The Matrix Metalloproteinase-9 Regulates the Insulin-like Growth
Factor-triggered Autocrine Response in DU-145 Carcinoma Cells*
Santos
Mañes
,
Mercedes
Llorente,
Rosa Ana
Lacalle,
Concepción
Gómez-Moutón,
Leonor
Kremer,
Emilia
Mira, and
Carlos
Martínez-A
From the Department of Immunology and Oncology, Centro Nacional de
Biotecnología, Consejo Superior de Investigaciones
Científicas, Universidad Autónoma de Madrid, Campus de
Cantoblanco, E-28049 Madrid, Spain
 |
ABSTRACT |
The androgen-independent human prostate
adenocarcinoma cell line DU-145 proliferates in serum-free medium and
produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF
type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins
(IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed
selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa.
By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect
surface receptor-bound IGF-I on serum-starved DU-145 cells, which
activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145
cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus
anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145.
Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3
also modifies the DU-145 cell cycle, decreases p34cdc2 levels,
and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since
des-(1-3)IGF-I, which does not bind to IGFBP-3, reverses its
inhibitory effect. DU-145 also secretes the matrix metalloproteinase
(MMP)-9, which can be detected in both a soluble and a membrane-bound
form. Matrix metalloproteinase inhibitors, but not serpins, abrogate
DNA synthesis in DU-145 associated with the blocking of IGFBP-3
proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits
80% of DU-145 cell proliferation that can be reversed by IGF-I in a
dose-dependent manner. Inhibition of MMP-9 expression is
also associated with a decrease in IGFBP-3 proteolysis and with reduced
signaling through the IGF-1R. Our data indicate an IGF autocrine loop
operating in DU-145 cells, specifically modulated by IGFBP-3, whose
activity may in turn be regulated by IGFBP-3 proteases such as
MMP-9.
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INTRODUCTION |
In normal cells, proliferation is a coordinated process involving
intercellular communication through soluble regulatory molecules known
as polypeptide growth factors (1). In contrast, neoplastic cells are
characterized by a relative autonomy of growth, a consequence of the
constitutive expression of growth factors and receptors involved in
autocrine loops (2). Examples of constitutive autocrine growth factor
loops have been reported for different cancer cells and growth factors,
such as transforming growth factor
, insulin-like growth factors
(IGF-I and IGF-II),1 and
platelet-derived growth factors, among others (3).
IGF-I and IGF-II are potent mitogens for several non-transformed and
cancer cell types, and viral and nonviral oncogenes appear capable of
interfering with the IGF autocrine loop (4). Indeed, c-MYB increases
IGF-I secretion and IGF type-1 receptor (IGF-1R) production. It has
also been suggested that the IGF-1R is critical in the establishment
and maintenance of the transformed phenotype. Mouse embryo cells with a
targeted disruption of the IGF-1R gene (5, 6) cannot be
transformed by SV40 large T antigen alone or in conjunction with
Ha-ras (7, 8). In addition, antibodies to the IGF-1R (9), as
well as antisense expression (10) and dominant negative mutants of this
receptor (11), reverse the transformed phenotype and/or inhibit tumorigenesis.
In serum and in extracellular fluids, both IGF-I and -II are
bound with high affinity to soluble IGF-binding proteins
(IGFBP), seven of which have been identified to date (12).
Many tumor cell types secrete one or more of these proteins (13). The
relevance of the IGFBP lies in their potential to modify the metabolic
and mitogenic effects of IGF. In fact, IGFBP may either inhibit and/or enhance IGF activity (14-17). The inhibitory effects of IGFBP have been attributed to competitive scavenging of IGF peptides away from the
IGF receptors (18). The enhancer mechanism is poorly understood,
however, and probably involves binding to the cell membrane or
extracellular matrix and/or processing into smaller molecular weight
species by limited proteolysis (15, 19-22). The result is a dramatic
reduction in IGFBP affinity for IGF, which enhances the availability of
the growth factors to the target cells (23). Possible direct effects
have also recently been suggested for some IGFBP, independent
of their IGF binding activity (12).
In this study, we characterize the role of IGF, IGF-1R, and IGFBP in
tumor cell proliferation using DU-145 cells, a human androgen-independent prostate adenocarcinoma cell line (24). Earlier
studies indicated that this cell line expresses several components of
the IGF axis (25, 26). Our results suggest the existence of an IGF
autocrine loop in DU-145 cells and its specific modulation by IGFBP-3
proteolysis. This proteolytic activity may be ascribed to the matrix
metalloproteinase (MMP)-9, which is also produced by this cell line in
an autocrine manner. Furthermore, we find MMP-9 both in soluble
form and in a membrane-associated form on the DU-145 cell surface. The
expression of an MMP-9 antisense cDNA leads to an 80% inhibition
in DU-145 proliferation, which is reversed by the addition of exogenous
IGF-I. This growth inhibition is associated with the abrogation of
IGFBP-3 proteolysis as well as with a decrease in IGF-1R-promoted cell
signals. MMP-9 therefore controls DU-145 cell proliferation by
interacting, at least partially, with the IGF-I autocrine loop in this
cell line.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
BALB/c 3T3 fibroblasts overexpressing the
human IGF-1R (3T3-IGF-1R, a gift of Drs. A. Ullrich and R. Lammers) and
DU-145 cells (ATCC HTB-81, American Type Culture Collection, Manassas,
VA) were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) supplemented with 10% fetal calf serum. Murine interleukin-3-dependent Ba/F3 cells (27) were cultured in
RPMI, 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM L-glutamine, and 10% conditioned medium
from the interleukin-3-producing cell line WEHI-3B.
Anti-IGF Monoclonal Antibodies--
The monoclonal antibodies
(mAb) KM5A1 and BB9E10 were raised in our laboratory by immunization
with the IGF-I-(55-70) synthetic peptide or recombinant human IGF-I,
respectively (28, 29). KM5A1 is IGF-I-specific, with less than 0.1%
cross-reactivity with IGF-II, and recognizes IGF-I when bound to
IGFBP or the IGF-1R. BB9E10 has approximately 3%
cross-reactivity with IGF-II and binds to an IGF-I epitope hidden by
IGFBP and the IGF-1R. We also used an anti-IGF-II mAb raised in our
laboratory, with an apparent affinity constant of 1011
M
1 and less than 0.1% cross-reactivity with
IGF-I, which is an IGF-II antagonist.2
Amplification of IGF-I, -II, and IGF-1R mRNA by
RT-PCR--
Total RNA, derived from either starved or control cultured
cells, was isolated by ultracentrifugation through a cesium chloride cushion (30). This material (5 µg) was then reverse-transcribed using
a first strand cDNA synthesis kit (Pharmacia AB, Stockholm, Sweden). One-tenth of the transcription product was amplified for
either 40 PCR cycles with hIGF-I-specific primers or for 30 cycles with
primers specific for hIGF-II or the IGF-1R. The primers for hIGF-I were
5'-GGTGGATGCTCTTCAGTTCGTGTGT-3' and 5'-GCAATACATCTCCAGCCTCCTTAGA-3'. The primers for hIGF-II were 5'-CTTACCGCCCCAGTGAGACCCTGTG-3' and 5'-CTCTCGGACTTGGCGGGGGAGCAC-3'. For amplification of the IGF-1R, the primers were used as described previously (31). Finally, 10 µl of
the PCR products were resolved on 2% agarose gels.
