From the Laboratories of Cellular and Molecular
Biology and § Medicinal Chemistry, Division of Basic
Sciences, NCI, National Institutes of Health,
Bethesda, Maryland 20892-4255
Received for publication, November 9, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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Hepatocyte growth factor (HGF) stimulates
mitogenesis, motogenesis, and morphogenesis in a wide range of
cellular targets during development, homeostasis and tissue
regeneration. Inappropriate HGF signaling occurs in several human
cancers, and the ability of HGF to initiate a program of protease
production, cell dissociation, and motility has been shown to promote
cellular invasion and is strongly linked to tumor metastasis. Upon HGF
binding, several tyrosines within the intracellular domain of its
receptor, c-Met, become phosphorylated and mediate the binding of
effector proteins, such as Grb2. Grb2 binding through its SH2 domain is
thought to link c-Met with downstream mediators of cell proliferation,
shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with
ED50 values of 30 nM or less, but only
modest inhibition of mitogenesis. These compounds are 1000-10,000-fold
more potent anti-motility agents than any previously characterized Grb2
SH2 domain antagonists. Our results suggest that SH2 domain-mediated
c-Met-Grb2 interaction contributes primarily to the motogenic and
morphogenic responses to HGF, and that these compounds may have
therapeutic application as anti-metastatic agents for tumors where the
HGF signaling pathway is active.
Hepatocyte growth factor
(HGF)1 stimulates
mitogenesis, motogenesis, and morphogenesis in a wide range of cellular
targets including epithelial and endothelial cells, hematopoietic
cells, neurons, melanocytes, as well as hepatocytes (reviewed in Refs. 1-3). These pleiotropic effects play fundamentally important roles
during development, organogenesis, and tissue regeneration. For
example, HGF is essential for the normal development of both liver and
placenta (reviewed in Ref. 4), contributes to neural development
(reviewed in Ref. 5) and branching morphogenesis in various organs
(reviewed in Ref. 6), and promotes kidney and lung regeneration (7,
8).
The biological responses to HGF are mediated by its cell surface
receptor, c-Met, a transmembrane tyrosine kinase. Upon HGF binding,
several tyrosines residues within the c-Met intracellular domain are
phosphorylated, some of which are essential for catalytic activity, and
some of which mediate the binding of signaling proteins such as the p85
subunit of phosphoinositide 3-kinase (PI3K), phospholipase C- In addition to its critical roles in normal development and adult
homeostasis, HGF signaling is strongly linked to cancer, including
colon, breast, lung, thyroid, and renal carcinomas; melanoma; and
several sarcomas; as well as glioblastoma (reviewed in Ref. 18). The
inappropriate expression of c-Met in certain mesenchymal cells can lead
to a carcinogenic transformation in which the tumor cells express both
mesenchymal and epithelial markers (19). Inherited mutations in c-Met
that result in constitutive activation of the HGF signaling pathway are
associated with human renal papillary carcinoma (20-22). Importantly,
in addition to its mitogenic activity, the ability of HGF to initiate a
program of cell dissociation and increased cell motility coupled with increased protease production has been shown to promote cellular invasion through extracellular matrix substrates, and is correlated with tumor metastasis in vivo (reviewed in Refs. 6, 9, and 18). Increased extracellular matrix proteolysis, cell dissociation, and
increased cell motility are essential for tumor metastasis, and HGF may
be unique among extracellular signaling molecules in its ability to
initiate all of these events.
Artificial inhibitors of protein and lipid kinases, phosphatases, and
protein-protein interactions have been used extensively to examine the
roles of individual intracellular effector molecules in specific
HGF-stimulated activities, and to explore their potential as anticancer
drugs. Many intracellular effectors interact with receptor tyrosine
kinases through conserved amino acid sequence modules, such as the Src
homology 2 (SH2) domain (reviewed in Refs. 23 and 24). SH2 domains
directly recognize phosphotyrosine (Tyr(P)) (25), with additional
secondary binding interactions within two or three amino acids
C-proximal to the Tyr(P) residue introducing differential affinity
toward SH2 domain subfamilies (26, 27). These and other observations
have led to the development of potent tripeptide inhibitors of SH2
domain interactions. Modification of the Tyr(P) residue to
phosphonomethyl phenylalanine, or related structures, protects this
moiety from phosphatases (28, 29), whereas other modifications have
been identified that increase inhibitor affinity and the potential for
cell membrane penetration (30).
