(Received for publication, October 17, 1995; and in revised form, November 22, 1995)
From the
MCF-7 human breast cancer cells are commonly used to model tissues responsive to estrogens and antiestrogens. We examined the effects of estradiol and the antiestrogen ICI 182780 on MCF-7 cell proliferation and insulin-like growth factor binding protein 3 (IGFBP-3) gene expression. ICI 182780-induced growth inhibition was associated with increased transcription of the IGFBP-3 gene, increased IGFBP-3 mRNA abundance, and increased IGFBP-3 protein accumulation in the conditioned medium. The growth stimulatory effect of estradiol was associated with opposite effects, and the correlation between cellular proliferation and IGFBP-3 mRNA abundance was strong (r = -0.91). Recombinant IGFBP-3 inhibited basal and estradiol-stimulated MCF-7 cell proliferation, and an IGFBP-3 antisense oligodeoxynucleotide abolished antiestrogen-induced growth inhibition. These results provide evidence for an estradiol and antiestrogen-regulated IGFBP-3 growth inhibitory autocrine pathway in MCF-7 cells.
Insulin-like growth factors I and II (IGF-I ()and
IGF-II) are potent mitogens and inhibitors of apoptosis for many normal
and neoplastic cell types, including normal and transformed breast
epithelial cells(1, 2) . Both IGF-I and IGF-II bind
with high affinity to specific IGF-binding proteins (IGFBPs), which
modulate their bioactivity. At least six IGFBPs have been
described(3, 4) . IGFBP-3 acts as a growth inhibitor
in many (but not all(5) ) experimental systems (reviewed in (6) ). Examples of data consistent with a growth inhibitory
role for IGFBP-3 include the growth inhibition associated with IGFBP-3
gene transfection (6) , the increased IGFBP-3 accumulation
associated with senescence-related reduction of cellular
proliferation(7) , the increase in IGFBP-3 production
associated with retinoid-induced growth inhibition(8) , and the
decrease in IGFBP-3 production associated with epidermal growth
factor-stimulated proliferation(9) . Early studies attributed
the growth inhibitory action of IGFBP-3 to the reduction of IGF-I
and/or IGF-II bioactivity resulting from competition for somatomedins
between IGFBP-3 and the type I IGF receptor(10) . However,
there is recent evidence that IGFBP-3 also has growth inhibitory
activity that is independent of its IGF binding properties (11, 12, 13, 14) .
Antiestrogens are widely used in breast cancer treatment, and it has been proposed that the inhibitory effect of these compounds on IGF-I expression contributes to their antiproliferative activity(15, 16) (reviewed in (17) ). Antiestrogens have significant trophic effects on the uterus(18) , and there is a negative correlation between uterine weight and uterine IGFBP-3 expression; the positive uterotrophic actions of both estradiol and the partial estrogen receptor antagonist tamoxifen are associated with suppression of uterine IGFBP-3 expression, while the pure antiestrogen ICI 182,780 (19) causes uterine involution and markedly enhances uterine IGFBP-3 expression(20) . We undertook the present study to examine the possibility that the anti-proliferative action of ICI 182780 on breast cancer cells is related to changes in IGFBP-3 gene expression induced by this drug.
The cDNA for human IGFBP-3 (21) and recombinant human
IGFBP-3 (22) were provided by Celtrix Pharmaceuticals (Santa
Clara, CA), the 18 S cDNA was a gift from Dr. C. Karatzas, and ICI
182,780 (19) was a gift from Dr. A. Wakeling (Zeneka
Pharmaceuticals). 17-Estradiol was purchased from Sigma and
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole from
Calbiochem.
Figure 1:
IGFBP-3 accumulation in MCF-7
cell-conditioned media during incubation with estradiol, ICI 182780,
and ICI 182780 plus estradiol. MCF-7 cells at 85-90% confluence
were grown in SEPF medium for 24 h. Cells were then treated with
indicated doses of estradiol (A), ICI 182780 (B), or
10M estradiol plus various doses of ICI
182780 (C) for 48 h. Conditioned media were collected and
concentrated, and ligand blot analysis was performed as described under
``Experimental Procedures.'' The bands corresponding to
IGFBP-3 were quantified densitometrically (D and E).
