From the Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
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ABSTRACT |
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Insulin-like growth factor binding protein-3
(IGFBP-3) inhibits proliferation and promotes apoptosis in normal and
malignant cells. In MCF-10A human mammary epithelial cells, 30 ng/ml
human plasma-derived IGFBP-3 inhibited DNA synthesis to 70% of
control. This inhibition appeared IGF-independent, since neither an
IGF-receptor antibody nor IGFBP-6 inhibited DNA synthesis. Malignant
transformation of MCF-10A cells by transfection with Ha-ras
oncogene abolished the inhibitory effect of IGFBP-3, concomitant with
an increase in IGFBP-3 secretion and cell association of approximately
60 and 300%, respectively. When mitogen-activated protein (MAP) kinase activation was partially inhibited using PD 98059, IGFBP-3 sensitivity in ras-transfected cells was restored, with a significant
inhibitory effect at 10 ng/ml IGFBP-3. PD 98059 had no effect on
IGFBP-3 secretion or cell association by ras-transfected or
parent MCF-10A cells. Hs578T, a tumor-derived breast cancer cell line
that expresses activated Ha-ras, similarly has a high level
of secreted and cell-associated IGFBP-3. In the absence of PD 98059, DNA synthesis by Hs578T cells was reduced to 70% of control by 1000 ng/ml IGFBP-3. PD 98059 increased sensitivity to IGFBP-3, so that this
level of inhibition was achieved with 100 ng/ml IGFBP-3. These results
suggest that MAP kinase activation by oncogenic ras
expression causes IGFBP-3 resistance, a possible factor in the
dysregulation of breast cancer cell growth.
The potent growth promoting effects of the insulin-like growth
factors (IGFs)1 are regulated
in all tissues by the six members of a family of structurally and
functionally related proteins, the IGF-binding proteins (IGFBPs), which
bind the IGFs with affinities of between 109 and
1011 liters/mol (1). While sharing considerable amino acid
homology, the IGFBPs undergo considerable post-translational
modification to yield differently glycosylated, phosphorylated, and
proteolytically cleaved products (2). IGFBP-3, a 45-kDa glycoprotein,
is the predominant carrier of the growth factors in the circulation
where, as a component of a 150-kDa complex consisting of IGFBP-3, IGF-I or -II, and the acid-labile subunit, it maintains a circulating reservoir of the IGFs, limits their insulin-like hypoglycemic potential, and helps to control their egress from the circulation to
the extravascular tissues (3). In addition to its presence in the
circulation, IGFBP-3 is found in the pericellular environment of many
tissues, where it functions as a paracrine or autocrine modulator of
the mitogenic effects of the IGFs.
There is now good evidence that IGFBP-3, a p53-inducible protein (4),
inhibits proliferation and promotes apoptosis in normal and malignant
cells by mechanisms that may be independent of its effects on IGF
bioactivity (5-9). An important role for IGFBP-3 in the modulation of
breast cancer cell growth is suggested by the observation that DNA
synthesis and cell proliferation in tumor-derived breast cell lines may
be reduced by treatment with recombinant IGFBP-3 (7). In addition, the
antiproliferative effects of agents as diverse as transforming growth
factor- Materials--
Tissue culture reagents and plasticware were from
Trace Biosciences (North Ryde, New South Wales, Australia) and Nunc
(Roskilde, Denmark). Bovine serum albumin (BSA), bovine insulin,
hydrocortisone, epidermal growth factor (EGF), and protein A were
purchased from Sigma. Cholera enterotoxin was purchased from ICN
Biomedicals Australasia (Seven Hills, New South Wales, Australia). The
MAP kinase inhibitor PD 98059, and monoclonal antibody against type 1 IGF receptor, Cell Culture--
The MCF-10A cell line, and its control
vector-transfected and Ha-ras-transfected derivatives, were
the kind gift of Drs. Robert Pauley and Herbert Soule at the Karmanos
Cancer Institute, Detroit, MI (15). The parental, vector-transfected
and oncogenic ras-transfected cells were used between
passages 158-162, 31-35 (post-transfection), and 26-30
(post-transfection), respectively. Cells were maintained in DMEM/F-12
containing 15 mM Hepes, 5% horse serum, 10 µg/ml bovine
insulin, 20 ng/ml EGF, 100 ng/ml cholera enterotoxin, and 0.5 µg/ml
hydrocortisone. The Hs578T cell line (used between passages 63 and 68)
was purchased from ATCC and maintained in RPMI containing 15 mM Hepes, 5% fetal bovine serum, and 10 µg/ml bovine insulin.
