JBC Papers in Press. Published on December 20, 2001 as Manuscript M109068200
Vascular Endothelial Growth Factor (VEGF) -induced migration of
Multiple Myeloma cells is associated with 1-integrin- and
PI3-kinase- dependent PKC activation
By Klaus Podar, Yu-Tzu Tai, Boris K. Lin, Radha P. Narsimhan, Martin Sattler, Takashi
Kijima, Ravi Salgia, Deepak Gupta, Dharminder Chauhan, and Kenneth C. Anderson *
From the Jerome Lipper Multiple Myeloma Research Center/ Dana-Farber Cancer
Institute, and Department of Medicine, Harvard Medical School, Boston, MA 02115
* Address correspondance and reprints requests to:
Kenneth C. Anderson, MD, Dana-Farber Cancer Institute,
44 Binney Street, Boston, MA 02115.
Phone: (617)-632-2144; FAX: (617)-632-2140
E-mail: kenneth_anderson@dfci.harvard.edu
Running Foot: MM cell migration
Key words: MM·VEGF·migration·PKC·1-integrin·PI3-kinase·CADTK
1
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Summary
In Multiple Myeloma (MM)1, migration is necessary for homing of tumor cells to the
bone marrow (BM), for their expansion within the BM microenvironment, and for their
egress into the peripheral blood. In the present study we characterize the role of VEGF
and 1-integrin (CD29) in MM cell-migration. We show that PKC is translocated to the
plasma-membrane and activated by adhesion of MM cells to fibronectin and VEGF. We
identify 1-integrin modulating VEGF-triggered MM cell-migration on fibronectin. We
show that transient enhancement of MM cell-adhesion to fibronectin triggered by VEGF
is dependent on both PKC- and 1-integrin-activity. Moreover, we demonstrate that
PKC is constitutively associated with 1-integrin. This data is consistent with PKC-
dependent exocytosis of activated 1-integrin to the plasma-membrane, where its
increased surface-expression mediates binding to fibronectin; conversely, catalytically-
active PKC-driven internalization of 1-integrin results in MM cell de-adhesion. We
show that the regulatory subunit of PI3-kinase (p85) is constitutively associated with
FMS-like-tyrosine-kinase-1 (Flt-1). VEGF stimulates activation of PI3-kinase, and both
MM cell-adhesion and -migration are PI3-kinase-dependent. Moreover, both VEGF-
induced PI3-kinase-activation and 1-integrin-mediated binding to fibronectin are
required for the recruitment and activation of PKC. Time-lapse-phase-contrast-video-
microscopy (TLVM) studies confirm the importance of these signaling components in
VEGF-triggered MM cell-migration on fibronectin.
2

Introduction
In Multiple Myeloma (MM), migration is necessary for homing of tumor cells to the bone
marrow (BM), for the expansion of malignant plasma cells within the BM
microenvironment, and for their egress enter the peripheral blood. It has been reported
that the ECM proteins laminin, microfibrillar collagen type VI, and fibronectin are strong
adhesive components for MM cells, and that adhesion to laminin and fibronectin is 1-
integrin (CD29) mediated (1). 1-integrins are typically expressed on MM cells,
specifically -integrins VLA-4 (41) and VLA-5 (51) (2,3). 1-integrin mediated
adhesion of MM cells to fibronectin confers protection against drug-induced apoptosis
and triggers NFB dependent transcription and secretion of IL-6, the major MM growth
and survival factor (4,5). Interestingly, chimeric mice (1 -/- wild-type (wt) chimaeras)
lack 1-null cells in blood and in hematopoietic organs such as spleen, thymus and BM
as a consequence of the inability of 1-null cells to invade the fetal liver (6). Besides
upregulation of cell surface expression and induction of surface-clustering, integrin
activity can be triggered by multiple agonists through "inside-out" signaling independent
of changes in integrin expression levels, e.g. ligand binding to growth factor receptors is
associated with changes in the way in which adhesion receptors on the cell surface
engage the extracellular matrix (ECM). This concept is illustrated in HUVE cells, in
which VEGF stimulates 1-integrins and leads to markedly enhanced movement (7).
Although VEGF induces migration as a key step in angiogenesis, the interplay between
VEGF and integrins is not restricted to angiogenesis. VEGF and VEGFR are expressed
by many tumor cell lines; moreover, elevated levels of VEGF are found in cancer patients
and inhibition of VEGF can suppress tumor growth (8). Indeed, clinical studies are under
3

way investigating VEGF as a novel therapeutic target (9). In MM, VEGF is expressed
and secreted by tumor cells as well as BM stromal cells (BMSCs) (10,11); moreover,
binding of MM cells to BMSCs enhances both IL-6 and VEGF secretion (11). Besides
stimulating angiogenesis, we recently showed that VEGF directly induces MM cell
proliferation via a protein kinase C (PKC)- independent MEK-ERK pathway, and triggers
MM cell migration on fibronectin via a PKC- dependent pathway (12). Members of the
PKC family mediate multiple physiological functions (13-18), including integrin-
mediated cell spreading and migration (19-22). To date, 11 isoenzymes of the serine/
threonine kinase PKC have been identified and classified into three subgroups based on
their structure and cofactor regulation: conventional PKCs (cPKCs), novel PKCs
(nPKCs), and atypical PKCs (aPKCs) (15,18). The cPKC isoforms participate in the
"inside-out" signaling activation of cell adhesion mediated by 1-, 2-, and 3-integrins
and are also required for cell spreading (23-25). Moreover, PKC associates with 1-
integrins, thereby regulating cell trafficking (26,27).
In the present study, we describe the close interrelationship between integrin and growth
factor-induced signaling pathways in MM. We identify PKC as the primary PKC
isozyme involved in VEGF-induced MM cell migration. By showing that VEGF-
mediated MM cell migration is associated with 1-integrin- and PI3-kinase-dependent
PKC activation, we further confirm the importance of tumor cell BM
microenvironment interaction as a pivotal process in the pathogenesis of MM. Moreover,
our studies identify several potential targets for novel therapies to improve outcome in
MM.
4

