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INTRODUCTION |
Retinoids induce growth inhibition and apoptosis in a variety of
tumor cells, including breast cancer cells (1). Recently, we proposed a
mechanism by which all-trans-retinoic acid
(atRA)1 synergizes with
interferon to inhibit the growth of both estrogen receptor-positive and
estrogen receptor-negative breast cancer cell lines (2). Here we
studied mechanisms by which atRA counteracts the growth-promoting
effects of insulin-like growth factors (IGFs) in breast cancer cells,
focusing on the involvement by retinoic acid receptors (RARs).
It is known that the molecular actions of retinoids are primarily
mediated by their nuclear receptors (RAR
,
and
, and the
retinoid X receptors (RXRs)
,
, and
), which function as liganded transcription factors (3). These receptors show both spatiotemporal patterns of expression during development and
tissue-specific distribution in adults, suggesting that the various
receptors play different roles in transducing retinoid signals. Among
the RARs, RAR
is expressed ubiquitously in adult tissues, RAR
is expressed mainly in skin, and RAR
is expressed primarily in
epithelial cells, including those in mammary tissue (4). Expression of RAR
is lost in the majority of breast cancer cell lines; it can be
induced by retinoic acid (RA) in estrogen receptor-positive breast
cancer cell lines but not in estrogen receptor-negative cancer cell
lines (4-7). The latter are believed to represent more advanced forms
of breast carcinoma. Induction of RAR
expression correlates well
with the growth-inhibitory and apoptotic effects of retinoic acid (8,
9), suggesting that loss of RAR
expression may be one of the
critical events involved in breast carcinogenesis/progression and in
responsiveness of breast cancer cells to retinoid chemotherapy. At the
same time, there is strong evidence that RAR
is the mediator of the
growth inhibition of breast cancer cells by retinoids (10, 11). In
general, RAR
expression is lower in estrogen receptor-negative breast cancer cell lines than in estrogen receptor-positive lines; this
corresponds to the responsiveness of these cell lines to RA. Taken
together, these observations raise the possibility that both RAR
and
RAR
are involved in the physiological action of retinoic acid in
breast cancer cells.
The insulin-like growth factor system includes IGF-I and IGF-II, their
corresponding receptors, six IGF-binding proteins (IGFBPs), and four
IGFBP-related proteins (12). IGF-I and IGF-II are thought to be
important growth factors for breast cancer. IGF-I and -II receptors and
IGFBP-2 and -4 proteins have been found in breast cancer cell lines and
in tissue specimens (13). Although IGF-I and -II proteins are not
expressed in breast cancer cell lines, they are expressed in breast
cancer specimens, possibly by stromal cells (13), suggesting that IGFs,
through a paracrine mechanism, promote breast cancer cell growth and
underscoring the importance of IGFBPs for their ability to modulate
IGF-I actions in the extracellular matrix.
In addition to the well established roles of IGFBPs in regulating IGF
bioavailability and IGF-I receptor responsiveness to IGF-I, IGFBP-3 has
also been recently proposed to function as a negative regulator of
growth, independently of the IGF-I receptor (14, 15). Supporting its
role as a growth inhibitory regulator, IGFBP-3 expression is
up-regulated by growth-inhibitory (and apoptosis-inducing) agents, such
as retinoic acid (16-19), vitamin D (20), transforming growth
factor-
(16, 21, 22), antiestrogens (23), tumor necrosis factor-
(24), and, most compellingly, the tumor suppressor gene p53
(25); IGFBP-3 expression is down-regulated by growth-promoting factors,
such as estrogen (26) and epidermal growth factor (27). All of this
information clearly indicates that IGFBP-3 is a common downstream
effector of many growth regulatory agents. We report here that both
RAR
and RAR
, by relaying the atRA signal in MCF-7 cells, are
involved in the induction of IGFBP-3, and our experiments suggest that
lack of RAR
expression in the majority of breast cancer cell lines
may result in the failure of IGFBP-3 induction and growth inhibition by retinoids.
