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INTRODUCTION |
Transforming growth factor-
(TGF-
)1 is a pleiotropic
polypeptide growth factor that is part of a superfamily of structurally related ligands that includes the TGF-
s, activins, and bone
morphogenetic proteins (BMPs) (1). TGF-
ligands play a critical role
in modulating cell growth, differentiation, plasticity, and migration. They elicit their biological effects by binding to a heteromeric complex of transmembrane serine/threonine kinases, the type I and type
II receptors. TGF-
ligands can also bind to a transmembrane proteoglycan referred to as the type III receptor, which is thought to
present ligand to the signaling type I and type II receptors. Following
ligand binding to the type II receptor, the type I receptor is
recruited to the complex. This allows the type II receptor, which is a
constitutively active kinase, to transphosphorylate and thereby
activate the type I receptor (2). Multiple pathways have now been
implicated in mediating TGF-
effects downstream of these receptors.
These include the extracellular signal-regulated kinase (ERK) (3, 4),
c-Jun NH2-terminal kinase (JNK) (5-7), p38
mitogen-activated protein kinase (MAPK) (8, 9), and phosphatidylinositol 3-kinase (PI3K) pathways (10, 11). Several small
GTPases can also be activated by TGF-
(12) and are involved in the
activation of many of the above-mentioned signaling pathways. However,
the Smad pathway was the first signaling pathway identified to mediate
TGF-
effects and remains the best characterized (reviewed in Ref.
1).
Signal transduction through the Smad pathway involves phosphorylation
of a set of intracellular signaling proteins termed receptor-regulated
Smads (R-Smads) by the activated type I receptor. Once phosphorylated,
R-Smads can associate with a common mediator Smad, Smad4, translocate
to the nucleus, and regulate gene transcription. In addition to the
R-Smads and the common mediator Smad, Smad4, there is a distinct,
structurally related class of antagonistic Smads, Smad6 and Smad7,
which inhibit TGF-
family signals. Smad6 preferentially inhibits BMP
signaling by either competing with Smad4 for binding to R-Smads (13) or
interfering with BMP receptor-mediated phosphorylation of Smads (14).
Smad7 has been reported to inhibit both TGF-
and BMP signaling by
binding to activated type I receptors and interfering with their
ability to phosphorylate R-Smads (15, 16).
Although TGF-
1 was originally identified for its ability to cause
reversible phenotypic transformation and anchorage-independent growth
of fibroblasts (17, 18), TGF-
can act as both a tumor suppressor and
a tumor promoter (19, 20). TGF-
elicits most of its tumor suppressor
activity by potently inhibiting the proliferation of most epithelial
cells. It is thought that escape from the growth inhibitory effects of
TGF-
through dysregulated expression or mutational inactivation of
various components of the TGF-
signaling pathway can contribute to
tumorigenesis (21-23). In addition, there is increasing evidence that
after cells lose their sensitivity to TGF-
-mediated growth
inhibition, autocrine TGF-
signaling may promote tumorigenesis. The
importance of autocrine TGF-
signaling in tumor progression has been
highlighted by several studies that have shown that expression of a
dominant-negative type II TGF-
receptor (dnT
RII) in various tumor
cells can prevent the conversion of cells from an epithelial to an
invasive mesenchymal phenotype, delay tumor growth, and reduce
metastases (24-27). These data suggest that TGF-
can act directly
on tumor cells to promote tumor maintenance and progression. In
addition to promoting epithelial to mesenchymal transformation of tumor
cells, TGF-
can stimulate the motility of many cell types in
vitro, suggesting that TGF-
production in vivo may
enhance migration of tumor cells and thus contribute to tumor
invasiveness and metastases. There is also evidence that TGF-
can
increase cellular motility without affecting proliferation, suggesting
that the effects on motility and proliferation may occur via different
biochemical pathways (28).
To understand the molecular mechanisms by which autocrine TGF-
may
selectively contribute to tumor cell motility, we have generated
MDA-MB-231 breast cancer cells stably expressing dnT
RII. MDA-MB-231
cells express TGF-
receptors (29), secrete TGF-
(30), and,
although they are resistant to the growth inhibitory effects of TGF-
(29), can respond to TGF-
with an increase in spreading (31) and
invasiveness (32). In addition, there is evidence that blocking TGF-
signaling by administration of a neutralizing TGF-
antibody can
inhibit MDA-MB-231 cell tumorigenicity and metastases in nude mice
(33). In this paper we show that expression of dnT
RII in MDA-MB-231
cells impairs their basal migratory potential. This impairment in
motility can be restored by expression of a constitutively active type
I TGF-
receptor (ALK5TD) but not by overexpression of
Smad2/4 or Smad3/4. In addition, the levels of ALK5TD
expression sufficient to restore motility in the cells expressing dnT
RII are associated with an increase in phosphorylation of Akt and
ERK1/2, but not Smad2, suggesting that Smad signaling is dispensable
for autocrine TGF-
-mediated motility and that this response depends
on alternative signaling pathways activated by TGF-
.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Reagents--
The MDA-MB-231 and MDA-MB-468
breast cancer cell lines were purchased from the American Type Culture
Collection (Manassas, VA) and were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum
(FCS). The SW480.7 clone 15.13 (34) was a gift from Dr. Joan
Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY)
and was maintained in Dulbecco's modified Eagle's medium supplemented
with 10% FCS, 0.3 mg/ml Zeocin, and 0.7 mg/ml G418. TGF-
1 and BMP2
were obtained from R&D Systems (Minneapolis, MN). Peter ten Dijke (The
Netherlands Cancer Institute, Amsterdam, The Netherlands) graciously
provided the rabbit polyclonal sera directed against activin-like
receptor kinases (ALKs) (35). Antibodies against the hemagglutinin (HA)
epitope (catalog number sc-7392), the type II TGF-
receptor (catalog
number sc-220), Smad4 (catalog number sc-7966), and p38 MAPK (catalog
number sc-7972) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Antibodies to fibronectin (catalog number F14420) and Smad2/3 (catalog number S66220) were from Transduction Laboratories (San Diego, CA). The
C-terminal phospho-Smad2 antibody (catalog number 06-829) was obtained
from Upstate Biotechnology, Inc. (Lake Placid, NY). The
C-terminal phospho-Smad1 antibody (catalog number 9511) and the
phospho-p38 MAPK antibody (catalog number 9211S) were from Cell
Signaling Technology (Beverly, MA). Monoclonal antibodies to actin
(catalog number A-4700) and the FLAG epitope (catalog number F3165), as
well as the polyclonal antibody to
-catenin (catalog number C2081),
were obtained from Sigma. Phalloidin-Texas Red and Hoechst 3342 were
from Molecular Probes (Eugene, OR). LY294002, SB202190, and JNKiII were
purchased from Calbiochem. The MEK inhibitor, UO126, was purchased from
Promega (Madison, WI).
