From the
Most estrogen receptor-negative breast cancer cells, including
Hs578T cells, express mRNAs encoding insulin-like growth factor-binding
protein (IGFBP)-3, as well as transforming growth factor (TGF)-
The insulin-like growth factor-binding proteins
(IGFBPs)
The
growth of human breast cancer cells can be inhibited in vitro by several factors, including TGF-
We first investigated whether treatment with TGF-
To more directly test
whether IGFBP-3 mediates TGF-
Previous studies have demonstrated that TGF-
We have reported previously that exogenously
added IGFBP-3 acts as a cell growth inhibitor, and that its inhibitory
action may be mediated through specific IGFBP-3
receptors
(4, 6) . In the present study, we demonstrate
that physiological concentrations of TGF-
In addition, recent studies
have indicated that retinoic acid and the anti-estrogen tamoxifen
stimulate production of IGFBP-3 and inhibit monolayer cell growth in
human breast cancer
cells
(8, 13, 24, 25) , suggesting that
IGFBP-3 may have a role in the growth-inhibitory action mediated by
those factors in human breast cancer. Our IGFBP-3 antisense ODN and
IGF-II analog data clearly demonstrate that TGF-
We thank C. Roberts (Oregon Health Sciences
University) and P. Cohen (University of Pennsylvania, Philadelphia, PA)
for critical reviews of the manuscript, Z. Gucev for technical
assistance, and A. Sommer (Celtrix Inc., Santa Clara, CA) for providing
IGFBP-3 and TGF-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
receptors. Our previous studies (Oh, Y., Muller, H. L., Lamson, G., and
Rosenfeld, R. G.(1993) J. Biol. Chem. 268, 14964-14971;
Oh, Y., Muller, H. L., Pham, H. M., and Rosenfeld, R. G.(1993) J.
Biol. Chem. 268, 26045-26048) have demonstrated a
significant inhibitory effect of exogenous IGFBP-3 on Hs578T cell
growth and existence of IGFBP-3-specific receptors that may mediate
those direct inhibitory effect of IGFBP-3. TGF-
is also a potent
growth inhibitor in human breast cancer cells in vitro and
regulates IGFBP-3 production in different cell systems, suggesting that
IGFBP-3 is a major anti-proliferative factor and a key element for
TGF-
-induced growth inhibition in human breast cancer cells. In
support of this hypothesis, we have demonstrated using Hs578T cells
that: 1) TGF-
stimulates IGFBP-3 gene expression and production
prior to its inhibition of cell growth, 2) treatment with an IGFBP-3
antisense oligodeoxynucleotide selectively inhibits TGF-
-induced
IGFBP-3 synthesis and cell growth inhibition, and 3) treatment with
IGF-II and IGF-II analogs diminish TGF-
effects by blocking
TGF-
-induced binding of IGFBP-3 to the cell surface. These
findings suggest that IGFBP-3 is a major anti-proliferative factor and
a key element in TGF-
-induced growth inhibition in human breast
cancer cells.
(
)
bind specifically to the insulin-like
growth factors (IGF-I and IGF-II) and modulate the mitogenic effects of
IGFs in various systems
(1) . IGFs are the most potent mitogens,
apart from estrogens, for human breast cancer cells (2) and exert their
mitogenic effect mainly through the IGF-I receptor, which possesses an
intrinsic ligand-activated tyrosine kinase activity (3). Recently, our
studies have demonstrated a significant inhibitory effect of exogenous
IGFBP-3 on the growth of Hs578T cells
(4) and
IGFBP-3-transfected mouse Balb/c fibroblast cells
(5) .
Furthermore, we have demonstrated the existence of IGFBP-3-specific
receptors that may mediate the direct inhibitory effect of IGFBP-3 on
Hs578T cells, and we have shown that IGFBP-3 can inhibit cell
proliferation by itself, even in the absence of IGFs
(6) .
(7) , retinoic acid
(8), and anti-estrogens
(9, 10) . It has been reported
that these anti-proliferative factors stimulate expression of IGFBP-3
in various cell systems
(8, 11, 12, 13) .