[3H]Thymidine Incorporation--
For analysis of
DNA synthesis, confluent cultures of cells in 24-well plates were
changed to Hepes-buffered DMEM/F-12 containing 0.1% BSA for 48 h
prior to addition of treatments. Spent media were replaced by fresh
serum-free medium containing additives as indicated for individual
experiments, and incubations were continued for 20 h. One
µCi/well [3H]thymidine (35 Ci/mmol, ICN) was added in
50 µl of medium for a further 4-h incubation at 37 °C. Monolayers
were rinsed twice with ice-cold saline and fixed with 1 ml/well
ice-cold methanol:acetic acid (3:1) at 4 °C for a minimum of 2 h. Cells were solubilized in 0.5 ml of 5 g/liter of sodium dodecyl
sulfate (SDS), and 250 µl of each lysate mixed with scintillant
(OptimaGold, Amersham Pharmacia Biotech) before counting for 2 min in a
Hewlett-Packard Determination of Secreted and Cell-associated
IGFBP-3--
IGFBP-3 concentrations in conditioned media were
determined by radioimmunoassay as previously reported (10).
Cell-associated IGFBP-3 was assayed on intact monolayers using an
immunological technique described in an earlier study (16). Briefly,
spent media from cells treated in 24-well plates were removed for assay and replaced by 1 ml of fresh medium containing anti-IGFBP-3 serum or
nonimmune serum (1:5000 dilution). After overnight incubation at
22 °C, media were aspirated, and 1 ml of medium containing 20,000 cpm 125I-labeled protein A was added for 2-3 h at
22 °C. Cells were then washed twice with serum-free medium,
solubilized with 5 g/liter SDS, and lysates counted for 2 min in a
Hewlett-Packard SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western
Blotting--
Cell-conditioned media from control and IGFBP-3-treated
cells were fractionated by 12% SDS-PAGE under nonreducing conditions as described previously (16). Separated proteins were transferred to
Hybond-C nitrocellulose (Amersham Pharmacia Biotech, Bucks, United
Kingdom) over 2 h at 250 mA using a Novablot transfer unit (Amrad,
Ryde, New South Wales, Australia). Blots were incubated in blocking
buffer (TBS, 10 mM Tris-HCl, pH 7.4, containing 9 g/liter
NaCl, 10 g/liter BSA, 0.05% Nonidet P-40, and 0.2 g/liter sodium
azide) for 3 h at 22 °C, then probed with
125I-labeled IGF-I (1 × 106 cpm) diluted
in blocking buffer, overnight at 22 °C. Blots were washed in TBS
containing 0.05% Nonidet P-40, before autoradiography using Hyperfilm
MP (Amersham Pharmacia Biotech) for 1 day at
Phosphorylated p44/42 MAPK was detected in cell lysates by
immunoblotting using Thr202/Tyr204 polyclonal
antibody (New England Biolabs). Cells treated with PD 98059 were lysed
in SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 2%
SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromphenol blue), sonicated for 15 s, and heated at 95 °C for 5 min. Fifty µL of each lysate was fractionated on a 10% gel, before transfer to
nitrocellulose. Blots were blocked using 5% skim milk in TBS containing 0.1% Tween 20, then probed with the
phospho-Thr202/Tyr204 p44/42 antibody (1:1000
dilution) in the same buffer overnight at 4 °C and proteins detected
using enhanced chemiluminescence (Supersignal ECL, Pierce). Blots were
then stripped by submersion in 62.5 mM Tris-HCl, pH 6.7, containing 100 mM 2-mercaptoethanol and 20 g/liter SDS for
30 min at 55 °C, then reprobed using p44/42 kinase antibody (1:1000
dilution) and ECL as before. Band densities on developed films were
measured using National Institutes of Health imaging software.
Statistics--
All experiments were conducted in triplicate or
quadruplicate at least twice; results shown are pooled data from all
similar experiments unless otherwise indicated. Statistical
significance was determined by analysis of variance and Fisher's PLSD
on pooled data using Statview software for Macintosh (SAS Institute, Inc).
The MCF-10A cell line is a spontaneously immortalized,
phenotypically normal breast epithelial line (17). When these cells were treated with human plasma-derived IGFBP-3 for 24 h, DNA
synthesis (determined by incorporation of tritiated thymidine) was
maximally inhibited to ~70% of control levels at 30 ng/ml IGFBP-3
(p < 0.01 compared with control), with a significant
effect apparent with 10 ng/ml IGFBP-3 (p < 0.05) (Fig.