Materials and methods
Materials
Recombinant human VEGF165 was purchased from R&D (Minneapolis, MN). Human
plasma fibronectin was obtained from GIBCO, Life Technologies (Grand Island, NY).
1-integrin-specific MoAb was purified from P4C10 ascites (Chemicon, Temecula, CA).
PKC isoforms were purchased from Transduction Laboratories. The goat polyclonal Ab
raised against the carboxy terminus of PYK2, the mouse MoAb against full length 1-
integrin (4B7R), and the rabbit polyclonal Ab directed against amino acids within the
extracellular domain of Flt-1 were purchased from Santa Cruz. Anti-phosphotyrosine
4G10 antibody was kindly provided by Dr. Tom Roberts (Dana-Farber Cancer Institute,
Boston, MA).
Cells and cell culture
The human MM cell line MM.1S (dexamethasone-sensitive) (28) was maintained in
Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-
inactivated fetal bovine serum (HI-FBS), 100U/mL penicillin, 10µg/mL streptomycin,
and 2mM L-glutamine.
Stimulation of cells.
Cells were starved for 15-18h in medium with 3% FBS overnight and then for 3h without
FBS prior to stimulation with indicated VEGF concentrations (Recombinant Human
VEGF, R&D) or 50nM/ 300 nM of PMA for 20-30 min at 37°.
5

Cell lysis, immunoprecipitation, and Western blot analysis.
Cells were washed three times with PBS and lysed with either lysis buffer (10mM Tris,
50mM
NaCl,
Na-pyrophosphate,
1%
triton,
1mM
sodium
vanadate,
1mM
phenylmethylsulfonyl fluoride (PMSF) and protein inhibitor cocktail; Boehringer
Mannheim) or RIPA lysis buffer (50mM Tris, pH 8.0, 150mM NaCl, 1% v/v NP-40,
0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1mM sodium vanadate, 1mM
PMSF and protease inhibitor cocktail). Insoluble material was removed by centrifugation
(15,000rpm for 30 min at 4°C).
Immunocomplexes were collected following overnight incubation at 4°C with 10-20µl
100% Protein A-Sepharose CL-4B beads (Amersham Pharmacia, Uppsala, Sweden).
For Western blotting, cell lysates (30-100µg/lane) or immunoprecipitates (500µg-1.5mg
total proteins) were separated by 8% or 10% SDS-PAGE prior to electrophoretic transfer
onto Hybond-C super nitrocellulose membranes (Amersham Life Science, Arlington
Heights, Illinois). After blocking with 5% nonfat milk in PBS-Tween 20 buffer at room
temperature for 1 h, membranes were sequentially blotted with the indicated specific
primary Abs and then with horseradish peroxidase-conjugated secondary mouse, rabbit or
goat Abs and developed using chemiluminescence (Amersham Pharmacia Biotech,
Uppsala, Sweden).
Cell fractionation
After washing three times with phosphate-buffered saline, cells were transferred into
200µl hypotonic lysis buffer (HLB: 10mM Tris-HCl, 1mM EDTA, 1mM sodium
vanadate, 1mM DDT, 1mM PMSF, and protease inhibitor cocktail) and incubated for 20
6

min on ice. The cells were then lysed by 80 strokes in a Dounce homogenizer, and
subjected to centrifugation at 1500g to pellet nuclei and unbroken cells, followed by
centrifugation of the supernatant at 100.000g for 20 min. The supernatant was collected
(S100 fraction) and the pellet resuspended in 70µl HLB containing 0.1% Triton-x (P100
fraction).
Measurement of PKC activity
PKC activity was measured with a PKC Assay Kit (Upstate Biotechnology, Lake
Placid, NY) according to the protocol. The phosphotransferase activity of PKC was
quantitated in a scintillation counter (Beckman LS 6500 Multi-Purpose Scintillation
Counter) measuring the amount of the -phosphate of [-32] ATP incorporated in a
specific PKC substrate peptide (QKRPSQRSKYL) bound to P81 phosphocellulose paper.
Endogenous phosphorylation of proteins in the sample was determined by substituting
the assay dilution buffer for the substrate cocktail. To assure equal amounts of PKC
used in the assay, immunoprecipitates were denaturated, eluted, separated by 10% SDS-
PAGE, electrophoretically transferred, and immunoblotted with PKC.
Phosphoinositide Kinase Assays
PI3-kinase assays were performed as described previously (29) in a total volume of 50µl.
The radioactivity was visualized and quantitated on a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
7

Cell Adhesion Assays
These were performed using the Vybrant Cell Adhesion Assay Kit (Molecular Probes,
Eugene, OR), according to the protocol. Briefly, after 18 h starvation in RPMI/ 2%FBS,
MM.1S cells (4x106 cells/mL) were harvested and labeled with Calcein-AM (5µM per
cell loading) for 30 min. After washing with prewarmed (37°C) RPMI 1640 (without
serum), the cells were preincubated with or without blocking 1-integrin neutralizing
MoAb, bisindolylmaleimide I (BIM I, Calbiochem, San Diego, CA), or LY294002
(Calbiochem, San Diego, CA), respectively, and then stimulated with VEGF. The cell
suspensions were immediately added to fibronectin- or polylysine (1µg/mL)- coated or
non-coated wells. After 90-120 min, nonadherent calcein-labeled cells were removed by
gently washing two times with RPMI 1640 by inversion of the plates. Adherent cells
were quantitated in a Fluorescence Multi-Well Plate Reader (Molecular Devices
Corporation, Sunnyvale, CA) and examined microscopically. All experiments were done
in triplicate.
Transwell migration assay.
Cell migration was assayed as described previously (12,30,31). After 2-5 h, cells that
have migrated into the lower compartment of a Boyden-modified chamber were counted
using a Coulter counter ZBII (Beckman Coulter).
Time-lapse video microscopy
MM.1S cells were starved in RPMI medium containing 2% FBS for 16h, and plated to
uncoated or fibronectin-coated tissue culture plates (35x10mm plates, Becton Dickinson
8

