1
Department of Stomatology, University of California San Francisco, San
Francisco, CA 94143, USA
2
Department of Surgery, University of California San Francisco, San Francisco,
CA 94143, USA
3
Department of Pathology, Washington University School of Medicine, St Louis,
MO 63110, USA
4
Departments of Anatomy and Cell Biology, SUNY Health Science Center, Syracuse,
NY 13210, USA
*
Author for correspondence (e-mail:
dramos{at}itsa.ucsf.edu
)
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SUMMARY |
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Key words: ß3 integrins, Melanoma, Focal adhesion kinase, Src, Motility
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INTRODUCTION |
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During invasion, tumor cells adhere to and penetrate the ECM. These
processes are mediated by the integrin family of adhesion receptors. Integrins
are heterodimeric cell surface receptors consisting of an and a ß
subunit (Hynes, 1992
). Several
studies have linked the expression of the
vß3 integrin with
invasion of various types of malignant tumors (Zheng et al.,
1999
; Seftor et al.,
1992
).
vß3 has
also been shown to be neo-expressed in melanomas when they switch from being a
superficially spreading to a vertically invasive phenotype (Albelda et al.,
1990
; McGregor et al.,
1989
). Additionally, it was
shown that overexpression of
vß3 in radial growth phase primary
melanoma cells initiates invasive growth in three dimensional skin
reconstructs and increases tumor growth in vivo (Hsu et al.,
1998
). We have also shown
previously that expression of
vß3 is associated with the invasive
phenotype in the K1735 melanoma system (Li et al.,
1998
).
Recent evidence from the field of integrin biology has elucidated some of
the signaling mechanisms involved in the regulation of cell motility and
proliferation by ECM (Schwartz and Shattil,
2000; Giancotti and Ruoslahti,
1999
). Two non-receptor protein
tyrosine kinases (NRPTKs), focal adhesion kinase (FAK) and Src, were found to
modulate integrin-mediated cell migration. FAK is a novel type of NRPTK that
becomes tyrosine phosphorylated and subsequently activated when integrins
adhere to various matrix proteins, including vitronectin (VN). Although
intensively studied, the exact sequence of events leading to FAK activation is
still not well defined. FAK can be phosphorylated in vivo at six or more
tyrosines - two within the N-terminal domain (Tyr397 and
Tyr407), two within the activation loop (Tyr576 and
Tyr577) and two within the C-terminal region (Tyr861 and
Tyr925) - and at several serines (Richardson et al.,
1997
; Calalb et al.,
1996
; Schlaepfer et al.,
1999
). It has been suggested
that (auto)phosphorylation of FAK at Tyr397 recruits the Src-family
members, Src or Fyn (Schaller et al.,
1994
). Binding of Src or Fyn
to Tyr397 further augments FAK tyrosine kinase activity (Schaller
et al., 1999
). Src binding to
FAK at Tyr397 is also required for phosphorylation of
Tyr925, which recruits the adapter molecule Grb2, linking integrins
to the Ras pathway (Schlaepfer and Hunter,
1996
). Integrin activation of
gene transcription and cytoskeletal reorganization may not always involve the
formation of FAK/Src complexes. A FAK-independent pathway, regulated by
another Src-family member, Fyn, has also been proposed (Giancotti and
Ruoslahti, 1999
; Wary et al.,
1998
).
Direct evidence linking integrin-ECM interactions with the phosphorylation
of FAK and tumor cell motility has been described by others. Specifically,
binding of vß3 to VN results in an increase in FAK tyrosine
phosphorylation during prostatic cell migration (Zheng et al.,
1999
). Activation of Src has
also been observed following melanoma cell-osteopontin interactions; an event
mediated by the
vß3 integrin (Duong et al.,
1998
). Tumor cell invasion
requires ECM remodeling, which occurs through the action of various matrix
metalloproteinases (MMPs). Both MMP-2 and MMP-9 are highly expressed in many
different types of tumors, and their expression has been correlated with
increased invasion (Kurschat et al.,
1999
; Pyke et al.,
1992
).