Generation of Stable Cell Lines--
To generate MDA-MB-231
cells stably expressing dnT
RII, we obtained a construct encoding a
kinase-inactive T
RII mutant in which the lysine at position 277 has
been mutated to arginine (pGABE-T
RII-K277R) (25) from Martin Oft
(UCSF, San Francisco, CA). Lysine 277 corresponds to an invariant
lysine found in the ATP-binding site of subdomain II in all protein
kinases, and even its substitution with arginine results in loss of
kinase activity (36). The pGABE vector is a modified version of the
commonly used retroviral vector pBABE in which the puromycin cassette
has been replaced by enhanced green fluorescent protein (GFP). In this
construct, T
RII-K277R is HA-tagged, and its expression is driven by
the viral long terminal repeat, whereas expression of GFP is driven by
the SV40 promoter. MDA-MB-231 cells were transfected with the control
pGABE vector or the pGABE-T
RII-K277R vector utilizing LipofectAMINE
reagent (Invitrogen) according to the manufacturer's instructions.
Following transfection, cells expressing GFP were sorted by flow
cytometry. Clones were then isolated by sorting individual cells from
the >95% positive GFP pool.
Affinity Labeling of Cells with 125I-TGF-
1 and
Immunoprecipitation of HA-tagged
T
RII-K277R--
125I-TGF-
1 was obtained from
PerkinElmer Life Sciences. Near confluent MDA-MB-231 cells, as well as
clones and pools stably expressing GFP alone (GABE) or GFP and
T
RII-K277R (dnT
RII) in 12-well plates, were washed three times
over 30 min with 500 µl of ice-cold 0.1% bovine serum albumin
dissolved in Dulbecco's phosphate-buffered saline (D-PBS)
containing Ca2+ and Mg2+. The cells were then
affinity labeled with 100 pM 125I-TGF-
1 as
described previously (37), with slight modifications. Briefly, after a
3-h incubation with 100 pM 125I-TGF-
1 at
4 °C, the cells were washed with 500 µl of ice-cold D-PBS, and the ligand-receptor complexes were cross-linked
with 400 µl of 1 mM bis(sulfosuccinimidyl)suberate
(BS3; Pierce) for 10 min on ice. The cross-linking reaction
was stopped with the addition of 100 µl of 500 mM
glycine. Cells were washed twice with 500 µl of D-PBS and
solubilized with 125 µl of 20 mM Tris buffer, pH 7.4, containing 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin, and 2 µg/ml soybean trypsin inhibitor. Solubilized material was centrifuged for 10 min at 4 °C
to pellet cell debris. The supernatants were transferred to one-fifth
volume of 5× electrophoresis sample buffer, boiled, and vortexed. All
samples were analyzed using 3-12% SDS-PAGE and visualized by
autoradiography. For immunoprecipitation experiments, the radiolabeled
cell lysate from a T75 flask was centrifuged at 5000 × g, and the supernatant was split into eight equal aliquots and incubated with antibodies directed against ALKs 1, 2, and 5, the
type II TGF-
receptor, or HA overnight at 4 °C. Aliquots of
radiolabeled cell lysates incubated with normal rabbit serum or no
antibody were used as controls.
Immunoblot Analysis--
Cells were washed twice with ice-cold
D-PBS and lysed with 50 mM Tris, 150 mM NaCl buffer containing 1% Nonidet P-40, 0.25% deoxycholate, 1 mM EDTA, 20 mM sodium fluoride,
1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin, and 2 µg/ml soybean trypsin
inhibitor. Protein content was quantitated utilizing the BCA
protein assay reagent (Pierce). Protein extracts were separated by
7.5% or 10% SDS-PAGE and transferred to nitrocellulose membranes at
100 volts for 2 h. Membranes were blocked with 5% nonfat dry milk
in TBS-T (20 mM Tris-HCl, pH 7.6, 150 mM NaCl,
0.1% Tween 20 (v/v)) for 1 h at room temperature and
incubated with primary antibodies diluted in TBS-T plus 2.5% nonfat
dry milk over night at 4 °C. The membranes were then washed four
times for 10 min with TBS-T, incubated with horseradish
peroxidase-conjugated secondary antibodies for 1 h at room
temperature, and rewashed four times for 10 min with TBS-T.
Immunoreactive bands were visualized by chemiluminescence (Pierce).
Immunofluorescence--
Cells grown on glass coverslips (22 × 22 mm) in 35-mm wells were washed twice with D-PBS,
fixed with 4% paraformaldehyde in D-PBS for 15 min,
permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 3%
nonfat dry milk in D-PBS for 60 min, all at room
temperature. Cells were then incubated with primary mouse monoclonal
antibodies diluted in 1% nonfat dry milk/D-PBS for 1 h at room temperature and washed three times with D-PBS, followed by incubation with a CY3-conjugated anti-mouse antibody in 1%
nonfat dry milk/PBS for an additional hour at room temperature. In some
experiments, cell nuclei were stained with 1 µg/ml Hoechst for 10 min
at room temperature. After three 10-min washes with D-PBS,
coverslips were mounted onto 25 × 75-mm microslides using AquaPolyMount (Polysciences Inc., Warrington, PA). Fluorescent images
were captured using a Princeton Instruments cooled CCD digital camera
from a Zeiss Axiophot upright microscope.