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EXPERIMENTAL PROCEDURES |
Cell Cultures and Retinoids--
Cells of the breast carcinoma
cell line MCF-7 (American Type Culture Collection, Manassas, VA) were
grown in phenol red-free Eagle's minimal essential medium (Sigma)
supplemented with 5% charcoal-stripped calf serum (Sigma). Cells from
<15 passages were used for experiments.
atRA was purchased from Sigma. The RAR-specific agonist Ro 13-7410, the
RXR-specific agonist Ro 25-7386, and the RAR
-selective antagonist Ro
41-5253 were generously provided by Hoffmann-La Roche. Retinoids
were dissolved in absolute ethanol under lights that were covered with
a UV-blocking film (CLHC, Sydlin, Inc., Lancaster, PA). The integrity
of atRA was routinely monitored by spectrophotometry.
Preparation of Conditioned Medium--
MCF-7 cells were grown as
described above for 24 h, washed with phosphate-buffered saline,
and then transferred to phenol red-free Eagle's minimal essential
medium supplemented with 2 µg/ml fibronectin and 2 µg/ml
transferrin (both from Sigma) for another 24 h before atRA
treatment. The conditioned medium was then harvested with the addition
of 0.2 mM phenylmethylsulfonyl fluoride and 10 µg/ml
aprotinin (both from Sigma), dried under speed vacuum, and resuspended
for analysis.
Cell Growth Inhibition Assay--
MCF-7 cells (4 × 103 cells/well) were cultured in the conditioned medium
described above in 96-well cell culture plates. Recombinant human
IGF-I, recombinant human IGFBP-3 (both generous gifts of Celtrix, Palo
Alto, CA), or medium from atRA-treated cell cultures was added alone or
in different combinations to the cell cultures for 2 days. Cells were
washed, fixed with 10% trichloroacetic acid for 1 h, and then
stained with 1% sulforhodamine B for 1 h. Cells were washed
again, and then 100 µl of 10 µM Tris-HCl, pH 10, was
added to release the dye (28). The absorbance was measured at 562 nm.
Immunodepletion--
Conditioned medium from atRA-treated or
untreated cells was incubated with 2 µg/ml of anti-IGFBP-3 antibodies
(goat polyclonal antibodies against human IGFBP-3; Santa Cruz
Biotechnology Inc., Santa Cruz, CA) or normal goat serum (Santa Cruz
Biotechnology) for 2 h. Protein A/Protein G PLUS-Agarose (Santa
Cruz Biotechnology) then was added, and the media were rocked at
4 °C overnight followed by filter sterilization of the supernatants.
Immunoprecipitates were boiled for 3 min in SDS gel loading buffer and
were used in a Western ligand blotting.
Western Immunoblotting and Western Ligand Blotting--
Fifty
µg of protein from cell lysates or conditioned medium was loaded onto
8-12% SDS-polyacrylamide gels under nonreducing conditions. After
transfer, nitrocellulose blots were incubated with rabbit polyclonal
antibodies against human RAR
(Santa Cruz Biotechnology). The blots
were then incubated with secondary antibodies and developed using an
ECL kit (Amersham Pharmacia Biotech). For Western ligand blotting,
nitrocellulose blots were initially washed in 3% Nonidet P-40 (Fluka
Chemical Corp., Ronkonkoma, NY) for 30 min, followed by blocking in 1%
bovine serum albumin (Sigma) for 2 h and 0.1% Tween 20 (Sigma)
for 15 min. Blots were then probed with 125I-labeled
recombinant human IGF-II (Bachem California Inc., Torrance, CA)
overnight followed by extensive washing with 1% Tween 20 before autoradiography.
Transient Transfection--
A luciferase reporter gene construct
under the control of a retinoic acid response element (DR5-tk-Luc,
provided by Dr. R. M. Evans, Gene Expression Laboratory, Salk
Institute for Biological Studies, La Jolla, CA) was used to measure
retinoid receptor-mediated gene activation. Ten µg of DR5-tk-Luc was
co-transfected into MCF-7 cells with 2 µg of
-galactosidase
expression vector (pCMV
; CLONTECH, Palo Alto,
CA) using Lipofectin reagent (Life Technologies, Inc.). Transfection
efficiency was normalized to
-galactosidase activity.
