intervals. Nuclear
extracts from each sample were prepared, and oligonucleotides
containing the consensus binding sequence of NF-B bound to agarose
beads were used to pull down nuclear NF-B. The resulting samples
were analyzed by Western blotting using an anti-p65 NF-B Ab. (B)
Serum-starved MM.1S cells were either left intact or pretreated with
PS1145 and SN50 at the indicated concentration for 90 minutes and 30 minutes, respectively, and then added to the upper chamber in a
transwell cluster plate for a transmigration assay. (C) MM.1S cells
were transduced with Ad dnAKT, Ad myrAKT, or control Ad -gal
adenoviruses. Cells were preincubated with or without PS1145 (10 µM)
and SN50 (1 µM), and a transmigration assay then performed triggered
by G28.5 (5 µg/mL) anti-CD40 mAb. (D) Serum-starved cells were
treated for 1 hour with act D or chx (1 and 10 µg/mL), and transmigration was then determined as described
in "Materials and methods." Data are the mean ±SD of
triplicate determinations. Similar results were obtained in at least 2 additional experiments.
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Since NF-B can activate many target genes, we speculated that gene
transcription and protein synthesis may modulate CD40-induced MM
migration. MM.1S cells were pretreated with act D and chx for 1 hour, and transmigration was determined as described in "Materials and methods." Both act D (1 and 10 µg/mL) and chx (1 and
10 µg/mL) significantly inhibit CD40-induced MM.1S migration in a
dose-dependent manner (Figure 8D), suggesting that cell migration is
dependent on transcription and de novo protein synthesis. These data
demonstrate that NF-B plays a pivotal role in mediating MM cell
migration stimulated by CD40.
CD40-induced uPA secretion is inhibited by PI3K and
NF-B blockade
We previously showed that CD40 induces VEGF secretion and
migration in MM cells7; however, NF-B-responsive
elements in the VEGF promoter have not yet been unidentified. We next
examined a potential downstream target of PI3K/AKT-dependent NF-B
activation by CD40 signaling in MM.1S cells, specifically testing
whether uPA is induced by CD40 in MM.1S cells. To address this
hypothesis, conditioned media from control and CD40-activated MM.1S
cells incubated for 12 hours in serum-free RPMI1640 media were
collected, concentrated, and subjected to Western blotting analysis
with anti-uPA mAb. As seen in Figure 9A,
uPA is not detectable in control MM.1S cell supernatants, but uPA
secretion is induced by CD40 activation. Treatment with NF-B
inhibitors PS1145 (10 µM) or SN50 (1 µM) inhibits the secretion of
uPA (Figure 9B). The PI3K inhibitors LY (30 µM) and Wort (0.2 µM)
also inhibit CD40-induced uPA expression and secretion from
CD40-stimulated MM.1S cells (Figure 9B). Inhibition of protein
synthesis by chx (5 µg/mL) has a similar effect. These data indicate
that uPA is induced and secreted in CD40-activated MM.1S cells, and
that uPA expression is dependent on CD40-induced PI3K and NF-B
activity.
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| Figure 9.
CD40 stimulates uPA secretion, which is blocked by PI3K
and NF-B inhibitors.
(A) Media from untreated ( ) and CD40-activated (+) MM.1S cells
cultured in serum-free medium for 12 hours were collected and
concentrated as described in "Materials and methods." Secretion of
uPA was detected by Western blot analysis with an anti-uPA mAb. (B)
Media from untreated or CD40-activated MM.1S cells, in the presence or
absence of PI3K inhibitors (0.2 µM Wortmannin, Wort; 30 µM
LY294002, LY), NF-B inhibitors (10 µM PS1145; 1 µM SN50), or 5 µg/mL chx for 48 hours, were concentrated for assay of uPA secretion
as described in "Materials and methods."
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Discussion |
We and others have defined the role of PI3K, AKT, and NF-B
signaling in mediating MM cell proliferation and
antiapoptosis.30,31,44,45 In the present study, we have
shown that PI3K/AKT signaling mediates CD40-induced transmigration of
MM.1S MM cells. We first showed a significant induction of AKT and PI3K
activity by CD40 stimulation; and, conversely, we demonstrated a
concentration-dependent inhibition of MM migration by PI3K inhibitors
Wort and LY. Using adenoviruses expressing dominant-negative
AKT and constitutively expressed AKT, we confirmed
the role of AKT in MM migration. Specifically, expression of
dominant-negative AKT blocks CD40-induced MM migration, whereas
expression of constitutively active AKT overcomes inhibition of
CD40-induced migration by LY. In addition, we found that constitutive AKT activity induces MM migration. We show that constitutively active
AKT promotes IB degradation, whereas expression of
dominant-negative AKT inhibits IB degradation, and went on to
identify NF-B as a downstream target of PI3K/AKT signaling. Finally,
the blockade of NF-B activation and transmigration using PS1145 and
SN50, respectively, abrogates CD40-induced MM cell migration. These studies confirm that PI3K/AKT/NF-B activation mediates CD40-induced MM cell migration. CD40-induced PI3K/AKT/NF-B activity also induces expression and secretion of uPA, suggesting a role of uPA in this migratory response.