Transcription Reporter Assays--
Cells were transiently
transfected with 1 µg per 35-mm dish of the
Smad-dependent heterologous promoter reporter construct p(CAGA)12-Luciferase (38) provided by Dr. Jean-Michel
Gauthier (Laboratoire Glaxo Wellcome, Les Ulis Cedex, France) along
with 0.01 µg per 35-mm dish of pCMV-Renilla using FuGENE 6 reagent (Roche Molecular Biochemicals) according to the manufacturer's protocol. The following day, cells were split into 24-well plates, and
~45 h post-transfection, cells were either left unstimulated or were
stimulated with 40 pM TGF-
1 for 16-20 h. All cells were then washed with D-PBS and lysed. Firefly and Renilla
reniformis luciferase activities were measured using Promega's
dual luciferase reporter assay system according to the manufacturer's
protocol. Luciferase activity was normalized utilizing the ratio of
Firefly to R. reniformis luciferase activity and
presented as -fold induction. All assays were done in triplicate wells,
and each experiment was repeated at least twice.
Wound Closure and Transwell Motility Assays--
For wound
closure assays, confluent cell monolayers were wounded by manually
scraping the cells with a pipette tip. Following wounding, wound size
was verified with an ocular ruler to ensure that all wounds were the
same width. The cell culture medium was then replaced with fresh
medium, and wound closure was monitored by microscopy at various times.
Transwell motility assays were performed utilizing 5-µm pore, 6.5-mm
polycarbonate transwell filters (Corning Costar Corp., Cambridge, MA).
For these assays, single cell suspensions were seeded in serum-free
medium containing 0.1% bovine serum albumin onto the upper
surface of the filters and allowed to migrate toward various
concentrations of FCS. After a 16-20-h incubation period, cells on the
upper surface of the filter were wiped off with a cotton swab, and the
cells that had migrated to the underside of the filter were fixed,
stained with 0.5% crystal violet, and counted by brightfield
microscopy at ×200 in five random fields.
Adenoviral Expression of ALKs and Smads--
The adenoviral
construct encoding FLAG-tagged Smad4 (39) was obtained from Dr. Harold
Moses (Vanderbilt University, Nashville, TN). All other adenoviral
constructs encoding FLAG-tagged Smads or HA-tagged constitutively
active mutants of the TGF-
(ALK5T204D), activin
(ALK2Q207D), and BMP (ALK3Q233D) type I
receptors (40) were generously provided by Dr. Kohei Miyazono (Japanese
Foundation for Cancer Research, Tokyo, Japan). Stocks of recombinant
viruses for each of these constructs were generated in 293 cells and
titered utilizing the Takara assay (Takara Biomedicals, Tokyo, Japan).
Cells were then infected with these or a control
-galactosidase
adenovirus at a multiplicity of infection (m.o.i.) that resulted
in >90% cell infection (~15 plaque-forming units/cell or less). The
efficiency of infection was evaluated by in situ staining of
cells for
-galactosidase activity 48 h following infection.
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RESULTS |
T
RII-K277R Is Expressed in MDA-MB-231 Cells--
To abrogate
TGF-
signaling in MDA-MB-231 cells, an expression vector encoding
GFP and kinase-inactive T
RII was transfected into cells. Expression
of the kinase-inactive T
RII-K277R mutant was verified by affinity
labeling cell surface receptors with 125I-TGF-
1 (Fig.
1A). Because T
RII-K277R has
an intact extracellular domain, it can still bind TGF-
and should
therefore co-migrate with endogenous T
RII. Cell surface labeling of
parental cells resulted in the labeling of three proteins corresponding
to the endogenous type I, II, and III TGF-
receptors. There was
little or no change in the amount of receptor labeling observed in the control cells expressing GFP alone (GABE 15 Pool) compared with parental cells. However, in the pool expressing T
RII-K277R
(dnT
RII 15 Pool), there was a significant increase in the amount of
labeled type II receptor, suggesting that the exogenous receptor was
expressed. Individual clones obtained from each of these pools
expressing either GFP alone (G15-5, -6) or GFP and T
RII-K277R
(dn15-2, -3, -5, -11) displayed a similar pattern of labeling.

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Fig. 1.
T RII-K277R is
expressed in MDA-MB-231 cells. MDA-MB-231 parental cells, as well
as clones and pools stably expressing GFP alone (G15-5,
G15-6, and G15 Pool) or GFP and T RII-K277R
(dn15-2, dn15-3, dn15-5, and
dn15-11 clones and dn15 Pool), were affinity
labeled with 100 pM 125I-TGF- 1 and
cross-linked with BS3. Labeled ligand-receptor complexes
were resolved by SDS-PAGE using a 3-12% gradient gel and visualized
by autoradiography (A) or lysed and incubated with a mouse
monoclonal anti-HA antibody for immunoprecipitation of HA-tagged
T RII-K277R (B). Immunoprecipitates were resolved by
SDS-PAGE using a 7.5% polyacrylamide gel and visualized by
autoradiography. Affinity labeled but non-immunoprecipitated
(NIP) G15-5 cells were loaded as a reference
(lane 1).
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To confirm that this increase in the labeling of T
RII was indeed
because of expression of the HA-tagged T
RII-K277R, extracts from
affinity labeled cells were immunoprecipitated with an HA antibody. As
shown in Fig. 1B, the HA antibody immunoprecipitated a
labeled type II receptor in the pool and clones expressing
T
RII-K277R but not in the control pool or clones expressing GFP
alone, confirming transgene expression. The type I TGF-
receptor
appeared to co-immunoprecipitate with T
RII-K277R in these
experiments. This was confirmed in subsequent co-immunoprecipitation
experiments (see Fig. 2).

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Fig. 2.
T RII-K277R can
associate with T RI. MDA-MB-231 pools
expressing GFP alone (GABE 15 Pool; left panel)
or GFP and T RII-K277R (dnT RII 15 Pool; right
panel) were affinity labeled with 100 pM
125I-TGF- 1, cross-linked with BS3, lysed,
and incubated with normal rabbit serum (NRS), polyclonal
rabbit antisera directed against various type I TGF- superfamily
receptors (ALK1, ALK2, and ALK5), the
type II TGF- receptor (T RII), or HA as indicated.