Stable Transfection--
Plasmid constructs for stable
transfection experiments were pRC/CMV-RAR
and pRC/CMV-antisense
RAR
(generous gifts from Dr. X.-K. Zhang, La Jolla Cancer Research
Center, La Jolla, CA). MCF-7 cells grown to 50% confluence were washed
with serum-free growth medium. Two µg of either empty vector or
construct was mixed with Lipofectin reagent and added to cells for
5 h. Selection was initiated with 400 µg/ml of G418 (Life
Technologies, Inc.) on the third day and continued for 17-21 days
until drug-resistant colonies emerged. Single colonies were cloned and
assayed for the expression of the inserted genes by Northern blotting,
and the expression of RAR
receptor protein was measured by Western blotting.
Northern Blot Analysis--
Total RNA was isolated using TRI
Reagent (Sigma). RNA was separated on 1% agarose/1.1 M
formaldehyde gels and then transferred and cross-linked to GeneScreen
nylon membranes (NEN Life Science Products). Hybridization was carried
out using the following probes: T4 polynucleotide
kinase-labeled 40-mer antisense RAR
, RAR
, or
-actin DNA
(Oncogene Research Products, Cambridge, MA) or random primer-labeled
IGFBP-3 cDNA (Genentech, Inc., South San Francisco, CA). The
results were analyzed with a phosphorimager (Bio-Rad).
Nuclear Run-on Assay--
MCF-7 cells were lysed in Nonidet P-40
lysis buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.5% (v/v) Nonidet P-40. Lysates were centrifuged for 5 min at 500 × g and 4 °C. Nuclei were resuspended in 150 µl of glycerol storage buffer containing 50 mM Tris-HCl, pH 8.3, 40% (v/v) glycerol, 5 mM MgCl2, and 0.1 mM EDTA and were
frozen in liquid nitrogen. Nuclear run-on assays were performed using
5 × 107 frozen nuclei and 10 µCi of
[
-32P]UTP (ICN Pharmaceuticals, Inc., Irvine, CA).
Newly transcribed RNA was hybridized to the IGFBP-3 cDNA that was
immobilized on nylon membranes. The results were analyzed using a PC
molecular imager.
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RESULTS |
IGFBP-3 Expression Is Induced by atRA in a Dose- and
Time-dependent Fashion in MCF-7 Cells--
To determine
the effects of atRA on the expression of IGFBP-3 in our experimental
system, MCF-7 cells were grown in the presence of 0, 10
9,
10
8, 10
7, or 10
6
M atRA for 72 h followed by Northern blotting analysis
of IGFBP-3 mRNA. As shown in Fig.
1A, MCF-7 cells did not
express IGFBP-3 message in the absence of atRA, but as little as
10
8 M atRA was effective in inducing the
expression of IGFBP-3 mRNA. Higher levels of IGFBP-3 mRNA were
detected with increasing concentrations of atRA (Fig. 1A).
Fig. 1B shows the temporal effect of 10
6
M atRA on the expression of IGFBP-3 message. IGFBP-3
mRNA was detected as early as 24 h after atRA treatment and
was maximal at 48 h.

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Fig. 1.
Induction of IGFBP-3 expression by atRA in
MCF-7 cells. Northern blotting of IGFBP-3 mRNA from MCF-7
cells treated with 0, 10 9, 10 8,
10 7, or 10 6 M atRA for 72 h (A) or with 10 6 M atRA for
different periods of time (B). -Actin mRNA was
measured to verify equal loading. The results represent at least three
independent experiments.
