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Department of Pharmacology and Cancer Biology (A.L.W., S.C.N., J.D.N., D.P.M.), Duke University Medical Center, Durham, North Carolina 27710; and Novalon Pharmaceutical Corporation (L.A.P., D.J.C., D.M.F.), Durham, North Carolina 27703
Address all correspondence and requests for reprints to: Donald P. McDonnell, Department of Pharmacology and Cancer Biology, Box 3813 Duke University Medical Center, Durham, North Carolina 27710. E-mail: mcdon016{at}acpub.duke.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) positive breast cancers; however,
the effectiveness of this therapy is limited by the almost universal
development of resistance to the drug. If antiestrogens are recognized
differently by the cell as it has been suggested, then in disease
conditions where tamoxifen fails to function effectively, a
mechanistically different antiestrogen might yield successful results.
Although many antiestrogens have been developed, a direct comparison of
their mechanisms of action is lacking, thus limiting their utility.
Therefore, to determine if there are mechanistic differences among
available antiestrogens, we have carried out a comprehensive analysis
of the molecular mechanisms of action of 4-hydroxy-tamoxifen (4OHT),
idoxifene, raloxifene, GW7604, and ICI 182,780. Using a novel set of
peptides that recognize different surfaces on ER, we have found that
following binding to ER, each ligand induces a distinct ER-ligand
conformation. Furthermore, transcriptional assays indicate that each
ER-ligand complex is recognized distinctly by the transcription
machinery, and consequently, antiestrogens vary in their ability to
inhibit estradiol- and 4OHT-mediated activities. Relative binding
assays have shown that the affinity of these ligands for ER is not
always representative of their inhibitory activity. Using this assay,
we have also shown that the pharmacology of each antiestrogen is
influenced differently by hormone binding proteins. Furthermore,
GW7604, like ICI 182,780, but unlike the other antiestrogens evaluated,
decreases the stability of the receptor. Overall, our results indicate
that there are clear mechanistic distinctions among each of the
antiestrogens studied. However, GW7604 and ICI 182,780 differ more
significantly from tamoxifen than idoxifene and raloxifene. These data,
which reveal differences among antiestrogens, should assist in the
selection of compounds for the clinical regulation of ER function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the recent discovery of ERß, a second estrogen receptor
isoform, has extended our understanding of ER-biology (4, 5, 6). As yet,
specific functions for ERß in tissues other than the ovary have not
been defined, suggesting that it may have a more specific role in
ER-action than its ER counterpart (7). Thus, we have limited this
study to an analysis of the pharmacology of ER.
In the absence of ligand, ER resides in a transcriptionally inactive
form within the nuclei of target cells (8). Upon binding ligand, it
undergoes an activating conformational change, an event that
facilitates the formation of receptor dimers (9). These ER dimers
bind specific estrogen response elements (EREs) contained within the
regulatory region(s) of target genes and modulate gene transcription
(10). The ultimate biological effect of this cascade of events is
determined by the cellular and promoter context of the DNA bound
receptor (11, 12).
Approximately 50% of all breast cancers express elevated levels of
ER (13), and prolonged exposure to estrogen(s) is a major risk
factor for breast cancer (14). Therefore, antiestrogens have become the
front-line therapy in the treatment and management of ER-positive
breast cancers, where it has been shown that they can inhibit the
mitogenic actions of estrogens (15, 16, 17). Tamoxifen is the most
effective antiestrogen used in the treatment of these breast cancers.
In the metastatic setting this translates to a tumorostatic activity
lasting on average 17 months, and in the adjuvant setting tamoxifen has
been shown to decrease the incidence of second primary tumors by 35%.
Although originally considered an antiestrogen, tamoxifen is now
classified as a selective estrogen receptor modulator (SERM) because it
functions in some tissues as an antagonist, while in others, like bone
and the cardiovascular system, it can manifest partial or full agonist
activity. The ability of tamoxifen to function as an agonist in some
settings may explain why resistance to the antiestrogenic actions of
this compound arises within 2 yr in the majority of patients with
metastatic ER-positive breast tumors. Specifically, it has been
proposed that there may be cells within the breast tumor that are able
to recognize tamoxifen as a mitogenic agent, giving these cells a
selective growth advantage over their inhibited neighbors.
Interestingly, recent clinical data have revealed that
tamoxifen-resistant ER-positive cancers respond successfully to
treatment with the ER pure antagonist ICI 182,780 (18), but
unsuccessfully when treated with the ER partial agonist raloxifene
(19). We hypothesize that cross-resistance occurs between antiestrogens
having similar mechanisms of action, and that tamoxifen-resistant
tumors will respond to mechanistically different antiestrogens. By
inference, different antiestrogens would also find utility in the
treatment of other estrogenopathies such as osteoporosis, uterine
fibroids, and endometriosis as well as ER-positive, yet tamoxifen
nonresponsive, breast cancers (20). Although a large number of SERMs
have been developed, a direct comparison among them to evaluate
mechanistic differences has not been performed. Furthermore, even
though some of the antiestrogens have been studied individually, most
of the information necessary for a comparison of differences among
antiestrogens is lacking due to differences in the systems, sources,
and the contexts in which these studies have been carried out.