Labware, Bedford, MA), respectively, in the presence or absence of VEGF (100ng/mL).
For image capturing, an Olympus IX70 inverted microscope (Olympus, Lake Success,
NY) with Hoffman optics (10/20/40x) equipped with a temperature- (Therm-Omega-
Tech, Warmington, PA) and CO2- (5%) controlled chamber was connected to an
Optronics Engineering DEI-750 3CCD digital video camera (Optronics, Galeta, CA).
Animation and export to Quick Time movie were performed using the QED Camera
Standalone 145 software at 2-min intervals. Images were analyzed with the NIH Image
1.62 software. To generate migration tracks, the position of centroid of individual cells on
each image were marked. The migratory speed was calculated based on the sum of
distances, divided by the time of observation. Migration of at least 15 cells was analyzed
for each experimental condition.
Statistical analysis.
Statistical significance of differences observed in VEGF-treated versus control cultures
was determined using an unpaired Student's t-test. The minimal level of significance was
p < 0.05.
9

Results and Discussion
Previously we showed that VEGF-triggered MM cell migration on fibronectin is
mediated via a PKC- dependent signaling pathway (12). In the present study we define
and characterize the interrelationship of VEGF- and integrin-signaling in activating PKC
that ultimately leads to MM cell migration.
PKC isoform expression in MM cells
Several PKC isoforms, including PKC (32), PKC (33), and PKC (34), have been
implicated in a cell migratory phenotype. Additionally, overexpression of PKC was
shown to enhance cell motility/ invasiveness of breast cancer cells (26). We have shown
previously that MM cell migration is PKC-dependent, since it can be selectively inhibited
by the PKC inhibitor BIM I (12). As a first step to identify the class of PKC mediating
VEGF-induced migration in MM cells, we examined the expression of the PKC isoforms
in MM cell lines and patient cells (Fig. 1). Immunoblot analysis revealed that PKC,
PKC, PKC, and PKC are significantly expressed in all human MM cell line and MM
patient cells investigated, in contrast to PKC (low expression), PKC (variable
expression) and PKC (no expression, data not shown). As in our previous studies we
chose the dexamethasone-sensitive (MM.1S) human MM cell line (28) as a representative
model system for this study.
10

VEGF-induced signaling pathways and adhesion to fibronectin mediate PKC
activation
Since the activation of PKCs occurs concomitant with their recruitment to the plasma
membrane, we next performed immunoblotting of cell fractions with isoform-specific
Abs against PKC, PKC, PKC, and PKC to delineate their intracellular distribution
after VEGF stimulation (Fig. 2). In non-treated MM cells, all PKC isoforms were
primarily detected in the cytosolic fraction. After VEGF stimulation of cells seeded on
fibronectin, PKC translocated into the membrane fraction after 30 minutes (Fig. 2a),
whereas no significant changes in distribution of PKC isoforms as PKC, PKC, and
PKC were observed. The activation of PKC after VEGF treatment of cells attached to
fibronectin was further supported by a 2-fold increase in PKC-IP kinase activity;
PKC-IP kinase activation by phorbol-12-myristate-13-acetate (PMA) served as a
positive control (Fig. 2b). Although a PKA/ CaMK inhibitor cocktail was used, this assay
(Upstate Biotechnology, Lake Placid, NY) may not exclude the phosphorylation of a
specific substrate peptide (QKRPSQRSKYL) by unknown PKC-coprecipitated kinases.
Neither plating of cells on fibronectin alone nor stimulation of suspended cells with
VEGF alone significantly changed the subcellular distribution of PKC (Fig. 2c and d).
In contrast, treatment with PMA, a major PKC activator, induced translocation of
responsive PKCs (PKC, PKC, PKC) into the detergentsoluble membrane fraction as
expected (Fig. 2d) (35,36)
11

VEGF enhances MM cell adhesion to fibronectin
Laminin, the microfibrillar collagen type VI, and fibronectin bind MM cells, and
adhesion to laminin and fibronectin is 1-integrin (CD29) mediated (1). 1-integrins
expressed on MM cells include VLA-4 (41) and VLA-5 (51) (2-5), which mediate
adherence to both the ECM and BM stromal cells. In HUVE cells, VEGF stimulates 1-
integrins via "inside-out" signaling, leading to significantly increased motility (7). As
migration is a dynamic process of cell adhesion formation and release, we next
investigated whether VEGF can modulate 1-integrin mediated MM cell adhesion. As
shown previously, MM cells spontaneously adhered to fibronectin, and this adhesion was
increased upon stimulation with VEGF (Fig. 3a). Maximal increments of VEGF-
mediated cell adhesion were observed at fibronectin-concentrations of 25-30µg/mL,
whereas adhesion decreased to baseline levels at higher fibronectin concentrations (Fig.
3b). Notably, VEGF-mediated increaments in adhesion were time-dependent, with
maximal binding observed after 75-90 min VEGF-treatment and decreasing after 120 min
(Fig. 3c).
We next examined whether the increment of adhesion observed at 90 min of VEGF-
stimulation was PKC- and 1-integrin- dependent. As shown in Figures 3 d-e, incubation
with the PKC-inhibitor BIM I, as well as with the 1-neutralizing MoAb, blocked dose-
dependent VEGF-induced cell adhesion to fibronectin. This involvement of PKC in MM
cell adhesion to fibronectin was further confirmed by a dose-dependent increment of
adhesion triggered by PMA stimulation (Fig. 3d), similar to that induced by VEGF.
Taken together, our results show that VEGF transiently enhances MM cell adhesion to
fibronectin, dependent on both PKC- and 1-integrin- activity.
12