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atRA Activates IGFBP-3 Gene Transcription, and RAR, Rather Than
RXR, Mediates This Process--
We next wished to determine whether
the retinoic acid-induced expression of IGFBP-3 in MCF-7 cells was
mediated by RAR or RXR and whether atRA directly activates the
transcription of the IGFBP-3 gene. The second point was of interest
because it is known that retinoids can regulate gene expression
posttranscriptionally (29, 30). For these experiments, MCF-7 cells were
incubated for 48 h with 10
6 M of either
atRA, the RAR-specific agonist Ro 13-7410, or the RXR-specific agonist
Ro 25-7386. Nuclei were isolated, and nuclear run-on assays were
performed. As indicated in Fig.
2A, both atRA and the
RAR-specific agonist Ro 13-7410 activated IGFBP-3 gene transcription,
but the gene was not transcribed in cells treated with vehicle only or
with the RXR-specific agonist Ro 25-7386. The
-actin gene was
transcribed normally under all of these experimental conditions. These
results indicate that 1) RAR but not RXR is involved in transducing the
atRA signal to induce IGFBP-3 expression, and 2) atRA and Ro 13-7410 directly activate IGFBP-3 gene transcription. IGFBP-3 mRNA was
measured in parallel experiments following treatment of MCF-7 cells
with the various retinoids for 72 h (Fig. 2B). IGFBP-3
mRNA was only present in cells treated with atRA and Ro 13-7410, the RAR-specific agonist.

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Fig. 2.
Activation of IGFBP-3 gene transcription by
various retinoids. A, nuclear run-on assay of nuclei
isolated from MCF-7 cells treated with 10 6 M
of various retinoids (atRA, the RAR-specific agonist Ro 13-7410, or the
RXR-specific agonist Ro 25-7386) for 48 h. -Actin gene
transcription was measured as the control. B, Northern
blotting of IGFBP-3 mRNA from MCF-7 cells treated with
10 6 M of various retinoids for 72 h.
-Actin mRNA was measured to verify equal loading. The results
represent at least three independent experiments. C,
luciferase activity of cell lysates prepared from MCF-7 cells
co-transfected with DR-tk-Luc and pCMV in the presence of
10 6 M of various retinoids for 72 h. The
results were normalized to -galactosidase activity and represent
three independent measurements.
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To verify the ability of the synthetic retinoids, Ro 13-7410 and Ro
25-7386, to activate retinoid receptors in our experimental system, a
luciferase reporter gene under the control of a DR5 element, the
canonical retinoic acid response element activated by RARs, was
introduced into MCF-7 cells. Luciferase activity was measured 72 h
later in the presence of 10
6 M of atRA, Ro
13-7410, or Ro 25-7386. As documented in Fig. 2C, Ro
13-7410, the RAR-specific agonist, activated the expression of
luciferase gene at a level similar to that of atRA, but Ro 25-7386, the
RXR-specific agonist, was not effective in activating the expression of
luciferase gene. These results validate the use of the synthetic
retinoids in our experimental system.
RAR
Expression Is Induced by atRA in an
RAR
-dependent Pathway, and RAR
Relays the atRA Signal
That Leads to the Induction of IGFBP-3 Expression in MCF-7
Cells--
It has been shown that the transcription of the RAR
gene
is induced rapidly after retinoid treatment, peaking by 6 h, and that it is independent of new protein synthesis (31, 32). Furthermore,
the level of RAR
expression in breast cancer cell lines appears to
be correlated with the induced levels of RAR
expression (5, 8, 9).
Thus, it is reasonable to postulate that RAR
is induced in MCF-7
cells by atRA through a signaling pathway mediated by RAR
. To test
this hypothesis, MCF-7 cells were grown in the presence or absence of
10
6 M atRA for 72 h. Total RNA was
extracted, and 30 µg was used to measure mRNAs for RAR
and
RAR
by Northern blotting. As documented in Fig.
3, the levels of RAR
expression in
MCF-7 cells were similar in the presence or absence of atRA, whereas
RAR
expression was detectable only after atRA treatment. These
results indicate that RAR
mediates the atRA-induced expression of
RAR
.

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Fig. 3.