Detection of IGFBP by Western Ligand and
Immunoblotting--
DU-145-conditioned medium (DU145-CM) was recovered
from subconfluent cells cultured for 5 days in serum-free DMEM (SFM)
and concentrated 10-fold using Centricon filters with a 3,000 molecular weight cut-off (Amicon, Danvers, MA). Ligand blots were performed basically as described (32). Briefly, 40 µl of concentrated DU145-CM
were fractionated in 12.5% SDS-PAGE under nonreducing conditions and
transferred to nitrocellulose (Schleicher & Schuell). IGFBP species
were detected with biotin-labeled IGF-II, followed by peroxidase
(PO)-labeled streptavidin (Sigma), and the ECL chemiluminescence detection system (Amersham, Aylesbury, UK). Immunoblots were performed as above, but IGFBP species were detected with specific anti-IGFBP-1 mAb or anti-IGFBP-3 (raised in our laboratory), anti-hIGFBP-2, hIGFBP-4, hIGFBP-5, or hIGFBP-6 (Austral Biologicals; San
Ramon, CA) polyclonal antibodies, followed by PO-labeled goat
anti-mouse or anti-rabbit IgG and ECL.
Detection of MMP-9 in DU145-CM--
Either DU145-CM or
conditioned medium from the phorbol 12-myristate 13-acetate-stimulated
human fibrosarcoma HT-1080 cell line was recovered from subconfluent
cultures after 2 days in serum-free DMEM supplemented with 0.5% BSA
(RIA Grade; Sigma; SFM/BSA) and concentrated using Centricon filters.
Zymography was performed in SDS-PAGE gels containing gelatin (1 mg/ml)
as previously reported (33). The same samples were electrophoresed in
SDS-PAGE under reducing conditions, blotted to nitrocellulose, and
probed with anti-MMP-9 Ab3 antibody (Calbiochem), followed by
PO-labeled goat anti-mouse antibody and ECL.
Growth Assays and Cell Cycle Analyses--
DU-145 cells were
detached using 0.05% trypsin, 0.02% EDTA (Life Technologies, Inc.)
and plated at several cell densities in 96-well plates (for
proliferation experiments) or in 24-well plates (for cell cycle
analysis). Twenty-four hours later, the cells were washed extensively
with PBS and cultured for 24 h SFM/BSA. Thereafter, medium was
renewed with SFM/BSA with or without recombinant human IGF-I (rhIGF-1,
Pharmacia & Upjohn), recombinant human des-(1-3)-IGF-I (kindly
provided by Dr. Pär Gellerfors, Pharmacia & Upjohn, Stockholm, Sweden), IGFBP-3 (Calbiochem), blocking anti-hIGF-I and -II mAb, anti-IGF-1R mAb
IR-3 (Oncogene Science, Uniondale, NY), aprotinin (Sigma), tissue inhibitor of metalloproteinases (TIMP)-2, or Batimastat (BB-94, kindly provided by Dr. F. Colotta, Pharmacia & Upjohn, Milan,
Italy). Cell lysates from IGFBP-3- or anti-hIGF-I mAb-treated DU-145
cells were prepared and analyzed for IGF-1R autophosphorylation as
described below.
For proliferation experiments, DU-145 cells were pulsed for 8 h
with 0.5 µCi/well of [3H]thymidine
([3H]TdR, Amersham Pharmacia Biotech) at various times
during the course of the experiment, and nuclei were harvested using a
cell harvester (LKB-Wallac, Sweden). [3H]TdR
incorporation was determined on a liquid scintillation counter. Ba/F3
cell proliferation assays were performed as described (28).
For cell cycle analysis, DU-145-treated cells were detached, washed
with PBS, and stained with propidium iodide using the DNA-Prep Stain
kit (Coulter Corp., Miami, FL). Cell cycle analysis was carried out in
a flow cytometer equipped with a pulse processing facility to enable
discrimination of cell doublets (Epics XL, Coulter). Cell number was
determined in some experiments by manual counting on a hemocytometer.
Detection of Cell Surface-bound IGF-I and MMP-9--
DU-145
cells were washed and cultured in SFM/BSA alone or supplemented with
different amounts of human insulin (Life Technologies, Inc.). Cells
were detached after 72 h, washed twice in ice-cold PBS, and
resuspended at 2 × 106 cells/ml in PBS plus 0.5%
BSA, 0.01% NaN3. Biotinylated anti-IGF-I, anti-MMP-9 Ab3,
or anti-MMP-2 (Calbiochem) mAb was added, followed by
phycoerythrin-labeled avidin or fluorescein isothiocyanate-labeled goat
anti-mouse IgG (Southern Biotechnologies, Birmingham, AL), respectively. An irrelevant isotype-matched mouse antibody was used as
control. Cell-associated fluorescence was visualized by flow cytometry.
To eliminate cell surface-bound IGF, detached cells received an acid
wash (34) prior to staining as above.
Autophosphorylation of the IGF-1R--
Subconfluent DU-145 cells
were cultured in SFM/BSA for 3 days and pulsed with IGFBP-3 for 24 h, with the anti-IGF-1R mAb
IR-3 for the times indicated, or with
IGF-I (10 nM) for 5 min at 37 °C. After washing with
ice-cold PBS, cells were lysed at 4 °C for 30 min using 20 mM Tris-HCl, pH 7.5, 130 mM NaCl, 1 mM MgCl, 1% Nonidet P-40, 10% glycerol, a proteinase
inhibitor mixture, 1 mM sodium orthovanadate, and 10 mM NaF. Lysates were centrifuged for 25 min at 4 °C, and
their protein concentration was determined using the micro-BCA kit
(Pierce). Cell lysates (50 µg) were immunoprecipitated with
IR-3
or anti-IRS-1 mAb (Upstate Biotechnology Inc., Lake Placid, NY) for
3 h at 4 °C, followed by goat anti-mouse IgG1-agarose (Sigma),
and then fractionated in 7.5% SDS-PAGE under reducing conditions.
Tyrosine-phosphorylated proteins were developed with the PY-20 mAb
(Santa Cruz Biochemicals, Santa Cruz, CA), and IRS-1 specifically with
anti-IRS-1 mAb, followed by peroxidase-labeled goat anti-rabbit Ig
antibody (ICN, Costa Mesa, CA) and ECL. A similar protocol was followed
for control 3T3-IGF-1R cells.
Detection of p34cdc2--
The level of cell
division cycle 2 (Cdc-2) was estimated in DU-145 cell extracts treated
with IGFBP-1, IGFBP-3, anti-IGF-I mAb, or BSA as described above.
Samples containing equivalent amounts of protein were fractionated in
12.5% SDS-PAGE under reducing conditions, transferred to
nitrocellulose, and incubated for 2 h with anti-Cdc-2
(Transduction Laboratories, Lexington, KY) or anti-p53 antibodies
(PharMingen, San Diego, CA). After washing, the filter was incubated
with a PO-labeled goat anti-mouse antibody and ECL.
Transfection of MMP-9 Antisense cDNA--
The entire MMP-9
cDNA was cloned in the XbaI site of pEFBOS in the
antisense direction, as determined by restriction analysis (pEFBOS-MMP-9AS). DU-145 cells were transfected with the pEFBOS-MMP-9AS or the pEFBOS empty vector using LipofectAMINE reagent (Life
Technologies, Inc.) according to the manufacturer's instructions.
Transfection efficiency was determined by cotransfecting an equal
amount of green fluorescent protein-pEFBOS plasmid and subsequent FACS
analysis determination of the percentage of cells expressing green
fluorescent protein at 48 h. Expression was maximal between 48 and
96 h.