Means ± S.E. of quadruplicate experiments are plotted. In E, the solid line joins data points for ICI 182780
alone, and the dashed line joins points for various
concentrations of the drug in the presence of 10
M estradiol. The time course of estradiol and ICI 182780
effects on IGFBP-3 accumulation in MCF-7 cell-conditioned media is
shown in F. MCF-7 cell-conditioned media were harvested at the
indicated times after incubation with 10
M estradiol (E) or 10
M ICI
182780 (I), or neither (C), concentrated, and
analyzed by ligand blotting.
Figure 2:
Regulation of IGFBP-3 mRNA levels by
estradiol and ICI 182780. MCF-7 cells at 85-90% confluence were
grown in SEPF medium for 24 h. Cells were then treated with indicated
doses of estradiol (A), ICI 182780 (B), or
10M estradiol plus various doses of ICI
182780 (C) for 48 h. Cytoplasmic RNA was isolated and Northern
blotting was performed as described under ``Experimental
Procedures.'' The 2.6-kilobase IGFBP-3 mRNA (top panel)
and 18 S ribosomal mRNA (lower panel) are shown. The bands
corresponding to IGFBP-3 were quantified densitometrically (D and E). Means ± S.E. of quadruplicate experiments
are plotted. In E, the solid line joins data points
for ICI 182780 alone, and the dashed line joins points for
various concentrations of the drug in the presence of 10
M estradiol.
Figure 3:
Effects of estradiol and ICI 182780 on
MCF-7 cell DNA synthesis. Cells were cultured as described under
``Experimental Procedures'' and then incubated for 48 h in
SEPF medium with indicated doses of estradiol (A), ICI 182780 (B, solid line), or ICI 182780 in combination with
10M estradiol (B, dashed
line), prior to measurement of thymidine incorporation. Means of
quadruplicate experiments are plotted. S.E. at each point was
14%.
The relationship between IGFBP-3 mRNA abundance and proliferation rate
of MCF-7 cells is shown in C. Points represent mean
values of each variable at the SEPF control condition (asterisk) or various concentrations of ICI 182780 (squares) or estradiol (crosses). The effect of
recombinant human IGFBP-3 on basal and estradiol-stimulated MCF-7 cell
proliferation is shown in D. Cells were cultured as described
under ``Experimental Procedures'' and then incubated for 48 h
in SEPF medium in the presence or absence of recombinant human IGFBP-3
(0.9 nM) and of estradiol (10
M)
for 48 h prior to measurement of thymidine incorporation. Means and
S.E. of quadruplicate experiments are plotted. In both the presence and
absence of estradiol, thymidine incorporation was significantly reduced
in the presence of IGFBP-3 (p < 0.01, Mann-Whitney U test). The effect of sense (S) and antisense (AS) on MCF-7 cell proliferation is shown in E.
Results are expressed relative to the control condition in the absence
of ICI 182780 and oligodeoxynucleotides. Means and S.E. of
quadruplicate experiments are plotted. Basal and ICI 182780-suppressed
proliferation rates were significantly increased in the presence of
antisense IGFBP-3 oligodeoxynucleotide (p < 0.001,
Mann-Whitney U test).
Fig. 3C summarizes the relationship between IGFBP-3 gene expression and proliferation in MCF-7 cells by relating the thymidine incorporation data to the mRNA abundance data from the experiment described in Fig. 2. The assays of thymidine incorporation and IGFBP-3 mRNA abundance were performed under identical conditions at the same concentrations of estradiol or ICI 182780. A significant negative correlation (r = -0.91) between IGFBP-3 mRNA abundance and proliferation rate was detected, and the relationship fit a negative log function as shown.
