From the Genetica, Dipartimento di Oncologia,
Biologia e Genetica, Università di Genova, 26 C. Europa and
§ Istituto per la Ricerca sul Cancro, Viale Benedetto XIV,
Genova 16132, Italy
Received for publication, July 25, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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Breast and prostatic carcinomas, melanoma, and
endothelial cell lines are chemoattracted by medium conditioned by
mature osteoblasts. The chemoattractant for endothelial cells was
identified with C3, carboxyl-terminal trimer of pro-collagen type I. We
report that C3 induces directional migration and proliferation, the
expression of tissue inhibitor of metalloproteinases-2,
pro-metalloproteinase-2 and -9, and their activation in MDA MB231
cells, without changing the expression of tissue inhibitor of
metalloproteinases-1 and of metalloproteinase-14. Antiserum against
metalloproteinase-2 or -9 or -14, tissue inhibitor of
metalloproteinases-1, or GM6001 inhibits the C3-induced migration.
Urokinase and its receptor are detected and unchanged upon exposure to
C3. The antibody against urokinase or addition of plasminogen activator
inhibitor inhibits migration. Blocking antibodies to integrins
Mature osteoblasts conditioned medium
(CM),1 collected when
sustained biosynthesis of collagen type I occurs during osteogenesis in vitro from differentiating cultures of rat, rabbit, human
osteoblasts, and conditionally transformed human osteoblast-like line
HFO, induces directional migration of endothelial and melanoma and breast and prostatic carcinoma cells but not of normal cells or of
transformed mesenchymal cells in the classical in vitro
double chamber Boyden assay (1-4).
The molecule chemoattractant for endothelial cells was purified from
rat osteoblast CM, and it identifies by sequence, immunoreactivity, and
molecular size with the trimer carboxyl-terminal of type I pro-collagen, C3 (4). C3 is a by-product of the processing of
pro-collagen by pro-collagenase C (or BMP-1), preliminary to the
formation of collagen fibrils (5).
We investigated whether MDA MB231 breast carcinoma cells, which are
stimulated by mature osteoblasts CM to directional migration, are
induced by C3 to activate migratory and proliferation responses. It is
recognized that the stroma of the tumors plays instructive and active
roles toward tumor cells growth and invasiveness and that
tumor-associated stromal fibroblasts undergo changes in the biosynthetic phenotype (6), assume the production of factors supportive
for the proliferation of initiated tumor cells (7-10), and undergo
precocious genotypic changes (11, 12).
We focused the study on a breast carcinoma-derived cell line because it
is known that stromal fibroblasts of breast tumors synthesize type I
collagen (6), and high expression of type I collagen in the stroma
correlates with worse prognosis and high invasiveness in
vivo (10). Molecules, produced and localized in the
microenvironment of the stroma shared by tumor and endothelial cells
and capable of inducing in both cell types directional migration and/or
cell proliferation and peri-cellular proteolysis, might behave as
promoters for the overall development of primary tumors and for the
establishment of metastasis. No such molecule has been yet described,
and C3 presents itself as a likely candidate for this role.
Tumor cells invasion and angiogenesis share mechanistic analogies and
the common feature of presenting high expression of metalloproteinases
(MMP) and/or serine proteinases, in particular the urokinase (uPA)
system, and/or their activation. In both tumor and endothelial cells
the enhancement of peri-cellular proteolysis and the acquisition of the
migratory phenotype can occur in response to various extracellular
matrix components or to factors stored within it, mediated through
engagement of integrins, growth factors receptors, and activation of
heterotrimeric G-proteins (13, 14). Increased expression and activation
of pro-MMPs play multiple roles during angiogenesis and tumor growth
and invasion as follows: modulation of tight cell-cell and cell-ECM
relationships, control of tissue homeostasis, proteolysis, release from
the ECM of growth factors, and of protein fragments with distinct
biological activities (15-17). MMPs regulate tumor cell growth, at
both primary and metastatic sites. Increased expression of pro-MMP-2
and -9 is associated to the metastatic behavior of most tumors,
including breast carcinoma, and in vitro the production of
active forms of MMPs is associated with the induction of migratory
behavior in cancer cells (18-22). The migration of mouse melanoma,
human breast, and prostatic carcinoma cell lines induced by CM from
mature rat and rabbit osteoblasts is accompanied by induction of MMPs
(2, 3). Tissue inhibitors of metalloproteinases (TIMP) -1 and -2 play a
regulatory role on proteolysis by MMPs (16-19, 23). Modulation of cell
adhesion and cell migration can depend on the direct interaction of
MMP-14 and MMP-2 with integrins and ECM components (24-26). The uPA
system determines the controlled proteolysis of ECM, regulated by a
complex network of feedback mechanisms and by the balancing in amount of proteinases (tPA and uPA), their inhibitors (PAIs), and the uPA
receptor (uPAR). It is involved in the activation of secreted and
ECM-associated growth factors, in cell adhesion, migration, cytoskeleton reorganization, in MMP-9 and -14 activation (27), and in
proliferation (28-29). The uPA system has an important and possibly
causal role in the development of the metastatic phenotype. Free uPA is
a prognostic marker for human breast malignancy (30-31), and the
expression of uPAR is associated with invasiveness in breast tumors and
is absent in normal breast tissue (32-36). Proteolytic and
non-proteolytic mechanisms have been implied for the effects of uPA on
cell migration. In prostatic carcinoma cells lines the uPA system was
shifted by the exposure to the CM collected from mature osteoblasts
toward a balance favoring proteolysis (3). The intrinsic basal mobility
in vitro of the breast carcinoma cell line MDA MB231 is
regulated by the level of uPA, via activation of phosphatidylinositol
3-kinase (PI3K) (37). Modulation of cell migration is dependent on the
direct interaction of uPAR with integrins and ECM components (28,
38).
