From the
Institute of Applied Biochemistry,
University of Tsukuba, 1-1-1 Tenno-dai, Tsukuba Science City, Ibaraki
305-8572, the ||Department of Obstetrics and
Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8655, **Taiho Pharmaceutical Co., Ltd.,
Cancer Research Laboratory, Hanno Research Center, 1-27-1 Misugidai, Hanno
City, Saitama 357-8527,
Institute of
Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-0034,
CREST, Japan Science
and Technology, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, and
SORST, Japan Science and Technology, 4-1-8
Honcho, Kawaguchi, Saitama 332-0012, Japan
Received for publication, January 30, 2003 , and in revised form, April 23, 2003.
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ABSTRACT |
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INTRODUCTION |
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The ligand-dependent activation of ER requires ligand-dependent
association of coactivator complexes
(1215).
The co-activator complexes for AF-2 contain histone acetylases p300/CBP
(16,
17), p300/CBP-associated
factor (pCAF) (18), p160
protein family members steroid receptor coactivator-1 (SRC-1)
(19,
20), transcription
intermediary factor 2 (TIF2)
(21), p300/CBP-interacting
protein (p/CIP)
(2224),
non-acetylase vitamin D receptor-interacting protein/TR-associated proteins
(DRIP/TRAP)
(2529)
or transformation/transcription domain-associated protein (TRRAP)
(3032),
and general control of amino acid protein-5 (GCN5)
(32,
33). AF-1 transcriptional
activity is enhanced by p300 and DEAD box protein p68/p72, which form a
protein complex with p160 family proteins and p300/CBP, and directly bind to
the A/B domain to potentiate AF-1 activity
(34,
35). The phosphorylation of
the serine residue at position 118 in the A/B domain stabilizes the complex
formation of ER
and the coactivator complex containing p68/p72 to
potentiate the AF-1 activity
(3436).
Estrogen is known to stimulate hormone-dependent tumors such as endometrial
cancer and breast cancer (37).
Recently, it was suggested that some endocrine disrupters may also contribute
to the development of hormone-dependent cancers
(38,
39). Several studies have
demonstrated that many endocrine disrupters, such as butylbenzyl phthalate
(BBP), a phthalate ester used as a plasticizer, are capable of interacting
with estrogen receptors and induce estrogen receptor-mediated responses,
suggesting that estrogenic or anti-estrogenic effects elicited by these
substances may be receptor-mediated
(3841).
For the endocrine therapy of these cancers, the development of inhibitory
ligands for ERs has yielded important therapeutic treatments, including the
use of tamoxifen (37,
42,
43). Tamoxifen exhibits a wide
range of estrogen-like and anti-estrogen actions according to the target
tissue examined (44). While
tamoxifen may exert anti-estrogenic activity by silencing the transcriptional
activity of AF-2, agonist activity of tamoxifen can be mediated through AF-1
in a cell- or tissue-dependent manner
(4549).
However, most patients undergoing long-term treatment of breast cancer with
tamoxifen eventually experience recurrence of tumor growth. One of the reasons
for this treatment failure is the acquisition by the tumor of the ability to
respond to tamoxifen as a stimulatory rather than inhibitory ligand
(5053).
Wolf et al. (54,
55) identified a mutant
ER from a tamoxifen-stimulated tumor that contained a point mutation
that led to a tyrosine for aspartate substitution at amino acid 351
(ER
(D351Y)), located within the LBD of ER
(54,
55).
Recent studies
(5660)
have suggested that tamoxifen promotes the binding of ER to nuclear
receptor corepressor (N-CoR) or related factors silencing mediator of retinoid
and thyroid receptors (SMRT), which mediate repression by recruiting histone
deacetylases (HDACs). We reported previously
(61) that ER
(D351Y)
exhibited reduced interaction with N-CoR and SMRT in the presence of
4-hydroxytamoxifen (OHT). These observations raised the possibility that
OHT-dependent interaction between ER
and corepressor complexes may be
essential for the anti-estrogenic effect of OHT and that abrogation of
OHT-dependent binding of corepressors to ER
may convert OHT from
antagonist to agonist to stimulate cancer growth.
In this paper, we identified BBP as an agonist for AF-1 of ER.