In the present study we show that overexpression of vß3 in
poorly invasive, ß3-negative K1735 melanoma cells enhances their
metastatic behavior in vivo. We also show that phosphorylation of FAK and Src
occurs as a result of post-
vß3-ligand interactions and is
important for K1735 cell motility.
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MATERIALS AND METHODS |
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Cell culture
The mouse K1735 melanoma cell lines, the highly metastatic K1735M2
(hereafter M2) and the poorly metastatic K1735C23 (hereafter C23) lines, were
a kind gift from I. J. Fidler (University of Texas M.D. Anderson Hospital and
Tumor Institute, Houston, TX). Their isolation and metastatic activity have
previously been described (Fidler et al.,
1981). All cell lines used in
this study were generated from these two K1735 melanoma lines and listed in
Table 1. Cells were routinely
cultured in 10% fetal bovine serum in Dulbecco's minimal essential medium
(DMEM) and passaged in log growth phase by a brief treatment with 0.25%
trypsin/2 mM EDTA. To minimize phenotypic drift, cells in culture were
replenished after 8-10 passages from frozen stocks.
|
Retroviral transduction experiments
Constitutively active Src (CA-Src), kinase dead Src (KD-Src), FAK-related
non-kinase (FRNK) or vector alone were stably expressed in either M2 or C23
melanoma cell lines using the Retro-X system (Clontech, Palo Alto, CA) (Li et
al., 1998;
Kova
i
et
al., 1998
). Cells with stable
expression of the sense or the antisense ß3 integrin subunit were
generated as described (Li et al.,
1998
).
Briefly, the mutant Src and FRNK cDNAs were inserted in the sense orientation into the multiple cloning site of the retroviral vector pLXSN. The retroviral vector was transfected into the PT67 packaging cell line by the lipofectin transfection method. Conditioned growth medium containing secreted retrovirus was collected and endpoint titers were determined on NIH3T3 cells. After G418 selection, the supernatant was supplemented with 8 µg/ml polybrene, sterile filtered, and incubated with the M2 cells (KD-Src and FRNK) or the C23 cells (CA-Src) for 24 hours. Transfected cells expressing the retrovirus-encoded cDNA were selected with G418 growth medium. Pooled cultures of G418-resistant cells were used for the described studies.
In vivo tumor growth and metastasis assays
To evaluate primary tumor growth, C3H/HeN syngeneic mice were anesthetized
with Avertin (0.015 ml of a 2.5% solution per gram of body weight).
2x105 tumor cells in 50 µl of DMEM were injected
transorally into the floor of the mouth. After 4 weeks, animals were
sacrificed by cervical dislocation and the extent of tumor formation was
evaluated. For metastasis assays, 2x105 cells were injected
into the hind flank of C3H/HeN mice. The animals were sacrificed after 12
weeks and analyzed for the presence of tumors in the lungs or other sites.
Immunohistochemistry
Metastatic cardiac lesions were isolated from two syngeneic mice and
directly embedded in OCT (optimal cutting temperature compound; Miles
Laboratories, Elkhart, IN) embedding medium and snap frozen, or first immersed
in 10% sucrose at 4°C for 4 hours and then embedded and frozen. Briefly,
4-µm cryostat sections of tissue samples were air dried for 30 minutes,
fixed in -20°C acetone for 5 minutes, and air dried again. Endogenous
peroxidase activity was blocked with Peroxoblock Solution (Zymed Laboratories)
for 45 seconds at room temperature. After rinsing in PBS for 15 minutes to
remove the OCT, sections were incubated for 30 minutes at room temperature
with 0.5% casein/0.05% thimerosal/PBS and then incubated for 2 hours at room
temperature with primary antibody (anti-ß3 integrin subunit) or without
primary antibody as a control. The sections were further processed using the
Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Some sections were
counterstained with hematoxylin and eosin (H and E) (Sigma, St Louis, MO).
Sections were mounted with Permount (Fisher Scientific, Pittsburg, PA) and
analyzed by microscopy.