1A). This effect of IGFBP-3 was not due to the binding protein sequestering endogenous IGFs and
inhibiting their mitogenic effects, because the monoclonal antibody
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
), retinoic acid, vitamin D analogs, and
anti-estrogens is accompanied by induction of IGFBP-3 (6, 10-12), and
prevention of this induction with antisense oligonucleotides restores
DNA synthesis. Very little is known of the mechanisms by which IGFBP-3
exerts these effects, however, and the factors involved in regulating
cell sensitivity to IGFBP-3 are completely uncharacterized. We now show
that breast epithelial cells expressing a constitutively active Ras
protein are resistant to IGFBP-3 and that abrogation of the MAPK/ERK
signaling pathway restores sensitivity to IGFBP-3. These findings
implicate the Ras-MAPK/ERK pathway in the development of IGFBP-3
insensitivity in breast cancer.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IR-3, were obtained from Calbiochem-Novabiochem (Alexandria, New South Wales, Australia). Polyclonal antibodies against
Thr202/Tyr204 phosphorylated and total p44/42
MAP kinase were from New England Biolabs (Beverley, MA). Recombinant
human TGF-
1 was purchased from Austral Biologicals (San Ramon, CA),
and recombinant human IGF-I was the gift of Pharmacia and Upjohn
(Stockholm, Sweden). Human IGFBP-3 was purified from Cohn fraction IV
of human plasma and IGFBP-6 from SV-40-transformed
fibroblast-conditioned medium, as described previously (13, 14).
Electrophoresis reagents were purchased from Bio-Rad and
Amrad-Pharmacia (Ryde, New South Wales, Australia). Protein A and IGF-I
were radiolabeled with 125I (ICN) using chloramine T.
counter.
counter. Cell-associated IGFBP-3 was determined
from the total amount of radioactive protein A present in cell lysates
and reflected the amount bound to IGFBP-3-antiserum complexes on the cells.
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IR-3, which acts as an IGF antagonist at the type 1 IGF receptor
(18), had no significant effect on basal DNA synthesis in MCF-10A cells
(Fig. 1B); at the concentration used (5 µg/ml) it was able
to fully reverse stimulation by 5 ng/ml IGF-I (Fig. 1B).
Similarly IGFBP-6, which binds both IGF-I and IGF-II with high
affinity, did not inhibit basal DNA synthesis when added at 100 ng/ml,
but blocked the stimulatory effect of IGF-I (Fig. 1B).
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Fig. 1.
IGFBP-3 inhibition of DNA synthesis in breast
cells. MCF-10A cells were treated with IGFBP-3 at the
concentrations shown for 24 h prior to assessment of DNA synthesis
by [3H]thymidine incorporation as described under
"Experimental Procedures" (A). In B, MCF-10A
cells were treated with anti-IGF-I receptor antibody IR-3 (5 µg/ml) or hIGFBP-6 (100 ng/ml), in the absence or presence of IGF-I
(5 ng/ml) as indicated for 24 h before [3H]thymidine
incorporation. C shows thymidine incorporation in
MCF-10Aras cells treated with IGFBP-3. D,
IGF-stimulated thymidine incorporation in MCF-10A (open
symbols) and MCF-10Aras cells (closed symbols)
was assessed over the concentration range indicated after 24-h
exposure. In E, media conditioned for 24 h by MCF-10A
and MCF-10Aras cells in the absence or presence of 100 ng/ml
IGFBP-3, as indicated, were analyzed by ligand blotting with
125I-IGF-I, as described under "Experimental
Procedures." In F, MCF-10A (open bars) and
MCF-10Aras (hatched bars) cells were treated with
TGF-
1 at the indicated concentrations for 24 h
prior to thymidine incorporation. For each panel (A-D and
F) results are expressed as percentage of the counts
incorporated in the absence of additions. Points shown are mean ± S.E. of combined data (determined in triplicate wells) from three or
four experiments in A, C, and D; in B and
F representative data from one of two experiments with similar
results are shown. In A and F, significant
decreases in DNA synthesis compared with control are indicated as *
(p < 0.05) and ** (p < 0.01). In
B, a significant increase compared with control is indicated
as ** (p < 0.001), and no other treatments were
significantly different from control. Statistical significance was
determined by analysis of variance and Fisher's PLSD test.