In this study we describe the effects of several artificial
tripeptide-based inhibitors of the Grb2 SH2 domain on HGF-stimulated mitogenesis, motogenesis, and extracellular matrix invasion in epithelial and hematopoietic target cell models. Grb2 is thought to
link HGF-stimulated c-Met activation with the activation of Rho, Ras,
and Rac (17), and to regulate critical steps in early embryonic
development, as well as in malignant transformation (31). We show that
these compounds potently block HGF-stimulated cell motility, matrix
invasion, and branching morphogenesis, but not HGF-stimulated
mitogenesis, with ED50 values of 1-30 nM. The anti-motility and anti-invasion effects were characterized using four
different assay systems, with no evidence of toxicity, or loss of
contractility required for cellular functions other than locomotion.
The active compounds did not alter HGF-stimulated c-Met tyrosine kinase
activation, and their potency for inhibiting HGF-stimulated motility
correlated well with their affinity for binding to Grb2 in
vitro. Overall, our results suggest that c-Met-Grb2 interaction
contributes primarily to the motogenic and morphogenic cellular
responses to HGF, and that these compounds may have therapeutic application as anti-metastatic drugs in tumors where the HGF signaling pathway is active.
Materials--
Human HGF protein and the cDNA for human
c-Met in the pMOG vector were gifts from Dr. G. Vande Woude. The
truncated HGF isoform HGF/NK1 was produced in a bacterial expression
system, purified, and refolded as previously described (32). HGF/NK1
produced by this method stimulates all of the major biological
responses of full-length HGF (33). The Grb2 SH2 domain antagonists
designated 1-4 (Fig. 1) were synthesized and purified as described
(30, 34).
Cultured Cell Lines and cDNA Transfections--
The human
mammary epithelial cell line 184B5 (35) was maintained in RPMI 1640 + 10% fetal bovine serum (FBS) and 5 ng/ml epidermal growth factor
(Becton Dickinson). Balb/MK keratinocytes (35) were maintained in low
calcium Eagle's minimal essential medium + 10% dialyzed FBS
and 5 ng/ml mouse epidermal growth factor (R&D Systems). The human
gastric carcinoma cell line Okajima (36) was maintained in RPMI + 15%
FBS. The murine interleukin-3-dependent cell line 32D (37)
was cultured in RPMI 1640 + 15% FBS and 5% WEHI-3B conditioned
medium. 32D/c-Met cells were generated by co-transfection of 32D cells
with pMOG/c-Met and pCEV27 encoding neomycin-resistance as described
(37). The human leiomyosarcoma cell line SK-LMS-1 and Madin-Darby
canine kidney (MDCK) cells were maintained in DMEM + 10% FBS. TAC-2
(38), a normal mammary gland epithelial cell line, was cultured in high
glucose DMEM (Life Technologies, Inc.) supplemented with 10% FBS.
Immunoprecipitation and Immunoblot Analysis--
c-Met
autophosphorylation was analyzed by immunoprecipitation and
immunoblotting as described (32). c-Met-Grb2 interaction was analyzed
by co-immunoprecipitation of c-Met with agarose-conjugated antibodies
against Grb2 (Santa Cruz Biotechnology), followed by immunoblot
detection using antibodies against c-Met (Santa Cruz Biotechnology),
phosphotyrosine (Tyr(P); Upstate Biotechnology), and Grb2 (Upstate
Biotechnology). Briefly, intact cells were serum-deprived for 16 h
in the presence or absence of Grb2 antagonists. Cells were then growth
factor-treated for 10 min as indicated, and lysed in cold buffer
containing non-ionic detergents and protease and phosphatase
inhibitors. After immunoprecipitation for 2 h on ice, immunocomplexes were washed, eluted with SDS sample buffer, resolved by
SDS-polyacrylamide gel electrophoresis, transferred to Immobilon P
(Millipore), and detected by ECL (Amersham Pharmacia Biotech). HGF/NK1-stimulated PI3K activation in intact cells after 16 h of
incubation in the presence or absence of Grb2 antagonists was analyzed
by co-immunoprecipitation of the p85 subunit of PI3K with
agarose-conjugated anti-Tyr(P), followed by immunoblot detection using
antibodies against PI3K (Upstate Biotechnology). Cells were treated
similarly to assess Akt and MAP kinase activation, except that
SDS-polyacrylamide gel electrophoresis-resolved whole cell lysates were
immunoblotted using anti-phospho-Akt and anti-Akt, or anti-phospho-MAP
kinase (New England Biolabs), respectively.
Mitogenicity Assays--
Mitogenic assays were performed using
184B5 cells or 32D/c-Met cells as described (35, 37). Briefly, cells
were serum-deprived for 24 h (184B5) or 4 h (32D/c-Met)
before the addition of growth factors and/or antagonists for 16 h
(184B5) or 36 h (32D/c-Met). Cells were incubated with
[3H]thymidine for 6 h, and DNA synthesis was
measured by liquid scintillation counting.