Western blotting
Cells were seeded at 70% confluence and deprived of serum for 16 hours. The
cells were then detached and replated on VN-coated dishes (20 µg/ml) for 30
minutes at 37°C. Cells were lysed in modified RIPA buffer (150 mM NaCl, 1
mM EDTA, 20 mM Tris-HCl, 1% Nonidet P-40) with freshly added proteinase (1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin)
and phosphatase (1 mM Na3VO4, 50 mM NaF) inhibitors.
Next, the lysates were spun down at 15,000 g for 15 minutes and precleared for 1 hour at 4°C with protein A-agarose. Protein concentrations were determined by BCA protein assay kit (Pierce, Rockford, IL). For immunoprecipitations, 300 µg of cell lysate was incubated at 4°C for 1 hour with the primary antibody. Immune complexes were captured with Protein A-Sepharose and washed with lysis buffer followed by wash buffer (10 mM Tris, pH 7.4, 0.01% sodium dodecyl sulfate (SDS), 1% glycerol, 0.1% ß-mercaptoethanol, 1 mM Na3VO4, 1 mM EDTA) and boiled in the presence of sample buffer. The samples were then separated by SDS-polyacrylamide gel electrophoresis (PAGE) followed by western blotting using commercially available anti-FAK, anti-pTyr397 FAK, anti-Fyn and anti-Src antibodies.
Migration assay
Cell migration was assayed in 24-well, 6.6-mm-internal-diameter Transwell
cluster plates (8.0 µm pore size; Costar, Cambridge, MA) as described
(Ramos et al., 1997; Ramos et
al., 1998
). Five random fields
of each Transwell filter were counted at x200 magnification, and the
cell numbers were calculated as total migrated cells per filter. Briefly, the
lower surfaces of the filters were coated with VN (10 µg/ml) overnight at
4°C. Then 5x104 cells were placed onto the upper surfaces
of the filters, and the chambers were incubated at 37°C for 2 hours. The
upper surfaces of the filters were wiped clean, the filter was fixed and
stained, and the number of cells that had migrated to the underside were
counted in triplicate.
Invasion of a reconstituted basement membrane
A thin layer of a reconstituted basement membrane (RBM) (Collaborative
Research, Bedford, MA) was overlaid onto a Transwell filter (8 µm pore
size) and allowed to polymerize at 37°C for 1 hour. Then
5x104 cells were seeded onto the RBM-coated filter and
allowed to invade for 16 hours. After the incubation period, the cells were
processed as in the migration assay.
Kinase assay
In vitro autophosphorylation assays were performed as described (Kanazawa
et al., 1996). Briefly, Src,
Fyn or FAK immunoprecipitates were washed once with kinase reaction buffer (20
mM 1,4-piperazine bis-(ethane sulfonic acid) (PIPES), pH 7.4, 10 mM
MnCl2) and labeled by addition of 0.5 ml
[
-32P]ATP (3000 Ci/mmol, 10 mCi/ml) in kinase buffer (total
volume 10 µl) for 30 minutes at 37°C. The reaction was terminated by
the addition of sample buffer. The labeled protein, separated by SDS-PAGE, was
transferred to a nylon membrane. The amount of Src protein on the membrane was
visualized with alkaline phosphatase-conjugated secondary antibodies and
BCIP/NBT substrate (Gibco BRL, Grand Island, NY). The membrane was then dried
and exposed to X-ray film to detect incorporated radioactivity.