After 24 h, DU-145-transfected cells were detached and plated in
96- or 24-well plates, allowed to adhere, washed three times with PBS,
and starved overnight in SFM/BSA. Cells were then washed extensively
with PBS and cultured for 24 h in SFM/BSA alone or supplemented
with different amounts of IGF-I or 20 nM IGFBP-3 and then
processed for proliferation experiments as described above.
DU-145-transfected cells in 24-well plates were maintained in SFM
without BSA for an additional 48 h, after which conditioned medium
was collected and analyzed for IGFBP-3 proteolysis by Western blot or
for MMP-9 activity by zymography.
To analyze the effects of MMP-9 antisense expression on IGF-induced
cell signaling, DU-145-transfected cells, either with the
pEFBOS-MMP-9AS or the empty vector, were plated on 35-mm2
dishes and treated as above. After 24 h starvation, dishes were incubated for 5 min in SFM/BSA with or without IGF-I (1 µg/ml). Cell
lysates were obtained and immunoprecipitated with
IR-3 as described
above and proteins resolved in 7.5% SDS-PAGE gels, and after blotting,
nitrocellulose membranes were incubated sequentially with the PY-20 mAb
and anti-IGF-1R
-subunit rabbit polyclonal antiserum (Santa Cruz
Biotechnology) and ECL.
Fluorometric Assay of DU-145 Cell-associated MMP
Activity--
DU-145 cells, untransfected or transfected with
pEFBOS-MMP-9AS or empty pEFBOS plasmid, were starved for 48 h, washed five times with PBS, and then incubated with the
fluorogenic peptide 2,4-dinitrophenol-Pro-
-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-N-methyl-aminobenzoyl) (Bachem, Bubendorf, Switzerland) at a final concentration of 5 µM, as described (33). The fluorescence increase after
incubation at 37 °C was evaluated at 360 and 460 nm excitation and
emission wavelengths, respectively. When inhibitors were tested, they
were preincubated for 30 min with DU-145 before addition of the
fluorogenic substrate.
Cross-linking of DU-145 Membrane Proteins--
DU-145 cell
monolayers were rinsed twice with minimal essential medium without
amino acids plus 20 mM HEPES, pH 7.3, and cell-surface proteins were cross-linked by adding 0.5 mM disuccinimidyl
suberate (Pierce). After 30 min at 4 °C, the cross-linking reagent
was removed, and the reaction was terminated by rinsing and subsequent cell incubation with 37.5 mM Tris-HCl, pH 7.4, plus 150 mM NaCl for 10 min. The cells were then lysed with 100 mM n-octyl glucopyranoside (Calbiochem) in 37.5 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM benzamidine hydrochloride, and a protease inhibitor
mixture as above. Cell lysates were resolved in 7% SDS-PAGE under
reducing conditions, blotted, and probed with anti-MMP-9 mAb
(Calbiochem) and, after stripping according to manufacturer's
specifications, with antibodies specific for different integrin chains.
 |
RESULTS |
DU-145 Cells Express IGF-I, IGF-II, IGF-1R, and IGFBP--
To
analyze DU-145 growth factor requirements, we tested the effect of
several serum concentrations and cell densities in proliferation experiments. As reported previously, DU-145 cells grow in serum-free medium (36), determined both by [3H]TdR incorporation and
the increase in the number of viable cells (data not shown). A
sensitive, specific RT-PCR assay shows that DU-145 cells express
mRNA coding for IGF-I and IGF-II and the IGF-1R (Fig.
1A).

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Fig. 1.
DU-145 produces IGF ligands, IGFBP, and
IGF-1R. A, RT-PCR detection of mRNA encoding IGF-I,
IGF-II, and IGF-1R in DU-145 cells. Total RNA from starved (lanes
2, 5, and 8) or serum-supplemented DU-145 cells
(lanes 1, 4, and 7) was reverse-transcribed, and
one-tenth of the transcription product was amplified in PCR using
oligonucleotides specific for each cDNA, as described under
"Experimental Procedures." Amplification is shown of IGF-I
(lanes 1-3), IGF-II (lanes 4-6), and IGF-1R
(lanes 7 and 8). Lanes 3 and
6 show the control amplification of non-reverse-transcribed
RNA from DU-145 cells. Migration of the 100-base pair (bp)
DNA ladder (Pharmacia AB) is shown at the left. The
predicted PCR fragments are 154 (IGF-I), 205 (IGF-II), and 354 bp
(IGF-1R). B, analysis of IGFBP secreted by DU-145 cells.
DU145-CM was recovered from subconfluent cultures grown in SFM for 5 days and concentrated 10-fold prior to analysis in SDS-PAGE under
non-reducing conditions (see "Experimental Procedures"). Ligand
blot was performed on nitrocellulose membranes by incubation with
biotinylated IGF-II alone (lane 1) or in combination with
unlabeled IGF-I at 1, 0.1, or 0.01 µg/ml (lanes 2, 3 and
4, respectively), and the reaction was developed with
PO-labeled streptavidin. Lane 5 shows the immunoblot
obtained by incubation with an anti-IGFBP-3 antiserum, indicating the
degradation of IGFBP-3. Prestained molecular mass standards (Bio-Rad)
are shown at the left.
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IGFBP secreted by DU-145 cells were analyzed by Western ligand and
immunoblot (Fig. 1B). Labeled IGF-II binds to three proteins in the DU145-CM as follows: a protein of approximately 40 kDa, corresponding to the 39-42-kDa doublet characteristic of IGFBP-3, and
two proteins of 34 and 25 kDa, identified in immunoblot as IGFBP-2 and
IGFBP-4, respectively. A weak band of approximately 30 kDa is also
occasionally visible, although no reactivity was observed with
anti-IGFBP-1, -IGFBP-5, or -IGFBP-6 antibodies. The anti-IGFBP-3
antibody confirmed that the 39-42-kDa doublet is IGFBP-3, but it also
developed a 31-kDa protein and, weakly, another of 19 kDa, which are
not detected in ligand blot. This suggests that IGFBP-3 is proteolyzed
in DU145-CM, as it is in other biological fluids (37). This proteolytic
processing was not observed for either IGFBP-2 or -4.
Secreted IGF Binds to the Cell Surface and Activates the IGF-1R in
DU-145 Cells--
To demonstrate that the IGF secreted by DU-145 cells
binds to cell-surface receptors, we used the IGF-I-specific mAb KM5A1, which recognizes the growth factor complexed either to the IGF-1R or to
IGFBP-1 or -3 (28). KM5A1 binds specifically to DU-145 cells that have
been starved for 72 h (Fig.
2A); KM5A1 mAb binding to
DU-145 cells is lost when the antibody is preincubated with IGF-I
before being added to the cells (Fig. 2A). Other anti-IGF-I mAb (BB9E10) recognizing an epitope occult in the IGF-I·IGF-1R complex (28) do not bind to DU-145 cells. As an additional specificity control, we performed an acid wash of cells to remove any
receptor-bound ligand; this wash completely abolishes KM5A1 reactivity
with the cells (Fig. 2B), which can be restored by
incubation of cells with IGF-I (Fig. 2C).

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Fig. 2.