Induction of RAR
expression by atRA in MCF-7 cells. Northern blotting of 30 µg of total RNA from MCF-7 cells treated with either 0 or
10 6 M atRA for 48 h. The probe was a
40-mer antisense RAR or RAR DNA. -Actin mRNA was measured
as an indicator of equal loading. The results represent at least three
independent experiments.
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These experiments led us to ask whether the signal leading to the
induction of IGFBP-3 expression was mediated by RAR
, or if the
induced RAR
mediates IGFBP-3 induction. In order to answer this
question, MCF-7 cells were cultured for 72 h in the presence of
10
7 M atRA plus 0, 10
8,
10
7, or 10
6 M of Ro 41-5253, an
RAR
-selective antagonist. A lower concentration of atRA was used
because we wanted to minimize the cytotoxicity of retinoids that is
observed at high concentrations. After incubation, 30 µg of total RNA
was used to assay RAR
mRNA. As documented in Fig.
4A, 1 molar excess of Ro
41-5253 blocked the induction of RAR
expression. With decreasing
concentrations of Ro 41-5253, RAR
expression increased, indicating
that the process is mediated by RAR
. IGFBP-3 expression was measured
in MCF-7 cells grown for 72 h in the presence of the same
combinations of retinoids by Northern blotting (Fig. 4B).
Paralleling the diminished expression of RAR
in the presence of
10
6 M of Ro 41-5253, IGFBP-3 expression was
also abolished, indicating that retinoid-induced IGFBP-3 expression is
correlated with RAR
expression.

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Fig. 4.
RAR mediates the
atRA-induced expression of IGFBP-3 (antagonist experiments).
A, MCF-7 cells were treated with 10 7
M atRA and different amounts of Ro 41-5253, an
RAR -selective antagonist, for 72 h. Total RNA was extracted and
assayed by Northern blotting for RAR mRNA expression using a
40-mer antisense RAR DNA as the probe. -Actin mRNA was
measured as an indicator of equal loading. The results represent at
least three independent experiments. B, Northern blotting of
IGFBP-3 mRNA expression from MCF-7 cells treated with different
combinations of atRA and Ro 41-5253 for 72 h. -Actin mRNA
was measured as an indicator of equal loading. The results represent at
least three independent experiments.
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In order to further document the direct involvement of RAR
in the
atRA-induced expression of IGFBP-3, RAR
sense and antisense cDNA
constructs were introduced into MCF-7 cells via expression vectors.
Positive colonies were identified, cloned, and tested for RAR
expression by Western immunoblotting (Fig.
5). Three clones with average levels of
expression of each sense (Fig. 5A,
3,
5,
and
6) and antisense (Fig. 5B, As-
4,
As-
6, and As-
9) RAR
were used for experiments
similar to those described above. As exemplified by the results shown
for
5 (Fig. 6A), the
RAR
-selective antagonist Ro 41-5253 was unable to block atRA-induced
IGFBP-3 expression in the three clones of RAR
sense transfectants,
indicating that RAR
is not directly involved in this process. In
contrast, in RAR
antisense transfectants, the induction of IGFBP-3
expression by atRA was totally blocked (Fig. 6B), indicating
that RAR
is directly involved in IGFBP-3 gene activation.

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Fig. 5.
RAR expression by
sense and antisense RAR transfectants.
MCF-7 cells were transfected with sense or antisense RAR constructs.
Stable clones were established for both sense transfectants
( 2-7) (A) and antisense transfectants
(As- 4-9) (B). RAR expression in these
clones was measured by Western blotting.
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Fig. 6.
RAR mediates the
atRA-induced expression of IGFBP-3 (transfection experiments).
Northern blotting of IGFBP-3 mRNA from total RNA extracted from
RAR sense cDNA-transfected MCF-7 cells ( 5) treated with
different combinations of atRA and the RAR -selective antagonist Ro
41-5253 for 72 h (A) or from three clones
(As- 4, As- 6, and As- 9) of MCF-7 cells
transfected with the RAR antisense cDNA and treated with
10 6 M atRA for 72 h (B).