CD40 activation in human MM.1S cells, either by anti-CD40 mAb or sCD40L
treatment, results in activation of PI3K, AKT, and NF-B. CD40
activation also induces ERK/MAPK, without significant activation of JNK
and p38. Since CD40 activation induces either p38 or JNK in B cells,
these data suggest distinct molecular sequelae of CD40 signaling in MM
cells versus normal B cells. An important sequelae of CD40
activation in MM.1S cells is migration, and we defined the signaling
pathways mediating this response. The MEK inhibitor PD098059 only
partially (10%-15%) blocked MM migration induced by CD40, suggesting
that ERK activation plays only a minor role. Importantly, PI3K activity
is significantly induced in MM.1S cells following CD40 activation; and
conversely, CD40-induced MM cell migration is inhibited by Wort and LY
in a dose-dependent manner. Therefore, PI3K activity mediates
CD40-induced MM cell migration.
Since CD40 activation also induces sustained AKT activity in MM.1S
cells, we next defined whether AKT mediated MM cell migration following
CD40 activation. CD40-induced migration was not observed in MM.1S
transfectants overexpressing dnAKT, confirming that induction of AKT
activity is required for MM.1S cell migration stimulated by CD40.
Conversely, overexpression of AKT in Ad myrAKT-transduced MM.1S induced
migration but did not overcome the inhibitory effect of PI3K inhibitor
LY, indicating that AKT is downstream of PI3K and mediates
CD40-triggered migration. Our results are consistent with the recent
demonstration that overexpression of constitutively active AKT in
bovine lung microvascular endothelial cells stimulates cytokinesis and
migration in the absence of VEGF.21 Our prior study shows
that AKT activation in MM cells stimulates
proliferation,30 whereas the present results suggest that
AKT activation also mediates MM migration. The present study therefore
indicates that AKT activation mediates migration even in primary
patient MM cells before onset of secondary plasma cell leukemia.
We next show that CD40-induced AKT activation mediates IB
degradation and NF-B activation in MM.1S cells. Using adenovirus transduction and 2 different classes of NF-B inhibitors, PS1145 and
SN50, our results confirm that the mechanisms whereby CD40 activates
NF-B involve consecutive activation of AKT and IKK, consistent with
the observed phosphorylation and degradation of IB and
translocation of NF-B p65 into the nucleus. Since both PS1145 and
SN50 completely inhibit CD40-induced migration in control as well as in
Ad myrAKT-transduced MM.1S transfectants, NF-B is downstream of AKT
in CD40-induced signaling mediating MM cell migration.
The role of NF-B activity in controlling cell survival and
conferring protection against drug-induced apoptotic stimuli is well
documented.44-47 Our prior studies have identified NF-B
as a novel target whereby PS341 (proteosome inhibitor) and IMiDs (thalidomide analog immunomodulatory drugs) target MM cells to inhibit growth, induce apoptosis, and overcome drug resistance in the
bone marrow microenvironment.44,45 To our
knowledge, however, the present study is the first demonstration of the
involvement of NF-B in cell migration. Coupled with evidence that
many tumor cells have constitutive NF-B site binding and
transactivation activity,46,47 the current results suggest
that enhanced NF-B activity may potentiate tumor cell migration and invasion.
Little is known about the role of uPA in MM pathogenesis; however, uPA
and uPA receptor are expressed in MM cells.48 uPA is only weakly expressed in unstimulated MM.1S cells, as reported recently in U266 MM cells.49 However, uPA is induced in
U266 as well as MM cells from patients following binding to vitronectin (VN) and fibronectin (FN) ECM proteins.49 uPA induced by
interaction with VN and FN interaction may enhance the ability of cells
to invade via stroma and subendothelial basement membrane. In the current study, we observed significant induction of uPA by CD40 activation in MM.1S cells. This is the first report of uPA induction by
CD40 and further supports a functional role of CD40 activation in MM
invasion and spreading. In addition, our data showed that induced uPA
secretion is dependent on NF-B activity and, to a lesser extent,
PI3K/AKT activity. These data confirm that uPA promoter contains
NF-B binding sites that mediate the induction of uPA expression.
Furthermore, these results suggest a possible role of uPA in MM cell
invasion and proteolytic digestion of bone matrix.
In summary, our data suggest that CD40-induced MM transmigration is
mediated by PI3K/AKT-induced transactivation of NF-B and related
induction of expression and secretion of uPA. These provide the
framework for novel therapeutic strategies targeting PI3K/AKT/NF-B
signaling and uPA secretion to inhibit migration and progressive
disease in MM.
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Acknowledgments |
We thank Dr Kenneth Walsh at St Elizabeth's Medical Center
(Boston, MA) for valuable reagents.
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Footnotes |
Submitted September 16, 2002; accepted November 7, 2002.
Prepublished
online as Blood First Edition Paper, November 14, 2002;
DOI 10.1182/ blood-2002-09-2813.
Supported by a Multiple Myeloma Research Founda |
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Abstract
Introduction