In particular, TGF-
is expressed by most human breast cancer
cells
(7, 14) and by stromal fibroblasts, regardless of
whether the fibroblasts originate from normal or malignant cells,
indicating that TGF-
is part of a tightly balanced negative growth
factor system in vivo(15) . The exact mechanisms by
which TGF-
inhibits breast cancer cell proliferation are not well
understood. In this study, we demonstrate that the inhibitory effect of
TGF-
on Hs578T cell growth is mediated, at least in part, by
enhanced expression of IGFBP-3.
Peptides and Proteins
Recombinant human IGF-II
was provided by Lilly. [Leu]IGF-II and
[Gln
,Ala
,Tyr
,Leu
,Leu
]IGF-II,
synthetic IGF-II analogs, were synthesized as described previously
(16). Recombinant TGF-
2 was the generous gift of Celtrix, Inc.
(Richmond, CA). Iodination of IGF-II to a specific activity of
350-500 µCi/µg was performed by a modification of the
chloramine-T technique.
IGFBP-3 Antisense Phosphorothioate Oligodeoxynucleotide
(ODN)
The IGFBP-3 antisense and sense ODNs used in this
experiment were prepared by OLIGOS Etc., Inc. (Guilford, CT). The
IGFBP-3 antisense ODN was complementary to 20 nucleotides that encode
the N terminus of human IGFBP-3
(17) and had the sequence 5`-CAT
GAC GCC TGC AAC CGG GG-3` (positions 2021-2040); the sequence of
the IGFBP-3 sense ODN was 5`-CCC CGG TTG CAG GCG TCA TG-3`. A search of
GenBank revealed no other matching sequences.
Cell Cultures
Hs578T human breast cancer cells
were maintained in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 4.5 g/liter glucose, 110 mg/liter sodium
pyruvate, and 10% fetal bovine serum as described
previously
(4) .
Preparation of Conditioned Medium (CM)
Cells were
grown until 95% confluent in serum-containing media and then switched
for 12 h to serum-free DMEM. Medium was aspirated again, and cells were
maintained in serum-free DMEM with or without TGF-2 or IGFBP-3 ODN
for various times indicated in the text. Conditioned media were
collected and centrifuged at 1000
g for 10 min to
remove cell debris. The harvested CM from triplicate wells within each
experiment were pooled and stored at -70 °C until assay.
Western Ligand Blots
Proteins from CM samples were
size-fractionated by SDS-polyacrylamide gel electrophoresis under
non-reducing conditions and electroblotted onto nitrocellulose filters
(0.45-µm pore size) using a BioTrans unit. Filters were incubated
overnight with 1.5 10
cpm of
I-labeled IGF-II, washed, dried, and exposed to film.
Monolayer Cell Replication Assay
Cells were grown
in 12-well plates until 60% confluent (0.2 10
cells/well) and changed to serum-free media for 12 h, and then
maintained in serum-free DMEM containing 0.25% bovine serum albumin in
the presence or absence of different concentrations of TGF-
2 or
IGFBP-3 ODN for various time points as indicated in the text. Cells
were then gently detached from plates by phosphate-buffered saline/EDTA
and cell number counted using either a hematocytometer or a Coulter
Counter (Coulter, Ltd., Beds., United Kingdom). In experiments
investigating the effect of IGFBP-3 ODN and IGF peptide on the
growth-inhibitory action of TGF-
2, cells were incubated with 5
ng/ml TGF-
2 in the presence of various concentrations of IGFBP-3
ODN, IGF-II, or IGF-II analogs for 5 days.
Northern Blots
Total cellular RNA was isolated
from cells by a modification of the guanidinium isothiocyanate-LiCl
precipitation method
(18) . Sample RNA was size-fractionated on
1.2% agarose-formaldehyde gels and then transferred to nitrocellulose.