Although BBP exhibited the same properties as tamoxifen in a transient
transfection assay, BBP-occupied ER
did not bind to corepressors and
enhanced the proliferation of the breast cancer cell line MCF-7. The
transcriptional activity of OHT-occupied ER
was modulated by the ratio
of the expression levels between AF-1 coactivators and corepressors. Moreover,
MCF-7 breast cancer cell lines expressing the dominant-negative type of N-CoR
exhibited a growth phenotype in the presence of OHT. These results indicate
that activation of AF-1 induces the stimulation of breast cancer growth and
that the ratio between AF-1 coactivators and corepressors plays a key role to
prevent proliferation of tumor by tamoxifen.
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EXPERIMENTAL PROCEDURES |
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Measuring IC50 Values of E2 and
BBPFor measurement of the binding constant value of BBP to
ER, IC50 measuring kit was purchased from Wako Chemicals
Co., and IC50 was examined according to the manufacturer's
protocol.
Plasmid ConstructionThe ER/
expression
plasmids (HEG0/ERG0
) and their deletion mutants (HE19/HE19
) were
described previously (3,
11,
61,
64). The p300, p68, p72,
SRC-1, TRAP220, and TRRAP expression plasmids were also described previously
(32,
35,
6264).
Human N-CoR cDNA was cloned into pEF1-V5-His A for V5-N-CoR. Reporter
constructs (17m5-luc, MH100-tk-luc, and ERE3-tk-luc) have been described
previously (35,
61,
64). The ligand binding domain
of ER
was inserted into the pM vector (Clontech) to generate GAL-DEF.
VP-SRC-1, VP-TRAP220, and VP-p300 were described previously
(11,
61,
64). Nuclear receptor
interaction region in TRRAP was inserted into pVP16 vector to generate
VP-TRRAP (32). C-terminal
fragments of N-CoR and SMRT (including the NR interaction domains ID1 and ID2)
were inserted into the pVP16 vector (Clontech) to generate VP-N-CoR, VP-SMRT,
and pGEX-2T vector to GST-ID1-2 of N-CoR and SMRT and pcDNA3 vector
(Invitrogen) for FLAG-N-CoR ID1-2. ER
mutation in amino acid
replacement D351Y was introduced into full-length ER
and GAL-DEF
plasmid by PCR-based point mutagenesis (Stratagene).
Transfection, Luciferase Assay, Mammalian Two-hybrid Assay, and
Repression Assay293T cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Two days
before transfection, medium was changed to phenol red-free DMEM containing 5%
charcoal-stripped FBS. Transfection was performed with Lipofectin reagent
(Invitrogen) according to the manufacturer's protocol. For luciferase assays,
250 ng of ERE3-tk-luc plasmid was cotransfected with 25 ng of ER expression
vector (HEG0/ERG0) or mutants. For mammalian two-hybrid assays, 250 ng
of ERE3-tk-luc or 17m5-luc vector was cotransfected with 250 ng of HEG0,
GAL-DEF, or GAL-DEF(D351Y) in combination with 250 ng of indicated
VP16-conjugated constructs and/or p300, p68, N-CoR, or N-CoR ID1-2 plasmids.
For repression analysis, 1 µg of MH100-tk-luc vector was cotransfected with
250 ng of GAL-DEF. As a reference plasmid to normalize for transfection
efficiency, either 5 ng of pRL-CMV vector (Promega) or 125 ng of pRSV
GAL
vector was cotransfected in all experiments. Six hours after transfection,
culture medium was replaced with fresh medium containing 0.2% FBS. At this
time, either E2 (10 nM), OHT (100 nM), ICI182,780 (100
nM), ethanolic vehicle, or 5 ng/ml trichostatin A (TSA) was added,
and cells were incubated for an additional 24 h. Preparation of cell extracts
and luciferase assays were performed following the manufacturer's protocol
(Promega).
-Galactosidase activity was measured to control the
efficiency for each transfection. Individual transfections, each consisting of
triplicate wells, were repeated at least three times. For establishing MCF-7
stable transfectant of N-CoR ID1-2, Lipofectin reagent was used for introduce
pcDNA-ID1-2, and transfectants were selected by 500 µg/ml G418 (Sigma), and
several clones were isolated.