MCF-10A cells transfected with the Ha-ras oncogene (MCF-10Aras) exhibit characteristics of malignant transformation (15), including 2-3-fold increased DNA synthesis in the absence of added growth factors (not shown). By contrast with the MCF-10A cell line, MCF-10Aras cells appeared resistant to the inhibitory effects of IGFBP-3 (Fig. 1C), with concentrations up to 100 ng/ml IGFBP-3 failing to inhibit DNA synthesis in these cells. Although IGF signaling pathways might be expected to be activated in these cells, they showed no loss of sensitivity toward stimulation by exogenous IGF-I; on the contrary, IGF-I sensitivity was increased 4-5-fold compared with MCF-10A cells (Fig. 1D).
As proteolysis of IGFBPs may decrease their bioactivity compared with
their intact counterparts (19), we investigated whether the absence of
IGFBP-3 inhibitory activity in MCF-10Aras cells was due to
increased degradation of the protein. Media conditioned by MCF-10A and
MCF-10Aras cells treated with IGFBP-3 were analyzed by IGF
ligand blot. By this method, increased proteolysis results in decreased
signal attributable to intact 43-kDa IGFBP-3 (20), as the proteolyzed protein has reduced binding to iodo-IGF-I (21). As shown in Fig.
1E, there was no loss of 43-kDa IGFBP-3 in the
MCF-10Aras cell-conditioned medium compared with the
untransfected MCF-10A cells by ligand blot, confirming that the lack of
an inhibitory effect of IGFBP-3 was not due to increased degradation of
exogenous IGFBP-3 by the MCF-10Aras cells. MCF-10Aras
cells also showed a similar sensitivity to the inhibitory effects of
TGF- as MCF-10A (Fig. 1F), indicating that resistance to
IGFBP-3 was not due to a general change in sensitivity to inhibitory
factors as a result of constitutive activation of the Ras signaling
pathway. Taken together, these data indicate that the inhibition of DNA synthesis in breast epithelial cells by IGFBP-3 is not a simple consequence of IGF binding by IGFBP-3 and that expression of oncogenic ras by these cells results in resistance to inhibition by
IGFBP-3.
Signaling via Ras involves the activation of intermediates in a number
of downstream pathways (22); we therefore investigated whether
sensitivity to IGFBP-3 in MCF-10Aras cells could be changed in
the presence of specific inhibitors of these pathways. The inhibitor
wortmannin (10 nM), which blocks the phosphatidylinositol
3-kinase pathway, had no effect on IGFBP-3 sensitivity in MCF-10A or
MCF-10Aras cells (not shown). However PD 98059, a synthetic
flavone that acts as a specific inhibitor of activation of the MAPK/ERK
(mitogen-activated protein kinase/extracellular signal-regulated
kinase) kinase MEK (23) had a marked effect on IGFBP-3 sensitivity in
ras-transfected cells. When exposed to 5 µM PD
98059, each cell line showed 30-40% inhibition of DNA synthesis over
24 h, an effect similar to that described in other cell types
(23). Addition of 10-100 ng/ml IGFBP-3 resulted in further inhibition
of DNA synthesis (Fig. 2A),
implying that sensitivity to IGFBP-3 in these cells is restored when MEK activation is inhibited.
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Increased signaling through Ras-dependent pathways, either as the result of mutations in ras itself as seen in a small proportion of breast tumors or via increased activity of factors that activate Ras, is believed to be an important contributing factor in the development and progression of breast malignancies (24). The tumor-derived Hs578T cell line expresses an activated Ha-ras gene (25), and we found that, like ras-transfected MCF-10A cells, Hs578T cells were relatively insensitive to growth inhibition by IGFBP-3, requiring 1000 ng/ml IGFBP-3 for significant inhibition of DNA synthesis (Fig. 2B). These results indicate that in either transfected or tumor-derived cell models, breast epithelial cells expressing constitutively active Ras are relatively resistant to the inhibitory effects of IGFBP-3. As was seen with the MCF-10Aras cells, however, co-incubation of Hs578T cells with PD 98059 (5 µM) and IGFBP-3 at a concentration of 100 ng/ml resulted in significant inhibition of DNA synthesis (Fig. 2B), indicating a 10-fold increase in sensitivity compared with IGFBP-3 treatment in the absence of PD 98059. To confirm that this reagent was inhibiting MAP kinase phosphorylation in the MCF-10Aras and Hs578T cells, lysates from cells treated for 24 h with PD 98059 were analyzed by immunoblot using antibodies against phosphorylated p44/42 MAPK (Fig. 2C). Incubation with 5 or 20 µM PD 98059 reduced phosphorylated MAPK by 50-60% (determined by densitometric scanning of the autoradiograms shown), in the absence of any change in total MAPK. Thus, partial blockade of Ras signaling by inhibiting MAPK/ERK activation restores sensitivity to IGFBP-3 in breast cells expressing oncogenic ras.