Integrin receptors mediate multiple cell responses to the ECM and the
peri-cellular microenvironment, adhesion, migration, apoptosis, and
proliferation (39). Different integrins interact on the cell surface
with multiple transmembrane proteins and with membrane-associated
receptors as well as and depending on interactions with the ECM (31,
38, 40, 41). Expression and utilization of the integrin chains
In endothelial cells the directional migration induced by C3 requires
the function of In this study we investigated if purified C3 causes the induction of
directional migration and proliferation and/or apoptosis of human
breast carcinoma cell lines. We used inhibitors and antibodies to
investigate which proteinases and receptors and which elements of the
molecular machinery of cell signaling are involved in the induction of
the migratory process by C3.
Cell Cultures--
Rat tibial osteoblasts cultures (ROB) were
obtained as described (1) and expanded in Coon's-modified F-12
supplemented with 10% fetal calf serum (FCS, Seromed, Italy). Cultures
at 46-70 cumulative population doublings were utilized for the
preparation of conditioned medium in differentiation medium (100 µg/ml ascorbic acid and 10 mM Conditioned Media from Osteoblasts, Purification of
C3--
Conditioned media were collected from mature rat osteoblasts
in the second phase of osteogenesis (after 7-10 days of culture in
differentiation medium) in vitro, and C3 was purified as
described (4). The fractions obtained from the heparin-Sepharose column were tested in chemotaxis with endothelial and MDA MB231 cells.
Cell Proliferation, Mitotic and Apoptotic Indexes--
Cells
were plated in the presence of serum, and after allowing cell
attachment, the medium was removed, and cultures were rinsed with
serum-free medium (SFM) and maintained in SFM, with or without purified
C3 (13 µg/ml), or 10% FCS. In the presence of C3, cells loosened
their attachment to the plastic. We therefore separately analyzed at
each time point cells easily detaching and detached and those adhering
to the plastic for the numbers of mitosis and apoptosis. Because no
significant differences were detected in their frequency in the two
fractions, we report the sum of the two fractions. Mitotic and
apoptotic indexes were determined, after 48 or 72 h, in cells
fixed and stained in 50 µg/ml Hoechst for 20 min. At least 1000 cells/sample were counted in-blind by two observers, and standard error
was calculated. Experiments were performed in duplicate.
Chemotaxis Assay and Treatment with C3--
Migration assay was
performed in Boyden chambers as described (4). 12 × 104 cells were placed in the upper compartment of each
Boyden chamber, and unfractionated CM, purified C3 (13 µg/ml), or SFM
for negative control were placed in the lower compartment. In the study
of the effects on migration of antibodies against the C3 chains
In other experiments tumor cells grown to confluence on plastic were
challenged with C3 (13 µg/ml) in SFM for 6 h or cultured for the
same time in SFM, and the CM were collected. In 6 h attached cells
do not lose their attachment to the plastic. Cells lysates were
prepared in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS).
Antibodies and Inhibitors--
Anti-human antibodies utilized
for inhibition of migration were to integrin Zymograms--
Samples of CM were concentrated by cold ethanol
precipitation (46:100, v/v) for 1.5 h on ice, collected by
centrifugation, and resuspended in sample buffer for electrophoresis.
Protein equivalent amounts (by Lowry) from CM were loaded on
SDS-acrylamide gel. The gels for detection of uPAs were cast with 1%
non-fat dry milk and 10 µg/ml human plasminogen and run at 6 Western Blotting--
50 µg of proteins from cells lysed in
RIPA buffer were run after reduction on SDS-acrylamide gels. The
samples were electrotransferred to nitrocellulose, and the membranes
were saturated at room temperature for 1 h in TTBS (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.01% Tween 20), 5% bovine serum albumin. The membranes were incubated 16 h
in TTBS added with monoclonal R4 anti-uPAR, polyclonal anti-MMP-14, polyclonal anti-MMP-2, polyclonal anti-TIMP-1, or monoclonal
anti-TIMP-2 antibodies, followed by 1 h in TTBS with horseradish
peroxidase-secondary antibody, and the bands were visualized by ECL
chemiluminescence (Amersham Biosciences).
C3, the inducer of directional migration specific for endothelial
cells, is purified from medium conditioned by mature rat osteoblasts.
Fig. 1A shows that the
chemoattractant for both endothelial (EA hy926) and breast carcinoma
(MDA MB231) cells is found in the same fractions of the last
purification step, a heparin-Sepharose column. A pool of these
fractions yields on SDS-PAGE a single band at 120 kDa, unreduced, and a
doublet between 33 and 35 kDa, upon reduction, corresponding to
purified C3 (not shown). Purified C3 is, therefore, the major single
inducer of directional migration present in the osteoblasts CM for both
EA hy926 and MDA MB231. To confirm further that C3 is the only inducer
of directional migration of MDA MB231 in these fractions, we have added
in the lower compartment of the Boyden chamber antibodies against
2,
6,
1, and
3 inhibit chemotaxis and do not change urokinase and
urokinase receptor expression. Blockage of
2,
1, and
3 integrins affect differently the
induction by C3 of pro-metalloproteinase-2 and -9 and of tissue inhibitor of metalloproteinases-2. Chemotaxis to C3 is also inhibited by genistein, by pertussis toxin, which also inhibits
C3-induced pro-metalloproteinase -2 and -9, but not urokinase
expression. Wortmannin partially inhibits C3-induced cell migration.