Evaluation and classification of antiestrogens/SERMs into distinct
groups, based on their mechanisms of action, will provide important
criteria that can be used in the selection of treatment methods for
clinical regulation of ER function.
We have established a series of in vitro and cell-based
assays to identify potential mechanistic differences among a diverse
group of clinically important antiestrogens. To this end, we have
analyzed, in a step-wise manner, differences exhibited by
antiestrogens. This includes measurements of competitive binding to
ER, analysis of the consequent changes that occur in ER
conformation, and determination of the mechanisms by which the
transcription machinery distinguishes among different ER-ligand
complexes. In addition, we have evaluated the influence of these
ligands on receptor stability and the manner in which serum binding
components modulate antiestrogenic activity. Our results indicate that
the mechanism of action of each antiestrogen evaluated is unique;
however, some antiestrogens differ more significantly from 4OHT than
others.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) was a gift from Dr. G. Greene (Ben May Institute,
University of Chicago, Chicago, IL). Secondary antibodies, Hybond-C
Extra transfer membranes, x-ray film and [35S]-methionine
was obtained from Amersham Pharmacia Biotech (Arlington
Heights, IL). Recombinant Human ER was purchased from PanVera Corp.
(Madison, WI). Anti-M13 antibody coupled to horseradish peroxidase
(HRP) was purchased from Pharmacia & Upjohn (Piscataway,
NJ). The biotinylated vitellogenin estrogen response element
(Biotin-GATCCCGCAGGTCACAGTGACCTG) was synthesized by Genosys
Biotechnologies Inc. (Woodland, TX). Immulon 4 plates were purchased
from VWR (Ocala, FL).
Cell culture and transient transfection assays
All transfections were performed using reagents and media
purchased from Life Technologies, Inc. (Grand Island, NY)
and sera were purchased from HyClone Laboratories, Inc.
(Logan, UT). MCF-7 cells were maintained in DMEM supplemented with 8%
FCS. HeLa and HepG2 cells were maintained in MEM supplemented with 8%
FCS. Ishikawa cells were maintained in DMEM-Ham’s F12 medium
supplemented with 8% FCS and 1% sodium pyruvate, glutaMAX-1 and
penicillin-streptomycin. Cells were plated in 24-well plates (coated
with gelatin for transfections of HepG2 cells) 24 h before
transfection. DNA was introduced into cells by transfection using
Lipofectin. Briefly, triplicate transfections were performed using 3 mg
of total DNA. For standard transfections, 100 ng pCMV-ßGAL
(normalization vector), 2000 ng reporter (either C3-Luc,
ERE3-TATA-Luc, or ERE-TK-Luc), and 900 ng receptor
(pRST7-hER (21), or ER-AF1 (22)) or control vector pBSII-KS
(Stratagene) were used for each triplicate. Cells were
transfected for 3 h, at which time medium was removed and induced
with the appropriate hormone diluted in phenol red-free medium
supplemented with 8% charcoal-stripped FCS. Incubation with hormone
continued for 24 or 48 h, after which cells were lysed
and assayed for luciferase and ß-actosidase activity as previously
described (23). Statistical analysis of data (t test-paired)
was done using StatView 4.5 program (Abacus Concepts, Inc., Berkeley,
CA.)
MCF-7 cell proliferation assay
5,000 cells were seeded per well in 24-well plates on day 0 in
estrogen-free maintenance medium. The cells were treated on days 3
through 6 with test media containing estradiol and antiestrogens at the
indicated concentrations. On day 7, cells were dissolved and assayed
for DNA content as described previously (24).
Relative binding affinity (RBA) assay in MCF-7 cells
RBA analyses were conducted as competition assays against 1
nM [3H]estradiol in serum-free medium (SFM)
and 2 nM [3H]estradiol in 8% CFS-containing
medium as described previously (25, 26). Briefly, MCF-7 cells were
plated in 24-well plates at least three days before assay in
estrogen-free medium. For each assay, cells were incubated with 0.5 ml
of test media per well at 37 C and 5% CO2 for 1 h
with shaking every 15 min. At the end of the incubation, the cells were
washed, resuspended in 1 ml of 10 mM EDTA (pH 12.5),
neutralized with 0.1 ml of 0.77 M
KH2PO4 (final pH, 7.2) and sonicated. Aliquots
were then taken for scintillation counting and measurement of DNA. DNA
was measured fluorometrically in an aliquot of the sonicate using
Hoechst dye 33258 as described previously (24). Calf thymus DNA was
used as the standard.