GST Pull-down AssayFor GST pull-down assays, bacterially expressed GST fusion proteins or GST bound to glutathione-Sepharose 4B beads (Amersham Biosciences) were incubated on ice with [35S]methionine-labeled proteins expressed by in vitro translation using the TNT-coupled transcription-translation system (Promega). After 1 h of incubation, free proteins were removed by washing the beads 5 times with phosphate-buffered saline containing 10% glycerol and protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µM phenylmethylsulfonyl fluoride). Specifically bound proteins were eluted by boiling in SDS sample buffer and analyzed by 6% SDS-PAGE. After electrophoresis, radiolabeled proteins were visualized by autoradiography.
Coimmunoprecipitation and Western Blotting293T cells were
transfected with the indicated plasmids, lysed in TNE (10 mM
Tris-HCl (pH 7.8), 1% Nonidet P-40, 0.15 M NaCl, 1 mM
EDTA, 1 µM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin)
buffer, and immunoprecipitated with anti-FLAG M2 monoclonal antibody (Sigma)
or anti-ER (Chemicon). Interacting proteins were separated by 6%
SDS-PAGE, transferred onto polyvinylidine difluoride membranes (Millipore),
and detected with anti-ER
, anti-p300 (Santa Cruz Biotechnology),
anti-FLAG M2, or anti-V5 tag (Invitrogen), and secondary antibodies were
conjugated with horseradish peroxidase. For detecting the expression of
ER
and N-CoR ID1-2, isolated clones were lysed in TNE, and each lysate
was detected by immunoblotting using anti-ER or anti-FLAG and secondary
antibodies.
Cell Proliferation AnalysisTwo days before assay,
MDA-MB-231 (ER-negative) and MCF-7 (ER
-positive) cells were
cultured in a 24-well plate in phenol red-free DMEM supplemented with 0.2%
charcoal-stripped fetal bovine serum. As experimental medium, either E2 (10
nM), OHT (1 µM), BBP (1 µM), or ethanol
vehicle was supplemented. Cells were harvested for the indicated times, and
the number of viable cells was counted with hemocytometer.
S-phase Entry AnalysisFor S-phase entry analysis, NIH3T3
cells were cultured in phenol red-free DMEM supplemented with 5%
charcoal-stripped FBS and seeded onto glass coverslips at 6070%
confluence. Transfection with 3 µg of wild-type or mutant ER
expression plasmid was performed by using Perfectin Reagent (Gene Therapy
Systems) according to the manufacturer's protocol. Incubation medium was
changed after 24 h into phenol red-free DMEM supplemented with 0.2%
charcoal-stripped FBS. Cells were left in this medium for 24 h and then
cultured with 100 µM 5-bromouridine 5'-triphosphate
(BrdUrd) in the presence of either E2 (10 nM), BBP (1
µM), or OHT (1 µM) for an additional 24 h. After
incubation, the cells were fixed for immunostaining.
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RESULTS |
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A Contrastive Effect between BBP and OHT on the Proliferation of Breast
Cancer CellsThus, we next examined the effects of BBP on
proliferation of two human breast cancer cell lines: MCF-7 and MDA-MB-231. The
MCF-7 cell line expresses ERs, and its proliferation is estrogen-dependent.
MDA-MB-231 cells, whose growth is not affected by estrogens, were used to
control for false-positive responses. Whereas E2 induced MCF-7 cell
proliferation at concentrations at 10 nM, OHT strongly inhibited
the proliferation of MCF-7 cells (Fig.
2C, right panel)
(41). In contrast with OHT,
BBP exerted an increase in MCF-7 proliferation at 1 to 10 µM,
and the maximum effect represented 70% of the presence of E2
(Fig. 2C, right
panel). None of the three compounds we tested affected proliferation of
MDA-MB-231, which does not express ER
(Fig. 2C, left
panel).
To investigate whether the AF-1 activity is necessary for the BBP-dependent
cell proliferation, newly synthesized DNA was evaluated by BrdUrd
incorporation method. NIH3T3 fibroblasts were transiently transfected with
expression plasmid encoding either ER (HEG0) or ER
mutant
(HE19). The transfected NIH3T3 fibroblasts were made quiescent and then
stimulated for 24 h with either E2, BBP, or OHT
(86). BrdUrd was added to the
medium together with the ligands, and its incorporation into DNA was analyzed.