Other, but not all, breast carcinoma lines tested responded to C3 with migration and pro-metalloproteinase-2 induction. Presently C3 is the
only agent known to induce migration specifically of both endothelial
and breast carcinoma cells. The mitogenic and motogenic role of C3
in vitro might prefigure a role in in
vivo carcinogenesis and in the establishment of metastasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
4 in breast epithelium controls cell mobilization, concomitant to ECM-derived and growth
factor-associated stimuli (42).
6
1
expression is associated with the migratory behavior of early
differentiating normal breast cells and invasive breast carcinoma, in
the regulation of mammary epithelial cells survival, and in the
apoptotic response of breast carcinoma cells (43-44).
6
4 is involved in the ECM-independent
migratory behavior and in the survival of breast epithelial cells (45).
3
1 (46) and
v
3 integrins interact with uPAR, uPA,
PAI, and ECM components (29).
v
3 enters
in complexes with MMP-14 and MMP-2, functional to the enhancement of
peri-cellular proteolysis and to inside-in signaling in a transfected
cell line of breast carcinoma (29, 40, 47) and in melanoma (24). The
effects of these interactions are the control of localized proteolysis,
through regulation of proteinase function and/or directional migration.
In ovarian carcinoma cells in collagen gels
1 integrin
controls the expression of MMP-14 and MMP-2 (48); in melanoma cells
3 integrin signaling regulates MMP-1 expression (24); in
MDA MB231 cells
3
1-tetraspanin complexes
regulate the expression of MMP-2 via PI3K-mediated signaling (41), and
in osteosarcoma cells plated in collagen,
2
1 integrin regulates the expression of
MMP-1 (49). In tumor cells different integrins interacting with
different ECM proteins can therefore regulate the expression of
secreted and transmembrane MMPs, and supermolecular complexes of
integrins with transmembrane and/or secreted MMPs, and/or uPA and uPAR,
can regulate in turn both peri-cellular proteolysis, cell migration,
and cell proliferation.
1,
1, and
3 integrins and is inhibited by pertussis toxin (PTX)
(4), and the cell signaling pathway(s) activated through these
integrins involve phosphotyrosine kinase and heterotrimeric G-proteins
(4, 39). In general, the signaling machinery involved in the induction
of cell migratory behavior includes pathways mediated through
activation of phosphotyrosine kinases (inhibited by genistein),
heterotrimeric G-proteins (13, 14) (inhibited by PTX) (4, 50), and
PI3K (inhibited by wortmannin) (37, 48). All of these signaling
pathways are also, directly or in a cooperative fashion, capable of
affecting the progression of the cell cycle and/or the cell death by
apoptosis (39, 52). The mechanistic aspects of this signaling are still largely unraveled.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate) (53).
MDA MB231, MDA MB435, MCF-7, and ZR75.1 breast cancer cell line
(courtesy of Prof. S. Toma, Università di Genova, Italy) and
endothelial cells, EA hy926, were cultured in Dulbecco's modified
Eagle's medium, 10% FCS.
1 and
2, these were added to the lower
compartment of the Boyden chamber. To study the effects on migration of
antibodies against MMPs, uPA, and integrins, and in that of TIMP-1,
GM6001, PAI, PTX, wortmannin, and genistein, these were added with the
cells in the upper compartment. The concentrations of antibodies
against
1,
3 integrin chains, against
MMP-2 and -9, and against the
1 and
2
chains of type I collagen were chosen as effective in the inhibition of
C3-induced endothelial cell migration. After migration five random
fields of the filters were counted for each sample; the standard
deviation was calculated and indicated in the figures. Duplicate
samples were run in each experiment, and the experiments were repeated
at least twice. To study the expression by cells of MMPs and uPA upon
induction with C3, the media conditioned during the chemotaxis by the
tumor cancer cells (the upper compartment of the Boyden chamber) were collected.
1
(monoclonal AIIB2, courtesy of Dr. C. Damsky, University of
California), integrin
1 (monoclonal antibody 6S6, Chemicon, Temecula, CA), integrin
3 (monoclonal antibody
B212, courtesy of Dr. G. Tarone, University of Torino, Italy), integrin
2 (monoclonal antibody A2-IIE10, Sigma), integrin
6 (monoclonal antibody 135-13C, courtesy of Dr. Kennel,
Ornel Life Science Division, Oak Ridge, TN),
1 and
2 chains of the COOH-terminal of procollagen type I
(polyclonal antibody, courtesy of Dr. A. Veis, Northwestern University,
Chicago), MMP-2 and -9 (polyclonal antibody, courtesy of Dr. W. Stettler Stevenson, National Institutes of Health, Bethesda), MMP-14
(polyclonal antibody, Biotrend, Koln, Germany), and uPA (monoclonal
antibody, Calbiochem). Anti-human uPAR (monoclonal antibody R4,
courtesy of Dr. F. Blasi, S. Raffaele, Milano, Italy), TIMP-1
(polyclonal antibody, Chemicon), and TIMP-2 (monoclonal antibody 13446, Chemicon) were utilized for Western blotting. Inhibitors utilized were
TIMP-1 (human recombinant, Chemicon), PAI (human recombinant,
Chemicon), and GM6001 (Chemicon). All other chemicals, unless otherwise
stated, were from Sigma.