Immunoprecipitates were resolved by SDS-PAGE using a 3-12%
polyacrylamide gel and visualized by autoradiography. Affinity labeled
but non-immunoprecipitated (NIP) cells were loaded as a
reference (lane 1).
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T
RII-K277R Is Functional in MDA-MB-231 Cells--
Having
ascertained that T
RII-K277R was expressed, we then examined whether
it was functional. Immunoprecipitation experiments revealed that when
affinity labeled cells expressing T
RII-K277R were precipitated with
an HA antibody, a labeled protein the size of a type I receptor
co-precipitated with T
RII-K277R (see Fig. 1B and Fig. 2,
right panel, lane 8). We confirmed that this was T
RI by precipitating similarly labeled cells with various TGF-
superfamily type I receptor antibodies, including ALK1, -2, and -5. Only the ALK5 (T
RI), but not the ALK1 or ALK2 antibodies, precipitated the cross-linked type I receptor (Fig. 2). Although the
T
RII antibody co-precipitated ALK5 efficiently, the ALK5 antibody
co-precipitated T
RII only weakly (see Fig. 2, lane 5, both panels). In the control GABE 15 Pool, the HA antibody
failed to precipitate any proteins, as expected. Immunoprecipitations with a T
RII antibody were carried out in both pools as a positive control, and both resulted in the co-precipitation of T
RI (Fig. 2,
lane 7, both panels). These data indicate that
T
RII-K277R associates with T
RI.
To determine whether T
RII-K277R prevented TGF-
signaling, we
examined its effect on the ability of T
RI to phosphorylate Smad2.
Immunoblot analysis of TGF-
1-treated cell lysates using a
phospho-specific Smad2 antibody revealed that although TGF-
1 could
induce phosphorylation of Smad2 in both GABE clones (G15-5 and
G15-6), its ability to do so in the T
RII-K277R clones (dn15-2, -3, -5, -11) was impaired (Fig.
3A). This impairment was not
because of a decrease in total Smad2 protein, as reprobing with an
antibody directed against total Smad2/3 did not reveal any significant change in protein levels. We next examined the effect of T
RII-K277R expression on TGF-
-induced Smad translocation to the nucleus by
immunofluorescence (Fig. 3B). In the GABE clone Smad2
staining was relatively diffuse, but upon TGF-
1 treatment for 60 min, Smad2 staining became concentrated in the nucleus. In contrast, in
the T
RII-K277R clones, there was little or no change in Smad2 staining following TGF-
1 treatment, suggesting impaired
TGF-
-mediated translocation of Smad2 to the nucleus. We then
examined the effect of T
RII-K277R on TGF-
1-induced transcription.
A reporter construct containing twelve Smad binding elements
repeated in tandem, p(CAGA)12-Luciferase, was transiently
transfected into the GABE and T
RII-K277R clones, along with
pCMV-Renilla. Normalized luciferase activity indicated that TGF-
1
could induce transcription of both reporter constructs in the GABE
clones, but its ability to do so in the T
RII-K277R clones was
impaired (Fig. 3C).

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Fig. 3.
Expression of
T RII-K277R impairs TGF-
signaling. A, near confluent clones expressing
GFP alone (G15-5 and G15-6) or GFP and T RII-K277R (dn15-2,
dn15-3, and dn15-11) were incubated overnight under serum-free
conditions, stimulated with 80 pM TGF- 1 for the times
indicated, washed, and lysed. Protein extracts (50 µg/lane) were separated by 10% SDS-PAGE followed by
immunoblot analysis for phospho-Smad2 (p-Smad2) and total
Smad2/3. B, cells were grown on glass coverslips for 48 h and serum-starved for 16 h followed by treatment with 40 pM TGF- 1 for 1 h. Cells were then prepared for
indirect immunofluorescence staining of Smad2. Nuclear localization of
Smad2 was confirmed by staining the same cells with Hoechst.
C, cells were transiently transfected with
p(CAGA)12-Luciferase along with pCMV-Renilla. The following
day, cells were split to six wells of a 24-well plate, treated with 40 pM TGF- 1 for 16 h, washed, and lysed. Firefly and
Renilla luciferase activities were measured using Promega's
dual luciferase reporter assay system. Fold induction of luciferase
activity (y axis) is based on the ratio of Firefly to
Renilla luciferase activities. Each data point
represents the mean ± S.D. of three wells. D, near
confluent pools expressing GFP alone (G15) or GFP and
T RII-K277R (dn15) were incubated overnight under
serum-free conditions, stimulated with 80 pM TGF- 1 for
the times indicated, and prepared for immunoblot analysis as in
A. The fibronectin blot (Fn) was probed with an
antibody directed against actin as a loading control, whereas the
phospho-Smad2 (p-Smad2) and phospho-p38 MAPK (p-p38
MAPK) blots were probed with antibodies directed against the
unphosphorylated forms of the respective proteins to verify equal
loading.
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To determine whether signaling pathways other than the Smad pathway
were also affected by expression of T
RII-K277R, we examined fibronectin expression, which has been reported to be induced by
TGF-
in a JNK-dependent but Smad4-independent manner
(6). We chose to perform these experiments in our pools as the results obtained in these cells are representative of those obtained in the
clones (compare phospho-Smad2 blots in Fig. 3, A and
D). Following TGF-
stimulation for 24 h, we observed
an increase in fibronectin expression in the GABE pool, but this
induction was decreased significantly in the pool expressing
T
RII-K277R, as was the basal level of fibronectin expression (Fig.
3D). We were unable to detect any induction of
phosphorylation of JNK in response to TGF-
in our GABE pools (data
not shown). However, we did observe an increase in phosphorylation of
p38 MAPK following TGF-
stimulation for 60 min, and this induction
of phosphorylation was slightly attenuated in the pool expressing
T
RII-K277R (Fig. 3D).