Flow cytometric detection of IGF-I binding to
the DU-145 cell surface. A, cells were cultured in SFM,
incubated with the biotinylated anti-IGF-I mAb KM5A1 or BB9E10 as
indicated, and developed with phycoerythrin-labeled avidin (Av-PE). The
cell-associated fluorescence intensity after incubation with an
irrelevant antibody (gray area) was used as the negative
control. KM5A1 binding specificity was assessed by preincubation of the
mAb with IGF-I (KM5A1+IGF-I). B, cells were
cultured as above, acid-washed before mAb incubation (see
"Experimental Procedures"), and the cell-associated fluorescence
after Av-PE development was determined. C, cells were
treated as in B and then incubated with IGF-I. After removal
of unbound IGF-I, biotinylated mAb were added and developed using
Av-PE. D, cells were cultured in SFM/BSA supplemented with
the indicated amounts of insulin for 72 h and then stained with
the biotinylated antibodies as in A. Non Sp.,
nonspecific antibody.
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To determine whether KM5A1 recognizes IGF-I bound either to IGF-1R or
to membrane-associated IGFBP, DU-145 cells were cultured for 72 h
in serum-free medium supplemented with BSA and then pulsed for 24 h with different amounts of human insulin (Fig. 2D). Since insulin binds to the IGF-1R and to insulin receptor with different affinities and does not bind to IGFBP, the effect of insulin dose on
KM5A1 mAb cell binding may indicate the receptor to which IGF-I ligates. KM5A1 mAb reactivity is completely abolished when the cells
are cultured in 1 µM insulin and significantly reduced
(but not lost) at 0.1 µM, indicating that
autocrine-secreted IGF-I binds mainly to the IGF-1R. We were not able
to analyze IGF-II binding in DU-145, although it probably is similar to
that of IGF-I.
Since secreted IGF (or at least IGF-I) binds to IGF-1R, we examined
IGF-1R tyrosine kinase activity in serum-starved DU-145 cells. After
incubation of 3T3-IGF-1R cells with IGF-I,
IR-3 immunoprecipitates a
98-kDa phosphoprotein band that represents the autophosphorylated
IGF-1R
-subunit, not observed in the absence of IGF-I (Fig.
3A). IGF-I-treated or -starved
DU-145 cells also show this phosphoprotein band. Upon
autophosphorylation, the IGF-1R
-subunit associates with the insulin
receptor substrate-1 (IRS-1), a docking protein with a key role in
IGF-1R signal transduction (38). The
IR-3-immunoprecipitated cell
lysates were assayed with anti-IRS-1 antibody (Fig. 3B),
showing that the IGF-1R is associated with IRS-1 in both IGF-I-treated
and -starved DU-145 cells. Similar cell lysates, immunoprecipitated
with an anti-IRS-1 antibody, show a 98-kDa phosphoprotein band
corresponding to the IGF-1R
-subunit (data not shown).

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Fig. 3.
IGF-1R is activated in DU-145 cells.
Cell lysates from IGF-I-treated and -starved cells were
immunoprecipitated with IR-3 mAb, and the blotted proteins were
developed with either anti-phosphotyrosine (A) or anti-IRS-1
(B) mAb. As positive control, 3T3 fibroblasts overexpressing
the human IGF-1R (3T3-IGF-1R) were used. Arrows in
A indicate the position of the 98-kDa phosphoprotein
representing the phosphorylated IGF-1R -subunit and in B,
the position of the 185-kDa band representing IRS-1.
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IGFBP-3 and Anti-IGF-1R mAb, but Not Inhibitory Anti-IGF-I and -II
Antibodies, Block DU-145 Cell Growth--
The relevance of the
autocrine IGF/IGF-1R loop in DU-145 cell growth was analyzed by
blocking the activation of IGF-1R induced upon IGF/IGF-1R interaction.
To block the IGF autocrine loop, we first used anti-IGF-I and
anti-IGF-II mAb, which inhibit the binding of both ligands to the
IGF-1R (29). Incubation of cells with these mAb at concentrations as
high as 100 µg/ml for 3 days does not inhibit DU-145 serum-free
growth, as measured by [3H]TdR incorporation (Fig.
4A). The failure of anti-IGF-I
mAb to inhibit DU-145 cell proliferation is not a consequence of lack of activity, since this mAb inhibits IGF-I-induced Ba/F3 cell proliferation at the same level as IGFBP-3 in Ba/F3 cells (Fig. 4C). The most probable explanation for the difference in the
results with the IGF mAb in DU-145 and Ba/F3 cell lines is the presence of IGFBP in the DU145-CM, which may compete with antibodies for IGF
binding.

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Fig. 4.
Effect of anti-IGF-I mAb, anti-IGF-I plus
anti-IGF-II mAb, IR-3, or IGFBP-3 on DNA
synthesis in DU-145 cells. A, DU-145 cells were
transferred for 24 h to SFM/BSA containing IGFBP-3 ( ), IR-3
( ), anti-IGF-I mAb BB9E10 ( ), anti-IGF-II mAb (×), or
anti-IGF-I + anti-IGF-II mAb ( ). Cells were pulsed for 8 h with
[3H]TdR, and DNA-incorporated radioactivity was
determined. All assays were carried out in triplicate. Where
error bars are not seen, the standard error is smaller than
the symbol. Data shown are representative of five independent
experiments. The basal [3H]TdR incorporation value was
12,558 ± 1,001 cpm/well; the cpm/well obtained at the largest
inhibitor dose used were IGFBP-3 (8,067 ± 307), IR-3
(6,902 ± 353), anti-IGF-I mAb (12,396 ± 1,121), anti-IGF-II
mAb (12,098 ± 524), and anti-IGF-I + anti-IGF-II mAb (12,234 ± 1,152). Intra- and interassay statistical significance was
calculated using the Kruskal-Wallis and the Mann-Whitney tests with all
data obtained (**, p < 0.01). Similar results were
obtained when cells were incubated for 48 and 72 h under the same
conditions. B, DU-145 cells starved as above were incubated
with IGFBP-3 ( ), IGFBP-3 preincubated with IGF-I ( ), or IGFBP-3
preincubated with des-(1-3)IGF-I ( ). Cells were then pulsed for
8 h with [3H]TdR, and radioactivity was incorporated
into DNA determined. As above, all assays were performed in triplicate
and where error bars are not seen, the standard error is smaller than
the symbol. The data shown are representative of five (in the case of
IGF-I) or three (for des-(1-3)IGF-I) independent experiments. The
cpm/well of [3H]TdR incorporation values were 17,839 ± 1,235 for basal incorporation, 10,014 ± 282 for IGFBP-3,
14,269 ± 260 for IGFBP-3/IGF-I, and 16,173 ± 501 for
IGFBP-3/des-(1-3)IGF-I. Statistical analyses were performed as in
A (*, p < 0.05; **, p < 0.01). C, Ba/F3 cells were withdrawn from interleukin-3 and cultured for 20 h with
IGF-I (250 ng/ml) alone (control, 100%) or in the presence of IGFBP-3
( ), IGFBP-1 ( ), or anti-IGF-I mAb BB9E10 (×) at the
concentrations indicated and then pulsed for 6 h with
[3H]TdR. In all cases, data given are the percentage
(n = 5) of [3H]TdR incorporated, divided
by the control (absence of competitor).
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The antagonistic anti-IGF-1R mAb
IR-3 inhibits cell proliferation by
40% at 10 nM (Fig. 4A). This contradicts
earlier data showing that
IR-3 blocks IGF-II-dependent
growth but not DU-145 serum-free proliferation (26). We also analyzed
the effect of exogenous hIGFBP-3 on DU-145 cell proliferation. IGFBP-3
at 20 nM causes a 40% inhibition of DNA synthesis (Fig.