8 °C
in a water-cooled box. After electrophoresis, gels were washed twice in
2.5% Triton X-100, incubated 16-18 h at 37 °C in 100 mM glycine, pH 8.0, stained with 0.2% Coomassie Blue in
50% methanol, 10% acetic acid, and destained in 50% methanol, 10%
acetic acid. The gels for detection of MMPs were cast with 0.28% w/v
gelatin (type A), run at 6-8 °C in a water-cooled box, rinsed twice
for 30 min in 2.5% Triton X-100, incubated 16-18 h at 37 °C
in 40 mM Tris-HCl, 0.2 M NaCl, 10 mM CaCl2, stained, and destained as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
2 COOH-terminal chains of
pro-collagen type I, at a concentration previously shown to block the
C3-induced migration of endothelial cells. The addition of the
antibodies causes full inhibition of the directional migration of MDA
MB231 cells (Fig. 1B).
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Fig. 1.
Purification of the chemoattractant from
medium conditioned by mature rat osteoblasts. A, Boyden
chamber assay of the fractions eluted from heparin-Sepharose column,
tested with endothelial (EA hy926, open column) and breast
tumor cells (MDA MB 231, dashed column). B,
Boyden chamber assay with MDA MB 231 cells of mature osteoblasts CM,
purified C3 (13 µg/ml), C3 in the absence or presence of antibodies
against 1, and
2 chains of the trimer
carboxyl terminus of pro-collagen type I, and SFM. Error
bars refer to S.D.
The induction of directional migration by CM and by C3 is
positively correlated with the enhancement in the expression of pro-MMP-2, of the 69-kDa form of MMP-2 and with its activation. This is
seen as a marked increase in the relative amounts of activated forms of
MMP-2 at lower molecular size in zymograms on gelatin of both the media
conditioned by the migrating cells (Fig.
2A) and those collected from
cells adhering to plastic (Fig. 2B) and identified by
Western blot (Fig. 2C).
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The basal level of expression of MMP-9, detected by zymograms on
gelatin as pro-enzyme at 92 kDa, is increased in the cells upon
exposure to C3 both during migration in a Boyden chamber (Fig.
3A) and while adhering to
plastic (Fig. 3B), and in both conditions the activated form
of MMP-9 at 82 kDa is detected. The level of TIMP-1 is unchanged (Fig.
3C) and that of TIMP-2 is up-modulated upon exposure to C3
(Fig. 3D), as detected by Western blot in the CM from
adhering cells and detected in the CM from migrating cells by
zymography and reverse zymography (not shown). MMP-14, the
transmembrane metalloproteinase activator of MMP-2, is expressed
constitutively in MDA MB231, and its level is not significantly changed
upon exposure to C3 and during induction of MMP-2 and -9 and their
activation, as measured in zymograms of cell lysates (not shown)
and in Western blots from cell lysates (Fig. 3E). Each
specific antiserum, against MMP-2 or MMP-9 or MMP-14, inhibits the
migration of MDA MB231 in Boyden assay. Migration is also inhibited by
a generic MMP inhibitor, GM6001, or by addition of exogenous TIMP-1
(Fig. 4), indicating that the induction
and activation of both pro-MMP-2 and -9 are required for the
acquisition of the migratory phenotype.
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MDA MB231 cells express constitutively, and in a fashion unaffected by
exposure to C3, the serine proteinases of the uPA system, here shown in
zymograms on plasminogen of the medium collected from cells exposed to
C3 while adhering to plastic (Fig.
5A) or collected after
chemotaxis with C3 (Fig. 5B). Lesser amounts of low
molecular forms of uPA are found in the media collected after chemotaxis, compared with that collected from adhering cells, possibly
due to diffusion to the lower chamber. MDA MB231 also express
constitutively the uPAR, shown by Western blot, and its expression is
unchanged upon treatment with C3 (Fig. 5C). The uPA system
is involved in the chemotactic response of MDA MB231 to C3, as shown by
the inhibition of directional migration obtained by addition of
antibodies against uPA or by addition of PAI during chemotaxis (Fig.
5D).
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A direct connection between the acquisition of migratory phenotype upon
exposure to C3 and the expression of pro-MMP-2 and pro-MMP-9 is
established utilizing PTX, an inhibitor of heterotrimeric G-proteins
and related signaling pathways. PTX inhibits the directional migration
of MDA MB231 cells (Fig. 6A);
the induction of pro-MMP-2, and its activation associated with the
exposure to C3 and the induction of pro-MMP-9 (Fig. 6B).
Nonetheless, PTX does not affect the expression of uPA (Fig.
6C). The inhibition occurs at concentrations of PTX that do
not affect the viability of the cells, as judged by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (not
shown). Other factors required for migration of MDA MB231 induced by C3
are integrin chains 2,
6,
1, and
3. Each of the corresponding
antibodies, utilized in standard Boyden assay, is inhibitory to the
migration to a different extent. Antibodies against the human integrin
chains
1 or
3, at a concentration that
severely inhibits migration of endothelial cells induced by C3, only
partially inhibit the migration of MDA MB231 but blocking antibodies to
2,
6, and to
1 chains
inhibit fully the C3-induced chemotaxis, whereas no inhibition is
observed with a nonspecific IgG (Fig.
7A). Blocking antibodies for
these specific integrins were utilized in the presence or absence of C3
also on cells adhering to plastic, and the expression of MMPs (Fig.