Calculations. The Ki was calculated as Ki = IC50 ÷ [1 + (F ÷ Kd)] where IC50 is the concentration of unlabeled competitor required to inhibit 50% of [3H]estradiol binding, F is the concentration of [3H]estradiol in the medium and Kd is the apparent Kd of estradiol measured in SFM or 8% serum containing medium (26). When calculating the Ki of an antiestrogen in SFM or in 8% serum containing medium, the Ki of estradiol measured in the same media in the same assay was used instead of the Kd. The RBA was calculated by dividing the IC50 for unlabeled reference estradiol by the IC50 for each antiestrogen, and this number was expressed as a percent; estradiol = 100%.
Phage enzyme-linked immunoabsorbent assay (ELISA)
Assays were conducted as previously described (27, 28). Briefly,
96 well plates were coated with streptavidin in 0.1 M
sodium bicarbonate and blocked with BSA. Biotinylated ERE (2 pmol) was
added to each well. Following a 1-h incubation, the plates were washed
five times with 10 mM Tris-HCl pH 8.0, 150 mM
NaCl containing 0.05% Tween 20, and 2 pmol ER was added to each
well. Following a 1-h incubation, the plates were washed again and the
ER ligands were added at a final concentration of 1
µM. Following a 5-min incubation period, phage were added
to the wells and incubated for 30 min at room temperature. The plates
were washed as described above, and the bound phage were detected with
an anti-M13 antibody coupled to horse radish peroxidase (HRP). Assays
were developed with ABTS (2,2'-azinobis(3-ethylbenzthiazoline-sulfonic
acid) and hydrogen peroxide for 10 min and the reactions were
terminated by the addition of 1% SDS. Absorbance was measured at 405
nm in a microplate reader.
Time resolved fluorescence (TRF) assay
Assays were conducted as previously described (27, 28). Briefly,
Costar high-binding 384 well plates were used to
immobilize ER as mentioned above (see ELISA assay). The ER
modulators were added at a range of concentrations. Following a 30-min
incubation with the modulators, 2 pmol of a europium-labeled
streptavidin (Wallac, Inc., Gaithersburg, MD)-biotinylated
peptide conjugate (prepared as described below) was added and incubated
for 1 h. The plates were then washed and the europium enhancement
solution was added. Fluorescent readings were obtained with a POLARstar
fluorimeter (BMG Lab Technologies) using a < 400 nm excitation
filter and a 620 nm emission filter. The europium-labeled
streptavidin-biotinylated peptide conjugate was prepared by adding 8
pmol biotinylated peptide to 2 pmol labeled streptavidin. After
incubation on ice for 30 min, the remaining biotin binding sites were
blocked with biotin before addition to the ER coated plate.
Western blot analysis
Cells were induced with ligand in culture media containing 8%
CFS for 48 h and nuclear extracts were prepared as described
previously (29). Either 10 or 100 µg of total protein was analyzed by
SDS-PAGE. Proteins were transferred to nitrocellulose membrane and
probed with monoclonal antibody H222 raised against the human ER.
Complexes were detected using ECL (Amersham Pharmacia Biotech). Densitometric quantitation of ER levels was done by
using the Image Quant software program (Molecular Dynamics, Inc., Sunnyvale, CA).
Northern blot analysis
Cells were treated with ligand for 48 h as described above
and total RNA was extracted using the Ultraspec RNA Isolation System
following the manufacturer’s protocol (Biotecx Laboratories, Inc.,
Houston, TX). Twenty micrograms of total RNA were separated by
electrophoresis in denaturing agarose gel (2.2 M
formaldehyde and 1% agarose), and transferred to a nylon filter by
capillary blotting and cross-linked by UV irradiation. The blot was
hybridized in QuikHyb Hybridization Solution following the
manufacturer’s protocol for double-stranded probes
(Stratagene, La Jolla, CA). The probe was prepared with 50
ng complementary DNA (cDNA) fragments randomly labeled with
[32P]dCTP and was added to the QuickHyb solution with 100
mg of salmon DNA. The following cDNA fragments were used to prepare
probes: the PCR product of the N terminus of pRST7-hER plasmid (600 bp)
and 36B4 cDNA fragment (708 bp) excised with PstI from the
pBR322 vector (provided by P. Chambon, Institut de Genetique et de
Biologie Moleculaire et Cellulaire, France). The membrane was washed
successively with 2 x SSC buffer and 0.1% (wt/vol) SDS solution
for 15 min at room temperature and with 0.1 x SSC buffer and
0.1% (wt/vol) SDS for 30 min at 60 C. The blot was exposed to film for
autoradiography and quantitated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(22); thus, the affinity
with which an antiestrogen binds ER is an important parameter in
competitive binding. However, our data indicate that antiestrogen
action is more complex. We compared the ability of specific
antiestrogens to competitively inhibit estradiol-mediated transcription
and proliferation with their affinities for ER. Previous studies
have primarily used in vitro systems with cellular extracts
or overexpressed exogenous ER to measure ER affinity, and
compared these data to the ability of the test compound to inhibit
estrogen-mediated transcription and/or proliferation in intact cells.