1-integrins modulate VEGF-triggered migration on fibronectin
We next sought to determine whether 1-integrin modulates VEGF-mediated MM cell
migration on fibronectin. As seen in Fig. 3f, 1-integrin neutralizing MoAb, but not
irrelevant IgG, mediated dose-dependent inhibition of VEGF-triggered MM cell
migration in a Boyden-modified microchemotaxis chamber. These data confirm that 1-
integrin (CD29) is the integrin primarily associated with VEGF-triggered MM cell
migration on fibronectin.
PKC associates with 1-integrin and CADTK
The control mechanisms leading to the various stages of the integrin receptor life cycle
are largely unknown. Propagation of cell movement is thought to be regulated by
distribution and redistribution of integrins through surface diffusion, internalization,
clustering at the leading front, as well as ripping release from the cell rear (37). In
mammary epithelial cells Ng et al. (26) have recently found that PKC interacts with
activated 1-integrin, which regulates its exocytosis to the plasma membrane; moreover,
catalytically active PKC is responsible for 1-integrin internalization through a Ca2+-
and PI3-kinase dependent, dynamin I-controlled endocytic pathway. PKC induced
upregulation of integrin-dependent cell migration of these cells, which was blocked under
conditions that prevented the internalization of the receptor complex. We therefore next
determined whether PKC and 1-integrin are associated in MM cells. Constitutive
complex-formation
between
these
two
proteins
was
demonstrated
by
co-
immunoprecipitation (Fig. 4a). Upon their activation and translocation, conventional
13

PKCs associate with proteins of the transmembrane-4 superfamily (TM4SF, or
tetraspans) linking PKC to several subsets of integrins (38), including 41 (39) and
51 (40). Specificity of binding to TM4SF dependent on the extracellular domain of the
integrin chain, and integrin-TM4SF-PKC complex formation results in phosphorylation
of integrin cytoplasmic tail (41). In ongoing studies, we are investigating the regulatory
role of these integrin-associated proteins on VEGF-induced MM cell migation.
VEGF stimulates tyrosine phosphorylation of CADTK in adherent MM cells
Besides influencing cell motility via controlling 1-integrin trafficking, catalytically
active PKC also regulates other components of the focal complexes, including the small
GTPases (Cdc42, Rho, Rac) and/ or actin cytoskeleton-binding proteins. Calcium-
dependent tyrosine kinase (CADTK), also known as Proline Rich Tyrosine Kinase 2
(PYK2), Calcium-Dependent Tyrosine-Kinase (CAK) and Related Adhesion Focal
Tyrosine Kinase (RAFTK), is a cytoplasmic tyrosine kinase homologous to Focal
Adhesion Kinase (FAK). Like FAK, CADTK is a platform kinase site for the coalescence
of signaling and adaptor molecules, thereby facilitating the transmission of surface
signals to the cytoskeleton and signaling pathways associated with cell growth, apoptosis
and migration. A number of studies have demonstrated tyrosine phosphorylation of
CADTK in cells of hematopoietic origin, e.g. T and B cells, monocytes, NK cells,
granulocytes, bone marrow progenitors, mast cells, megakaryocytes, and platelets.
Stimuli that activate CADTK in these cells are associated with cell motility or at least
cytoskeletal rearrangement; e.g. CADTK activation is required for cytoskeletal
reorganization and monocyte motility (42).
14

In MM.1S cells, we have previously shown that CADTK is activated upon dex-treatment,
suggesting its role in dex-induced apoptosis (47). Notably, CADTK has been reported to
link calcium- and integrin-mediated signaling to the cytoskeleton in brain and
hematopoietic cells (43-46). Our results show that VEGF induces the association of
CADTK
with
the
1-integrin/ PKC- protein complex (Fig.4a). VEGFR
immunoprecipitation and immunoblotting studies using anti-Flt-1 and anti-CADTK Abs
showed their specific constitutive association (Fig. 4b). We next investigated whether the
VEGF-induced formation of a membrane complex containing PKC, 1-integrin and Flt-
1 changes the activity of the associated CADTK in adherent MM cells. As shown in
Figure
4d,
VEGF
increased
(2-fold)
fibronectin-induced
CADTK
tyrosine-
phosphorylation in MM.1S cells; equivalent loading of proteins was confirmed by
stripping and reprobing the membrane with anti-CADTK Ab. The PKC activator PMA
enhanced constitutive phosphorylation of CADTK (Fig 4c). Finally, VEGF-triggered
CADTK-activation was blocked by both the PKC-inhibitor BIM I and a 1-integrin
neutralizing MoAb (Fig. 4e). These results show that the activation of CADTK in
adherent MM cells is influenced by both PKC- and 1-integrin- mediated signaling
pathways. Moreover, a recent report shows that CADTK in hematopoietic cells is also
associated with the cytoskeletal protein paxillin (48). We therefore postulate that VEGF
may facilitate signal transduction to the cytoskeleton in MM cells and modulate MM cell
migration and /or adhesion on fibronectin via CADTK. Ongoing studies are directed
toward understanding this role of CADTK in MM cells in more detail.
15