7B), TIMP-2 (Fig. 7C), uPA (Fig. 7D),
and uPAR (Fig. 7E) was detected. The exposure to antibody to
2 reduces slightly only the induction of pro-MMP-9 and
exposure to anti-
3 antibodies, and one of the two
anti-
1 monoclonal antibodies tested inhibits induction
of pro-MMP-2 and -9 upon exposure to C3, whereas the other
anti-
1 monoclonal antibody has no effect on MMPs
expression, also confirmed by Western blot for MMP-2 (not shown). The
conditioned medium of MDA MB 231, analyzed by zymograms after exposure
to
6 antibody, also in absence of C3, showed MMPs in
large amounts, and testing of the antibody preparation showed these
were present in the antibody itself as contaminants (Fig.
7B). TIMP-2 level in the medium is unaffected by treatment
with anti-
2 antibody, and it is decreased upon treatment with anti-
6, both the anti-
1 monoclonal
and anti-
3 antibodies, in the presence and absence of
C3. uPA and uPAR expression are unaffected by exposure to blocking
antibodies. Addition to the cells during Boyden chamber assay of
genistein inhibits, in dose-dependent fashion, cell
migration indicating that PTK-mediated signaling is required in the
induction of the migratory phenotype by C3. Induction of migration by
C3 also requires the function of PI3K, as shown by the inhibitory
effect, exerted in dose-dependent fashion, by addition of
wortmannin during Boyden chamber assay (Fig.
8). Parallel cultures were treated with
genistein or wortmannin for 6 h to certify the absence of
cytotoxicity (not shown).
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The effects of C3 on cell growth and death has been analyzed at time
intervals over 5 days in MDA MB231 cultured in SFM, without or with C3
or 10% FCS. Over five days, after a lag, cell numbers increase in the
cultures supplemented with serum and less in the cultures in SFM,
with or without C3 (not shown). Hoechst-stained cells, cultured
in the three conditions utilized above, were analyzed at successive
time points over 3 days for morphological evidence of mitotic and
apoptotic cells. C3 has a mitogenic effect in the first 48 h of
culture comparable with that of 10% FCS. During this length of time in
culture the cells in the presence of C3 become loosely attached and
easily detach from the plastic. Both adhering and detached cells were
collected and analyzed. The frequency of mitotic and apoptotic cells in
the two populations are not significantly different, and the data from
the two have been pooled in Table I. At
72 h the serum-supplemented cultures maintain the same frequency
of mitosis as at 48 h, and it decreases in the C3-supplemented
cultures, indicating that addition of C3 alone is not sufficient to
support in time the proliferation and that other factors are required
to maintain over time a level of cell duplication similar to that
supported by 10% FCS. Apoptotic cells are more frequently found
in cultures supplemented with serum than in SFM or SFM + C3 at 48 h. At 72 h the frequency of apoptotic cells in SFM + C3 becomes
not significantly different from that of the cultures in FCS and
exceeds that of the control in SFM. The estrogen receptor (ER)
expression varies in different lines of breast carcinoma, and its loss
has been often associated with the progression to malignancy of the
tumor cells. We have tested for the migratory response to C3 and for
the induction of MMPs three other breast carcinoma-derived cell lines,
beside MDA MB231, in order to assess whether a correlation existed
between C3-inducible migratory behavior, MMPs induction, and ER
expression. The human breast carcinoma MDA MB435 (ER) and MCF-7 (ER+)
lines challenged with C3 are induced to migrate directionally by C3
(Fig. 9A), and the induction
of directional migration by CM and by C3 is positively correlated with
the enhancement of expression of MMP-2 and with its activation, as
shown in zymograms on gelatin of the media conditioned by the migrating
cells (Fig. 9B). Each cell line has a different basal
pattern of expression of pro-MMP-2 and a different extent of induction.
The increase in the expression of pro-gelatinase and of the 69-kDa form
of MMP-2, and a marked increase in the relative amounts of activated
forms of MMP-2, commonly detected in the three cell lines
chemoattracted by CM and by purified C3 so far identified, associate
migratory phenotype with MMP-2 activity. Nonetheless, the line ZR75.1
(ER+), which has undetectable basal levels of expression of pro-MMP-2
and is not induced by C3 to migrate and to express pro-MMP-2, upon
exposure to CM is induced to express pro-MMP-2 and activated forms of
MMP-2 but not to migrate (Fig. 9, A and B),
suggesting that MMP-2 induction and activation are not the only
required factor for the acquisition of the migratory phenotype.
Moreover, other factors must contribute or be responsible for the MMP-2
induction by CM in ZR75.1 cells.