Cell Migration Assays--
The migration of Okajima, 184B5, and
SK-LMS-1 cells in modified Boyden chambers was measured using Biocoat
Cell Environment control inserts (8-µm pore size; Becton Dickinson).
Lower chambers contained DMEM + 0.1% bovine serum albumin, to which
HGF (50 ng/ml) and/or Grb2 inhibitors were added. Cells were
trypsinized, washed in DMEM + 0.1% bovine serum albumin, added to
upper chambers (2 × 105 cells/ml) with growth factors
and/or inhibitors, and incubated for 16 h at 37 °C. Cells on
the upper surface of each filter were removed with a cotton swab,
whereas cells that had traversed to the bottom surface of the filter
were fixed and stained using Diff-Quik (Dade Diagnostics), and counted
using brightfield microscopy. Mean values from 10 randomly selected
unit areas were calculated for each of triplicate wells. The ratio of
growth factor-treated to control migrating cells is designated on the
y axis as "migration (-fold increase)." 32D/c-Met cell
migration was assayed using a modified Boyden chamber with 5-µm pore
size Nucleopore filters (Corning). HGF/NK1 (300 ng/ml) and/or Grb2
inhibitors were added to both chambers, and cells were applied to the
upper chamber at a final density of 2 × 106 cells/ml.
After incubation for 8 h at 37 °C, cells that migrated to the
lower chamber were counted with an automated cell counter (Coulter,
Inc.), and migration was expressed as described for Okajima cells.
Extracellular Matrix Invasion Assays--
SK-LMS-1 cell invasion
was analyzed using Matrigel Invasion Chambers (Becton Dickinson) and
quantitated essentially as described for Okajima cell migration across
uncoated membranes. Briefly, SK-LMS-1 cells were seeded at 1 × 104 cells/chamber in DMEM + 5% FBS. Chambers were placed
into 24-well culture plates containing DMEM + 5% FBS alone, or with
HGF (10 ng/ml) and/or compounds 1-4 as indicated for 40 h at
37 °C. The ratio of growth factor-treated to control invading cells
is designated on the y axis as "invasion (-fold increase)."
MDCK cell invasion into three-dimensional collagen gels was
analyzed as described (39). Briefly, type I collagen (1.5 mg/ml; Cohesion Technologies) was mixed with 10× minimal essential medium and
sodium bicarbonate (11.76 mg/ml) at a ratio of 8:1:1 (v/v/v) on ice,
and 0.4-ml aliquots were dispensed into 16-mm tissue culture wells
and allowed to gel at 37 °C for 20 min. Cells were seeded onto gels
(1 × 104 cells/well) in 0.4 ml of growth medium
containing HGF and/or compounds 1 or 4 as indicated. After 5 days,
cells were fixed in situ, and cells that had invaded the gel
below the surface monolayer in 10 randomly selected fields (1 × 1.4 mm) were counted microscopically using a 20× phase contrast
objective. Depth of cellular invasion into the collagen gel was
quantitated in the same 10 fields per treatment group using a
calibrated fine focusing micrometer. Values shown in Table I are the
mean number of invading cells/field or mean invasion depth/cell in
micrometers ± S.E.
MDCK Cell Scatter Assays--
MDCK cell scatter, observed as the
dispersion of single cells from tightly grouped colonies, was assayed
as described (32). Briefly, MDCK cells were seeded into 24-well plates
(2 × 10 4 cells/well) in DMEM containing various
concentrations of inhibitors and/or HGF (30 ng/ml). Cells were
incubated for 16 h at 37 °C. The scatter of fixed and stained
cells was observed by brightfield microscopy; images were captured at
12.5× total magnification.
Branching Morphogenesis Assays--
TAC-2 cells were suspended
in three-dimensional collagen gels as described (38) at 1 × 104 cells/ml in collagen and incubated in complete medium
containing HGF and/or Grb2 inhibitors as indicated. After 3 days, the
cultures were fixed with 2.5% glutaraldehyde in 0.1 M
cacodylate buffer, and brightfield microscopic images of three or more
randomly selected fields per experimental condition were digitally
recorded in each of three separate experiments. The total length of the
tubular structures (cord length) in each colony present in each optical field was measured using IPLab software (Scanalytics, Inc.). Cord length was considered "0" for spheroid colonies and for elongated colonies with length to diameter ratios less than 2. The mean values
for each experimental condition were compared with controls using
Student's unpaired t test.