Blotted RNAs were hybridized sequentially with cDNA probes labeled with
[P]dCTP by random priming (Pharmacia, Uppsala,
Sweden); bands were visualized by autoradiography after exposure to XAR
film with intensifying screens at -70 °C. RNA ladder size
markers (Life Technologies, Inc.) were used to obtain size estimates of
specific transcripts. The human IGFBP-3 probe was a 1082-base pair
EcoRI-PvuII fragment of the human IGFBP-3 cDNA that
includes the entire coding region sequence (cDNA kindly provided by Dr.
W. Wood, Genentech, South San Francisco, CA). Band densities were
analyzed using the area under the curve, as calculated by a LKB
densitometer. The relative density of the bands corresponding to
IGFBP-3 on autoradiography were expressed as absorption units per
millimeter.
Statistical Analysis
Data were analyzed with a
two-tailed Student's t test, using the software program
Statview (Abacus Concepts, Inc.)
2 would
stimulate expression of IGFBP-3 by Hs578T cells. As in previous studies
(7), 5 days of treatment with 1 ng/ml TGF-
2 inhibited cell growth
by 40% relative to control cells (p < 0.05), as shown in
Fig. 1A. A time-course experiment with 5 ng/ml
TGF-
2 showed that the inhibitory effect of TGF-
2 on monolayer
growth occurs during days 4-6 of treatment, with maximal
inhibition at day 5 (Fig. 1B). We next examined the
effect of TGF-
2 on IGFBP-3 mRNA levels and the levels of secreted
protein. Hs578T cells secrete IGFBP-3 (shown as 41-kilodalton (kDa) and
39-kDa IGF-binding species) and IGFBP-4 (24-kDa species) as major
IGFBPs. Treatment with 5.0 ng/ml TGF-
2 for 2 days increased
IGFBP-3 levels in conditioned media by 3.0-fold, whereas CM IGFBP-4
levels were not changed significantly by the same concentrations of
TGF-
2 (Fig. 2A). As reported previously,
IGFBP-3-specific proteases are major regulators of secreted and cell
surface-associated IGFBP-3 levels
(19) . To determine if the
effect of TGF-
2 on IGFBP-3 resulted from changes in IGFBP-3
protease activity, we performed IGFBP-3 protease assays as described
previously
(20) . No IGFBP-3 protease activity was detectable
either in the presence or absence of TGF-
2 (data not shown).
Figure 1:
Effect of TGF-2 on Hs578T cell
growth. A, dose response of TGF-
2 effect. Hs578T cells
were grown until 60% confluent (0.2
10
cells/well)
and then changed to serum-free media containing 0.25% bovine serum
albumin with various concentrations of TGF-
2. After 5 days of
treatment, cell numbers were counted with a Coulter Z1 counter
(Coulter, Ltd). Results represent means ± S.E. of two separate
experiments performed in triplicate. *, two-tailed t test;
p < 0.05. B, time course of growth inhibition with
5 ng/ml TGF-
2. Experiments are similar to those shown in
A. The data are expressed as the percent of control cell
numbers at each time point.
Figure 2:
Effect of TGF-2 on IGFBP-3 production
in Hs578T cells. A, IGFBP-3 concentrations in CM from
TGF-
2-treated cells. Representative Western ligand blot of CM from
cells incubated in DMEM alone (lane1) or with
0.05-5.0 ng/ml TGF-
2 (lanes 2-6) for 2 days.
Arepresentative gel from one of the two experiments is shown.
B, effect of TGF-
2 on IGFBP-3 mRNA levels. Cells were
grown until 90% confluent and incubated with serum-free media
containing 1 or 5 ng/ml TGF-
2 for 8 h. Total RNA was extracted and
analyzed by Northern blot. A representative blot from two separate
experiments is shown.