In this study, we have evaluated these activities in intact breast and
endometrial cancer cells, which express endogenous ER, in an effort
to compare antiestrogens under conditions that are more representative
of bona fide target cells. This parallel approach has the
advantage that it equalizes the effects of the following: 1) the
ability of the hormone to penetrate the cell and reach the receptor; 2)
the effect of hormone binding proteins within serum on the pharmacology
of the ligands; and 3) the effects of target cell metabolism on both
physiological antagonism and ER binding. We have included in these
mechanistic studies most of the new antiestrogens that are currently
under development (Fig. 1
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in the human breast cancer
cell line MCF-7 (Fig. 2A). For this
purpose, a reporter construct containing three copies of vitellogenin
ERE (ERE3-TATA-Luc) was transiently transfected into MCF-7
cells and induced with 10 nM estradiol in the presence of
increasing concentrations of each antiestrogen. Because endogenous
levels of ERß have not yet been detected in the cell lines that we
used, this assay measures the ability of the compounds to inhibit
through ER in an endogenous setting. ICI 182,780 was the most potent
inhibitor of ER-mediated transcriptional activity (Fig. 2A), and the
relative order of potencies obtained from this study was ICI
182,780 > GW7604, raloxifene, 4OHT > idoxifene. This same
order of potencies was observed in assays performed in the human
endometrial adenocarcinoma Ishikawa cell line (data not shown).
Furthermore, the order of potencies with which these antiestrogens
inhibit estradiol mediated MCF-7 cell proliferation (Fig. 2B), in a
completely endogenous system, was similar to the above order of
potencies.
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using whole cell binding assays. This study was
designed to compare in parallel the relative affinities with the
relative potencies of antiestrogens under the same culture conditions
in intact MCF-7 cells on endogenous ER levels. MCF-7 cells were
incubated with a fixed concentration (2 nM) of
[3H]estradiol in the presence of increasing
concentrations of each of the antiestrogens. In this RBA assay,
raloxifene exhibited the highest affinity for ER relative to
estradiol (Fig. 2C
, Table 1). The RBA
values obtained for each ligand can be summarized as raloxifene >
GW7604, 4OHT, ICI 182,780 > idoxifene. For some antiestrogens,
potency differed from that expected based upon binding affinity alone.
For instance, although ICI 182,780 is the most potent inhibitor of
ER-mediated transcriptional activity and cell proliferation,
raloxifene shows the highest affinity for ER (the RBA values we
obtained differ from published values (30, 31) most likely due to
differences in the methodology employed for this measurement). These
data indicate that antagonist potency is influenced not only by
receptor binding affinities, but also by processes that permit the cell
to distinguish among different ER-ligand complexes.
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. Hence, as an additional step, we focused on
how hormone-binding proteins in serum affect the ability of ligands to
reach intracellular ER. To assess this effect, we measured the RBA
in the presence (culture media) and absence of serum as described
above. Our data indicate that the potency of GW7604 (4OH metabolite of
GW5638) was greatly reduced in the presence of serum in the amounts
present in tissue culture medium (Table 1; compare columns 2 and 3.)
This may be due to the presence of the -COOH group on GW7604 and the
ability of serum albumin to bind to acidic compounds (32, 33).
Conversely, the RBAs of other compounds were increased in the presence
of serum, suggesting that their entry into the cell from serum is
greater than that of estradiol, presumably due to less binding to serum
proteins. However, the magnitude of change from the presence to the
absence of serum for each antiestrogen is distinct, implying different
levels of binding to these serum proteins. We conclude from this
experiment that binding factors contained within serum have distinct
effects on the overall pharmacological profile of antiestrogens, and
that comparison of potency in live cells to ER affinity must control
for these effects.