Grb2 Antagonists Inhibit Grb2/c-Met Interaction in Intact
Cells--
The structures of the Grb-2 SH2 domain binding antagonists
used in this study are shown in Fig. 1.
The affinity of these compounds for binding to Grb2 SH2 domains
in vitro have been described (30, 34). Each compound
contains a common backbone structure corresponding to the invariant
Grb2 SH2 domain binding motif pY-X-N, with distinct modifications to the Tyr(P)-mimetic moiety that render each resistant to phosphatases, but maintain the net charge (
To rule out the possibility that the Grb2 antagonists might interfere
with HGF binding or receptor activation, we examined HGF-stimulated
c-Met autophosphorylation in intact Okajima cells in the presence and
absence of various concentrations of compound 1 (Fig.
2). As shown in panel
A, HGF/NK1-stimulated tyrosine phosphorylation of the
145-kDa c-Met
Direct evidence that these compounds blocked c-Met-Grb2 interaction was
obtained from co-immunoprecipitation/immunoblot analysis (Fig.
2B). Intact Okajima cells were treated with Grb2 inhibitors for 16 h, then briefly stimulated with HGF/NK1 before lysis,
immunoprecipitation with anti-Grb2 antibodies, and immunoblotting with
anti-c-Met, anti-Tyr(P), or anti-Grb2 (Fig. 2B). Consistent
with previous studies describing the
phosphorylation-dependent binding of Grb2 to Tyr-1356 in
c-Met (14), HGF/NK1 stimulated the co-immunoprecipitation of the 145 kDa c-Met
Despite complete inhibition of Grb2-c-Met interaction by the active
compounds in intact cells, other SH2 domain-mediated interactions such
as HGF-stimulated PI3K activation were not affected (Fig. 2C). The ability of HGF/NK1 to stimulate
anti-Tyr(P)-immunoprecipitability of the PI3K p85 subunit, as well as
activation of the downstream effector Akt, was identical in 32D/c-Met
cells treated with compound 1 at 300 nM for 16 h and
control cells (Fig. 2C). Consistent with their known
dependence on PI3K, HGF-stimulated activation of the MAP kinases ERK1
and ERK2 was also completely unaffected by compound 2 after incubation
of intact 32D/c-Met cells for 16 h with antagonist concentrations
as high as 1 µM (Fig. 2D). Together, our data
demonstrate that these antagonists act selectively on Grb2 SH2 domain
binding interactions in vivo at concentrations more than
100-fold over biologically effective doses.
To characterize the mechanism by which the Grb2 SH2 domain antagonists
entered intact cells, we analyzed HGF/NK1-stimulated Grb2-c-Met
interaction by co-immunoprecipitation following treatment of cells with
compound 1 for varying time periods (Fig. 2E). Very short
(10 min) exposure of cells to 300 nM compound 1 had no
effect on Grb2-c-Met interaction (data not shown). With increasing time of exposure to this treatment, Grb2-c-Met association diminished gradually over 6 h; association was inhibited by more than 50% after 2-4 h (Fig. 2E). Similar experiments using 10-fold
lower concentrations of compound 1 also revealed a gradual increase in
inhibition, reaching 50% inhibition after 8 h (data not shown). Although these experiments are not definitive, the simple time- and
concentration-dependent inhibition of Grb2-c-Met interaction observed is consistent with passive diffusion of these compounds across
the plasma membrane.
Grb2 Antagonists Fail to Block HGF-stimulated
Mitogenesis--
Three well established cultured cell systems
representing hematopoietic and epithelial HGF targets were used to
evaluate the effects of the Grb2 SH2 domain antagonists on
HGF-stimulated DNA synthesis: 32D/c-Met cells (37), 184B5 human mammary
epithelial cells (35), and Balb/MK mouse keratinocytes (35). Although substantial inhibition of Grb2-c-Met interaction was observed with as
little as 30 nM compound 1, none of the compounds blocked HGF or HGF/NK1-stimulated DNA synthesis in 32D/c-Met cells or either
epithelial cell line at concentrations up to 300 nM (data not shown). When tested over a much broader concentration range of
1-100 µM, compound 2 had little significant effect on
32D/c-Met cells, but at 100 µM, 50% inhibition of DNA
synthesis was observed in 184B5 mammary epithelial cells, and 20%
inhibition in Balb/MK keratinocytes. Results with compounds 1 and 3 were similar to those observed for compound 2. Even at doses in the
micromolar range, no adverse effects of the compounds on cell
morphology or proliferation rate were observed for up to 3 days.