4A), and proliferation is partially restored when IGFBP-3 is
preincubated with a molar excess of IGF-I (Fig. 4B). The
des-(1-3)-IGF-I variant, which binds to the IGF-1R as does IGF-I, but
does not bind to IGFBP, reverses IGFBP-3-induced DU-145 growth
inhibition almost completely (Fig. 4B). This suggests that
IGFBP-3 does not exert IGF-independent effects in these cells.
In accordance with the reduction in [3H]TdR
incorporation, incubation with IGFBP-3 promotes a significant reduction
in the percentage of cells in the G2/M peak (8 ± 0.5%) when compared with addition of anti-IGF-I plus anti-IGF-II mAb
(17 ± 1.3%) or controls (20 ± 0.8%) (Fig.
5A). This is associated with
an increase in the sub-G0/G1 peak (20 ± 0.3% compared with 12.3 ± 2.1% in controls). Furthermore,
IGFBP-3 promotes a drastic decrease in p34cdc2 levels compared
with control or anti-IGF antibody-treated cells (Fig. 5B).
This suggests that cell treatment with IGFBP-3 interferes with the
pathway regulating Cdc-2 synthesis or stability. The same blot was
reprobed with an anti-p53 mAb as an internal protein loading
control, exploiting the constitutive overexpression of a mutant p53
form by DU-145 cells (39) (Fig. 5C).

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Fig. 5.
Exogenous IGFBP-3 modifies cell cycle
parameters. A, analysis of the cell cycle distribution
of DU-145 cells incubated with SFM/BSA (No additions) or
SFM/BSA supplemented with anti-IGF-I plus anti-IGF-II antibodies or
IGFBP-3 (20 nM). DU-145 cells were incubated for 72 h
with additions as indicated in the upper right corner and
then harvested and stained with propidium iodide as described under
"Experimental Procedures." Cell number is depicted on the
ordinate and DNA content, as measured by propidium iodide
intensity after doublet exclusion, on the abscissa. The
percentage of cells in each phase of the cycle is also indicated.
B, p34cdc2 expression in DU-145 cells. DU-145 cells
cultured in serum-free medium were incubated for 72 h with IGFBP-3
(lane 1), anti-IGF-II mAb (lane 2), anti-IGF-I
mAb BB9E10 (lane 3), anti-IGF-I plus anti-IGF-II mAb
(lane 4), or an irrelevant antibody (lane 5).
Cell lysates were prepared as described under "Experimental
Procedures," and Western blot analyses were performed. C,
as an internal control, the filters in B were incubated with
an anti-human p53 antibody. Similar results were obtained in three
independent experiments.
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To ascertain that
IR-3 and IGFBP-3 effects on DU-145 cell
proliferation are mediated by interference with the IGF-1R signaling pathway, we measured the IGF-1R autophosphorylation level. Treatment of
DU-145 cells with
IR-3 mAb promotes a biphasic effect on IGF-1R autophosphorylation; at short incubation times,
IR-3 significantly increases the IGF-1R
-subunit phosphorylation level, which is progressively reduced when incubation time is increased (Fig. 6A). The final balance is
that, after 20 h of incubation,
IR-3 significantly reduces the
IGF-1R-mediated signaling in DU-145. On the other hand, incubation of
DU-145 cells with IGFBP-3 (20 nM) results in a decrease in
IGF-1R
-subunit phosphorylation as compared with untreated cells;
autophosphorylation can be recovered by co-addition of IGF-I with
IGFBP-3 (Fig. 6B). As expected from cell growth assays, the
anti-IGF-I or anti-IGF-I plus anti-IGF-II mAb treatment does not induce
changes in
-subunit autophosphorylation (data not shown).

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Fig. 6.
Analysis of IGF-1R autophosphorylation in
DU-145 cells treated with anti-IGF-1R mAb or IGFBP-3.
A, effect of IR-3 mAb on IGF-1R signaling. Equal amounts
of cell lysates from untreated or IR-3-treated cells for the times
indicated were resolved in SDS-PAGE, and tyrosine-phosphorylated
(PY) proteins were detected by Western blot
(w.b.) as described in Fig. 3. After stripping, the membrane
was reprobed with an anti-IGF-1R -subunit antibody (lower
panel). Arrow indicates the position of the 98-kDa
phosphoprotein representing the phosphorylated IGF-1R -subunit.
Densitometric measurements of anti-phosphotyrosine (solid
bars) or anti-IGF-1R -subunit blots (hatched bars)
are displayed in arbitrary units in the right panel. Data
are representative of three independent experiments; the error
bars in the densitometric analysis represent the standard
deviation of all data obtained. B, IGFBP-3 interferes with
IGF-1R autophosphorylation. Serum-starved DU-145 cells were transferred
for 24 h to SFM/BSA without additions or with 20 nM of
IGFBP-3, 35 nM IGF-I, or IGFBP-3 plus IGF-I, as indicated.
After washing, cell lysates were prepared, immunoprecipitated
(i.p.) with IR-3, and blotted proteins were sequentially
developed either with anti-phosphotyrosine (PY) or
anti-IGF-1R -subunit antibodies. The densitometric analysis of the
anti-phosphotyrosine (solid bars) or the anti-IGF-1R
-subunit blots (hatched bars) is displayed in arbitrary
units in the right panel. The data represent two independent
experiments, and the standard deviation represented was calculated from
both experiments. In both A and B, * indicates a
significant difference between the intensity of the phosphotyrosine and
the IGF-1R bands.
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MMP-9 Regulates DU-145 Proliferation by IGFBP-3
Proteolysis--
To analyze the consequences of IGFBP-3 proteolysis on
cell proliferation, we tested the effect of various protease inhibitors on serum-free DU-145 cell growth (Fig.
7A). Both BB-94 and tissue inhibitor of metalloproteinase (TIMP)-2, two MMP inhibitors, produce a
significant reduction of DNA synthesis in a dose-dependent
fashion, whereas the serine protease inhibitor aprotinin does not
modify DU-145 cell proliferation. Addition of IGF-I together with the MMP inhibitors restores DU-145 cell proliferation (Fig. 7B).
MMP inhibitor-promoted DU-145 growth abrogation correlates with the absence of IGFBP-3 proteolytic fragments detected in the conditioned medium of these cells (Fig. 7C), thus linking the
antiproliferative effect of the MMP inhibitors and their interference
in the IGF/IGF-1R axis by preventing IGFBP-3 proteolysis. Cell cycle
analysis of DU-145 cultured with protease inhibitors revealed that, as
in the case of IGFBP-3 addition, TIMP-2 and BB-94 treatment reduce the
percentage of cells in G2/M compared with untreated or
aprotinin-treated cells (Fig. 7D).

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Fig. 7.
An MMP controls DU-145 cell
proliferation. A, serum-starved DU-145 cells were
transferred for 24 h to SFM/BSA containing different
concentrations of TIMP-2 ( ), BB-94 ( ), or aprotinin ( ). Cells
were pulsed for 8 h with [3H]TdR, and
DNA-incorporated radioactivity was determined. All assays were carried
out in triplicate. Where error bars are not seen, the standard error is
smaller than the symbol. The data shown are representative of five
independent experiments. The basal [3H]TdR incorporation
value was 17,718 ± 1,330 cpm/well; the cpm/well obtained at the
largest inhibitor dose used were as follows: aprotinin (17,288 ± 1,954), BB94 (7,863 ± 1,508), and TIMP-2 (4,228 ± 256).