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RESULTS |
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Tumor growth was subsequently evaluated in vivo. The highly invasive, ß3-positive M2 cells and the descendant M2-Tß3 cells were injected into the floor of the mouth of C3H/HeN syngeneic mice (12 mice per cell line). After 4 weeks, large primary tumors were formed in mice injected with the M2 cells (Fig. 1A, left, arrow), whereas tumors formed by the M2-Tß3 cells were extremely small (Fig. 1A, right), suggesting that expression of ß3 enhances K1735 tumor formation in vivo. The average weight of the tumors produced by the M2 cells (black histogram) was 5.0 g (±1 g), whereas the tumors formed by the M2-Tß3 cells averaged approximately 1.2 g (±0.4 g) (Fig. 1B).
|
To evaluate a possible link between expression of the ß3 integrin and
metastatic potential in these two melanoma cell lines, we performed in vivo
metastasis assays. M2 or M2-Tß3 cells were injected into the hind flank
of C3H/HeN syngeneic mice (12 animals/cell line). After 12 weeks, the mice
were sacrificed and analyzed for the formation of metastases. On average, 100
lung lesions (±20 colonies) were formed by the M2, whereas none were
formed by the M2-Tß3 cells (Fig. 2A
and B, respectively). The ß3-negative C23 and the
C23-mß3 cells, stably expressing the ß3 subunit, were also evaluated
by spontaneous metastasis assays. The C23-mß3 cells formed an average of
40 lung lesions per animal (±10 colonies)
(Fig. 2D), whereas the C23
cells did not metastasize (Fig.
2C). These results indicate that expression of vß3
potentiates metastatic colonization. Quantitation of experiment is included
(Fig. 2E).
|
Metastatic lesions formed by the C23-mß3 cells retain expression
of ß3 integrin
C23-mß3 cells produced not only lung metastases but also cardiac
muscle lesions in two of the 12 animals
(Fig. 3A, arrows).
Immunostaining of frozen tumor tissue sections with anti-ß3 antibodies
localized the integrin to the tumor cell membrane
(Fig. 3B, arrow). These results
indicate that the C23-mß3 cells continue to express the ß3 integrin
in vivo.
|
ß3 ligand-binding induces phosphorylation of FAK and activation
of Src
Integrin engagement triggers a variety of intracellular signaling cascades.
One of the foremost events is the phosphorylation of FAK on Tyr397
(Schlaepfer et al., 1999). The
K1735 cell lines (M2, M2-Tß3, C23, C23-mß3) were plated on VN at 70%
confluence. Serum was withdrawn for 16 hours to avoid the confounding effects
of exogenous growth factors. The cells were detached and then replated on VN
for an additional 30 minutes in the absence of serum. FAK was
immunoprecipitated from the cell lysates. Tyr397 phosphorylation
was examined by western blotting of the FAK immunoprecipitates with
phospho-specific anti-pTyr397 antibodies
(Fig. 4A, top). Levels of
pTyr397 in the M2 and the C23 cells were comparable; however,
higher levels of pTyr397 were detected in the M2 and the
C23-mß3 cell lysates when compared directly with the M2-Tß3 or C23
lysates, respectively (Fig. 4A,
top). The membrane was stripped and reblotted with anti-FAK antibodies to
confirm that our results were not due to unequal amounts of immunoprecipitated
protein (Fig. 4A, bottom).
Results were further confirmed by comparison of
-32P
incorporated into FAK in FAK (auto)phosphorylation assays (data not shown).
Phosphorylation of FAK on Tyr397 appeared in part to be associated
with expression of the ß3 integrin.
|
The predominant partners of FAK in integrin-mediated signaling are Src and
Fyn (Schlaepfer et al., 1999).
Both Src and Fyn are known to bind to FAK and augment its kinase activity. We
assessed both Src and Fyn kinase activity in our system to attempt to make a
physiological distinction between Src and Fyn activity and metastatic
potential (Fig. 4B). We
detected Src but not Fyn kinase activity in the cell lysate
(Fig. 4B). Greater
incorporation of
-32P into Src corresponded to samples that
also showed higher levels of pTyr397 FAK (M2 and C23-mß3)
(Fig. 4B, top). Results of Src
and Fyn autophosphorylation assays were confirmed by blots with clone 28, an
antibody that specifically recognizes the active form of Src and Fyn
(Kawakatsu et al., 1996
) (data
not shown). These data suggest that Src, rather than Fyn, may be involved in
the ß3-mediated invasive phenotype of K1735 cells.