Compounds 1-3 bind to the Grb2 SH2 domain with affinities in the low
nanomolar range in vitro, whereas compound 4 binds with at
least 100-fold lower affinity (30, 34). The results of immunoprecipitation and immunoblot analyses demonstrated that, within
16 h, effective concentrations of these compounds gain access to
the cytosol and bind selectively to intracellular Grb2 protein. In
light of this, our mitogenicity studies suggest that SH2-domain-mediated Grb2-c-Met interaction is not crucial for HGF-stimulated DNA synthesis in epithelial or hematopoietic HGF target
cells. The modest inhibitory effects of high doses of these compounds
on 184B5 cells and Balb/MK keratinocytes may reflect a limited
dependence of mitogenic signaling on Grb2-c-Met interaction in these
cell types. Alternatively, it may have resulted from the less selective
antagonism of SH2 domain-mediated interactions other than those of Grb2
that might be anticipated at doses 1000-10,000-fold above their
affinity for the Grb2 SH2 domain.
The role of SH2-domain-mediated c-Met-Grb2 interaction in
HGF-stimulated mitogenesis is controversial. Our conclusions differ from certain prior studies in which mutation of c-Met Tyr-1356, or
neighboring residues was used to assess HGF signaling through Grb2 (13,
40). It is important to note that several downstream effectors of HGF
signaling interact with c-Met in the region of Tyr-1356, and thus
mutation of this site may block c-Met interaction with effectors other
than Grb2 that are required for mitogenesis. Moreover, different
oncogenic isoforms of c-Met show varying dependence on Grb2 for
transforming activity (41), which is often taken as an index of
mitogenic pathway activation. In particular, Tpr-Met has been used
extensively to assess the role of Grb2 in HGF mitogenic signaling, and
shows the greatest dependence on Grb2 for its transforming activity
(41). The different subcellular localizations of Trp-Met and c-Met, as
well as other possible differences such as biological half-life, could
contribute to differences in their relative dependence on Grb2 for
mitogenic signaling. Our observations of unaltered HGF-stimulated PI3K
and MAP kinase activation in the presence of the Grb2 SH2 domain
antagonists are consistent with the well established dependence of HGF
mitogenic signaling on these intracellular effectors. Clearly the Grb2
SH2 domain antagonists described here afford a valuable independent
approach for further investigation of the role of Grb2 in growth factor
and cytokine signaling generally.
Grb2 Antagonists Inhibit Cell Motility, Matrix Invasion, and
Tubulogenesis--
The effect of the various Grb2 SH2 domain
antagonists on the migration of Okajima human gastric carcinoma cells
is shown in Fig. 4A. Grb2 SH2 domain antagonists 1-3 each
reduced HGF-stimulated Okajima cell migration in a
dose-dependent manner, with approximate ED50
values in the range of 1-10 nM (Fig. 4A). In
contrast, compound 4 was as much as 100-fold less potent in blocking
ligand-stimulated cell migration, consistent with its more than
100-fold lower binding affinity for Grb2 in vitro (Fig.
3A). The active compounds were equally effective in blocking HGF-stimulated migration by human mammary
184B5 cells, as well as Balb/MK keratinocytes (data not shown).
Similar to the effects of the active Grb2 SH2 domain antagonists on the
HGF-stimulated migration of epithelial cells, potent blockade of
HGF/NK1-stimulated 32D/c-Met cell migration was also observed (Fig.
3B). As reported previously, HGF/NK1 stimulated cell
migration in this system almost 20-fold over untreated control cells
(Ref. 37 and Fig. 3B). Compounds 1-3 each reduced
HGF/NK1-stimulated cell migration in a dose-dependent
manner with ED50 values in the 1-10 nM range,
whereas compound 4 was far less potent in blocking 32D/c-Met cell
migration (Fig. 3B). Interestingly, we noted that the
various basal migration levels observed among the different cell types
analyzed were not suppressed by the active SH2 domain antagonists,
i.e. only HGF-stimulated cell migration was blocked by these
compounds. This suggests that steady-state levels of cell motility are
independent of SH2 domain-mediated Grb2 interactions, and that the
compounds studied here act selectively on the link between c-Met and
the locomotive machinery.
Consistent with their effects on HGF and HGF/NK1-stimulated cell
migration, compounds 1-3 potently inhibited invasion through Matrigel
by SK-LMS-1 cells (Fig. 3C). This highly invasive
leiomyosarcoma cell line exhibits a complex response to HGF that
includes increased protease production (42), decreased tissue inhibitor
of metalloproteinase-3 expression (43), and increased motility, and has
been exploited as a model of HGF-stimulated metastasis in
vivo (42). Compounds 1- 3 displayed ED50 values in the
range of 10-30 nM, whereas compound 4 was only modestly
effective at 300 nM (Fig. 3C). The 3-10-fold higher doses of compounds 1-3 needed to effectively block matrix invasion compared with cell migration alone suggest that certain aspects of matrix invasion may be less Grb2-dependent than
cell motility per se. Nonetheless, the overall potency of
compounds 1-3 in this cultured cell model system warrants further
evaluation of their potential use as anti-metastatic drugs in
vivo.