BrdUrd-positive cells expressing either ER
or HE19 were counted. The
NIH3T3 cells transfected with control vector did not respond to ligand
treatments. Although E2 and BBP strongly stimulated progression of
ER
-transfected NIH3T3 cells toward S-phase, BrdUrd incorporation into
HE19-transfected cells was enhanced by E2 but not by BBP
(Fig. 2D). These
results indicate that the BBP-dependent S-phase entry requires the AF-1
activity of ER
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BBP and OHT Induce the Binding of ER to AF-1
Coactivators but Not to AF-2 CoactivatorsOur results indicate that
both BBP and OHT are selective agonists for ER
AF-1 and show an
antagonistic effect on AF-2, whereas BBP exhibits an estrogenic effect and OHT
shows an anti-estrogenic effect on the cell proliferation. In order to reveal
a reason for this discrepancy, we tested the interaction between ER
and
coactivators or corepressors in the presence of either BBP or tamoxifen. By
using a mammalian two-hybrid assay, we first evaluated the BBP- or
OHT-dependent binding of AF-1 coactivators to ER
. HEG0 was
cotransfected with either VP16 transactivation domain-fused p72 (VP-p72), p68
(VP16-p68), or p300 (VP-p300) constructs into 293T cells. All of the three
ligands we tested induced the interaction of ER
with either p68, p72,
or p300 (Fig. 3A). The
BBP-dependent interaction between ER
and AF-1 coactivators was
confirmed using a coimmunoprecipitation method. By using an anti-ER
antibody, ER
was immunoprecipitated from a nuclear extract of 293T
cells that were cotransfected with ER
and either FLAG-tagged p72,
FLAG-tagged p68, or p300. Strong anti-FLAG or anti-p300 antibody binding was
observed on immunoblots of anti-ER
immunoprecipitates from
cotransfectants treated with either E2, BBP, or OHT
(Fig. 3B).
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We then examined the binding of the DEF region of ER with AF-2
coactivators. A GST pull-down assay showed that whereas E2 induced the direct
binding of ER
(GAL-DEF) to AF-2 coactivators SRC-1, TRAP220, or TRRAP,
BBP and OHT abrogated the interaction between GAL-DEF and AF-2 coactivators
(Fig. 3C). In a
mammalian two-hybrid experiment, E2-bound GAL-DEF exhibited significant
binding to either SRC-1, TRAP220, or TRRAP
(Fig. 3D, left
panel). Conversely, neither BBP nor OHT induced the interaction between
GAL-DEF and AF-2 coactivators (Fig.
3D, middle and right panels). These
results were in good agreement with the observation that BBP and OHT act as
antagonists for AF-2.
OHT but Not BBP Induces the Interaction between ER and
Corepressor ComplexesIn a previous paper, we showed that OHT
induces the binding between GAL-DEF and corepressor complexes to repress the
basal transcriptional activity of TK promoter located downstream from
GAL4-binding elements (17 x 5 m)
(61). This OHT-induced binding
of ER
to corepressor complexes is reduced by the amino acid
substitution at position 351 in ER
(ER
(D351Y)), which is derived
from tamoxifen-induced tumor (Fig.
4A) (54,
55). Consistent with previous
studies, OHT-bound GAL-DEF repressed the transcriptional activity of the TK
promoter (Fig. 4B,
lane 2). This repressive activity was inhibited by the addition of
TSA, a specific histone deacetylase inhibitor
(Fig. 4B, lane
5). The D351Y mutation, which exhibited decreased corepressor
association, impaired the tamoxifen-dependent repression by GAL-DEF
(Fig. 4B, lane
8). In the presence of BBP, the repressive activity of GAL-DEF was not
observed (Fig. 4B,
lane 3), indicating that BBP-bound ER
would not interact with
corepressor complexes.