Different ligands induce distinct conformational changes in
ER
It has been demonstrated that the ability of estrogens and
antiestrogens to manifest different biological activities in different
cells is determined in part by the ability of individual compounds to
affect ER-conformation (9, 10). It is believed that different
ER-ligand conformations may recruit different cofactors that
ultimately determine the pharmacology of each ligand. Indeed, recent
crystallographic studies of ER have demonstrated that raloxifene and
4OHT contact a different set of amino acids in the hormone binding
domain than does estradiol, and evoke a unique conformational change
that prevents certain cofactors from binding (34, 35). These results
expand upon earlier biochemical data, obtained from partial
proteolysis, indicating that ER agonists and antagonists have
different effects on ER-structure (9, 10). Even though these
crystallographic studies and partial proteolysis experiments have shown
differences among agonist and antagonist bound ER, neither study has
been able to discern significant conformational differences among
various antiestrogen-bound ER complexes. We have developed a
different approach to assess ER-conformations in the presence of
different ligands (28). Specifically, we have previously reported the
use of phage display technology, to identify eight classes of
bacteriophage that express surface peptides whose ability to interact
with ER is dependent on the conformation of a specific ER-ligand
complex. Although some of the phage identified clearly interact with
bona fide coactivator and/or corepressor binding sites on
ER, each of the peptide binding sites may represent a potential
protein-protein interaction surface.
We have assessed the binding of each of the eight classes of
phage-expressed peptides to ER-ligand complexes as a probe for the
effect of various ligands on ER-conformation. This was accomplished
by using an ELISA assay that measures the interaction of individual
phage with each ligand-ER complex (Fig. 3A). In this manner a specific
"fingerprint" was obtained for each compound (Fig. 3B) indicating
that the different SERMs evoke unique conformational changes within
ER. For instance, the phage probe /ß III interacts well with
ER in the presence of 4OHT, but interacts poorly with raloxifene- or
idoxifene-ER. The same probe shows no detectable binding to ICI
182,780- or GW7604-ER complexes, differentiating these two compounds
from the other antiestrogens. Further differentiation was accomplished
using the /ß V probe where the probe bound only 4OHT-ER,
separating the raloxifene- and idoxifene-ER conformations from
4OHT-ER conformation. Because 4OHT, GW7604, and idoxifene have the
same triphenylethylene core structure, the unique fingerprint of each
compound suggests that even minor modifications in the structure of the
ligand can have a profound influence on ER conformation. However, it
was interesting that in 3/5 instances, the peptides that interacted
with 4OHT-ER, also interacted with idoxifene- and raloxifene-ER.
This observation suggests that peptide interacting surfaces on these
ligand-conformations may be similar. Using the III probe, it is
possible to discriminate between the ICI 182,780- and GW7604-ER
where the probe binds to the GW7604 bound receptor but not the ICI
182,780 bound receptor. (We have also included the nonspecific binding
pattern of I probe to contrast between specific and nonspecific
binding). Each ligand studied induced a distinct ER conformation and
a distinct pattern of interaction with peptides.
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-ligand conformations suggested that for each
ER-interacting probe, a different surface of interaction is exposed.
Therefore, we sought to quantitate the extent to which a
peptide-binding surface was exposed upon formation of the ER-ligand
complex. For this purpose, we selected the II probe that interacts
with ER in the presence of each of the ligand used in this study.
This analysis was performed using time-resolved fluorescence
spectroscopy as described by Paige et al. (28), where the
interaction between the labeled peptide and ER was measured in the
presence of increasing concentrations of ligand. The II binding
capacity of estradiol-bound ER was set at 100. Our results (Table 2) indicate that the exposed binding
surface [as represented by relative binding capacity (RBC)] of ER
for II is different for each ligand. These results suggest that,
upon binding different ligands, ER exposes different
peptide-interacting surfaces, some of which may enable the receptor to
associate differentially with various coregulatory proteins.
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-antiestrogen complexes in a ligand- and cell-specific manner-ligand complex, it remained to be determined if, in classical
ER-responsive systems, the transcriptional machinery is capable of
distinguishing among the distinct conformations. To this end, we
measured the transcriptional activity of endogenous ER in MCF-7
cells (Fig. 4A) following treatment with
various ligands. This cell line was transiently transfected with
ERE3-TATA-Luc promoter and treated with solvent or a
specific ligand. Under these conditions, we observed that all
antiestrogens, with the exception of GW7604 and ICI 182,780, act as
partial agonists exhibiting approximately 10–15% of the agonist
activity of estradiol (at saturating concentrations, when compared with
basal response in the absence of ligand, each compound displayed
statistically significant partial agonist responses (P
< 0.05)). A parallel analysis in Ishikawa cells gave similar results
except that GW7604 acted as a partial agonist in these cells (data not
shown). Although it is not possible to predict from these in
vitro studies if the minimal level of agonist activity observed is
physiologically relevant, it may partially explain why ICI 182,780 is a
better inhibitor than the other antiestrogens in inhibiting
estradiol-mediated activity, as described above. The observation that
different ligands manifest varying levels of activity in the cell lines
used here indicates that the transcription machinery does not recognize
all ligand-induced ER conformations equally.