HGF exerts a unique and potent effect on the morphology, dispersion,
and movement of MDCK epithelial cells, an effect known as "scatter"
(Ref. 17; reviewed in Ref. 4). During this process compact cell
colonies initially spread centrifugally; the subsequent disruption of
intercellular junctions allows membrane ruffling, lamellipodia
extension, and increased cell motility (17). As shown in Fig.
4, HGF-stimulated MDCK cell scatter was
blocked by the Grb2 SH2 domain antagonists 1-3 at 10 nM
each, whereas compound 4 had no detectable effect at this
concentration. Although the active compounds did not appear to block
the initial HGF-stimulated spreading of MDCK cells, they appeared to
dramatically reduce the number of single cells, i.e. those
assumed to have the highest level of motility (Fig. 4). The ability of
MDCK cells to invade three-dimensional collagen matrices, a
prerequisite for HGF-stimulated branching morphogenesis, was also
assessed in the presence of compounds 1 and 4 (Table
I). After 5 days in culture in the
absence of HGF, MDCK cells remain as a monolayer on the surface of the collagen gel, but HGF stimulates a high proportion of these cells to
invade the gel (35-µm mean depth of invasion; Table I), as reported
previously (39). Compound 1 (100 nM) reduced both the number of invading cells, as well as the mean depth of invasion per
cell, by at least 50%, whereas compound 4 had no significant effects
(Table I). MDCK cell viability throughout the 5-day culture period was
unchanged in the absence or presence of the Grb2 SH2 domain
antagonists.
In a variety of epithelial and endothelial HGF target cells, increased
migration and extracellular matrix invasion also contribute to
HGF-driven branching (or tubular) morphogenesis (reviewed in Ref. 4).
To determine whether Grb2 SH2 domain antagonists might inhibit this
process, we used an in vitro model of ductal morphogenesis in which mammary gland-derived epithelial (TAC-2) cells grown within a
three-dimensional collagen gel are induced to form branching duct-like
structures by HGF (38). When grown in collagen gels for 3 days under
control conditions, TAC-2 cells formed small, irregular cell aggregates
(Fig. 5A, left
panel). Treatment for 3 days with HGF (20 ng/ml) resulted in
the formation of long, branching tubular structures (Fig.
5A, middle panel). In marked contrast,
co-addition of compound 1 (30 nM) and HGF to TAC-2 cultures blocked the elongation and branching of ductlike structures (Fig. 5A, right panel). Quantitative
analysis of tube formation revealed that, in the presence of compounds
1-3, HGF-induced elongation of epithelial tubes was significantly
inhibited (p < 0.0001) in a dose-dependent
manner, with submaximal inhibitory effects observed at 30 nM inhibitor, and maximal effects at 3 µM
(Fig. 5B and data not shown). Compound 4 had no significant
effect on tubulogenesis at these concentrations (Fig. 5B and
data not shown). Together, these data demonstrate that Grb2 SH2 domain
antagonists 1-3 potently block HGF-stimulated migration and matrix
invasion by a variety of cell types derived from both normal and tumor
tissues, as well as HGF-driven branching morphogenesis in a well
characterized mammary epithelial cell model.
Like its role in HGF-stimulated mitogenesis, the role of Grb2-c-Met
interaction in HGF-stimulated cell motility is controversial. Consistent with the findings reported here, loss of HGF-stimulated cell
motility has been associated with abrogation of Grb2-c-Met interaction
achieved through mutation of c-Met Tyr-1356 (14). However, loss of
motility has not been consistently associated with mutagenesis of the
c-Met Grb2 binding site (41, 44, 45). We also note that each of the
aforementioned reports were limited to a single cell line (14, 41, 44,
45). We observed that Grb2 SH2 domain antagonists inhibited
HGF-stimulated motility of Okajima, 184B5, SK-LMS-1, 32D/c-Met, and
MDCK cells at concentrations close to their affinity for binding Grb2
in vitro. This analysis clearly demonstrates that SH2
domain-mediated Grb2-c-Met interaction is required for HGF-stimulated
cell motility.