-Actin mRNA was measured as an indicator of equal loading. The
results represent at least three independent experiments.
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IGFBP-3 Is a Downstream Effector of RAR
in the Inhibition of
Breast Cancer Cell Growth by atRA--
The IGF growth factor system is
believed to be actively involved in the growth of breast cancer (14).
IGFBP-3 is a secreted protein that has been thought to primarily
regulate the biological activities of IGFs extracellularly. In order to
investigate the functional integrity of atRA-induced IGFBP-3 in
modifying the actions of IGF-I, we first assayed IGFBP-3 secretion by
MCF-7 cells after induction by atRA. For this purpose, MCF-7 cells were grown in conditioned medium for 6 days in the presence or absence of
10
6 M atRA. Conditioned medium was harvested
at 2, 4, and 6 days, concentrated, and analyzed for IGFBP-3 secretion
by Western ligand blotting. As shown in Fig.
7A, IGFBP-3 protein was
secreted into the conditioned medium at a measurable level on day 2 of
atRA treatment; higher amounts of IGFBP-3 were secreted on days 4 and 6. In addition to IGFBP-3, IGFBP-2 and IGFBP-4 were also secreted into
the conditioned medium, in both the presence and absence of atRA (Fig.
7A).

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Fig. 7.
The secretion and biological activity of
atRA-induced IGFBP-3 in MCF-7 cells. A, atRA-induced
secretion of IGFBP-3 into the conditioned medium. MCF-7 cells were
grown in conditioned medium in the presence or absence of
10 6 M atRA for 6 days. Conditioned medium was
harvested at 2, 4, and 6 days, concentrated, and analyzed for IGFBP-3
secretion by Western ligand blotting. B, growth effects of
exogenous IGF-I and IGFBP-3 in MCF-7 cells. Recombinant human IGF-I or
IGFBP-3 (BP3) was added alone or in different combinations
to MCF-7 cells for 4 days; cell growth was measured by the
sulforhodamine staining. The results represent the mean ± S.D.
for quadruplicate experiments. C, growth effects of
atRA-induced endogenous IGFBP-3 in MCF-7 cells. MCF-7 cells were grown
in the conditioned medium containing 10 6 M
atRA for 4 days. The medium (CM/RA/BP3) was collected,
immunodepleted of IGFBP-3 (CM/RA) and applied to untreated
MCF-7 cell cultures in the presence of 1 nM IGF-I. Medium
from untreated cell cultures (CM/CTL) and from cultures that
were treated but immunodepleted with nonspecific antibodies
(CM/RA/BP3/S) were used as the control. The growth of cells
treated with CM/CTL was taken as 100%, and that of cells treated with
CM/CTL plus 10 nM rhIGFBP-3 (CM/CTL+1 nM
IGF-I+10 nM BP3) was taken as 0%. The results
represent the mean ± S.D. for quadruplicate experiments.
D, Western ligand blot to confirm the occurrence of IGFBPs
in atRA-treated conditioned medium (lane 1),
IGFBP-3-immunodepleted medium (lane 2), and
immunoprecipitates from IGFBP-3-immunodepleted medium (lane
3). The results represent at least three independent
experiments.
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We next tested the responsiveness of MCF-7 cells to the IGF system.
Exogenous IGF-I or IGFBP-3 was added alone or in different combinations
to MCF-7 cells for 4 days, and cell growth was measured by
sulforhodamine staining. As documented in Fig. 7B, MCF-7
cells were sensitive to the mitogenic effects of IGF-I, and recombinant human IGFBP-3 (rhIGFBP-3) inhibited such activity in a
dose-dependent manner (0-10 nM).
To investigate the biological activity of atRA-induced endogenous
IGFBP-3, MCF-7 cells were maintained in conditioned medium for 4 days
in the presence or absence of 10
6 M atRA, and
the conditioned medium was collected. IGFBP-3 protein was
immunodepleted in half of the conditioned medium from atRA-treated cells. The medium was then filter-sterilized and added to MCF-7 cells
for 2 days in the presence of 1 nM IGF-I. Cell growth was measured by sulforhodamine staining and expressed as percentage of
absorbance relative to control MCF-7 cell cultures treated with control
medium supplemented with 1 nM IGF-I (100%) or 1 nM IGF-I plus 10 nM rhIGFBP-3 (0%) (Fig.