Expression of ß3 integrin correlates with an increase in FAK/Src
complex formation in K1735 melanoma cells
We wished to determine if FAK formed a complex with Src or Fyn in these
cell lines (M2 and C23-mß3). Src and Fyn were immunoprecipitated from the
lysates of cells plated on VN for 30 minutes and prepared as in the previous
experiments. The immune complexes were resolved by SDS-PAGE and probed with
anti-FAK antibodies. A complex containing both FAK and Src was found to be
several times higher in the M2 and C23-mß3 cells than in the M2-Tß3
and the C23 cells (Fig. 4C). A
complex containing FAK and Fyn was not detected in any of the four cell lines
(Fig. 4C). Reblotting with
antibodies to Src or Fyn indicated that our observations were not due to
differences in overall protein level (Fig.
4C; bottom). These results indicate that, in K1735 cells, a
FAK/Src complex is formed that may be important for ß3-integrin-mediated
invasion. This finding represents one of the first insights into phenotypic
differences that may be a result of association between FAK/Src and not
FAK/Fyn.
Phosphorylation of FAK on Tyr397 is required for K1735
cell motility
The independently expressed C-terminal region of FAK, termed FAK-related
non-kinase (FRNK), can serve as a dominant-negative regulator of FAK function
by competing with full-length FAK for a site in focal adhesions (Richardson
and Parsons, 1996). We
examined whether phosphorylation of FAK on Tyr397 is required for
the transduction of signals initiated by the binding of the ß3 integrin
to VN and subsequent motility. We introduced FRNK fused with green fluorescent
protein (GFP) into the K1735M2 cell line using a retroviral vector. The
resulting cell line was termed M2GFP-FRNK. The expression level of GFP-FRNK
was verified by western blotting with antibodies to GFP (data not shown).
Expression of GFP-FRNK was also verified by western blotting using antibodies
to the C-terminus of FAK (Fig.
5A). A decrease in phosphorylation of FAK at Tyr397 was
detected in the M2GFP-FRNK cell lysate when compared with the M2 cell lysate
(Fig. 5A, top). No change in
overall FAK protein expression was detected
(Fig. 5A, middle). Western blot
of whole cell lysate with antibodies to the C-terminus of FAK clearly detected
the FRNK construct in the M2GFP-FRNK cell line.
|
We next evaluated a possible role for FRNK in cell motility (Fig. 5B). We found that migration of GFP-FRNK cells was reduced by approximately 90% when compared with the M2 cells (Fig. 5B). On average, 238 (±11.5) M2 cells migrated on VN, whereas 11.5 (±1) M2GFP-FRNK cells migrated. Similarly, invasion of an RBM was suppressed approximately 80% by expression of FRNK (Fig. 5B). On average 46 (±19) M2 cells invaded, whereas only 8 (±1) M2-GFP-FRNK cells invaded. These results suggest that phosphorylation of FAK on Tyr397 is required for maximum K1735 cell motility.
Src activity affects K1735 melanoma cell motility
Having demonstrated increased activation of Src and increased FAK/Src
complex formation in the ß3 positive cells, we next addressed the effect
of Src activation on cell motility. We expressed a kinase dead Src in the
highly invasive M2 cells and derived the M2KD-Src cell line. We also expressed
a constitutively active Src in the non-invasive C23 cells to generate the
C23CA-Src cell line (Fig. 6A).
When examined by immunoprecipitation/western blot or western blotting of whole
cell lysates, the M2KD-Src and C23CA-Src cells showed slightly higher
expression of total Src levels (paxillin blot was used as a loading control).
A Src autophosphorylation assay showed markedly higher incorporation of
-32P into Src immunoprecipitates from M2 vs M2KD-Src cells.
The amount of
-32P incorporated into Src in C23CA-Src cells
was 20-fold that seen in the original C23 cells
(Fig. 6A).
|
We next evaluated the effect Src activation plays in K1735 cell migration. The C23CA-Src cells were four times as motile on VN as the original C23 cells. The expression of a KD-Src reduced M2 cell line migration by two thirds (Fig. 6B). These results suggest that activation of Src is required for K1735 cell migration.