Intra- and interassay statistical significance was calculated using the
Kruskal-Wallis and the Mann-Whitney tests with all data obtained (**
p < 0.01). B, serum-starved DU-145 cells
were treated for 24 h with different amounts of IGF-I, in the
presence of BB-94 (2 µM) or TIMP-2 (5 µM)
and processed as in A. The [3H]TdR
incorporation measured in the absence of protease inhibitors and IGF-I
was considered as 100%. All assays were carried out in triplicate.
Data shown are representative of five independent experiments.
C, DU145-CM was recovered from subconfluent DU-145 cultures
grown for 2 days in SFM containing aprotinin, TIMP-2, or BB-94 and
concentrated 10-fold prior to analysis in SDS-PAGE under reducing
conditions. Western blot was performed using anti-IGFBP-3 antibody as
described in Fig. 1. Arrows indicate the position of the
intact IGFBP-3, as well as the 30- and 19-kDa IGFBP-3 proteolytic
fragments. This experiment is representative of two independent assays.
D, cells cultured as in C were harvested and
stained with propidium iodide, and the cell cycle distribution was
analyzed. Cell number is depicted on the ordinate and DNA
content, as measured by propidium iodide intensity after doublet
exclusion, on the abscissa. The percentage of cells in each
phase of the cycle is also indicated.
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To identify the MMP activity responsible for DU-145 proliferation,
DU145-CM was analyzed by gelatin zymography. Zymograms indicate the
presence of a 92-kDa gelatinolytic activity in DU145-CM, identified
with specific antibodies as MMP-9 (Fig.
8A). The localization of
soluble MMP to the surface of normal and tumor cells has been reported
previously (40, 41). We thus examined whether MMP-9 secreted by DU-145
cells can also be localized to the membrane. Fig. 7B shows
the kinetics of a labeled MMP substrate degradation in starved DU-145
cells. This fluorogenic substrate, specific for several MMP, has been
designed such that its fluorescence remains >98% quenched in the
uncleaved product (42); a fluorescence increase thus indicates MMP
activity. Degradation of the substrate increases with time (Fig.
8B), and this MMP activity is partially inhibited by the
specific MMP inhibitor BB-94 but not by aprotinin. Affinity
cross-linking of cell-surface proteins was performed, and cell lysates
were analyzed in Western blot using anti-MMP-9 mAb (Fig.
8C). A major band of 150 kDa and another minor 130-kDa band
are observed in the cross-linked cells, in addition to the 92-kDa form
that is detected in both cross-linked and non-cross-linked DU-145
cells. To establish further the association of MMP-9 to the DU-145 cell
surface, non-cross-linked cells were stained with anti-MMP-9 and
analyzed by flow cytometry (Fig. 8D). The incubation of
DU-145 with an anti-MMP-9 mAb promotes a shift in the fluorescence intensity associated to the cells compared with that obtained using
either irrelevant or anti-MMP-2-specific antibodies. These results
indicate the presence of a membrane-bound form of MMP-9.

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Fig. 8.
DU-145 cells secrete MMP-9 that is anchored
to the DU-145 cell surface. A, DU145-CM from
subconfluent cultures was obtained, concentrated, and analyzed by
gelatin zymography and Western blot using anti-MMP-9 as a primary
antibody. For zymography, the human fibrosarcoma HT-1080 was used as
control, since it produces large amounts of both MMP-2 and MMP-9
following phorbol 12-myristate 13-acetate stimulation (74).
B, DU-145 monolayer was washed five times with PBS, and
DU-145 cell-associated MMP-like proteolytic activity was measured in
the presence of 0.5 µM BB-94 ( ), 10 µg/ml aprotinin
( ), or without additions ( ) using a fluorogenic substrate. The
values are expressed as a percentage of the signal obtained at time
0 h (100%). C, affinity cross-linking of DU-145
membrane proteins. DU-145 cells starved for 72 h were washed three
times and affinity cross-linked as described under "Experimental
Procedures." Cell lysates at equal protein concentrations were
electrophoresed in SDS-PAGE, transferred to nitrocellulose, and
incubated with an anti-MMP-9 antibody. Non-cross-linked DU-145 cell
lysate was run in parallel. DSS, disuccinimidyl suberate.
D, subconfluent DU-145 cells, starved for 48 h, were
harvested with EDTA and washed with sterile PBS containing 1 mM CaCl2 and 2 mM
MgCl2. The cells were incubated with antibodies as
indicated, followed by a secondary fluorescein isothiocyanate-labeled
anti-mouse IgG antibody. The binding of an irrelevant isotype-matched
antibody or an anti-MMP-2 mAb was considered background (solid
area).
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To specifically inhibit MMP-9 activity in DU-145 cells, antisense
expression studies were performed. pEFBOS-MMP-9AS-transfected DU-145
cells showed drastically reduced proliferation compared with
untransfected cells or cells transfected with the empty vector; furthermore, the inhibition caused by antisense expression can be
reversed by addition of exogenous IGF-I in a dose-dependent manner (Fig. 9A). Addition of
IGFBP-3 to pEFBOS-MMP-9AS transfected DU-145 cells does not result in
increased growth inhibition (Fig. 9B), suggesting that
IGFBP-3 inhibitory effects are dependent on MMP-9 activity. In fact,
pEFBOS-MMP-9AS-transfected DU-145 cells show a drastic reduction in MMP
membrane-associated activity (Fig. 9C), indicating that the
MMP-9 antisense effectively inhibits MMP-9 expression. A reduction in
gelatinolytic activity is also observed in MMP-9 antisense DU145-CM
(data not shown).

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Fig. 9.
MMP-9 specifically controls DU-145 cell
proliferation. A, DU-145 cells were transiently
transfected with pEFBOS-MMP-9AS ( ) or with the empty vector ( ),
were serum-starved, and DNA incorporated [3H]TdR in the
absence or presence of IGF-I was determined, as described under
"Experimental Procedures." The [3H]TdR incorporation
in untransfected DU-145 cells cultured at similar cell density under
the same conditions was considered as 100%. All experiments were
performed in triplicate, and data are representative of five
independent experiments. B, cells as in A were
cultured alone (solid bars) or with IGFBP-3 at a final
concentration of 20 nM (hatched bars), and the
[3H]TdR incorporation into DNA was measured as in
A. C, MMP-like proteolytic activity associated to
DU-145 cell membrane in cells transfected with pEFBOS-MMP-9AS ( ) or
the empty vector ( ) was determined as in Fig. 8B.
D, DU145-CM from pEFBOS-MMP-9AS- or empty vector-transfected
cells was collected after 72 h in SFM culture was concentrated
10-fold and analyzed for IGFBP-3 proteolysis in Western blot, as in
Fig. 7. Arrows indicate the position of the intact IGFBP-3,
as well as the 30- and 19-kDa IGFBP-3 proteolytic fragments.
E, DU-145 transfected with pEFBOS-MMP-9AS or the empty
vector were starved for 24 h and then treated with IGF-I (1 µg/ml) for 5 min or left untreated. Cell lysates were
immunoprecipitated (i.p.) with IR-3 mAb, and the blotted
proteins developed with anti-phosphotyrosine (PY) mAb
(upper panel) and then stripped and rehybridized with an
antibody specific for the IGF-1R -subunit (lower panel).
The position of the 98-kDa band shown phosphoprotein represents the
phosphorylated IGF-1R -subunit. The image shown is representative of
three independent experiments. w.b., Western blot.