7C). Similar to the results described in Fig. 7B,
the conditioned medium from atRA-treated MCF-7 cells was able to block
the growth promotion of MCF-7 cells by IGF-I (Fig. 7C,
CM/RA/BP3). When IGFBP-3 was depleted (Fig. 7C, CM/RA),
the medium was no longer effective in blocking the growth promotion by
IGF-I, whereas the normal goat serum-treated control (Fig. 7C,
CM/RA/BP3/S) did not remove the growth inhibition effect,
suggesting that atRA-induced inhibition of IGF-I-stimulated cell growth
is mediated rather specifically by IGFBP-3, not IGFBP-2 or IGFBP-4,
because IGFBP-3-depleted medium (CM/RA) was unable to
counteract IGF-I even when IGFBP-2 and IGFBP-4 were still present (Fig.
7D, lane 2).
As shown in the Western blots in Fig. 7D, conditioned medium
from the 4-day atRA-treated MCF-7 cells contained the IGFBPs 3, 2, and
4 (Fig. 7D, lane 1). After immunodepletion, only IGFBP-2 and
IGFBP-4 were present (Fig. 7D, lane 2); when the
immunoprecipitate was examined, only IGFBP-3 was found (Fig. 7D,
lane 3). These experiments clearly demonstrate that atRA-induced
IGFBP-3 is able to function as a downstream effector of RAR
to block
the growth promotion by IGF-I in MCF-7 breast cancer cells. A
semiquantitative Western blot analysis utilizing rhIGFBP-3 as a
standard indicated that the concentration of IGFBP-3 in atRA-treated
conditioned medium was ~3 nM (data not shown). This
result is consistent with a partial block of IGF-I action by
recombinant IGFBP-3, which resulted in ~50% inhibition at 2 nM concentration (Fig. 7B).
 |
DISCUSSION |
The tissue-specific distribution of retinoic acid receptors in
adults and the spatiotemporal patterns of expression during development
indicate that these receptors may play different roles. Yet the
coexistence of two or three retinoic acid receptor subtypes in a
specific tissue also suggests that some type of
compensation/coordination may exist among retinoic acid receptors in
transducing retinoid signals. Such a compensation/coordination of RARs
in breast cancer cells was demonstrated in our experiments that showed
that RAR
expression can be induced in MCF-7 cells by atRA via RAR
mediation. The levels of RAR
expression were similar in the presence
or absence of atRA in MCF-7 cells, but RAR
expression, which was undetectable in the absence of atRA, was strongly induced by atRA. When
the RAR
-selective antagonist Ro 41-5253 was used, the induction of
RAR
expression by atRA was blocked, indicating that RAR
induction is dependent on RAR
.
Both RAR
and RAR
have been implicated in tumor development. For
example, it is well documented that acute promyelocytic leukemia is
caused by a reciprocal chromosome 15:17 translocation in which the
t(15:17) breakpoint occurs in the RAR
gene (33). An involvement of
RAR
in cancer development was originally suggested by the finding
that RAR
is integrated by the hepatitis B virus in human hepatoma
(34). Moreover, defective RAR
expression is believed to be an early
event in epithelial carcinogenesis (35). Recently, it has been observed
that RAR
expression is lost in many epithelial tumors and tumor cell
lines, including breast cancer and breast cancer cells (5, 6, 35-39).
Furthermore, transgenic mice carrying antisense RAR
2 develop
carcinoma 14-18 months after birth (40), strongly supporting a role of
RAR
in tumorigenesis. By demonstrating an
RAR
-dependent RAR
induction, our experiments further
stress the importance of RAR
, which eventually becomes noninducible
with progression of the tumor, as in estrogen receptor-negative breast
cancer cells. The results of these experiments also allow us to clarify
why both RAR
and RAR
have been implicated in retinoid-induced
growth inhibition of breast cancer cells.