T
RII-K277R Impairs the Motility of MDA-MB-231 Cells--
Next
we examined the effect of T
RII-K277R expression on the motility of
MDA-MB-231 cells in a wound closure assay. In the GABE clones, cells
migrated into the wounded area and closed the wound within 24 h,
whereas in the T
RII-K277R clones the wound remained open at 24 h (Fig. 4A). This difference
in motility did not appear to be because of an effect on proliferation,
because when the experiment was performed in the presence of mitomycin C, a compound that inhibits cell division, the same results were obtained (data not shown). Thus, expression of T
RII-K277R in MDA-MB-231 cells appears to impair their motility, independent of
changes in proliferation. As an alternative measure of cell motility,
we also examined the effect of T
RII-K277R on the ability of cells to
migrate in a transwell assay system. We observed a 3- to 4-fold
reduction in the ability of cells expressing T
RII-K277R to migrate
toward FCS, as compared with control cells expressing GFP alone (Fig.
4B).

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Fig. 4.
Expression of
T RII-K277R impairs motility.
A, confluent cell monolayers of clones expressing GFP
alone (G15-5 and G15-6) or GFP and
T RII-K277R (dn15-2, dn15-3, and
dn15-11) were wounded with a pipette tip. Following
wounding, cell culture medium was replaced with fresh medium, and wound
closure was monitored by microscopy at the times indicated.
B, single cell suspensions of pools expressing GFP alone
(G15 Pool) or GFP and T RII-K277R (dn15 Pool)
in serum-free medium containing 0.1% bovine serum albumin were
seeded onto 5 µM polycarbonate transwell filters and
allowed to migrate toward increasing concentrations of FCS, as
indicated. After 20 h, cells on the underside of the filters were
fixed, stained, and counted. The results are represented quantitatively
in the bar graph below the representative filter
micrographs. Each data point represents the mean ± S.D. of two wells.
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The Impaired Motility of T
RII-K277R Cells Is TGF-
Type I
Receptor-specific--
Because the impaired motility of T
RII-K277R
cells was observed in the absence of exogenous TGF-
stimulation, we
wished to determine whether this impairment was TGF-
-specific. To do
so, we chose to restore TGF-
signaling in T
RII-K277R cells by
expressing a constitutively active mutant of T
RI. Mutation of
threonine 204 in ALK5 (T
RI) to aspartic acid leads to constitutive
activation of the type I receptor kinase, allowing it to induce signals
in the absence of ligands or type II receptors (41). Likewise, mutation
of corresponding threonine and glutamine residues in the activin (42)
and BMP (43) type I receptors to aspartic acid also leads to
constitutive activation of these kinases. To test for TGF-
specificity, cells expressing T
RII-K277R were infected with
adenoviruses encoding HA-tagged constitutively active mutants of the
TGF-
(ALK5TD), activin (ALK2QD), and BMP
(ALK3QD) type I receptors (40). Uninfected cells or cells
infected with a
-galactosidase adenovirus at the same m.o.i. were
used as controls. The efficiency of infection was >90% as evaluated by in situ staining of cells for
-galactosidase activity
48 h following infection (data not shown). At this time,
expression of the mutant type I receptors was confirmed by immunoblot
analysis utilizing an HA antibody (Fig.
5A), and their effect on
motility was assessed in wound closure assays. Motility was only
restored in cells expressing ALK5TD (Fig. 5C).
Although ALK2QD and ALK3QD were expressed and
functional, as evidenced by their ability to induce Smad1
phosphorylation (Fig. 5B), they failed to restore motility
in cells expressing T
RII-K277R (Fig. 5C). These results suggest that the impaired motility of T
RII-K277R cells is T
RI (ALK5)-specific.

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Fig. 5.
Expression of
ALK5TD/T RI restores motility in
T RII-K277R cells. Clone dn15-2 was
infected with adenoviruses encoding HA-tagged constitutively active
mutants of the activin (ALK2QD), BMP
(ALK3QD) or TGF-
(ALK5TD) type I receptors at an m.o.i. of 15. Uninfected cells and cells infected with a -galactosidase
( GAL) adenovirus at a similar m.o.i. were used as
controls. Approximately 48 h following infection, ALK expression
(A) and function (B) were verified by immunoblot
analysis utilizing an anti-HA or a phospho-specific Smad1 antibody, as
indicated. The blots were also probed with an actin antibody to verify
equal loading. The effect of ALK expression on wound closure was
monitored by microscopy at the times indicated (C).
|
|
Restoration of Smad Signaling Does Not Rescue the Impaired Motility
of T
RII-K277R Cells--
Although there is evidence that Smad
signaling is critical for the anti-proliferative effects mediated by
TGF-
(44, 45), it is unclear whether TGF-
-mediated motility
requires Smad signaling. We reasoned that if Smads are required for
TGF-
-mediated motility, blockade of Smad signaling with
dominant-negative Smad mutants or with the inhibitory Smad, Smad7,
should impair motility. However, expression of either dominant-negative
Smad4 or Smad7 in the MDA-MB-231 parental cells resulted in cell death
(data not shown). Therefore, it was not possible to address whether
TGF-
-mediated motility requires Smad signaling utilizing this
approach. Instead, we chose to overexpress the TGF-
R-Smads, Smad2
or Smad3, along with Smad4, in cells expressing T
RII-K277R to
determine whether reconstitution of Smad signaling could restore
motility. Cells were infected with adenoviruses encoding FLAG-tagged
Smad2 and Smad4 or FLAG-tagged Smad3 and Smad4, and exogenous Smad
expression was confirmed by immunoblot analysis utilizing an anti-FLAG
antibody (Fig. 6A). The
ability of Smad2/4 and Smad3/4 to activate Smad-dependent signaling was examined utilizing the Smad-dependent
transcription reporter construct, p(CAGA)12-Luciferase.
Expression of Smad3/4 resulted in a marked increase in basal
transcription (Fig. 6B). Stimulation with TGF-
1 did not
cause any further increase in transcription, suggesting that Smad
signaling was activated maximally. Despite this, Smad3/4 failed to
restore motility in the cells expressing T
RII-K277R (Fig.