p85 interacts with Flt-1
We next sought to identify the "inside-out" signaling components mediating regulation of
1-integrin function by VEGF in MM cells. PI3-kinases phosphorylate PI(4,5)P2 and
PLC hydrolyses PI(4,5)P2 to diacylglycerol and inositol 1, 4, 5- triphosphate, thereby
mediating intracellular calcium release and PKC activation (49). PLC activity is
enhanced by 3-phosphoinositides, both indirectly via Btk-related tyrosine kinases and
directly through binding of PI(3,4,5)P3 to the amino-terminal PH domain and the tandem
SH (Src homology) 2 domains of PLC (50-53). We have shown recently that Flt-1
VEGFR is expressed in human MM cell line and patient MM cells and is tyrosine-
phosphorylated upon VEGF stimulation (12). Since Flt-1 and p85 subunit of PI3-kinase
were associated when overexpressed in yeast cells (54), we next determined whether this
interaction is biologically significant in MM cells. Flt-1 was immunoprecipitated from
VEGF-treated and -non-treated MM.1S cells, followed by immunoblotting with anti-p85
Ab. As illustrated in Fig. 5a, p85 is constitutively associated with Flt-1, suggesting that
PI3-kinase participates in Flt-1- mediated VEGF signal transduction.
VEGF mediates PI3K-activation in MM cells
To confirm this hypothesis, we next examined whether PI3-kinase activation is an early
event in VEGF - induced signaling in MM cells. VEGF induced tyrosine phosphorylation
of the regulatory p85 subunit of PI3-kinase, evidenced by immunoblotting of anti-p85
immunoprecipitates with an anti-pY mAb (Fig. 5b). Moreover, immunoblotting of anti-
pY immunoprecipitates with specific p85 mAb showed an increased association of p85
with tyrosine-phosphorylated proteins, which was maximal after 5min of VEGF-
16

treatment (Fig. 5c). To determine whether VEGF-induced p85 phosphorylation regulates
p110, the catalytic counterpart of PI3-kinase, we performed in vitro kinase assays, using
PI-4,5-diphosphate as a substrate. As shown in Fig. 5d, VEGF induced peak activation
(6-fold) of PI3-kinase activity at 5 min. Taken together, these data show that activation of
PI3-kinase is an early event in the VEGF-triggered Flt-1 signaling cascade.
VEGF-induced PI3-kinase activation and adhesion to fibronectin mediate PKC
translocation to the plasma membrane
We next examined whether activation of PI3-kinase is required for activation of PKC.
When PI3-kinase activation in MM cells seeded on fibronectin was inhibited by
LY294002, PKC recruitment to the plasma membrane was also abrogated (Fig. 6).
These results indicate that VEGF-induced PI3-kinase activation along with 1-integrin
binding to fibronectin, activate PKC.
MM cell migration and cell adhesion is PI3-kinase-dependent
To determine whether activation of PI3-kinase is necessary for MM cell migration, we
next used a Boyden-modified chamber to assay the effect of VEGF on transfilter
migration activity of MM.1S cells seeded on membranes pre-coated with fibronectin or
polylysine as a control for ECM specificity. As seen in Fig. 7a, VEGF added to the
conditioned medium in the lower chamber induced a dose-dependent migration of
growth-factor-deprived MM.1S cells seeded on fibronectin in the upper chamber, which
was totally inhibited by the PKC inhibitor BIM I. Pre-incubation with the PI3-kinase
inhibitor LY294002 (20µM) at 37°C for 45min prior to VEGF stimulation similarly
17

inhibited MM cell migration. In contrast, VEGF did not induce migration of MM.1S cells
seeded on polylysine in the upper chamber. We next examined whether VEGF-induced
increments in adhesion were also PI3-kinase dependent. As shown in Fig. 7b, incubation
with the PI3-kinase inhibitor LY294002 (20µM) blocked VEGF-induced MM cell
adhesion to fibronectin. In contrast, VEGF did not effect MM cell adhesion towards
polylysine. Taken together, these results demonstrate that PI3-kinase activation during
MM cell migration on fibronectin regulates PKC. These observations are similar to the
Ca2+- and PI3-kinase- dependent PKC-catalysed endocytosis reported in breast cancer
cells (26).
TLVM
Migration is a complex dynamic process of cell adhesion formation and release organized
and coordinated both in time and space. To enhance our understanding of the above
results and to link them with the dynamic changes in MM cell morphology that ultimately
mediate cell migration, we used time-lapse phase contrast video microscopy (TLVM)
(Fig. 8 and 9). During migration filopodia and lamellipodia together with new adhesions
are formed at the leading edge, whereas detachment of adhesions are concomitantly
observed at the trailing cell edge. In time-course experiments, MM.1S cells were seeded
on either fibronectin pre-coated or control tissue culture plates, in the presence or absence
of VEGF. Representative cells were investigated for 30min starting at 3 h after plating,
and composite time-lapse phase-contrast micrographs (magnification, x40) acquired at 2
min intervals tracked with the NIH Image 1.62 software (Fig. 8). MM cells adherent to
non-coated tissue culture plates failed to polarize, and their random movement was
18