RAR
has been suspected as a tumor suppressor for a long time, and
loss of RAR
expression has been thought to be a critical event in
the development of breast cancer. We suggest that RAR
functions as a
tumor suppressor by regulating the expression of other critical cell
growth regulatory factors. Our experiments show that the regulation
(induction) of IGFBP-3 in MCF-7 cells is mediated by RAR
, because
blocking RAR
expression by an RAR
antagonist, Ro 41-5253, also
blocked the expression of IGFBP-3. When MCF-7 cells were transfected
with the sense cDNA of RAR
, IGFBP-3 was expressed, even in the
presence of Ro 41-5253. At the same time, when the antisense cDNA
of RAR
was transfected into MCF-7 cells, those cells were no longer
able to respond to atRA by expressing IGFBP-3.
Using nuclear run-on assays, we showed that atRA directly activates
IGFBP-3 gene transcription, supporting the recent finding that a major
consensus sequence for retinoic acid is present in the promoter region
of the IGFBP-3 gene (41). Whereas MCF-7 cells synthesize and secrete
IGFBP-2 and IGFBP-4 into conditioned medium, the application of atRA
not only induces the messenger for IGFBP-3, but also results in the
appearance of secreted protein in the conditioned medium. IGFBP-3
secretion seems to occur while secretion of IGFBP-2 decreases and
secretion of IGFBP-4 increases.
Among their diverse biological activities, IGFBPs are able to
negatively modulate the actions of IGF by binding IGFs and preventing them from binding to the type 1 receptor (12, 42). Here, we demonstrated that the application of IGF-I stimulates cell growth in MCF-7 cells and that the application of exogenous rhIGFBP-3 can
totally reverse this action. When we tested the biological activity of
IGFBP-containing conditioned media in their ability to inhibit the
IGF-I-stimulated growth of MCF-7 cells, we expected that changes in all
of the IGFBPs (that is, IGFBP-3 induced by atRA along with an increase
in IGFBP-4 and a decrease in IGFBP-2) would contribute to the growth
inhibition effect. However, immunodepletion of IGFBP-3 from the
conditioned medium removed all growth inhibitory activity, suggesting
an IGFBP-3-specific growth inhibitory mechanism. The significance of
atRA-induced changes in IGFBP-2 and IGFBP-4 and the reason why these
changes do not help counteract the IGF-I stimulation effect are not
clear. Although IGFBP-3 induction by retinoids has been consistently
observed, inconsistencies exist about retinoid-induced changes in
IGFBPs 2 and 4 (17-19). In addition, as mentioned earlier, the
biological activities of IGFBPs are not limited to negative effects on
IGFs. Thus, changes in IGFBP-2 and IGFBP-4 may be germane to other, as
yet unidentified mechanisms. In fact, the co-presence of IGFBPs 2, 3, and 4 in the cell culture medium may well indicate that these proteins
possess different functions rather than simply representing functional
redundancy. Another explanation is that IGFBP-3 may act through an
IGF-independent pathway. IGF-independent actions of IGFBP-3 have been
reported (14, 15, 43), and underlying mechanisms are being pursued vigorously. Of particular interest is the recent observation that IGFBP-3 can be translocated into the cell nucleus (44-46). Both exogenous (47) and endogenous
IGFBP-32 have been shown to
be translocated into the nucleus of breast cancer cells. Given the
extremely selective nature of nuclear protein localization, it is
reasonable to speculate that IGFBP-3 exerts profound biological
activity in the nucleus.
In summary, our experiments show that both RAR
and RAR
are
involved in the growth inhibitory activity of retinoids by mediating the induction of IGFBP-3 expression. By linking IGFBP-3 to RAR
, our
experiments have pinpointed an intersection between retinoid and IGF
signals. This information also expands knowledge of the downstream
effectors of RAR
and explains how RAR
might act as a tumor suppressor.