Northern blot analysis of total RNA from Hs578T cells revealed a
single, 2.6-kilobase mRNA species that hybridized to a human IGFBP-3
cDNA probe. Treatment with 5 ng/ml TGF-2 for 8 h resulted in a
2.0-fold increase in IGFBP-3 mRNA, after correction for
-actin
mRNA (Fig. 2B). Similar results were obtained with
TGF-
1 (data not shown). The observed increase in IGFBP-3 mRNA
following TGF-
treatment thus appears to account for the increase
in IGFBP-3 levels in conditioned media. The picture that emerges from
these data and other studies is as follows; TGF-
induces increases
in IGFBP-3 mRNA (less than 8 h) and secreted IGFBP-3 levels (less than
2 days) as early events, followed by inhibition of monolayer cell
growth (after 4 days) as a late event. These data are consistent with
the hypothesis that TGF-
2 inhibits proliferation, at least in
part, by increasing IGFBP-3 expression.
action, we employed two different
strategies to block the IGFBP-3 effect: 1) blocking TGF-
2-induced
IGFBP-3 expression by IGFBP-3 antisense ODN and 2) preventing IGFBP-3
binding to the cell surface by treatment with IGFs and IGF-II analogs,
thereby blocking IGFBP-3-mediated action
(4) . Cells were treated
with antisense and sense phosphorothioate ODN corresponding to
nucleotide positions 2021-2040 of the human IGFBP-3 cDNA
sequence. Treatment with 10 µg/ml antisense, but not sense, ODN
reduced IGFBP-3 protein concentrations by 80% and IGFBP-3 mRNA levels
by 60% (Fig. 3A), but had no effect on IGFBP-4
production (data not shown). We next investigated the effects of
antisense ODN on TGF-
2-induced IGFBP-3 production. As in previous
experiments, TGF-
2 alone increased IGFBP-3 levels by as much as
4-fold. In the presence of 20 µg/ml antisense ODN, however,
TGF-
2-induced IGFBP-3 production was inhibited by as much as 80%,
and this was correlated with 70% inhibition of TGF-
2-induced
IGFBP-3 mRNA expression at an ODN concentration of 20 µg/ml
(Fig. 3B).
Figure 3:
Effect of IGFBP-3 antisense ODN on IGFBP-3
protein and mRNA levels in control and TGF-2-treated cells.
Upperpanel, Western ligand blots of Hs578T CM from
cells treated with 1-20 µg/ml ODN on day 0 and again on day
1.5 without changing media in the absence (A) or presence
(B) of 5 ng/ml TGF-
2. CM was harvested after 3 days of
treatment from triplicate wells within each experiment, pooled, and
electrophoresed. Lowerpanel, Northern blots
demonstrating the effects of ODN on IGFBP-3 mRNA levels in control
(A) or TGF-
2-treated (B) Hs578T cells. Cells
were grown until 90% confluent and pretreated with 1-20 µg/ml
ODN for 4 h. Cells were then washed and treated with ODN in the absence
(A) or presence (B) of 5 ng/ml TGF-
2 for 12 h. A
representative gel from one of two experiments is
shown.
After initially ascertaining the ability
of the IGFBP-3 antisense ODN to specifically suppress
TGF-2-induced IGFBP-3 production, we next investigated the effect
of antisense ODN on TGF-
2-induced growth inhibition by treating
cells with antisense or sense ODN for 5 days. No significant growth
effect was observed by IGFBP-3 antisense ODN in the absence of
TGF-
2 despite inhibition of IGFBP-3 expression, indicating that
basal IGFBP-3 levels were not sufficient to exert a growth-inhibitory
effect (data not shown). These findings are similar to our previous
report that shown that IGFs had no effect on cell growth, despite their
ability to block cell surface binding of endogenous IGFBP-3
(4) .
However, when antisense ODN were employed in the presence of
TGF-
2, antisense, but not sense, ODN attenuated the
TGF-
2-induced cell growth inhibition by approximately 60% (p < 0.05) (Fig. 4).
Figure 4:
Effect
of antisense IGFBP-3 ODN on TGF-2-induced growth inhibition. Cells
were treated with 5 ng/ml TGF-
2 in the presence of antisense or
sense ODN at concentrations ranging from 1 to 50 µg/ml. The
inhibitory effect of TGF-
2 was determined by cell counts after 5
days of treatment. Results represent means ± S.E. of two
separate experiments performed in triplicate. *, p <
0.05.