One possible mechanism by which PD 98059 might increase sensitivity to
inhibition by IGFBP-3 would be through increasing the responsiveness to
endogenous IGFs. However, in the presence of PD 98059, neither IR-3
nor IGFBP-6 caused further inhibition of DNA synthesis in
MCF-10Aras or Hs578T cells (not shown), indicating that
restoration of IGFBP-3 inhibitory activity was unrelated to its binding
of endogenous IGFs. Furthermore, Hs578T cells, which show no response
to exogenous IGF-I (27), did not show increased IGF sensitivity in the
presence of PD 98059 (not shown).
It has been proposed that the intracellular actions of IGFBP-3 are initiated through its interaction with a cell-surface receptor (26), although no signaling receptor has been identified. In common with other peptide receptors, the cell-surface expression of such a receptor might be down-regulated by a high concentration of its ligand. In contrast with many other breast cancer cells which secrete little or no IGFBP-3 (10), Hs578T cells secrete relatively large amounts of IGFBP-3, and ras transfected MCF-10A cells similarly secreted higher levels than their nontransfected counterparts (Table I). To determine whether the restoration of sensitivity to IGFBP-3 in response to PD 98059 was the result of decreased production of IGFBP-3, and thereby release from down-regulation of a putative signaling receptor, IGFBP-3 concentrations in medium conditioned by cells treated with PD 98059 were measured. As shown in Table I, PD 98059 treatment, while restoring sensitivity to exogenous IGFBP-3, had no significant effect on IGFBP-3 secretion in any of the cell lines tested.
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Cell association of IGFBP-3 has been shown in some breast cancer cell
lines to correlate with its growth inhibitory effect (26). We therefore
investigated whether the amount of cell-associated IGFBP-3 changed
under conditions where sensitivity to its inhibitory effects was
increased. In the absence of IGFBP-3 or PD 98059, cell-associated
IGFBP-3 levels were similar in the MCF-10Aras and Hs578T cells
and 2-3-fold higher than in either the parent or control
vector-transfected MCF-10A cell lines (data not shown). As shown in
Fig. 3A for MCF-10Aras
cells, addition of IGFBP-3 significantly increased the amount of
IGFBP-3 associated with the cell at all doses tested. However, co-addition of 5 µM PD 98059 had no effect on
cell-associated protein in the absence or presence of
concentrations of IGFBP-3 shown previously (in Fig. 2) to inhibit DNA
synthesis. In the Hs578T cells, a significant increase in the amount of
cell-associated IGFBP-3 was seen with 100 ng/ml dose of exogenous
IGFBP-3 only, and this did not change in the presence of PD 98059 (Fig.
3B). These observations argue against a direct link between
the degree of cell association of IGFBP-3 and its inhibition of DNA
synthesis.
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DISCUSSION |
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Although antiproliferative and pro-apoptotic effects of IGFBP-3
have been reported in a variety of breast cancer cell lines, its
actions in nonmalignant breast cells have not been described previously. The data presented in this report indicate that normal breast epithelial cells are considerably more sensitive to IGFBP-3 than
tumor-derived breast cancer cells, which require 500-1500 ng/ml
IGFBP-3 to achieve a similar level of inhibition to that seen with
10-100 ng/ml in the nontransformed cells (7, 27). Our findings also
indicate that resistance to IGFBP-3 is induced in breast cells
transfected with oncogenic ras, thereby implicating increased activity of Ras-dependent signaling pathways in
the development of IGFBP-3 insensitivity. In contrast, sensitivity toward the growth inhibitor TGF- was unaffected by ras
transfection, suggesting some specificity in the IGFBP-3 effect.