While this work was in progress, another study reported the inhibition
of growth factor stimulated cell motility by Grb2 SH2 domain
antagonists with structures similar, but not identical, to those used
here (46). The compounds characterized in that study were biologically
active in the range of 10-100 µM (46); thus, the
compounds described here appear to be 1000-10,000-fold more potent
anti-motility agents than any Grb2 SH2 domain antagonists described to
date. Moreover, the antagonists described here do not require prodrug
derivatization for cellular bioavailability, which has been a major
obstacle to the potential use of Grb2 SH2 domain antagonists in
animals. Compounds 1 and 3 also have dicarboxylic acid moieties in
place of a phosphate group. These moieties confer resistance to
cellular phosphatases and are the most potent non-phosphorous containing Tyr(P) mimetics yet reported for the Grb2 SH2 domain (34).
The overall potency of these compounds in intact cells, and their
apparent lack of cytotoxicity, suggest that they maybe useful for the
treatment of certain cancers where the role of HGF in stimulating tumor
invasiveness and metastasis is well-established.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, Shc,
Gab1, and Grb2 (reviewed in Ref. 9). In epithelial cells, PI3K activity
is required for both HGF-stimulated scatter and mitogenesis (10, 11),
Gab1 is sufficient for tubulogenesis (12), and Grb2 binding appears to
be required for HGF-stimulated cell motility and branching
tubulogenesis (13-15). The small GTP-binding proteins Ras, Rho, and
Rac are required for HGF-stimulated cytoskeletal rearrangements and subsequent cell motility (16, 17).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2) of this moiety. Compound 4 also has these features, but differs from the others in that
it has greater than 100-fold lower affinity for binding to Grb2 SH2
domains in vitro (30). The similar charge and overall structure of compound 4 to compounds 1-3 thus made it an excellent negative control throughout the course of the biological
characterization of the effects of these compounds on HGF
signaling.
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Fig. 1.
Chemical structures of Grb2 SH2 domain
binding compounds 1-4. Note that, despite its similar chemical
structure, compound 4 has at least 100-fold lower affinity for Grb2 SH2
domain binding in vitro (30), and was used as a negative
control throughout the biological characterization of these
compounds.
subunit was well above the level observed in control,
untreated cells (compare left and right
upper panels). The lower
panels confirm that equal amounts of c-Met protein were present in each lane (Fig. 2A). No differences in the level
of HGF/NK1-stimulated c-Met autophosphorylation were observed in cells
treated with up to 1 µM compound 1 (Fig. 2A).
Identical results were obtained for compound 2, as well as for both
compounds on HGF-stimulated c-Met activation in intact 32D/c-Met cells
(data not shown). Additional evidence that these compounds did not
block HGF-c-Met interaction or subsequent c-Met activation is provided in the analysis of HGF-stimulated mitogenesis described below.
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Fig. 2.
Immunoprecipitation and immunoblot analyses
of the effects of Grb2 SH2 antagonists on HGF/NK1 pathway activation
and c-Met-Grb2 interaction. A, serum-deprived Okajima
cells were pre-treated with various concentrations of compound 1 for
16 h, then stimulated briefly with HGF/NK1 (right
panels, NK1), or left untreated (left
panels, control). Upper panels show
the detection of tyrosine-phosphorylated c-Met, whereas the amount of
c-Met protein present in each lane is shown in the lower
panels. B, serum-deprived Okajima cells were
pre-treated with various concentrations of compound 1 for 16 h,
then stimulated briefly with HGF/NK1 (right
panels, NK1), or left untreated (left
panels, control). The amount of c-Met that was
co-immunoprecipitated with Grb2 is shown in the upper
panel; the middle panel shows
anti-Grb2-precipitated tyrosine-phosphorylated c-Met, whereas the
lower panel shows the level of Grb2 protein
present in all lanes. C, serum-deprived 32D-c-Met cells were
left untreated or pretreated with compound 1 for 16 h, then
stimulated briefly with HGF/NK1 (right two
lanes, NK1), or left untreated (left
two lanes, control). The amount of
anti-phosphotyrosine-precipitable PI3K p85 subunit is shown in the
upper panel. The middle
panel shows the amount of active Akt kinase present in whole
cell lysates by immunoblotting with anti-phospho-Akt, whereas the
bottom panel shows the level of Akt protein
present in all lanes of the middle panel only.
D, serum-deprived 32D-c-Met cells were left untreated or
pretreated with various concentrations of compound 2 for 16 h,
then stimulated briefly with HGF/NK1 or left untreated, as indicated.