slightly increased by VEGF.
In contrast, MM cells adherent to fibronectin rapidly
polarized and exhibited increased continuous membrane ruffling. Although additional
stimulation with VEGF did not significantly change membrane ruffling, the migration
rate was markedly increased. Importantly, in the presence of the PKC inhibitor BIM I,
migration rate was again decreased to levels obtained in MM cells adherent to fibronectin
alone.
Average speed of MM cell migration was next measured as described in Materials and
Methods (Fig. 9). The baseline migration rate of MM cells seeded on tissue culture plates
not pre-coated with fibronectin corresponded to the spontaneous motility of resting cells.
The average speed remained steady for the whole period of observation and never
exceeded 15-20µm/hr. Similar results were observed using VEGF- treated cells in the
absence of fibronectin. Importantly, binding of MM cells to fibronectin induced a marked
acceleration of cell motility, peaking 2-3 h after seeding and remaining higher compared
with conditions described above. VEGF induced an additional increase in motility
peaking at 3 h at 100µm/hr and remaining significantly higher compared with non-
stimulated cells seeded to fibronectin. This acceleration was inhibited to baseline
migration rates in the presence of 2µM of the PKC inhibitor BIM I.
This study therefore demonstrates that VEGF-mediated MM cell migration is associated
with 1-integrin- and PI3-kinase-dependent PKC activation. To further verify the role
of PKC as a potential new therapeutic target in MM, ongoing studies are investigating
the effect of PKC-antisense both in vitro and in murine MM models.
19

References
1.
Kibler, C., Schermutzki, F., Waller, H. D., Timpl, R., Muller, C. A., and Klein, G.
(1998) Cell Adhes Commun 5(4), 307-23.
2.
Damiano, J. S., Cress, A. E., Hazlehurst, L. A., Shtil, A. A., and Dalton, W. S.
(1999) Blood 93(5), 1658-67.
3.
Jensen, G. S., Belch, A. R., Mant, M. J., Ruether, B. A., Yacyshyn, B. R., and
Pilarski, L. M. (1993) Am J Hematol 43(1), 29-36.
4.
Uchiyama, H., Barut, B. A., Chauhan, D., Cannistra, S. A., and Anderson, K. C.
(1992) Blood 80(9), 2306-14.
5.
Uchiyama, H., Barut, B. A., Mohrbacher, A. F., Chauhan, D., and Anderson, K.
C. (1993) Blood 82(12), 3712-20.
6.
Fassler, R., and Meyer, M. (1995) Genes Dev 9(15), 1896-908.
7.
Byzova, T. V., Goldman, C. K., Pampori, N., Thomas, K. A., Bett, A., Shattil, S.
J., and Plow, E. F. (2000) Mol Cell 6(4), 851-60.
8.
Ferrara, N. (1999) J Mol Med 77(7), 527-43.
9.
Ferrara, N., and Alitalo, K. (1999) Nat Med 5(12), 1359-64.
10.
Bellamy, W. T., Richter, L., Frutiger, Y., and Grogan, T. M. (1999) Cancer Res
59(3), 728-33.
11.
Dankbar, B., Padro, T., Leo, R., Feldmann, B., Kropff, M., Mesters, R. M., Serve,
H., Berdel, W. E., and Kienast, J. (2000) Blood 95(8), 2630-6.
12.
Podar, K., Tai, Y. T., Davies, F. E., Lentzsch, S., Sattler, M., Hideshima, T., Lin,
B. K., Gupta, D., Shima, Y., Chauhan, D., Mitsiades, C., Raje, N., Richardson, P.,
and Anderson, K. C. (2001) Blood 98(2), 428-35.
20

13.
Asaoka, Y., Nakamura, S., Yoshida, K., and Nishizuka, Y. (1992) Trends
Biochem Sci 17(10), 414-7.
14.
Nishizuka, Y. (1992) Science 258(5082), 607-14.
15.
Nishizuka, Y. (1995) Faseb J 9(7), 484-96.
16.
Nishizuka, Y. (1986) Science 233(4761), 305-12.
17.
Dekker, L. V., and Parker, P. J. (1994) Trends Biochem Sci 19(2), 73-7.
18.
Newton, A. C. (1995) J Biol Chem 270(48), 28495-8.
19.
Woods, A., and Couchman, J. R. (1992) J Cell Sci 101(Pt 2), 277-90.
20.
Klemke, R. L., Yebra, M., Bayna, E. M., and Cheresh, D. A. (1994) J Cell Biol
127(3), 859-66.
21.
Yebra, M., Filardo, E. J., Bayna, E. M., Kawahara, E., Becker, J. C., and Cheresh,
D. A. (1995) Mol Biol Cell 6(7), 841-50.
22.
Timar, J., Trikha, M., Szekeres, K., Bazaz, R., Tovari, J., Silletti, S., Raz, A., and
Honn, K. V. (1996) Cancer Res 56(8), 1902-8.
23.
Shattil, S. J., Cunningham, M., and Hoxie, J. A. (1987) Blood 70(1), 307-15.
24.
Dustin, M. L., and Springer, T. A. (1989) Nature 341(6243), 619-24.
25.
Shimizu, Y., Van Seventer, G. A., Horgan, K. J., and Shaw, S. (1990) Nature
345(6272), 250-3.
26.
Ng, T., Shima, D., Squire, A., Bastiaens, P. I., Gschmeissner, S., Humphries, M.
J., and Parker, P. J. (1999) Embo J 18(14), 3909-23.
27.
Ng, T., Parsons, M., Hughes, W. E., Monypenny, J., Zicha, D., Gautreau, A.,
Arpin, M., Gschmeissner, S., Verveer, P. J., Bastiaens, P. I., and Parker, P. J.
(2001) Embo J 20(11), 2723-2741.
21