In order to determine if IGFBP-3 could mimic
the antiproliferative action of ICI 182780 on MCF-7 cells, we examined
the effect of recombinant human IGFBP-3 (rhIGFBP-3) on MCF-7 cell
proliferation under experimental conditions identical to those used to
assay effects of estradiol and ICI 182780. Fig. 3D shows 30% inhibition of both basal and estradiol-stimulated
proliferation by 0.9 nM (25 ng/ml) rhIGFBP-3. Concentrations
of rhIGFBP-3 up to 250 ng/ml did not further inhibit proliferation.
These data raised the possibility that expression of IGFBP-3 is not
merely a marker that correlates with actions of estradiol and
antiestrogens but that it plays a role in mediating these actions.
To determine if the antiproliferative action of ICI 182780 is
mediated by its effect on IGFBP-3 expression, we used an IGFBP-3
antisense oligodeoxynucleotide (24) to examine the consequences
of reducing basal and ICI 182780-stimulated IGFBP-3 secretion. Western
blotting confirmed that this oligodeoxynucleotide reduced IGFBP-3
accumulation in MCF-7-conditioned media (data not shown). Basal MCF-7
thymidine incorporation increased with increasing concentration of the
antisense IGFBP-3 oligodeoxynucleotide and was 3-fold higher than
control at 5 µg/ml. Sense IGFBP-3 oligodeoxynucleotide was without
significant effect (Fig. 3E). This result was
consistent with our prior observation that in the absence of serum and
estrogens, MCF-7 cells secrete IGFBP-3 and suggests that the low
proliferation rate under these conditions is not entirely attributable
to lack of mitogenic stimulation but is in part a consequence of
autocrine growth inhibition mediated by IGFBP-3 expression. In the
presence of 10M ICI 182780, thymidine
incorporation by MCF-7 cells was 57 ± 11% of control values, and
we observed that this antiestrogen-induced inhibition was completely
abolished by 1 µg/ml IGFBP-3 antisense oligodeoxynucleotide, while
an equal concentration of the sense oligodeoxynucleotide had no
significant effect. While these results imply that in our experimental
system the early inhibitory action of ICI 182780 can be entirely
attributed to stimulation of IGFBP-3 gene expression, the importance of
this mechanism relative to other mechanisms underlying the action of
antiestrogens(18, 29) in vivo remains to be
determined.
Our data provide evidence for a previously unrecognized
aspect of estrogen and antiestrogen action: estradiol-stimulated
proliferation involves relaxation of an autocrine IGFBP-3 inhibitory
influence, and the antiproliferative effect of ICI 182780 is related at
least in part to up-regulation of IGFBP-3 gene expression (Fig. 4). A precedent for antiestrogen up-regulation of
autocrine growth inhibitory pathways comes from the demonstration that
tamoxifen up-regulates expression of TGF (reviewed in (30) ). As TGF
increases IGFBP-3 accumulation in media
conditioned by fibroblasts (31, 32) and recent data
suggest that IGFBP-3 plays a role in mediating TGF
-induced growth
inhibition in Hs578T cells(24) , we considered the possibility
that ICI 182780 stimulation of IGFBP-3 expression might be mediated via
changes in TGF
expression but observed no significant changes in
TGF
expression in MCF-7 cells by ICI 182780 (data not shown). In
our experimental system, exogenous IGFs are absent, and autocrine
expression of IGF-I and IGF-II could not be detected (data not shown).
Therefore, the observed inhibitory action of IGFBP-3 may involve a
direct growth inhibitory signal transduction pathway (11, 12) rather than reduction of bioavailability of
IGFs for cell surface receptor binding. However, these mechanisms are
not mutually exclusive, and both may be relevant to the in vivo actions of estrogens and antiestrogens.
Figure 4: Regulation of an autocrine IGFBP-3 inhibitory loop in estrogen receptor positive human breast cancer cells by estradiol and antiestrogens. Estradiol inhibits and antiestrogens stimulate IGFBP-3 production. Maximal stimulation of IGFBP-3 gene expression by the pure antiestrogen ICI 182780 is approximately 8-fold higher than achievable with tamoxifen, an antiestrogen commonly used in breast cancer treatment. IGFBP-3, in turn, may inhibit proliferation by a direct pathway (11, 12, 13, 14) and/or by competing with IGF-I receptors for IGF-I or IGF-II.