Activation of the MAPK/ERK pathway, whether by ras mutation
or other mechanisms such as growth factor receptor activation, may be a
common feature of the malignant growth of breast epithelial cells (24),
and our results would indicate that in these situations resistance to
the tumor-suppressive activity of endogenous IGFBP-3 would be one
result of such activation. Several lines of evidence indicate that
changes in sensitivity to IGFBP-3 resulting from oncogenic
ras expression or MAPK blockade are unrelated to effects on
IGF signaling or sensitivity. First, MCF-10A cells are not inhibited by
IGFBP-6 or the type 1 IGF receptor antagonist, IR-3, despite their
sensitivity to IGFBP-3. Second, transfection of MCF-10A cells with
oncogenic ras actually increased their sensitivity to IGF-I,
so that loss of IGF-I responsiveness could not explain their loss of
inhibition by IGFBP-3. Third, the insensitivity of Hs578T cells to
IGF-I is not altered in the presence of PD 98059.
Increased proteolysis of exogenous IGFBP-3 in the MCF-10Aras cell line was also investigated as a possible cause of the lack of response to its inhibitory effects. In other cell systems, limited degradation of IGFBP-3 has been demonstrated to affect its IGF-independent (28) and -dependent (29) actions; however, there was no significant loss of intact IGFBP-3 over the 24-h treatment period for the MCF-10Aras cells. We also investigated the possibility that a high level of endogenous IGFBP-3, which is characteristic of both the Hs578T and MCF-10Aras cell lines, was leading to down-regulation of a putative signaling receptor, but found that sensitivity to IGFBP-3 could be reinstated in the absence of any change in the level of secreted IGFBP-3.
It has been reported that the antiproliferative effects of IGFBP-3
correlate with its presence on the cell surface of the cell (27). This
observation has been interpreted as evidence for the existence of a
signaling receptor (26), which has been suggested recently to be the
type V TGF- receptor (30) despite a lack of specific evidence that
this receptor mediates either TGF-
or IGFBP-3-dependent
events. Although we identified IGFBP-3 in association with the surface
of MCF-10A and MCF-10Aras cells, its concentration did not
correlate with sensitivity to its effects. In IGFBP-3-insensitive
MCF-10Aras cells, addition of IGFBP-3 resulted in an increase
in the amount of cell-associated IGFBP-3 without inhibiting DNA
synthesis, while treatment with PD 98059, which restored sensitivity to
the growth inhibitory effects of IGFBP-3 in both Hs578T and
MCF-10Aras cells, did not alter the amount of cell-associated
IGFBP-3. This suggests that cell binding of IGFBP-3 per se
does not lead to inhibition of DNA synthesis and implies the existence
of other factors, either intracellular or extracellular, involved in
the regulation of IGFBP-3 inhibitory signaling. Our findings would also
indicate that the expression or activity of such factors is
Ras-dependent.
We showed recently that in T47D breast cancer cells transfected to
overexpress IGFBP-3, the cell population changed over several passages
from a state of relative cell cycle arrest by IGFBP-3 to a state of
insensitivity to its inhibitory effect, where cell growth was
uninhibited by high levels of secreted and cell-bound IGFBP-3 (31).
Although the mechanism for this transition is unknown, we have now
demonstrated that the expression of oncogenic ras, either in
a transfected cell line or in breast tumor-derived cells, is associated
with resistance to IGFBP-3 inhibitory signaling. IGFBP-3 is abundant in
the human circulation (32), where a high concentration is a negative
risk factor for the development of breast and prostate cancer (33, 34).
We propose that the development of resistance to IGFBP-3 may be a key
step in the dysregulation of breast cancer cell growth, raising the
possibility that future therapies could be directed toward maintaining
or restoring sensitivity to the growth inhibitory effects of this protein.
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FOOTNOTES |
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* This work was supported by the National Health and Medical Research Council of Australia (Grant 950199), the Faculty of Medicine of the University of Sydney, and the Sydney University Medical Foundation.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.: 61-2-9926-8486;
Fax: 61-2-9926-8484; E-mail: janetlm{at}med.usyd.edu.au.
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ABBREVIATIONS |
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The abbreviations used are:
IGF, insulin-like
growth factor;
IGFBP-3, insulin-like growth factor binding protein-3;
TGF-, transforming growth factor-
;
PAGE, polyacrylamide gel
electrophoresis;
MAP, mitogen-activated protein;
MAPK, MAP kinase;
ERK, extracellular signal-regulated kinase;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase;
BSA, bovine serum
albumin;
EGF, epidermal growth factor;
DMEM, Dulbecco's modified
Eagle's medium;
TBS, Tris-buffered saline;
PLSD, protected least
significant differences.
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