The amount of active MAP kinase present in whole cell lysates is shown
by immunoblotting with anti-phospho-MAP kinase antibody. E,
serum-deprived 32D-c-Met cells were left untreated or pretreated with
compound 1 (300 nM) for various time periods, then
stimulated briefly with HGF/NK1 or left untreated, as indicated. The
amount of c-Met that was co-immunoprecipitated with anti-Grb2 is shown
in the upper panel, whereas the lower
panel shows the amount of Grb2 protein present in each
sample.
subunit with Grb2 (Fig. 2B). When cells were
pretreated for 16 h with 30 nM compound 1 prior to
HGF/NK1 stimulation, the amount of HGF receptor that was
co-immunoprecipitated with Grb2 was reduced by ~50% (Fig.
2B, right lanes). Thus, the effective
concentration of this compound for blocking c-Met/Grb2 interaction was
almost identical to its relative affinity for Grb2 SH2 domain binding
measured in vitro (34). At 300 nM compound 1, Grb2-c-Met interaction was completely abolished (Fig. 2B). The lower panel of Fig. 2B confirms
that equal amounts of Grb2 protein were present in each lane. Similar
results were obtained using compound 2 (data not shown).
View larger version (28K):
[in a new window]
Fig. 3.
Effects of Grb2 SH2 domain
antagonists 1-4 on the HGF-stimulated migration of Okajima cells
(A) or 32D/c-Met cells (B), and on
HGF-stimulated Matrigel invasion by SK-LMS-1 cells
(C). For panels A and
B, unfilled bars represent the
migration of cells in the absence of HGF (or HGF/NK1 for 32D/c-Met
cells), whereas shaded bars represent the
migration of cells in the presence of HGF (or HGF/NK1 for 32D/c-Met
cells). Inhibitor concentrations are indicated on the x axis
in nM. For panel C,
unfilled bars represent matrix invasion by
SK-LMS-1 cells in the absence of HGF, whereas shaded
bars represent matrix invasion by cells in the presence of
HGF. Results are representative of three or more experiments. In all
panels, values are expressed as the ratio of migrating or invading
cells in HGF-treated wells to control wells treated with inhibitor
alone at the indicated concentrations. Error bars
indicate S.E.; where no error bars are visible,
the error is too small to be shown.
View larger version (104K):
[in a new window]
Fig. 4.
Effects of Grb2 SH2 domain antagonists 1-4
on MDCK cell scatter. Photomicrographs show representative areas
from one of triplicate samples for each condition. Panels on
the left side show cells not treated with HGF,
whereas panels on the right show cells treated
with HGF at 30 nM (final concentration). Grb2 SH2 domain
antagonists (indicated by number or C (for
control) at the left of each set) were added at 10 nM final concentration. Results are representative of three
experiments.
MDCK cell invasion into collagen matrices
View larger version (88K):
[in a new window]
Fig. 5.
Effects of Grb2 SH2 domain antagonists on
HGF-stimulated branching tubulogenesis by TAC-2 cells.
A, TAC-2 cells were left untreated or pretreated with
compound 1 (30 nM) for 18 h, then harvested and
resuspended in collagen gels as described under "Experimental
Procedures." After 3 days, control cells formed small colonies of
irregular cell aggregates with short, poorly defined branching
structures (left panel), whereas cells incubated
with HGF (20 ng/ml) for 3 days (middle panel)
formed elongated tubular structures characterized previously (38).
Compound 1-pretreated cells maintained in compound 1 and HGF for 3 days
were similar to control cells in overall appearance (right
panel). Phase contrast images are shown (original
magnification, ×125). B, quantitative analysis of TAC-2
tubulogenesis. Cells pretreated for 18 h with various
concentrations of compounds 2, 3, or 4 as indicated on the x
axis (nM) were suspended in collagen gels and further
incubated with the same concentrations of compounds and in the presence
(filled bars) or absence (open
bars) of HGF (20 ng/ml). After 3 days the cultures were
fixed and colony cord length was measured as described under
"Experimental Procedures." Values on the y axis are mean
cord length per field ± S.E. Values for all concentrations of
compounds 2 and 3 are significantly different (p < 0.001) from cultures treated with HGF alone; three or more experiments
were performed for all conditions.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Jeffrey Rubin and Angelina Felici for helpful discussions, and Nelson Ellmore for technical assistance.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Present address: EntreMed, Inc., 9640 Medical Center Dr., Rockville, MD 20850. Tel.: 301-517-5547; Fax: 301-217-9594; E-mail: donb@entremed.com.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M010202200
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ABBREVIATIONS |
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The abbreviations used are: HGF, hepatocyte growth factor; SH2, Src homology 2; PI3K, phosphoinositide 3-kinase; FBS, fetal bovine serum; MAP, mitogen-activated protein; MDCK, Madin-Darby canine kidney; DMEM, Dulbecco's modified Eagle's medium.
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