We have previously identified
IGFBP-3 receptors on Hs578T cells and demonstrated that blocking the
interaction of exogenously added IGFBP-3 with these receptors by IGF
peptides resulted in attenuation of IGFBP-3 inhibition of Hs578T cell
growth
(4) . Therefore, we tested the effect of IGF-II and IGF-II
analogs on TGF-2 action (Fig. 5). Whereas TGF-
2 alone
resulted in a 40% inhibition of cell growth, when coincubated with
IGF-II, the inhibitory effect was blocked in a dose-dependent manner,
with a 90% attenuation of the TGF-
2 effect at an IGF-II
concentration of 200 ng/ml (p < 0.05). Similar results were
observed using [Leu
]IGF-II, which has full
affinity for IGFBPs, but significantly reduced affinity for the IGF-I
receptor
(16) . In contrast, coincubation with similar
concentrations of
[Gln
,Ala
,Tyr
,Leu
,Leu
]IGF-II
([QAYLL]IGF-II), an IGF analog with 100-fold reduced affinity
for both IGFBPs and the IGF-I receptor, did not result in attenuation
of the TGF-
2 inhibitory effect. These results suggest that the
attenuating effect of IGF-II and [Leu
]IGF-II on
TGF-
2 inhibition of cell growth was not mediated through the IGF-I
receptor, but instead reflects the ability of IGF peptides to prevent
IGFBP-3 binding to the cell surface
(4) .
Figure 5:
Effect
of IGF-II and IGF-II analogs on TGF-2 action. Cells were treated
with IGF-II, [Leu
]IGF-II, or
[QAYLL]IGF-II at concentrations ranging from 5 to 200 ng/ml
for 5 days. Results represent means ± S.E. of three separate
experiments performed in triplicate. *, p <
0.05.
is a potent
anti-proliferative factor in a series of human breast cancer cells
in vitro(7, 21, 22) . Estrogen receptor
(ER)-negative cells in particular, including Hs578T cells, appear to
possess receptors specific for TGF-
, and TGF-
1 and TGF-
2
appear to be equipotent in inhibiting cell
growth
(21, 22) . In addition, TGF-
is produced and
hormonally regulated in some ER-positive cells; anti-estrogens such as
4-OH-tamoxifen induce biologically active TGF-
in MCF-7 cells, and
this anti-estrogen-induced TGF-
inhibits the growth of ER-negative
cells
(7) . Furthermore, a recent report has demonstrated that
anti-estrogens, including 4-OH-tamoxifen, induce IGFBP-3 expression in
MCF-7 cells
(13) . The exact mechanism by which TGF-
inhibits breast cancer cell proliferation is not well understood,
despite the fact that TGF-
receptors, at least type I and II
receptors, are known to exhibit serine/threonine kinase
activity
(23) . It has been also demonstrated that stromal
fibroblasts express TGF-
, regardless of whether the fibroblasts
originated from normal or malignant cells, indicating that TGF-
is
a tightly regulated negative growth factor in
vivo(15) .
specifically stimulate
the expression of IGFBP-3 mRNA and protein. Furthermore, increased
expression and production of IGFBP-3 precede the inhibition of cell
growth by TGF-
2 in Hs578T breast cancer cells. This result
suggests that stimulation of IGFBP-3 expression is one of the earliest
effects of the TGF-
and, therefore, may be necessary for
TGF-
-induced cell growth inhibition. Indeed, TGF-
-induced
growth inhibition can be prevented by addition of IGFBP-3 antisense ODN
or IGF peptides, but not by addition of sense ODN or IGF-II analogs
that have reduced affinity for IGFBP-3.
-induced growth
inhibition is mediated, at least in part, through IGFBP-3 action. To
the best of our knowledge, these studies constitute the first
demonstration that IGFBP-3 is required for a growth-inhibitory effect
that is induced by a different class of growth factors. A fuller
elucidation of the mechanisms underlying these IGF-independent actions
of IGFBP-3 in human breast cancer will allow us to understand how the
growth of breast cancer cells can be modulated by the IGF/IGFBP system,
and how other growth factors can interact with this system.
2.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.