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-ligand complexes, we used a specific ER mutant. Previous
studies have indicated that receptor conformation may affect
ER-transcriptional activity by modulating cooperative interactions
between the two major transcriptional activation functions within the
receptor, AF1 in the amino terminus and AF2 in the carboxy terminus.
These activation domains can function independently in some contexts
but, for the most part, full ER agonist activity requires both
activation domains. Based on previous studies from our laboratory and
others, we propose that all of the known ER antagonists inhibit
ER-AF2 activity (22, 36, 37). Additionally, it was determined that
the residual partial agonist activity manifested by antagonists like
tamoxifen, is due to their ability to facilitate activation by AF1 in
contexts where a functional AF2 is not required. It is likely,
therefore, that the ability to regulate AF1 activity is a major
discriminator among antiestrogens. Hence, evaluation of the ability of
a compound to regulate ER-transcriptional activity in contexts where
AF1 alone is functional, is likely to serve as a surrogate for
molecular differences among ER-antagonists. To test this hypothesis,
we have developed an assay to evaluate AF1 transcriptional activity.
Specifically, we have determined that ER-AF1, but not AF2, activity
is required for ER transcription on the complement 3 promoter (C3)
when assayed in the liver hepatocellular carcinoma cell line HepG2. In
this AF1-dependent environment, we assessed the response of the
antagonists under evaluation following transfection with a vector
expressing either ER-wt or a mutant protein (ER-AF1) in which AF2
has been mutated. Our results indicate that, when assayed on ER-wt,
4OHT acts as a partial agonist exhibiting approximately 30% of the
efficacy of estradiol (Fig. 4B). Under these conditions, raloxifene and
ICI 182,780 function as inverse agonists, suppressing the basal
transcriptional activity of the C3 promoter below the solvent control.
On the other hand, GW7604 and idoxifene yield a biphasic response by
exhibiting marginal partial agonist activity at 1 nM
concentration (P < 0.05 at 1 nM compared
with basal value) and by acting as inverse agonists at higher
concentrations.
A parallel analysis of ER-AF1, in which ER-AF2 has been
disrupted by three specific mutations, gave significantly different
results from that obtained using wild-type ER (Fig. 4C).
Specifically, although raloxifene and ICI 182,780 behaved as inverse
agonists on ER, on ER-AF1 raloxifene exhibited about 25% of the
agonist activity of estradiol, whereas ICI 182,780 showed no agonist
activity. The switch in the transcriptional activity of raloxifene
probably indicates that in addition to inhibiting AF2, this compound
can inhibit AF1 by a mechanism(s) that requires a functional AF2. This
ER mutant clearly differentiates raloxifene and ICI 182,780 and
highlights their mechanistic differences. The ER-AF1 mutant also
separated GW7604 from idoxifene, two compounds with similar activity on
ER. Specifically, on ER-AF1 idoxifene exhibited about 15% of the
activity of estradiol, whereas GW7604 was inactive. In contrast, 4OHT
was active on both ER and ER-AF1, as previously shown (9). We
conclude that all the antiestrogens tested here use unique mechanisms
of action to modulate transcription through ER. These mechanistic
differences of antiestrogens are likely to manifest distinct activities
in different cells and function differently when assessed in
vivo.
The partial agonist activity of 4OHT can be inhibited by
mechanistically distinct antiestrogens
The series of analyses we have done so far have provided
compelling evidence to support the hypothesis that all antiestrogens
tested in this study have unique effects on ER-conformation and
ER-mediated transcription, and distinct efficacies as competitive
inhibitors of estradiol function. Similar to previous studies (9), we
also demonstrated that all antiestrogens are capable of delivering
ER-ligand complexes to DNA (data not shown). Next, we wanted to
determine if the partial agonist activity displayed by tamoxifen could
be competitively inhibited by the other antiestrogens tested. The
rationale for this study was the observation that tamoxifen-resistant
cancers display a dependence on tamoxifen for growth (30), indicating a
cellular alteration that allows 4OHT to act as a partial agonist and
not as an inhibitor of estradiol action. A successful cell-based,
tamoxifen-resistant system had not yet been developed; therefore, we
selected two systems in which 4OHT acts as a partial agonist on
endogenous ER. Specifically, MCF-7 and Ishikawa cells were
transiently transfected with ERE3-TATA-Luc reporter and
induced with a saturating concentration (10 nM) of 4OHT in
the presence of increasing concentrations of each test compound. Under
these conditions, in both cell types, ICI 182,780 was the most potent
inhibitor of 4OHT partial agonist activity (Fig. 5, A and B), and the only antiestrogen
capable of completely inhibiting this partial agonist activity. GW7604
displayed better inhibition of 4OHT partial agonist activity than
idoxifene and raloxifene in both cell lines studied. Although idoxifene
and raloxifene were not very successful as antitamoxifen agents in
MCF-7 cells, they showed better inhibitory effects in Ishikawa cells.