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DISCUSSION |
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The heterogeneity of tumors is well documented (Nicolson,
1988). Within the primary
tumor, there is a subset of cells that may become metastatic. These cells
acquire specific characteristics that may promote the invasive phenotype.
Acquisition of specific integrin receptors may be one such characteristic.
In this study we demonstrated that expression of the ß3 integrin subunit increased K1735 melanoma cell invasion and metastasis. We also show that phosphorylation of FAK at Tyr397, and Src activity are strongly correlated with enhanced K1735 cell invasion.
In vivo, K1735 cells expressing the ß3 integrin (M2 and C23-mß3)
formed multiple metastatic foci in syngeneic mice; whereas ß3-negative
cells did not. Therefore, we suggest a specific mechanism by which the K1735
melanoma cells become metastatic: expression of the vß3
integrin.
Adhesion to the ECM `clusters' the integrin, generating intracellular
signals, similar to those demonstrated by growth factors, resulting in
cytoskeletal rearrangements (Juliano,
1996).
Two NRPTKs, FAK and Src, have been reported to act as signaling molecules
linking the integrin to cytoskeletal reorganization and subsequent migration
(Schlaepfer et al., 1999). In
our model system, adhesion to VN resulted in the phosphorylation of FAK at
Tyr397 and an increase in Src activity. This suggests that FAK and
Src are responsive to clustering of the
vß3 integrin on VN. To
explore this further, we perturbed Src function by expressing either a
constitutively active or a kinase-inactive Src mutant. Expression of a
constitutively active Src increased K1735 cell motility, whereas expression of
a kinase-dead mutant Src significantly suppressed cell movement.
We expressed FRNK in the M2 cell line to evaluate the role of FAK in K1735
tumor cell motility. FRNK, a naturally occurring C-terminal domain of FAK,
acts as a dominant-negative regulator of FAK function. The stable expression
of FRNK in the highly metastatic M2 cells decreased their invasive behavior
and the phosphorylation of FAK at Tyr397. These results indicate
that phosphorylation of Tyr397 of FAK is a contributing factor to
the invasive potential of K1735 melanoma cells. The suggestion that FAK is
important for tumor progression is not novel. Several other groups have
reported that the expression of FAK is upregulated in a variety of different
cancer cells (Judson et al.,
1999; Zheng et al.,
1999
; Tremblay et al.,
1996
; Owens et al.,
1995
; Owens et al.,
1996
; Weiner et al.,
1993
). However, its function
in tumor cell invasion and metastasis is still unclear. Recently, data from
three independent laboratories demonstrated that the phosphorylation of FAK
may also be involved in cell proliferation, a characteristic central to tumor
progression (Wang et al.,
2000
; Zhao et al.,
1998
; Oktay et al.,
1999
). In our system we also
observed a slight decrease in K1735 cell proliferation when FAK
phosphorylation was suppressed by expression of FRNK.
Phosphorylated Tyr397 of FAK serves as a binding site for the
Src-family kinases, Src and Fyn. It has also been shown by several groups that
formation of a FAK/Src or FAK/Fyn complex can lead to further phosphorylation
of FAK by Src or Fyn, and thereby increase FAK kinase activity. However, to
date, there is no strong evidence to indicate which biological events may be
mediated through a FAK/Src vs a FAK/Fyn complex (Schlaepfer et al.,
1999; Parsons and Parsons,
1997
; Thomas and Brugge,
1997
). Our data suggest that
the specific complex of FAK with Src, but not with Fyn, will lead to increased
cell migration, invasion and metastasis.
In summary, we detected in a single, well-characterized system that (1) the
specific clustering of vß3 results in phosphorylation of FAK at
Tyr397 and activation of Src, not Fyn; and (2) expression of Src
mutants affects K1735 cell motility. Therefore, we suggest that suppressing
the function or expression of
vß3, or targeting one or more of its
downstream signaling molecules, will have a significant impact on melanoma
progression in a clinical setting.
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ACKNOWLEDGMENTS |
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