28.
Moalli, P. A., Pillay, S., Weiner, D., Leikin, R., and Rosen, S. T. (1992) Blood
79(1), 213-22.
29.
Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989)
Cell 57(1), 167-75.
30.
Kundra, V., Anand-Apte, B., Feig, L. A., and Zetter, B. R. (1995) J Cell Biol
130(3), 725-31.
31.
Kuzuya, M., and Kinsella, J. L. (1994) Exp Cell Res 215(2), 310-8.
32.
Platet, N., Prevostel, C., Derocq, D., Joubert, D., Rochefort, H., and Garcia, M.
(1998) Int J Cancer 75(5), 750-6.
33.
Kiley, S. C., Clark, K. J., Goodnough, M., Welch, D. R., and Jaken, S. (1999)
Cancer Res 59(13), 3230-8.
34.
Tang, S., Morgan, K. G., Parker, C., and Ware, J. A. (1997) J Biol Chem 272(45),
28704-11.
35.
Kraft, A. S., and Anderson, W. B. (1983) Nature 301(5901), 621-3.
36.
Kraft, A. S., Anderson, W. B., Cooper, H. L., and Sando, J. J. (1982) J Biol Chem
257(22), 13193-6.
37.
Lauffenburger, D. A., and Horwitz, A. F. (1996) Cell 84(3), 359-69.
38.
Hemler, M. E. (1998) Curr Opin Cell Biol 10(5), 578-85.
39.
Mannion, B. A., Berditchevski, F., Kraeft, S. K., Chen, L. B., and Hemler, M. E.
(1996) J Immunol 157(5), 2039-47.
40.
Rubinstein, E., Le Naour, F., Billard, M., Prenant, M., and Boucheix, C. (1994)
Eur J Immunol 24(12), 3005-13.
22

41.
Zhang, X. A., Bontrager, A. L., Stipp, C. S., Kraeft, S. K., Bazzoni, G., Chen, L.
B., and Hemler, M. E. (2001) Mol Biol Cell 12(2), 351-65.
42.
Watson, J. M., Harding, T. W., Golubovskaya, V., Morris, J. S., Hunter, D., Li,
X., Haskill, J. S., and Earp, H. S. (2001) J Biol Chem 276(5), 3536-42.
43.
Avraham, S., London, R., Fu, Y., Ota, S., Hiregowdara, D., Li, J., Jiang, S.,
Pasztor, L. M., White, R. A., Groopman, J. E., and et al. (1995) J Biol Chem
270(46), 27742-51.
44.
Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M.,
Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376(6543), 737-45.
45.
Sasaki, H., Nagura, K., Ishino, M., Tobioka, H., Kotani, K., and Sasaki, T. (1995)
J Biol Chem 270(36), 21206-19.
46.
Li, J., Avraham, H., Rogers, R. A., Raja, S., and Avraham, S. (1996) Blood 88(2),
417-28.
47.
Chauhan, D., Hideshima, T., Pandey, P., Treon, S., Teoh, G., Raje, N., Rosen, S.,
Krett, N., Husson, H., Avraham, S., Kharbanda, S., and Anderson, K. C. (1999)
Oncogene 18(48), 6733-40.
48.
Salgia, R., Avraham, S., Pisick, E., Li, J. L., Raja, S., Greenfield, E. A., Sattler,
M., Avraham, H., and Griffin, J. D. (1996) J Biol Chem 271(49), 31222-6.
49.
Rhee, S. G., and Bae, Y. S. (1997) J Biol Chem 272(24), 15045-8.
50.
Bae, Y. S., Cantley, L. G., Chen, C. S., Kim, S. R., Kwon, K. S., and Rhee, S. G.
(1998) J Biol Chem 273(8), 4465-9.
51.
Rameh, L. E., Rhee, S. G., Spokes, K., Kazlauskas, A., Cantley, L. C., and
Cantley, L. G. (1998) J Biol Chem 273(37), 23750-7.
23

52.
Falasca, M., Logan, S. K., Lehto, V. P., Baccante, G., Lemmon, M. A., and
Schlessinger, J. (1998) Embo J 17(2), 414-22.
53.
Scharenberg, A. M., and Kinet, J. P. (1998) Cell 94(1), 5-8.
54.
Cunningham, S. A., Waxham, M. N., Arrate, P. M., and Brock, T. A. (1995) J
Biol Chem 270(35), 20254-7.
24

1 Abbreviations used in this paper: MM, Multiple Myeloma; BM, bone marrow; VEGF/R
vascular endothelial growth factor/ receptor; PKC, protein kinase C; Flt-1, fms-like
tyrosine kinase; VLA, very late antigens; IL-6, interleukin-6; ECM, extracellular matrix;
HUVE cells, human umbilical vein endothelial cells; BMSC, bone marrow stromal cells;
MoAb, monoclonal antibody; FBS, fetal bovine serum; BIM I, bisindolylmaleimide I;
TLVM, time lapse video microscopy; CADTK, Ca-dependent tyrosine kinase; PMA,
phorbol-12-myristate-13-acetate; IP, immunoprecipitation
We thank Dr. Frank Gesbert (Dana-Farber Cancer Institute), Dr. Werner Lubitz (VBC,
Vienna), and Dr. Heinz Ludwig (Wilhelminenspital, Vienna) for helpful discussions.
This work was supported by an IMF/ Brian D. Novis/ Benson Klein Research Grant
Award (to K-Podar), the National Institutes of Health grant PO-1 78378, and the Doris
Duke Distinguished Clinical Research Scientist Award to KC-Anderson.
25