F, the Western blot membranes described in E were
analyzed by densitometry. The densitometric units measured for the
anti-phosphotyrosine (solid bars) or the anti-IGF-1R
-subunit blots (hatched bars) are displayed in arbitrary
units (a.u.) (** p < 0.01).
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The inhibition promoted by MMP-9 antisense expression correlates with a
decrease in IGFBP-3 proteolysis (Fig. 9D). Furthermore, serum-starved pEFBOS-MMP-9AS-transfected cells show reduced
IGF-triggered cell signaling, observed as a decrease in the tyrosine
phosphorylation of the 98-kDa IGF-1R
-subunit as compared with empty
vector-transfected cells (Fig. 9E). Densitometric analysis
of Western blots demonstrates that, although the immunoprecipitated
IGF-1R levels are comparable, pEFBOS-MMP-9AS DU-145 cells show a 4-fold
decrease in tyrosine-phosphorylated IGF-1R
-subunit (Fig.
9F). Tyrosine phosphorylation at control levels is recovered
in pEFBOS-MMP-9AS DU-145 cells by addition of exogenous IGF-I,
indicating that MMP-9 antisense treatment interferes with the
bioavailability of IGF to DU-145 cells.
 |
DISCUSSION |
Autocrine growth control has been considered to be a mechanism by
which tumor cells proliferate autonomously (2). Recent reports have
claimed a role for IGF ligands and receptors in the establishment and
maintenance of the tumor phenotype (43), and studies dealing with IGF
expression in human cancer cell lines have revealed that IGF autocrine
loops may be operating in the majority of epithelial cancer cell lines
(44). In addition to ligands and IGF-1R, the concurrent secretion of
IGFBP, usually in a large excess over IGF ligands, has been also
reported in the same cell lines (45); however, the physiological
significance of IGFBP expression by cancer cells is still unclear.
Although several groups have studied the role of the IGF autocrine loop
using neoplastic prostate cells, there are substantial contradictory
results regarding the expression and mitogenic effects of IGF ligands
and IGFBP in DU-145 cells. For example, Pietrzkowski et al.
(36) reported both IGF-1R and IGF-I immunoreactivity in DU145-CM.
Conversely, Iwamura et al. (25) observed that IGF-I is a
potent DU-145 mitogen but failed to establish the presence of IGF-I in
conditioned medium from these cells. Connolly and Rose (46) found
neither IGF-I nor IGF-II expression by DU-145 cells but
demonstrated IGFBP production. With an RNase protection assay, Figueroa et al. (26) detected mRNA encoding
IGF-II, IGF-1R, IGF-2R, and IGFBP-2-6 but not that coding for IGF-I.
Employing a highly specific RT-PCR, we find mRNA expression for
both IGF-I and IGF-II as well as for IGF-1R, and we also encounter the
IGF-1R on the cell surface. In addition, the IGF-I D-domain-specific mAb KM5A1, which binds to IGF-I when complexed either with IGFBP or
IGF-1R, detects IGF-I bound to the surface of starved DU-145 cells.
This indicates that DU-145 cells secrete IGF-I. The loss of KM5A1
binding to cells cultured in the presence of insulin implies that
autocrine IGF-I binds mainly to IGF-1R on DU-145. We have no similar
IGF-II-specific antibody to monitor the binding of this growth factor
to the DU-145 membrane, but it is assumed to be similar to IGF-I.
DU-145 cells are reported to grow in the absence of external growth
factors (25, 26, 36, 46, 47). To ascertain whether the serum-free
growth of DU-145 is due to the autocrine action of secreted IGF, these
cells were exposed to the anti-IGF-1R mAb,
IR-3. This antibody is
reported to mimic IGF activity in some cell types that overexpress the
IGF-1R (48, 49), although it has been widely reported to be an IGF
antagonist in a large number of cell lines (50-57). In addition,
IR-3 inhibits tumor growth in vivo, probably by blocking
the IGF-1R (58-60). In agreement with this latter set of publications,
incubation of DU-145 cells with
IR-3 significantly inhibited DNA synthesis.
To analyze further the inhibitory effect of
IR-3 on DU-145 cells, we
examined IGF-1R
-subunit phosphorylation status. A short incubation
of the cells with
IR-3 results in increased IGF-1R
autophosphorylation, suggesting receptor activation. When the cells are
exposed to the antibody for longer periods, however,
IR-3 reduces
the IGF-1R autophosphorylation level compared with the basal conditions
(i.e. absence of exogenous factors).
IR-3 is reported to
stimulate IGF-1R autophosphorylation (49), although this
"activation" does not necessarily lead to a cellular response (53).
Moreover, Steele-Perkins et al. (48, 61) reported that
IR-3 alone induced cell proliferation and IGF-1R
autophosphorylation, although when
IR-3 is added with IGF-I or
IGF-II, the proliferation and IGF-1R autophosphorylation induced by
these factors are blocked in this cell line. It is therefore possible
that
IR-3 binding to the IGF-1R induces IGF-1R autophosphorylation
by cross-linking the receptor at the cell surface but, as a consequence
of this binding, a conformational change occurs that locks the receptor into a state that incapable of further signaling (61). This also may
account for the result obtained in DU-145 cells, which constitutively
produce both IGF-I and IGF-II. Nonetheless, further experiments are
required to confirm this viewpoint.
Incubation of DU-145 cells with several anti-IGF-I mAb, alone or in
combination with an anti-IGF-II mAb, failed to block the serum-free
growth of DU-145 cells, although these mAb are effective inhibitors of
IGF-induced cell survival and proliferation in several cell lines (28,
29); the interference by IGFBP in the DU145-CM might abolish the
inhibitory capacity of these anti-IGF mAb.
Normal prostate epithelial cells secrete IGFBP-2 and
IGFBP-4, whereas stromal fibroblasts produce IGFBP-2, -3, and
-4 (62, 63). IGFBP-3 has been found, however, in human prostate
epithelial cell culture medium by others (64). Another
androgen-independent prostate adenocarcinoma, PC-3, was reported to
secrete IGFBP-3 and other IGFBP species (54). We observe secretion of
at least three different IGFBP species (IGFBP-2, -3, and -4) in DU-145 cells, but only IGFBP-3 is proteolyzed. Moreover, the addition of
exogenous hIGFBP-3 to DU-145 inhibits the serum-free growth of this
cell line; co-addition with exogenous IGF-I partially reverses this
effect. Concurring with the reduction in DNA synthesis, exogenous
IGFBP-3 promotes a decline in the number of cells in G2/M
phases and a decrease in p34cdc2 levels. In vivo
treatment of human rhabdomyosarcoma with an antagonist anti-IGF-1R
antibody has been reported to down-regulate levels of p34cdc2
(9), a protein required for G2 transition to mitosis (65). The IGFBP-3 inhibitory effect is thus probably mediated by
interference with IGF-induced IGF-1R activation. This view concurs with
our data showing that the des-(1-3)IGF-I variant reverses
IGFBP-3-induced DU-145 growth inhibition and that IGFBP-3 treatment
reduces the level of IGF-1R
-subunit autophosphorylation.