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C3, the major chemoattractant component for endothelial cells in the conditioned medium collected from mature osteoblasts (4), induces directional migration in breast tumor cells, associated with induction of pro-MMP-2 and its activation but not unequivocally associated with their ER status. Recent studies (54) by microarray of the gene expression of breast cancer also suggested that there is not an absolute association between the ER expression by tumor cells and the expression of an invasive phenotype. The induction of migration by C3 is associated in all responding lines with the induction of MMP-2 activity within the 6 h of the experimental time and, in the line MDA MB231 where it was sought with the induction of transient proliferation, of MMP-9 activity and of TIMP-2. The induction of MMPs and TIMP-2 occur upon the exposure of MDA MB231 cells to C3 while adhering to plastic or in suspension during chemotaxis, and it is a direct effect of the cell interaction with C3. The induction of pro-MMP-2 and -9 requires G-proteins activity, because it is abolished in presence of PTX. In similar fashion, migration of endothelial cells induced by C3 is also inhibited by PTX (4). The induction of pro-MMP-2 and -9 and their functionality is a prerequisite for the acquisition of the migratory behavior, and migration is inhibited by the MMPs inhibitor GM6001, by TIMP-1, and by each of the specific antibodies against MMP-2, -9, and -14. The results obtained by inhibition experiments with these antibodies are consistent with the hypothesis that active MMP-9, possibly the end product of an activation cascade involving pro-MMP-14 and pro-MMP-2 (or alternatively activated by uPA), plays an essential role in migration of the tumor cells, because inhibition of migration by antibodies against MMP-9 is total. These results are also compatible with the possibility not the alternative that MMP-2, -9, and -14 are all required at the same time (or place) for the promotion of migratory phenotype. Similar conclusions with regard to the requirement for the essential function of MMP-9 can be drawn also from the fact that ZR75.1 cells, although induced by osteoblasts CM to express activated forms of MMP-2 but not of MMP-9 (not shown), do not activate a migratory phenotype, thus indicating that production and activation of pro-MMP-2 (by MMP-14) can be dissociated from or is not sufficient in itself to activate migration. The induction of pro-MMP-2 and -9 occurs in MDA MB231 in a background where MMP-14 is constitutively produced and active. This is not changed by exposure to C3 of cells both adhering on plastic and during migration. Given that MMP-14, the proteolytic activator of pro-MMP-2, as well as its potential extracellular activator uPA are constitutively expressed in MDA MB231 cells, the increase in activated forms of MMP-2, observed upon exposure to C3, might be simply consequent to the induction of the expression of the pro-enzyme. In addition, upon exposure to C3 we observed up-modulation of TIMP-2, the co-factor of pro-MMP-2 activation. The change in levels of TIMP-2 might suffice to enhance activation of secreted pro-MMP-2 by pre-existent MMP-14. Activated MMP-2 can activate pro-MMP-9, also induced by exposure to C3. The fact that inhibition of migration is obtained with antibodies against either MMP-2 or MMP-14 suggests that the migratory phenotype requires the simultaneous function of all three MMPs. Experiments of inhibition with GM 6001 and TIMP-1 also confirm that the functions of MMPs are required for the directional migration of MDA MB231 in response to C3. In establishing the requirement for MMPs, our data agree with those showing that MMP-2 and -9 functionality is required for primary and metastatic carcinogenesis in breast tumors (17) and enhances both primary and metastatic tumor growth (18). Nonetheless, pro-MMP-9 might also be processed by uPA, which is produced constitutively by MDA MB231 and is required for the induction of the migratory phenotype, as discussed below.
After 48 h of exposure of MDA MB231 to C3, we observe a mitogenic effect, which is not sustained for more prolonged culture times. The association of a growth-promoting effect to the induction of the activity of the endogenous MMPs, which is sustained for at least 24 h (the longest time tested by us, data not shown), suggests the possibility that these last might be involved in the mitogenic effect of C3, directly or through the production of mitogenic stimuli by proteolysis of the ECM, as current evidence suggests to occur in vivo (18). If the mitogenic effect of C3 was indeed mediated by proteolysis of ECM, its transience in vitro might be due to the presence of lesser and different ECM than that surrounding tumor cells in vivo. Other pathways for the mitogenic effect of C3 are equally possible, because the machinery that controls cell proliferation is at the end point of all the signaling pathways implied in the response to C3 by the experiments with inhibitors discussed below.
uPA and uPAR are constitutively expressed, both unchanged upon exposure to C3, by MDA MB231. We have not investigated if upon exposure to C3 the levels of expression of the specific inhibitors of the uPA system, PA-1 and -2, change. Their levels might regulate the availability of uPA and its interactions with the receptor (31), and this point deserves further studies.
The migratory phenotype is inhibited partially by treatment with up to 20 µg/ml of PAI and to about 80% by anti-uPA antibody, showing that uPA is required for the acquisition of the migratory phenotype in response to C3. uPA might be required for the activation of pro-MMP-9 and -14 or it might affect signaling via uPAR. One of the known signaling mechanisms affected by uPAR occupancy involves PI3K function. Studies with the PI3K inhibitor wortmannin indicate that C3 induction of the migratory phenotype is partly dependent on PI3K function. The maximum inhibition we could obtain with wortmannin at concentrations up to 600 nM (not shown) was 55%, suggesting that the PI3K pathway is not the only one that cells utilize for the acquisition of the migratory phenotype. Signaling through uPAR is known to involve multiple interactions between members of the uPA system and with integrins and ECM components (28). Although the signaling pathways involved in the response to occupancy of uPAR are complex and still not fully untangled (33, 34), evidence has accumulated that they also include heterotrimeric G-proteins and PTK (34), which are also required by MDA MB231 cells to migrate in response to C3.
Taken together, the inhibition experiments mentioned above, and those with genistein, show the involvement of all these pathways in the acquisition of the migratory phenotype induced by C3 in MDA MB231. They indicate that the function of both the uPA system and of at least 3 members of the MMPs family converge to activate fully the migratory phenotype of MDA MB231 in response to C3. The complementary action of the families of serine and metalloproteinase for the manifestation of migratory phenotype was reported previously (55, 56).