6C, bottom panel) and had no effect on the
motility of control cells expressing GFP alone (Fig. 6C, top panel). Activation of basal transcription was not as
marked with Smad2/4, as expected, because Smad2 itself cannot bind DNA (46). However, despite nearly 100-fold induction of transcription following infection at the maximally tolerated m.o.i., this combination also failed to restore motility in the cells expressing T
RII-K277R (Fig. 6C, bottom panel) and had no effect on the
motility of control cells expressing GFP alone (Fig. 6C,
top panel). These results indicate that reconstitution of
Smad signaling alone is not sufficient to restore autocrine
TGF-
-mediated motility in cells expressing T
RII-K277R, nor is it
sufficient to enhance the motility of control MDA-MB-231 cells.

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Fig. 6.
Expression of Smad2/4 or Smad3/4 does not
restore motility in T RII-K277R cells.
Cells were infected with both FLAG-tagged Smad2 and Smad4
(S2/4) or FLAG-tagged Smad3 and Smad4 (S3/4)
encoding adenoviruses at an m.o.i. of 3 for Smad2/3 and an m.o.i. of 15 for Smad4. Uninfected cells (Uninf.) and cells infected with
a -galactosidase ( GAL) adenovirus were used as
controls. Approximately 48 h following infection, Smad expression
was verified by immunoblot analysis utilizing an anti-FLAG antibody
(A). The ability of Smads to restore TGF- signaling in
dn15-2 was evaluated in transcription reporter assays utilizing the
TGF- responsive transcription reporter
p(CAGA)12-Luciferase (B). The effect of Smad
expression on wound closure in the control G15 Pool (top
panel) and dn15-2 clone (bottom panel) was monitored
by microscopy at the times indicated (C).
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|
Re-expression of Smad4 in Smad4-defective Cancer Cells Does Not
Enhance Motility--
To determine whether Smads are required for
cancer cell migration, we examined whether activation of TGF-
signaling could promote motility in the absence of Smad signaling
utilizing Smad4 null MDA-MB-468 breast cancer cells (47). Smad4 and
ALK5TD were expressed, either alone or in combination, by
adenoviral transduction, and their effects on the motility of
MDA-MB-468 cells were examined in wound closure assays. Expression of
HA-tagged ALK5TD and FLAG-tagged Smad4 was confirmed by
immunoblot analysis (Fig. 7A),
and their ability to activate Smad-dependent signaling was examined in transcription reporter assays utilizing the
Smad-dependent p(CAGA)12-Luciferase reporter
construct (Fig. 7B). As expected, in the absence of Smad4
(uninfected,
-galactosidase, ALK5TD alone), TGF-
1 was
unable to stimulate transcription in these cells. However, upon
re-expression of Smad4 a marked increase in both TGF-
1-mediated and
ALK5TD-mediated transcription was observed, indicating that
both Smad4 and ALK5TD were indeed functional in these
cells. Despite this, neither Smad4 nor ALK5TD had any
effect on cell motility, whether they were expressed alone or in
combination (Fig. 7C). The fact that ALK5TD
could not promote motility, even when Smad4 was co-expressed with it,
suggests that MDA-MB-468 cells are not responsive to the pro-migratory
effects of TGF-
.

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Fig. 7.
Effect of ALK5TD on the motility
of MDA-MB-468 cells /+ Smad4. Cells were infected with
adenoviruses encoding FLAG-tagged Smad4 (S4), HA-tagged
ALK5TD (A5), or both (A5+S4) at an
m.o.i. of 15. Uninfected cells (Uninf.) and cells infected
with a -galactosidase ( GAL) adenovirus were used as
controls. Approximately 48 h following infection, exogenous Smad4
and ALK5TD expression was verified by immunoblot analysis
utilizing anti-FLAG and anti-HA antibodies, respectively. The blots
were also probed with an actin antibody to verify equal loading
(A). The ability of Smad4 and ALK5TD to activate
Smad-dependent signaling was evaluated in transcriptional
reporter assays utilizing the Smad-dependent
p(CAGA)12-Luciferase reporter construct (B), and
their effect on motility was examined in wound closure assays
(C).
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|
To determine whether TGF-
could induce migration in the absence of
Smad signaling in other cells, the Smad4 defective SW480.7 colorectal
cells, conditionally expressing Smad4 via an ecdysone-inducible system
(34), were utilized. Cells were stimulated with increasing concentrations of TGF-
1 in the absence or presence of 3 µM ponasterone to induce Smad4 expression. Smad4
expression in ponasterone-treated cells was confirmed by immunoblot
analysis (Fig. 8A), and its effect on TGF-
-mediated motility was examined in wound closure assays. Again, as in the MDA-MB-468 cells, these cells failed to
respond to TGF-
both in the absence and presence of Smad4 (Fig.
8B). Taken together, these data indicate that reconstitution of Smad signaling alone is not sufficient to promote migration of
cancer cells.

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Fig. 8.
Effect of exogenous
TGF- 1 stimulation on the motility of SW480.7
cells /+ Smad4. Cells in 6-well plates were either left
untreated (left panel) or treated with 3 µM
ponasterone (right panel) for 40 h to induce Smad4
expression. Cells were then wounded, washed, and incubated with
serum-free medium in the presence of 0, 4, 20, or 100 pM TGF- 1 for 24 h. Wound closure was monitored by
microscopy at the times indicated (B). At the conclusion of
the wound closure experiment, cells were lysed, and Smad4
expression was examined by immunoblot analysis utilizing a monoclonal
antibody directed against Smad4 (A). The blots were also
probed with an actin antibody to verify equal loading. Ponasterone was
maintained in the culture medium of selected wells throughout the
experiment.
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|
Different Signaling Pathways Require Different Thresholds of
TGF-
Activation--
To determine whether Smad signaling is
actually required in addition to other pathways activated by TGF-
to
promote migration, we examined what signaling pathways were activated
under conditions where motility was restored following
ALK5TD expression in cells expressing T
RII-K277R (see
Fig. 5C and Fig. 9B). For these experiments,
expression of ALK5TD was confirmed by HA immunoblot (Fig.
9A), and the activation status
of candidate signaling pathways was examined utilizing phospho-specific
antibodies. Under these conditions, we observed an increase in the
phosphorylation of Akt and ERK1/2, with little or no change in the
phosphorylation of JNK or p38 MAPK (Fig. 9A). Interestingly,
the levels of ALK5TD expression that were sufficient to
restore motility were not sufficient to induce phosphorylation of
Smad2. Additional experiments indicated that ~4-fold greater
ALK5TD expression was required for induction of Smad2
phosphorylation (Fig. 9C). At that m.o.i., the viral load
per se interfered with the ability of the cells to migrate.