We conclude that ICI 182,780 and GW7604 are more efficient inhibitors
of the partial agonist activity of 4OHT. This observation suggests that
the partial agonist activity of tamoxifen that is thought to
limit its long-term chemotherapeutic efficacy, may be
circumvented by the use of mechanistically distinct
antiestrogens.
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expression and stability-signal transduction, we next focused on
the effect of each ligand on the stability of the receptor. It has been
shown previously, that ER is targeted for proteolysis upon binding
the pure antiestrogen ICI 182,780 (38). Thus, we assessed whether the
stability of ER is influenced similarly by mechanistically different
antiestrogens. We examined the effects of the test compounds on
endogenous ER expressed in either MCF-7 or Ishikawa cells. The cells
were incubated for 48 h in the absence or presence of compound,
and then the relative nuclear ER content was measured by
immunoblotting using the H222 monoclonal antibody. The results of a
typical experiment performed in MCF-7 cells (Fig. 6A) indicated that relative to solvent
control, there was an increase in ER levels in cells treated with
idoxifene or 4OHT. Raloxifene did not appear to have an effect on ER
levels. However, treatment with estradiol, GW7604 or ICI 182,780
greatly reduced the receptor levels. These changes in ER content
were not due to a redistribution of the receptor within target cells,
because similar results were observed when ER content was measured
in total cell extracts (data not shown). The ability of different
antiestrogens to affect ER expression was not restricted to the
breast cancer MCF-7 cells as we obtained similar results when the
analysis was performed in the endometrial Ishikawa cell line (data not
shown). However, the extent to which estradiol, GW7604, and ICI 182,780
decreased the ER level in Ishikawa cells was consistently less than
that observed with MCF-7 cells. These results are consistent with the
hypothesis that the stability and/or expression level of ER is
affected by the nature of the bound ligand and the cellular milieu.
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measurements performed thus far provided an assessment of the
effects of ligands on endogenously expressed ER protein. They did
not, however, address the mechanisms by which the observed changes in
receptor levels are achieved, i.e. altered receptor
stability, messenger RNA (mRNA) stability, or an alteration in the rate
of transcription of the ER-gene promoter. To examine this issue, we
extracted total RNA from MCF-7 cells under the treatment conditions
used for Western analysis, and used Northern analysis to measure the
impact of each antiestrogen under investigation on the expression of
ER-mRNA. After normalization (Fig. 6C), whereas estradiol decreased
ER mRNA levels, idoxifene, raloxifene, and 4OHT increased ER-mRNA
levels (Fig. 6B). ICI 182,780 and GW7604 did not show a significant
effect on ER-mRNA levels, implicating a posttranscriptional
mechanism(s) in the regulation of ER expression levels. We conclude
from these studies that the reduced ER expression level observed in
cells treated with ICI 182,780 and GW7604 is most likely due to
instability and subsequent degradation of the ligand-ER complex.
However, whether receptor down-regulation is required for the
antiestrogenic action of ICI 182,780 or GW7604 remains to be
determined. Cumulatively, these experiments indicate that ER
antagonists can be split into two families: those which decrease ER
protein levels (ICI 182,780 and GW7604), and those which increase or
have no effect on receptor protein levels (idoxifene, 4OHT, and
raloxifene). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-action suggest that the role of
ligand is that of a switch converting ER from an inactive to an
active form, implying a very simple pharmacology. It is thus considered
that all agonists are quantitatively the same and, when corrected for
affinity, should be indistinguishable. By inference, these models also
suggest that all antagonists are the same, functioning solely by
competitively blocking agonist access to the receptor. The results of
our studies, however, indicate that not all antiestrogens are the same
and that the cellular environment of the ER-antagonist complex
determines efficacy. These findings have important clinical
implications. For instance, with reference to the development of
tamoxifen-resistance in breast cancer, we propose that during the
course of treatment, tumors that initially respond to tamoxifen undergo
specific alterations that allow them to recognize tamoxifen as an
agonist, and display a selective growth advantage over their unaltered
neighboring cells (39). This may occur because either a specific
enabling cofactor is overexpressed, the expression level of a
corepressor decreases, or either class of protein is modified (40).
Whatever the reason, the realization that different antiestrogens are
not affected equally by receptor-associated proteins lends support to
the hypothesis that tamoxifen-resistant tumors will respond to
mechanistically different antiestrogens. To this effect, in a recent
clinical trial, 69% of advanced tamoxifen resistant breast cancer
patients responded favorably to treatment with ICI 182,780 (18).