The extracellular interactions that modulate the proteolytic and signaling functions of the uPA system include the activation of uPAR by specific MMPs (55). Basal cell mobility of MDA MB231 is regulated by MMPs and uPA levels via PI3K (37). Notwithstanding the implications mentioned above, we are not yet in a position to trace a mechanistic picture of the events involved in signaling induced by C3.
Fibroblasts bind to C3 via integrins 1
1
and
2
1 (57, 58), and the directional
migration of endothelial cells in response to C3 requires
1 and
3 integrins (4). MDA MB231 cells do not normally express the
1 integrin chain. The
expression of migratory phenotype through gelatin of MDA MB231 in
response to C3 is here shown, by inhibition studies with the specific
antibodies, to involve
2,
6,
1, and
3 integrin chains. In Boyden
chambers experiments it is nonetheless impossible to distinguish
whether integrins are required for the recognition of the
chemoattractant or for the adhesion to and traversing the gelatin
coating. The inhibition of migration upon treatment with genistein is
consistent with either or both possibilities.
Integrins play a role in determining the regulation of expression and
activation of MMPs in many tumor cells and in the regulation of the
function of uPA system in concurrence with the interaction with the ECM
(29). These interactions, in general, are functional to enhanced
localized proteolysis and are often associated with acquisition of a
migratory phenotype. The usage of integrins by each cell type is
dependent on the nature of the ECM and varies between different cell
types for the same ECM protein. Through the dissection of new and/or
cell-specific mechanistic responses of different cell types to
different ECM components, to growth factors, to transmembrane and
peri-cellular proteinases and their receptors (39), different signaling
pathways can be activated. Signals activated through integrins are
shown to be of a great complexity. 6 integrin expression
is involved in the migratory process in breast carcinoma cells and was
specifically associated with the metastatic phenotype (42, 43).
Engagement by ligands of dimers containing the integrin
1 (including
6
1) and the
3 chains causes the activation of signaling through PTK
but can also involve, according to the ECM components engaged and to
the presence of growth factors, different signal transduction
molecules, whose activation (as for most if not all integrins, except
6
4) converges in the activation of a core
signaling machinery affecting the actin cytoskeleton and activating the
c-Jun NH2-terminal kinase and thus regulating cell cycle
and ultimately cell growth (39). This signaling machinery involves Rac
and Ras, whose activation is required also for induction and activation
of MMPs and for the acquisition of invasiveness in transformed cell
lines (40, 52, 60). Exchange toward Ras and Rac is increased by the
activity of PI3K, a kinase involved in the processes of focal adhesion disassembly and migration. Substrate-independent signaling through
6
4 is wortmannin-sensitive, involves the
functionality of PI3K, and the activation of SOS exchange toward Rac
and Rho GTPases (48), and ultimately also affects growth regulation
(52).
4 integrin is also uniquely capable of direct
activation of Shc, which couples integrins to the control of cell cycle
(61).
The evidence gathered to this time by inhibition of signaling pathways establishes that the acquisition of the full migratory phenotype induced by C3 in MDA MB231 is mediated by signaling requiring the activities of heterotrimeric G-proteins, PTK, also shown to inhibit the MMPs induction by C3, and PI3K. The requirement for PI3K (perhaps through its role of increasing the exchange between SOS and Rho family members) represents a potential link between uPA-responsive and integrin-PTK-mediated pathways. The activation of PI3K can also play a role in the control of growth regulation through its effect on the p70S6K and cdk2-cycline-E pathway (51), an aspect presently under study in our system.
Experiments utilizing blocking antibodies to 2,
6,
1, and
3 integrin
chains show that these inhibit cell migration through gelatin and have
no effect on the level of the secreted uPA and of the uPAR. The pattern
of expression of pro-MMPs and TIMP-2 varied with the antibody utilized
from inhibition of the induction by C3 of the MMPs to no effect. The
MMPs detected upon the block of cell migration determined by exposure
to the anti-
6 antibody were shown to be contaminants of
the antibody preparation. We cannot therefore know if the blocking of
6 integrin chain has an effect on MMP expression,
although obviously exogenous MMPs added with the antibody do not seem
to interfere with its blocking the migration of cells.
In general, there is an intrinsic difficulty in the interpretation of the data of inhibition of function with antibodies, due to the impossibility to dissect the effect of treatment with each antibody on the interaction of the cells with the gelatin necessary for enacting migration, from the effect on the interaction with C3 necessary for initiating the migratory phenotype. Moreover, as well known, inhibition with antibodies of proteins with multiple bindings are intrinsically complex to decline mechanistically without further studies with deletion mutants and/or panels of monoclonal antibodies targeted to multiple epitopes. Such a dissection will have to be done.