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To test the interaction between ER and corepressors, a mammalian
two-hybrid assay was performed. Either GAL-DEF or GAL-DEF(D351Y) was
cotransfected into 293T cells with either VP, VP-SMRT, or VP-N-CoR. Whereas
the interaction between GAL-DEF and corepressors was observed in the presence
of OHT (Fig. 4C,
middle panel), BBP-bound GAL-DEF exhibited no binding to corepressors
as expected (Fig. 4C,
right panel). Consistent with previous studies, the D351Y mutation
reduced the OHT-induced interaction with corepressors
(Fig. 4C, lanes
1012) (61). The
corepressor binding was further investigated by a coimmunoprecipitation
method. By using an anti-ER
antibody, ER
was immunoprecipitated
from nuclear extracts of 293T cells that were cotransfected with ER
and
V5-tagged N-CoR. In the presence of OHT, anti-V5 antibody binding was observed
on immunoblots of anti-ER
immunoprecipitates from cotransfectants
(Fig. 4D, lane
5). In contrast, N-CoR was not coprecipitated with E2- or BBP-bound
ER
(Fig. 4D,
lanes 4 and 6).
The Ratio of AF-1 Coactivator/Corepressor Complexes in Cells Is an
Essential Determinant for the Transcriptional Activity of OHT-occupied
EROur results indicated that although OHT induced
the binding of ER
to both AF-1 coactivators and corepressors, BBP-bound
ER
selectively associated with AF-1 coactivators. Thus we next examined
the effect of AF-1 coactivators and corepressors on transcriptional activity
of ER
induced by OHT or BBP. The expression of p300 and p68 enhanced
the transcriptional activity of ER
in the presence of E2, OHT, or BBP
(Fig. 5A). On the
contrary, N-CoR reduced OHT-dependent transactivation function of ER
(Fig. 5B, middle
panel) but not E2- and BBP-dependent activation
(Fig. 5B,
left and right panels). The stimulation of OHT-dependent
transcriptional activity of ER
(D351Y) by the expression of AF-1
coactivators was much higher than that of wild-type ER
(Fig. 5A, compare
left to middle panel), suggesting that the association of
endogenous corepressor complexes with OHT-bound ER
reduces AF-1
activity. The maximum OHT-dependent transcriptional activity induced by
coactivator expression was comparable with the BBP-dependent activity
(Fig. 5A, compare
lanes 1416), raising the possibility that the overexpression
of coactivators may convert OHT from antagonist into agonist to stimulate
cancer growth. In addition, as shown in
Fig. 5D, enhancement
of transcriptional activity by the coactivator expression was reduced by the
expression of corepressor, N-CoR. These findings indicate that the ratio of
coactivator/corepressor in cells determines the transcriptional activity of
OHT-bound ER
and that the inhibition of corepressor binding to
ER
may convert OHT from partial agonist to full agonist for AF-1 to
stimulate cell growth of MCF-7.
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Inhibition of the Interaction between OHT-bound ER and
Corepressors Stimulates Proliferation of Breast Cancer Cells in the Presence
of OHTTo investigate this hypothesis, we first determined the
regions in N-CoR/SMRT, which are responsible for the interaction between
N-CoR/SMRT and OHT-occupied ER
. Recently, it was shown that retinoid X
receptor and TR LBDs associate with N-CoR/SMRT via the N-CoR/SMRT domains ID1
and ID2
(6669).
Therefore, we assessed whether GST-ID1-2 fusion proteins could associate with
in vitro translated ER
. A GST pull-down assay showed both
GST-ID1-2 derived from N-CoR and SMRT directly bound to ER
in the
presence of OHT (Fig.
6B). We then studied whether the expression of the ID1-2
region abrogates the interaction between OHT-occupied ER
and N-CoR/SMRT
using a mammalian two-hybrid assay. In this assay, the binding of N-CoR/SMRT
to OHT-bound ER
was inhibited by the expression of the ID1-2 region
derived from N-CoR (Fig.
6C). Coexpression of the ID1-2 region with ER
enhanced the OHT-dependent transactivation function of ER
(Fig. 6D, left
panel) but not that of ER
(D351Y)
(Fig. 6D, right
panel), indicating that the inhibition of corepressor binding enhances
the AF-1 activity of ER
.
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Next, we examined the effect of the expression of the ID1-2 region on the
E2-dependent growth phenotype of the MCF-7 breast cancer cell line. The
expression vector containing FLAG-tagged ID1-2 region was transfected into
MCF-7 cells, and several stable cell lines constitutively expressing the ID1-2
region in MCF-7 cells (MCF-7(ID1-2)) were established. We picked up four
independent clones, clones 14, and we examined the expression of
ER and ID1-2 by Western blot using specific antibodies against
ER
and FLAG epitope. In all four clones we tested, the expression of
FLAG-ID1-2 was observed (Fig.