These data indicate that different signaling pathways require different
thresholds of TGF-
activation, as do different biological effects
mediated by TGF-
(27), and suggest that TGF-
may promote motility
through mechanisms independent of Smad signaling, possibly involving
activation of the PI3K-Akt and/or MAPK pathways. Consistent with this,
the ability of TGF-
to promote migration was impaired in the
presence of the PI3K inhibitor, LY294002 (Fig.
10A), as well as in the
presence of JNK, MEK, and p38 MAPK pathway inhibitors (Fig.
10B).

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Fig. 9.
Different signaling pathways require
different thresholds of TGF- activation.
Clone dn15-2 was infected with an adenovirus encoding an HA-tagged
constitutively active mutant of the TGF- type I receptor
(ALK5TD) at m.o.i. values of 0, 5, 10, or 15, as
indicated. Approximately 48 h following infection, cells were
wounded, and the effect of ALK5TD expression on wound
closure was monitored by microscopy at the times indicated
(B). At the conclusion of the migration assay, cells were
lysed, ALK5TD expression was confirmed by HA immunoblot,
and the activation status of candidate signaling pathways was examined
utilizing phospho-specific antibodies, as indicated
(A). Actin was examined as a loading control. In
C, clone dn15-2 was re-infected with ALK5TD at
m.o.i. values of 0, 5, 15, 30, or 60, as indicated, and the activation
status of candidate signaling pathways was re-examined utilizing
phospho-specific antibodies as in A.
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Fig. 10.
TGF- -induced
motility requires activation of multiple signaling pathways.
Confluent MDA-MB-231 cell monolayers were wounded with a pipette tip.
Following wounding, cells were washed, the cell culture medium was
replaced with serum-free medium, and the cells were stimulated with 80 pM TGF- 1 in the absence or presence of the PI3K
inhibitor, LY294002 (A) or the p38 MAPK
(SB202190), MEK (UO126), and JNK
(JNKiII) inhibitors, as indicated (B). Wound
closure was monitored by microscopy at the times indicated.
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|
 |
DISCUSSION |
In this study, abrogation of autocrine TGF-
signaling in
MDA-MB-231 breast cancer cells resulted in an impairment in basal cell
migration, which could not be restored by reconstituting Smad
signaling, suggesting that Smad signaling alone is not sufficient for
autocrine TGF-
-mediated motility. Consistent with this,
reconstitution of Smad signaling in the Smad4-defective MDA-MB-468 and
SW480.7 cells did not promote migration. In addition, restoration of
migration following restoration of TGF-
signaling in cells
expressing T
RII-K277R was associated with an increase in
phosphorylation of Akt and ERK1/2 but not Smad2. These results indicate
that Smad signaling is dispensable for TGF-
-mediated motility and
that this response is instead mediated through alternative pathways
activated by TGF-
. In support of this, the ability of TGF-
to
promote migration was blocked in the presence of pharmacological
inhibitors of the PI3K, p38 MAPK, MEK, and JNK pathways.
Although Smads have been implicated as critical mediators of many
TGF-
responses (48-52), the role of Smads in cancer cell migration
has, to the best of our knowledge, not been reported. Previous studies
in non-transformed cells have generated conflicting data on the
requirement of Smad signaling for cell migration (50, 53-55). In one
study, expression of dominant-negative Smad3 in non-transformed murine
mammary cells had no effect on TGF-
-mediated motility even though it
blocked the anti-mitogenic effect of TGF-
(53). This suggests that
Smad3 is not required for this response or that residual Smad3
signaling, not blocked by expression of dominant-negative Smad3, is
sufficient to mediate motility. This would be consistent with the idea
that different biological responses require different thresholds of
TGF-
signaling (27). Thus, complete abrogation of Smad3 signaling
might be required to observe an impairment in TGF-
-mediated motility
whereas partial blockade of Smad function might be sufficient to block
the anti-proliferative effects of TGF-
.
Interestingly, expression of either dnSmad4 or antagonistic Smad7 in
MDA-MB-231 cells resulted in cell death. Although overexpression of
Smad7 has been reported to sensitize various cell types to cell death
(56), expression of dnSmad4 has not been associated with such a
response. There is, however, increasing evidence that TGF-
can
promote the survival of both transformed (7, 57, 58) and
non-transformed (59-61) cells. Whether Smad signaling is required for
these pro-survival effects of TGF-
is not known. In addition,
re-expression of Smad4 in Smad4-defective SW480 has been reported to
induce a more adhesive and flat phenotype (62). These results suggest
that blockade of Smad signaling could potentially lead to loss of
adhesion and result in anoikis. This could explain our inability to
express dnSmad4 and Smad7 in MDA-MB-231 cells.
Because we were unable to assess the requirement for Smads in autocrine
TGF-
-mediated motility by abrogating Smad signaling, we chose to
address this question by activating Smad signaling in cells expressing
T
RII-K277R. Having ascertained that the impaired motility of
T
RII-K277R cells was indeed T
RI-specific, we overexpressed the
TGF-
R-Smads, Smad2 or Smad3, each with Smad4, to determine whether
autocrine TGF-
-mediated motility was Smad-dependent. Despite their ability to activate Smad-dependent
transcription, neither Smad combination restored the impaired motility
of the T
RII-K277R cells. We (data not shown) and others (62) have observed an increase in cell spreading following Smad overexpression. It is tempting to speculate that this increased cell spreading may be
associated with increased adhesion, which interferes with cell
migration. This could potentially explain why restoration of Smad
signaling in T
RII-K277R cells failed to restore migration. These
data suggest that in breast cancer cells, autocrine TGF-
signaling
mediates motility in a Smad-independent manner or that alternative
pathways, in addition to the Smad signaling pathway, are required for
these effects.