So we have evidence that for the two different 1
monoclonal antibodies utilized, the epitope that they interact with
might elicit different responses in the MDA MB231. The blocking by one antibody blocks completely the expression of migratory phenotype not
accompanied by inhibition of the C3-mediated induction of pro-MMP-2
(only a slight inhibition of the induction of pro-MMP-9 and of TIMP-2
is observed), as if the interaction with the antibody still allowed the
interaction with C3 and the consequent induction of MMPs although
creating a contest where denatured collagen (gelatin) is not recognized
as substratum. A different monoclonal antibody only partially inhibits
the expression of migratory phenotype, and therefore we assume that it
permits recognition by the cells of gelatin, whereas it seriously
inhibits pro-MMPs and TIMP-2 modulation by C3, and therefore it
possibly affects the binding of C3. These data are consistent with
involvement of
1 chains in mediating the signaling from
C3 that induces modulation of the MMP system and its relevance in the
enactment of migration through gelatin. A monoclonal antibody to
3 blocks both cell migration and the C3-mediated
induction of MMPs. The involvement of
3 chains in the
recognition of C3 is in analogy with results in inhibition studies in
endothelial cells (4). A blocking antibody to
2 acts
similarly to the first mentioned monoclonal antibody to
1, inhibiting the expression of migratory phenotype and
not affecting C3-induced pro-MMPs and TIMP-2 modulation, leaving open
the possibility that
2 chains might not be critical for the C3-mediated induction of the MMPs-TIMP system. In summary, these
data support that
1 and
3 integrins are
involved in the signaling to the cells of the presence of C3 which
causes changes not involving the uPA system, but affecting the
MMPs-TIMP system, consistent with reports that implied
chain
interaction with collagenous ECM in modulation of MMPs in this and
other cell lines (24, 41, 48). Treatment with anti-
6
antibody is highly inhibitory for cell migration, even in presence of
contaminating exogenous MMPs in the antibody preparation (which also
make irrelevant the data on TIMP-2 level). Blocking of the
6 chain is capable to block migration through gelatin,
but we cannot presently unravel if the functionality of the cell
motility machinery (such as mobility signaling molecules and
phosphorylation of structural proteins of the cytoskeleton), known to
be directly affected by
6 occupancy, might be blocked by
antibody binding independently from MMP induction.
The multistep progression that epitomizes carcinogenesis is presently viewed as involving genetic alteration in the epithelia as well as epigenetic contributions from the surrounding stromal tissue. Collagen type I, from which C3 originates, is a major product of tumor stroma fibroblasts, and high synthesis of collagen in stromal fibroblasts is positively correlated to the metastatic outcome of breast carcinomas (6). The production by tumor-associated fibroblasts of processed collagen fragments and the half-life of C3 in the stroma associated with tumors has not been yet investigated, although it is known that carcinoma-associated fibroblasts acquire a different biosynthetic phenotype than their normal counterpart, involving ECM proteins, abnormal actin expression, and inappropriate secretion of proteolytic molecules (6, 8, 9). They also acquire the capability to stimulate tumor progression in initiated epithelial prostatic cells in vivo and to induce in vitro an increase in cell proliferation and a decrease in cell death (59).
Although quite aware that caution must be used in extending results obtained in vitro to the in vivo situation, and for the value these considerations might have in directing further investigations, we hypothesize that regulation of the level of C3 might be one of the early epigenetic changes in tumor stromal fibroblasts associated with permissivity to the growth of primary tumors. The relevance of C3 in this process is its unique capability to promote the migration and to attract both endothelial and primary tumor cells and thus to favor their encounter within the stroma and to allow for the known reciprocally inductive interactions to occur (e.g. vascular endothelial growth factors are produced by tumor cells that promote proliferation of endothelial cells and morphogenesis of vessels, and organization of new vessels is permissive for tumor growth (15)). The adjacency of the two cell types would establish a productive cycle toward the successive steps of malignancy. MMPs, induced by exposure to C3 in tumor cells, could contribute to the acquisition of the migratory phenotype of both kinds of cells and to the enhancement of growth of tumor cells (17).
The production of C3 in tissues with high levels of collagen
biosynthesis, as in bone, could attract preferentially circulating breast carcinoma cells and eventually other tumor cells that are attracted by C3, such as
melanoma2 and possibly
prostatic carcinoma cells. The endogenous production of MMPs in tumor
cells would be sustained by the presence of C3 in the homing site and
this might favor their growth and the neoangiogenesis, in a fashion
similar as that hypothesized above for primary tumors and with the end
effect of promoting the colonization by C3-responsive cancer cells of
C3-rich homing sites. This hypothesis is consistent with the current
opinion that the preferential homing site of different tumors depends
on the local composition of the ECM and on the molecules produced upon
its proteolysis and gives a testable rationale for the preferential
homing of breast carcinoma cells to bone (59). In an experimental set
up where C3 with breast carcinoma cells was implanted subcutaneously in
nude mice, we obtained a preliminary confirmation of the pro-angiogenic
role of C3 and of its promoting role on tumor growth.2 A
consequence of our hypothesis, presently under test, is that a subset
of tumors, those that preferentially metastasize to collagen-rich tissues like bone, will be most chemoattracted in vitro by
C3 and induced to the synthesis and activation of MMPs.
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ACKNOWLEDGEMENTS |
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We acknowledge the generosity of all the colleagues that have supplied us with cell lines and antibodies. We thank Dr. S. Bonatti, CNR, Genova, Italy, for critical reading of the manuscript and Nicola Palmieri for technical support.
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FOOTNOTES |
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* This work was supported by MUIR grants, Italy, and CNR Biotecnologie, Italy (to P. M.).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. Tel./Fax: 39-010- 3538240; E-mail: man-via@unige.it.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M207483200
2 P. Manduca, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CM, conditioned medium; C3, carboxyl-terminal trimer of pro-collagen type I; ER, estrogen receptor; MMP, metalloproteinase; TIMP, tissue inhibitor of MMP; uPA, urokinase-uPA; uPAR, urokinase receptor; PAI, plasminogen activator inhibitor; PTX, pertussis toxin; ECM, extracellular matrix; PI3K, phosphatidylinositol 3-kinase; FCS, fetal calf serum; PTK, protein-tyrosine kinase; SFM, serum-free medium.
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