7A). The expression level of ER
in these clones
was unchanged when compared with the control MCF-7 cells
(Fig. 7A). In the
absence of E2, ID1-2 stable clones exhibited normal cell growth similar to the
control MCF-7 (Fig.
7B). However, either E2 or BBP treatment enhanced the
growth rate of the control and clones. OHT inhibited the growth of control
MCF-7 cells but stimulated the growth of ID1-2 stable transformants
(Fig. 7B), indicating
that the inhibition of corepressors binding to ER
converts OHT from
anti-estrogenic to an estrogenic effect on the proliferation of breast cancer
cells.
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DISCUSSION |
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The Structure of LBD Induced by BBP Is Different from That Induced by
OHTAccumulating evidence suggests that the differential ability of
partial antagonists to modify gene expression cannot be accounted for by
alterations in the ligand-receptor complex alone but also must take into
consideration coregulator (coactivator and corepressor) proteins that regulate
ER interaction with the general transcriptional machinery and chromatin
(15,
32,
59,
7275).
Therefore, coactivators and corepressors of the ER were tested to
determine whether these coregulators interact with ER
in the presence
of these compounds. All of the three compounds we tested induced the
interaction between ER
and AF-1 coactivators p68/p72 and p300 as
expected. E2 induced the binding of LBD to AF-2 coactivators but not to
corepressors (32,
59,
74,
75,
78). Consistent with previous
reports (70,
71), OHT induced the
interaction with corepressors instead of AF-2 coactivators. In contrast,
BBP-occupied LBD bound to neither AF-2 coactivators nor corepressors.
The crystal structures of the LBDs of several nuclear receptors have been
determined and described as a sandwich of 12 -helices (H1H12)
with a central hydrophobic ligand-binding pocket
(70,
71,
76,
77). In the presence of
ligands, the hinge region between H11 and H12 is moved closer to H3 and H5,
and H12 is positioned over the ligand-binding pocket formed by H3, H4, and H5.
The repositioned H12 releases the corepressors from the LBD and forms a
hydrophobic groove with H3 and H5
(70,
71). This hydrophobic groove
is known to be important for interaction with LXXLL motifs found in
p160 family members (SRC-1, transcription intermediary factor 2, and
p300/CBP-interacting protein) as well as in other coactivator molecules
(49,
71,
79,
80). The structures of
tamoxifen-bound ER
revealed that the position of H12 differed compared
with that of H12 in E2-bound ER
and did not form a coactivator
interaction surface but a recognition surface for corepressor complexes
(70,
71). We observed that BBP
induced the binding of LBD to neither AF-2 coactivators nor corepressors,
indicating that the position of H12 in BBP-bound ER
is different from
that of H12 in E2- or OHT-bound ER
. These results also suggest that the
structural differences induced by compound binding would affect the binding
affinity of ER
to the coregulators.
The Ratio between AF-1 Coactivator and Corepressor Complexes in Cells
Is a Major Determinant of the Transactivation of OHT-occupied
EROur results indicated that OHT induced the
binding of ER
to both AF-1 coactivators and corepressors. The
enhancement of transcriptional activity induced by coactivator expression was
reduced by the corepressor expression, indicating that the ratio of
coactivator/corepressor in cells determines the transcriptional activity of
OHT-bound ER
. It is well known that p300 possesses histone
acetyltransferase activity that modifies local chromatin structure into a
transcriptionally permissive state
(16,
17). N-CoR/SMRT complexes
contain histone deacetylase activity
(62,
8183),
suggesting that AF-1 coactivator and corepressor complexes may act and/or bind
competitively to tamoxifen-bound ER
. If the OHT-occupied ER
simultaneously binds coactivators and corepressors under these conditions, the
repressor domain of corepressors may inhibit ER
transcriptional
activity by blocking the activation function of coactivators. Alternatively,
OHT may induce competitive binding between AF-1 coactivators and corepressors
to abrogate full AF-1 activity. OHT may induce an LBD conformation that
enables the receptor to retain its ability to interact with corepressor to
decrease the accessibility of AF-1 coactivators to the A/B region such that
AF-1 activity is not efficiently activated.