Our data therefore support an inhibitory role for exogenous IGFBP-3 in
DU-145 cell proliferation. The question that arises is why DU-145
secretes an inhibitory IGFBP. Autocrine production of IGFBP-3 by DU-145
cells may modulate ligand-receptor interactions at the cell surface,
thus regulating cell responses to locally produced IGF. This modulation
is probably more puzzling than simple interference with the IGF/IGF-1R
interaction, however, since the anti-IGF-I and anti-IGF-II antibodies
used here have no effect on DU-145 proliferation. IGF-independent
effects of IGFBP-3 have been reported for other tumor cell lines (35,
66), and receptors for this binding protein are also found in some
tumor cells (20). However, des-(1-3)IGF-I reverses IGFBP-3-induced
DU-145 growth inhibition, suggesting that IGFBP-3 has no
IGF-independent effect on this cell line.
The IGFBP enhancer mechanism is poorly understood but usually involves
their proteolytic processing, leading to a diminished affinity for IGF
ligands (22, 45). We found that DU-145 secretes MMP-9, a
metalloproteinase that degrades IGFBP-3 (67). The control of
DU-145 serum-free growth by MMP-9 is relevant, since overexpression of
an antisense cDNA for MMP-9 blocks almost 80% of DNA synthesis, an
effect also observed by the addition of nontoxic concentrations of MMP
inhibitors such as BB-94 or TIMP-2. Our findings indicate that the
antiproliferative effect obtained by inhibiting MMP-9 expression is
related to the inaccessibility of IGF to the IGF-1R, since (i) addition
of IGF-I to either pEFBOS-MMP-9AS-transfected or MMP inhibitor-treated
cells restores DU-145 proliferation in a dose-dependent
manner; (ii) MMP-9 antisense expression reduces autocrine IGF-triggered
cell signaling through the IGF-1R, which is recovered by incubation
with exogenous IGF-I; (iii) the growth inhibition correlates with the
disappearance of IGFBP-3 proteolytic fragments, and (iv) IGFBP-3 does
not promote an additive growth inhibitory effect on
pEFBOS-MMP-9AS-transfected DU-145 cells.
By using PC-3 cells, Angelloz-Nicoud and Binoux (54) documented
inhibition of cell proliferation by adding a serine protease inhibitor
able to block urokinase-type plasminogen activator activity detected in
the conditioned medium. In our study, incubation of DU-145 with
aprotinin results neither in growth inhibition nor in the abolition of
IGFBP-3 proteolysis. The involvement of kallikrein-type proteases in
this process cannot be ruled out, but DU-145 does not produce
prostate-specific antigen (68).
We demonstrate the existence of MMP-9 activity associated to the DU-145
cell surface using a fluorogenic substrate and FACS analysis. Affinity
cross-linking also suggests the physical association of MMP-9 to a
cellular receptor of about 60 kDa. Recent reports show cell-surface
localization of proteinases, including urokinase-type plasminogen
activator and MMP-2, on a variety of cell types both in
vitro and in vivo (41, 69, 70). Little is known,
however, regarding the biochemical interactions between MMP and
cell-surface receptors. The association of MMP-2 with
v
3 integrin has been described (41), but
reprobing of Western blots from DU-145 cross-linked cells with several
anti-
and -
integrin chain antibodies failed to show this
MMP-9/integrin association. A membrane-type MMP, called MT-MMP, has
recently been identified (71) and has been implicated in the
localization and activation of MMP-2 on the cell surface, in
conjunction with TIMP-2 (40). MMP-9 binding to the DU-145 cell surface
may thus be a step in MMP-9 activation, after which IGFBP-3 is degraded.
Our data support the idea that IGFBP-3 is an important regulator of
prostate cancer cell growth. It can be conjectured that autocrine IGF-I
and IGF-II are bound to IGFBP-3, which regulates liberation of growth
factors in close proximity to the IGF-1R following MMP-9 proteolysis.
The novelty of this report is the demonstration that optimal DU-145
growth is the result of a balance between autocrine secretion of
IGF ligands, IGFBP, and MMP levels. When this equilibrium is
upset, for example by addition of exogenous IGFBP-3 or inhibition of
endogenous MMP-9, a reduction in cell proliferation results. Exogenous
IGFBP-3-induced growth inhibition is thus not due to competitive
scavenging of IGF ligands away from the IGF-1R but to the competition
of MMP-9 activity with "unfilled" IGFBP-3.
This concurs with previous results in which androgen-induced growth
inhibition of PC-3 cells transfected with a constitutively activated
androgen receptor coincides with the complete absence of IGFBP-3 in the
culture media of these cells (72). Treatment of these transfected PC-3
cells with IGFBP-3 and IGF-I or IGF-II resulted in a proliferation rate
greater than that observed with IGF-I or -II alone. Accumulated
evidence indicates, however, that IGFBP-3 cell-surface association is
required for the enhancement of IGF action, and factors increasing this
membrane association also intensify the IGFBP-3 potentiating effect
(73). Furthermore, IGFBP-3 that adhered to the cell surface was
processed to lower molecular weight forms with decreased affinity for
IGF (19). Membrane-bound MMP-9 may therefore act as an IGFBP-3 receptor enabling this enhancing mechanism.
In summary, we show compelling evidence for an autocrine loop,
operative in DU-145 cells, for both IGF-I and IGF-II. This loop seems
not only to implicate both IGF ligands and IGF-1R but also IGFBP as
well as MMP-9, which specifically cleave IGFBP-3. Anchorage of MMP-9 to
the cell surface may thus provide a mechanism to coordinate IGFBP-3
proteolysis with increased IGF availability in close proximity to the
IGF-1R. The study of this pathway could lead to the design of new
anti-tumor agents for tumors resistant to other therapies and to a
better understanding of complex processes such as tumor growth and metastasis.
 |
ACKNOWLEDGEMENTS |
We thank M. C. Rodríguez for
preparing TIMP-2; Dr. P. Gellerfors for providing des-(1-3)-IGF-I; Dr.
F. Colotta for BB-94; Drs. A. Ullrich and R. Lammers for 3T3-IGF-1R
cells; Dr. G. del Real and M. Obrero for synthesis of oligonucleotides
used in PCR; and I. López-Vidriera, I. Poveda, and C. Mark for
extraordinary FACS, photographic, and editorial assistance. The
Department of Immunology and Oncology was founded and is supported by
the Spanish Research Council (CSIC), Pharmacia & Upjohn.
 |
FOOTNOTES |
*
This work was supported in part by the European Community
Human Capital and Mobility Program Number CHRX-CT94-0556.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: Dept. of Immunology
and Oncology, Centro Nacional de Biotecnología, CSIC,
Universidad Autonoma de Madrid, Campus de Cantoblanco, E-28049 Madrid
Spain. Tel.: 34 91-585-4660; Fax: 34 91-372-0493; E-mail
smanes{at}cnb.uam.es.
2
S. Mañes, M. Llorente, R. A. Lacalle,
C. Gómez-Moutón, L. Kremer, E. Mira, and C. Martínez-A., unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
IGF, insulin-like
growth factor;
IGFBP, IGF-binding protein;
IGF-1R, IGF type-1 receptor;
3T3-IGF-1R, 3T3 fibroblasts overexpressing the human IGF-1R AS
antisense;
CM, conditioned medium;
DMEM, Dulbecco's modified Eagle's
medium;
mAb, monoclonal antibody;
MMP, matrix metalloproteinase;
PBS, phosphate-buffered saline;
PO, horseradish peroxidase;
PAGE, polyacrylamide gel electrophoresis;
SFM, serum-free medium;
TIMP, tissue inhibitor of metalloproteinase;
TdR, thymidine;
BSA, bovine
serum albumin;
RT-PCR, reverse transcriptase-polymerase chain reaction;
FACS, fluorescence-activated cell sorter;
h, human.
 |
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