To address this question, we examined what signaling pathways were
activated under conditions where motility was restored in T
RII-K277R
cells following expression of a constitutively active type I TGF-
receptor. In these experiments, we observed an increase in the
phosphorylation of Akt and ERK1/2 but not Smad2. These data further
imply that Smad signaling is not required for TGF-
-mediated
motility. Although expression of ALK5TD may induce a low
level of Smad phosphorylation, which cannot be detected by immunoblot
analysis, it is unlikely that Smads are required for autocrine
TGF-
-mediated motility as reconstitution of Smad signaling in both
MDA-MB-468 and SW480.7 cancer cell lines failed to promote motility,
and expression of both low and high levels of either Smad2/4 or Smad3/4
failed to rescue the impaired motility of MDA-MB-231 cells expressing
dnT
RII. Thus, alternative signaling pathways activated by TGF-
are more likely to be important for migration. Indeed, blockade of the
PI3K, p38 MAPK, MEK, and JNK pathways with pharmacological inhibitors
impaired TGF-
-stimulated migration. The fact that inhibitors of p38
MAPK and JNK interfered with TGF-
-induced migration even though
ALK5TD failed to alter their phosphorylation status
suggests that these signaling pathways, though not activated further by
TGF-
in our experimental system, are required for basal cell
migration. In agreement with this, we have indeed observed an
impairment in the basal migratory potential of these cells in the
presence of these inhibitors (data not shown).
The observation that different levels of ALK5TD expression
resulted in differential activation of downstream targets (Fig.
9C) indicates that different signaling pathways require
different thresholds of TGF-
activation. In agreement with this,
others have reported that expression of dnT
RII in NMuMG mammary
cells impairs TGF-
-mediated Smad-dependent inhibition of
proliferation but not TGF-
-mediated activation of p38 MAPK (9). In
addition, there is evidence that different biological responses
mediated by TGF-
also require different thresholds of TGF-
signaling. For example, expression of dnT
RII in squamous carcinoma
cells has been reported to block the growth inhibitory effects of
TGF-
but not its ability to induce EMT (26). Likewise,
expression of dnT
RII in 4T1 murine mammary cancer cells impairs
TGF-
-mediated transcription but fails to block motility (27).
Because TGF-
signaling was not completely abrogated in the squamous
and mammary cancer cells (26, 27), the molecular mechanisms by which
autocrine TGF-
may selectively contribute to tumor progression could
not be fully addressed in those studies. Because we have expressed T
RII-K277R at levels high enough to block both Smad and non-Smad pathways in MDA-MB-231 cells, the model we have generated should prove
useful in dissecting the signaling pathways required for the diverse
effects elicited by TGF-
in cancer.
Our data indicate that autocrine TGF-
-mediated motility of cancer
cells is Smad-independent. This implies that non-transformed cells and
transformed cells utilize different mechanisms to promote motility as
others have reported that Smad3 null monocytes and keratinocytes
exhibit significantly reduced migration to TGF-
1 in transwell
motility assays (54). Moreover, Smad3 appears to be required for
TGF-
-mediated monocyte chemotaxis in vivo, as mice
lacking the Smad3 gene display a blunted monocyte chemotactic response
following cutaneous wounding (54). Studies in Drosophila also suggest that Smads may be required for cell migration as mutations
in Mad, the Drosophila receptor-activated Smad, impair migration of the epidermis during dorsal closure (55). Finally, recent
studies in endothelial cells have indicated that TGF-
acting through
ALK1 stimulates migration in a Smad-dependent manner, whereas TGF-
acting through ALK5 inhibits cell migration in a Smad-dependent manner (50). Taken together, these studies
highlight the importance of Smads in TGF-
-regulated migration of
non-transformed cells.
Despite compelling evidence for the role of Smads in non-transformed
cell migration, a lack of requirement for Smad signaling in
TGF-
-mediated cancer cell migration is consistent with previous studies that have shown that TGF-
can increase cellular motility of
prostate cancer cells without affecting proliferation, suggesting that
the effects on motility and proliferation may occur via different biochemical pathways (28). Likewise, expression of Smad7 in pancreatic
cancer cells has been shown to abrogate the anti-proliferative effects
of TGF-
but enhance matrix-associated transcriptional responses,
highlighting a dissociation between the matrix and anti-proliferative
effects induced by TGF-
(63). If the biological effects of TGF-
that can contribute to tumor progression were Smad-independent, it
might be possible to selectively disrupt those pathways, while ensuring
that the tumor suppressive, Smad-dependent pathways are
maintained. The signaling pathways currently implicated in mediating
the various pro- and anti-tumorigenic effects of TGF-
indicate that
this may in fact be possible. For example, recent studies aimed at
identifying the mechanisms by which TGF-
1 elicits EMT in mammary
cells have indicated that the PI3K, RhoA, and p38 MAPK pathways are
involved in this process (8, 9, 11, 53). However, whether Smad
signaling, which has been implicated in both the anti-proliferative
(44, 45) and pro-apoptotic (64, 65) effects of TGF-
, is also
required for TGF-
-mediated EMT is unclear. In one study, adenoviral
expression of low levels of constitutively active ALK5 induced EMT only
if Smad2/4 or Smad3/4 were co-expressed (66). In contrast, other
investigators have reported that inhibition of Smad signaling either by
overexpression of Smad7 or dominant-negative Smad3 did not affect the
transdifferentiation, arguing against the involvement of Smads in EMT
(53). Because epithelial transdifferentiation to a mesenchymal
phenotype is often associated with acquisition of motile properties,
the mechanisms through which TGF-
mediates EMT may be similar to
those required for TGF-
-mediated motility. Indeed, the PI3K, RhoA,
and p38 MAPK signaling pathways, which are required for
TGF-
-mediated EMT, have also been implicated in TGF-
-mediated
motility (8, 11, 53). Likewise, we have observed that blockade of these
and other pathways interfere with TGF-
-induced motility (Fig. 10),
suggesting that multiple pathways cooperate to elicit this effect.
It will be of interest to determine whether Smad signaling is
required for other effects mediated by TGF-
, as a dissociation
between the pathways required for the tumor suppressive
versus the tumor promoting effects of TGF-
could lead to
opportunities to selectively inhibit the non-desirable effects of
TGF-
without compromising its tumor suppressive function.