Enhancement of the AF-1 Activity Stimulates Cancer Cell
GrowthIn a previous paper, we showed that the ER(D351Y)
mutant, which is derived from an MCF-7 breast tumor cell line that showed
stimulated growth rather than inhibition by tamoxifen, exhibited reduced
OHT-dependent interaction with corepressors to increase OHT-induced AF-1
activity compared with wild-type ER
(61). The AF-1 activity
induced by BBP was comparable with that of OHT-bound ER
(D351Y), since
BBP induced the binding of ER
to AF-1 coactivators but not to
corepressors. These results raise the possibility that fully activated AF-1
induces the proliferation of breast cancer cells and that the binding of
corepressors to ER
is essential for the antagonistic effect of OHT to
inhibit proliferation of breast cancer cells. Therefore, we generated cell
lines stably expressing ID1-2 region of N-CoR to abrogate OHT-dependent
binding of ER
to corepressors. The stable transformants expressing the
ID1-2 region exhibited the OHT-stimulated growth phenotype, indicating that
the binding between OHT-occupied ER
and corepressors plays a key role
in inhibition of tumor growth by tamoxifen. In addition, these results also
indicate that fully activated AF-1 stimulates cancer growth.
Tamoxifen is an effective treatment for all stages of hormone-responsive
breast cancer and can prevent breast cancer in high-risk women
(44,
84). However, tamoxifen has a
partial estrogenic activity in the uterus and is associated with an increased
incidence of endometrial hyperplasia and cancer. Recently, Brown and
co-workers (75) showed that
the expression level of SRC-1, a coactivator for ER, is higher in an
endometrial cancer cell line, Ishikawa, than in MCF-7. SRC-1 silencing by
small interfering RNA in Ishikawa cells resulted in inhibition of
tamoxifen-stimulated cell cycle progression. Hayashi and co-workers
(85) reported that the
relative transcriptional activity of AF-1 of ER
compared with that of
AF-2 was 4-fold higher in Ishikawa cells than MCF-7 cells. They also mentioned
that mitogen-activated protein kinase, which phosphorylates the serine residue
at position 118 in ER
A/B domain, was constitutively activated in
Ishikawa cells but not in MCF-7 cells. We reported previously
(3436)
that the phosphorylation of serine 118 by mitogen-activated protein kinase
stabilized the complex formation between ER
and p68/p72 DEAD box
proteins, which are components of the p300/SRC-1 coactivator complex, to
stimulate AF-1 activity of ER
. Together with these observations, our
results suggest that the ratio between ER
AF-1-coactivator complex and
ER
-corepressor complex in cells is an important determinant of AF-1
activity of OHT-occupied ER
and that the proliferation of cancer cells
is regulated by AF-1 activity of OHT-bound ER
. From these observations,
it is suggested that the tissue and cell type specificity of tamoxifen action
is due to the difference of AF-1 activity of tamoxifen-bound ER
in
various tissues and cells. Our results shed light on the molecular mechanism
underlying tamoxifen-dependent inhibition of cancer growth and ring an alarm
against endocrine disrupters.
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FOOTNOTES |
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¶ Both authors contributed equally to this work.
¶¶ To whom correspondence should be addressed. Tel.: 81-29-853-6632; Fax: 81-29-853-4675; E-mail: junny-tky{at}umin.ac.jp.
1 The abbreviations used are: ER, estrogen receptor; AF, activation function;
BBP, n-butylbenzyl phthalate; N-CoR, nuclear receptor core-pressor;
SMRT, silencing mediator for retinoid and thyroid hormone receptor; LBD,
ligand binding domain; ID, interaction domain; OHT, 4-hydroxytamoxifen; ICI,
ICI182,780; TSA, trichostatin A; GST, glutathione S-transferase; TK,
thymidine kinase; ERE, estrogen-responsive element; DMEM, Dulbecco's modified
Eagle's medium; FBS, fetal bovine serum; BrdUrd, 5-bromouridine
5'-triphosphate; E2, 17-estradiol; SRC-1, steroid receptor
coactivator-1; TRAP, TR-associated proteins; TRRAP,
transformation/transcription domain-associated protein.
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ACKNOWLEDGMENTS |
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REFERENCES |
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