Figure Legends:
Figure 1. PKC isoform expression in MM cells. Cell lysates were prepared from MM
cell lines and MM patient cells as described in Materials and Methods. Aliquots of cell
extracts (80µg) were separated on 10% polyacrylamide gels by SDS-PAGE and
transferred to nitrocellulose; blots were stained with the isozyme-specific Abs indicated.
Cell lysate from rat brain were used as a positive control for PKC expression. Molecular
weight standards in kiloDaltons (kDa) are shown.
Figure 2. Effects of VEGF and adherence to fibronectin induce PKC subcellular
localization and activation. (a) MM.1S cells were either untreated or treated with
100ng/mL VEGF for indicated intervals, placed in suspension (S), or plated on
fibronectin (FN). (b) PKC activity was determined using a PKC kinase assay as
described in Materials and Methods. Stimulation of MM.1S cells with 300nM PMA for
20 min was used as a positive control. L, rat brain lysate; C, IP control. Results of a
representative experiment are shown. (c) MM.1S cells in suspension were either
untreated or treated with 100ng/mL VEGF for indicated time points. Treatment with
300nM PMA was performed as a positive control for PKC membrane translocation. (d)
MM.1S cells were either placed in suspension (S) or plated on fibronectin (FN) for the
indicated time points. (a, c, d) SP100 cell fractionation was performed as described in
Materials and Methods. Immunoblots of cytosolic (C) and membrane (M) fractions were
probed for expression of the indicated PKC isoforms. Molecular weight standards in
kiloDaltons (kDa) are shown.
26

Figure 3. VEGF enhances MM cell adhesion to fibronectin. (a) MM cell adhesion to
fibronectin is enhanced in dose-dependent manner by VEGF. contr, adhesion of cells in
suspension. (b) VEGF-mediated MM cell adhesion to various concentrations of
fibronectin. (c) VEGF-mediated MM cell adhesion is transient. (d) Dependency of MM
cell adhesion on PKC, PMA
(50nM, 300nM) was used as a positive control. (e)
Dependency of VEGF-mediated MM cell adhesion on 1-integrin, shown by using a 1-
integrin neutralizing MoAb (dilution: 1:1000, 1:500, 1:100). S, soluble; neg c, cells
seeded to fibronectin without stimulation; pos c, VEGF (100ng/mL); IgG, non-immune
IgG (f) Dependency of VEGF-mediated MM cell migration on 1-integrin, shown by
using 1-integrin neutralizing MoAb (anti-1, 1:1000 and 1:100). Neg c, migration
without chemoattractant; pos c, VEGF (10ng/mL); IgG, non-immune IgG. The data
shown are the means +/- SD of three separate experiments, adhesion assays were
performed in triplicate.
Figure 4. VEGFR-1/Flt-1 associates with 1-integrin and CADTK and enhances
CADTK-phosphorylation, dependent on PKC- and 1-integrin activation. MM.1S cells
were serum starved overnight in 2% FBS and an addition 2 h without serum, detached
from the culture dish, and either maintained in suspension (S) or incubated on
fibronectin-coated plates (FN) for the indicated intervals. Immunoprecipitation was
performed as described in Materials and Methods. (a) Co-immunoprecipitation of
CADTK and 1-integrin with PKC. (b) constitutive association of Flt-1 and CADTK.
(c) activation of CADTK upon treatment with PMA (300nM), 20 min. (d) Additional
increase of CADTK-phosphorylation in the presence of VEGF (100ng/mL). (e)
27

Dependency of VEGF-induced CADTK activation on PKC and 1-integrin. 1, 1-
integrin; pY, phosphotyrosine; C, IP control
Figure 5. VEGF- induced PI3-kinase activation is required for MM cell migration.
Serum-starved cells were stimulated with VEGF for indicated intervals, lysed, and
immunoprecipitated
with
anti-Flt-1
Ab
(a),
anti-p85
(b),
or
anti-pY
(c).
Immunoprecipitates were analyzed by SDS-PAGE, followed by immunoblotting with the
indicated antibodies.
(d) For the PI3-kinase assay, equal amounts of lysates were immunoprecipitated with
anti-phosphotyrosine MoAb, and immunocomplexes were assayed for their ability to
phosphorylate PIP2. PIP3, phosphatidylinositol-(3,4,5)-P3. C, control indicates a PI3-
kinase assay done on protein-A alone.
Figure 6. Subcellular localization of PKC isoforms and activation is PI3-kinase
dependent. MM.1S cells placed in suspension (S) or plated on fibronectin (FN) were
either pre-treated with LY 294002 (LY, 20µM) or left untreated prior to stimulation with
100ng/mL VEGF for 30 min. SP100 cell fractionation was performed as described in
Materials and Methods. Immunoblots of cytosolic (C) and membrane (M) fractions were
detected with PKC and reprobed with PKC. Molecular weight standards in kiloDaltons
(kDa) are shown.
Figure 7. VEGF- mediated MM cell adhesion and migration are PI3-kinase dependent.
(a) MM cell migration was performed as described in Materials and Methods. no inh,
28

VEGF (10ng/mL); results shown are representative of 3 independent experiments (b)
MM cell adhesion. S, soluble; neg c, cells seeded to fibronectin or polylysine without
stimulation; pos c, VEGF (100ng/mL); LY, LY294002 (5µM, 20µM). The data shown
are the means +/- SD of three separate experiments, adhesion assays were performed in
triplicate.
Figure 8. Morphology and velocity of migrating cells. Top left, average speed of
representative cells shown in a-e. (a-e) Top, Composite time-lapse series of phase-
contrast micrographs (magnification, x40) acquired at 2 min intervals were outlined and
tracked with the NIH Image 1.62 software. Images show migration of representative cells
during 30 min starting 3 h after plating. Cells were seeded on tissue culture plates pre-
coated (c-e) or not pre-coated (a,b) with fibronectin in the presence (b, d, e) or absence (a,
c) of VEGF (100ng/mL). Bottom, graphs represent surface area and length as a
description of morphological changes. Bar, 20µm.
Figure 9. Quantitative evaluation of MM cell motility. Average speed of MM cells was
measured as described in Materials and Methods. In time-course experiments, MM.1S
cells were seeded on fibronectin pre-coated or not pre-coated tissue culture plates, in the
presence or absence of VEGF.
29

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