A recent clinical trial (33) demonstrates that ICI 182780 can induce clinical responses
even in breast cancers resistant to tamoxifen, an antiestrogen widely
used clinically. In addition, a recent in vivo study
demonstrated that tamoxifen is a less potent inhibitor of in vivo MCF-7 cell proliferation than ICI 182780(34) . Tamoxifen
is a partial antagonist to the estrogen receptor and exhibits
estrogenic or antiestrogenic activities in a species-specific and
tissue-specific manner(18) . For example, in the
oophorectomized rat model (as well as in clinical use) tamoxifen has an
estrogen-like stimulatory effect on uterine growth, while ICI 182780
causes uterine involution. It has been demonstrated that these uterine
actions of antiestrogens are well correlated with effects on IGFBP-3
expression, assuming a growth inhibitory action of this protein; the
positive uterotrophic effect of tamoxifen is associated with
down-regulation of uterine IGFBP-3 expression, while ICI 182780-induced
uterine involution is associated with up-regulation of uterine IGFBP-3
expression(20) . In view of these results, we determined the
effect of tamoxifen on MCF-7 cell proliferation and IGFBP-3 gene
expression under our in vitro conditions. We observed that
tamoxifen was 2 orders of magnitude less potent than ICI 182780 in
up-regulating IGFBP-3 mRNA abundance and that maximum stimulation was
only to 125% of control values, as compared with the 8-fold
stimulation seen with ICI 182780 (data not shown). Taken together,
these data demonstrate a consistent relationship between the effects of
antiestrogens on proliferation and on IGFBP-3 expression, with respect
to both the degree of growth inhibition in the case of MCF-7 cells and
to the direction of proliferative change in the case of the uterus.
We have noted (data not shown) that estrogen receptor negative
breast cancer cell lines exhibit high rates of base-line proliferation
relative to estrogen receptor positive cells despite the fact that they
constitutively express IGFBP-3. In view of the inhibitory effect of
estradiol on IGFBP-3 expression in estrogen receptor positive cells,
the constitutive expression of IGFBP-3 in estrogen receptor negative
breast cancer cell lines is not unexpected and may be a consequence of
the absence of function of the signal transduction pathway that links
the estrogen receptor to the expression of the IGFBP-3 gene. While the
high proliferation rate of estrogen receptor negative breast cancer
cells in the presence of abundant autocrine IGFBP-3 appears paradoxical
in the context of the action of IGFBP-3 on MCF-7 cells reported here,
it is possible that this is a consequence of defects in the signal
transduction pathways that mediate the ``direct'' growth
inhibitory actions of IGFBP-3 (11, 12) and/or of
unrelated aspects of neoplastic progression that bypass the inhibitory
influence of IGFBP-3. The influence of agonist and antagonist estrogen
receptor ligands on IGFBP-3 expression in MCF-7 cells appears greater
in magnitude than the effect of TGF on IGFBP-3 expression by an
estrogen receptor negative breast cancer cell line, which
constitutively secretes this protein (24) .
In summary, expression of the IGFBP-3 gene by MCF-7 cells is detectable under serum-free conditions, enhanced by ICI 182780 in the presence or absence of estrogens, suppressed by estradiol, and negatively correlated with proliferation. This demonstrates that ICI 182780 does not merely act as a competitive antagonist to block estrogen action but rather actively regulates IGFBP-3 gene expression and cellular proliferation in a direction opposite to that of estradiol. Furthermore, estrogen-stimulated MCF-7 cell proliferation is attenuated by IGFBP-3, and the antiproliferative action of the antiestrogen ICI 182780 is reduced by blocking IGFBP-3 translation. While antiestrogens have previously been noted to have effects on IGF-I expression (15, 16, 17) , these data provide evidence for a novel mechanism by which estrogens and antiestrogens influence IGF physiology and cellular proliferation, suggest novel mechanisms that may be involved in the important clinical problem of antiestrogen resistance, and raise the possibility that in general steriod hormone action involves modulation of expression of IGF binding proteins.