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Prepublished online as a Blood First Edition Paper on November 14, 2002; DOI 10.1182/blood-2002-09-2813.
NEOPLASIA
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.
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)- 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 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- A large number of stimuli can activate NF- 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- In this study, we examined PI3K/AKT/NF- Cells and stimulation
Cell migration assay
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-I B 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.7PI3K 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 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.
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).
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.
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.
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.
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
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 Inhibition of 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.
To directly define the role of CD40-induced AKT activation in
downstream NF-
We next examined CD40-induced nuclear translocation of NF-
Since NF- CD40-induced uPA secretion is inhibited by PI3K and
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.
We and others have defined the role of PI3K, AKT, and NF- CD40 activation in human MM.1S cells, either by anti-CD40 mAb or sCD40L
treatment, results in activation of PI3K, AKT, and NF- 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 I The role of NF- 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- In summary, our data suggest that CD40-induced MM transmigration is
mediated by PI3K/AKT-induced transactivation of NF-
We thank Dr Kenneth Walsh at St Elizabeth's Medical Center (Boston, MA) for valuable reagents.
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 Foundation Fellow Research Award (Y.-T.T.); National Institutes of Health grants RO-1 50947 and PO1-78378; and the Doris Duke Distinguished Clinical Research Scientist Award (K.C.A.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kenneth C. Anderson, Department of Adult Oncology, Dana-Farber Cancer Institute, M557, 44 Binney St, Boston, MA 02115; e-mail: kenneth_anderson{at}dfci.harvard.edu.
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B signaling
Top
Abstract
Introduction
2,
); 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 (
) 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-