Accordingly, treatment of tamoxifen-resistant cancers as well as those
conditions where the occurrence of adverse effects upon treatment with
tamoxifen are evident (such as uterine hyperplasia), with antiestrogens
that are mechanistically distinct from tamoxifen, may yield favorable
results. The success of this approach relies on our ability to
distinguish among available antiestrogens. Therefore, to facilitate
drug evaluation and selection, we performed a head-to-head analysis of
the molecular mechanisms of action of a series of clinically important
SERMs/antiestrogens. The results of our studies revealed, that not all
antiestrogens are the same. In addition however, they permitted an
evaluation of the differences among these antiestrogens, an activity
that will be useful in predicting the clinical utility of specific
compounds.
Previous studies have shown that it is the ability of the cell to
distinguish among different receptor conformations that occur upon
ligand binding that ultimately defines the tissue-specific activity of
a ligand (21). To this end, in this study we have shown that: 1) ER
conformation is influenced differently by each ligand; 2) distinct
peptide binding surfaces are exposed in ER upon binding different
ligands; and 3) the affinity with which peptides interact with these
different ER conformations is affected by the nature of the bound
ligand. These observations suggest that different ER-ligand
complexes may interact with different coactivator/corepressor proteins
within the cell. Indeed, a series of different ER-associated
coactivators and corepressors has already been identified. These
coactivators, such as SRC-1 and GRIP1, potentiate the activity of ER
when coexpressed in cells (41, 42, 43, 44). Furthermore, SRC-1 when
overexpressed will convert tamoxifen from a pure antagonist to a
partial agonist, whereas overexpression has no affect on the
pharmacology of ICI 182,780 (45). This observation probably reflects
the affinity with which SRC-1 interacts with different ER-ligand
complexes and/or the ability of the ER-ligand conformation to
recruit the coactivator. The corepressors NCoR and SMRT have been shown
to associate with ER and, although it remains to be demonstrated for
ER, previous studies have shown that PR-antagonist efficacy
correlates with the relative affinity of the PR-ligand complex for
either NCoR or SMRT (46, 47). Importantly, from the perspective of this
study, it was shown that receptor conformation is the most important
single factor regulating these interactions. These observations suggest
that ER conformation strongly influences the ability of the receptor
to interact with ER-associated cofactors. Emerging from these latest
findings is a model in which we propose that the agonist/antagonist
activity of a given compound is influenced by the cellular expression
level of these receptor-associated proteins. For instance, it has been
shown that decreased levels of NCoR correlate with the development of
tamoxifen resistance in a mouse model system for human breast cancer
(40). We believe that this is a key finding that may explain the
tissue-selective agonist/antagonist activity of tamoxifen, and also the
reason why cells that originally recognize tamoxifen as an antagonist
can switch to recognizing the compound as an agonist.
We have also shown that ER stability within target cells is
influenced by the nature of the bound ligand. We speculate that certain
ER-ligand complexes such as those formed by ICI 182,780 and GW7604
target ER for degradation. The conformation of the ligand-receptor
complex may both regulate ER turnover and contribute to antagonist
efficacy. It must be noted that we have limited this study of ER
stability to two different cell lines. It is possible that the
ligand-induced receptor degradation pattern will differ in other target
cells. For instance, because GW7604 has been shown to be bone
protective in vivo (48), contrary to ICI 182,780 (49), it
would be interesting to see the effect of these ligands on ER in
bone. Whatever is the case, these results suggest that ligand- ER
structure and stability are integrally linked.
Based on our overall results (as summarized in Table 3), GW7604 and ICI 182,780 appear to be
two functionally unique antiestrogens whose mechanisms of action are
distinct from tamoxifen. Therefore, alterations in receptor associated
proteins that affect the function of ER-4OHT may not have a
significant impact on the pharmacology of GW7604 or ICI 182,780.
Therefore, we believe that these compounds may be effective in the
treatment of ER positive, tamoxifen-resistant, breast cancers.
Similarly, we predict that additional responders to treatment will be
found among the 30% ER positive, tamoxifen-nonresponsive breast
cancers. Although functionally unique, raloxifene and idoxifene act in
a manner similar to tamoxifen; therefore, the same cellular alterations
that limit the efficacy of 4OHT-function could have an impact on the
function of these agents. We would add as a cautionary note, however,
that the separation of compounds into mechanistically different groups,
based on their molecular mechanisms of action, has been assessed using
the parameters we understand at the current time. Thus, as our
understanding of antagonist action evolves, we may need to fine-tune
and modify these classifications. Although it has proven difficult in
the past to relate in vitro findings to clinical outcomes,
it appears that cross-resistance is most likely to occur among
compounds that are mechanistically similar. Thus, studies that help to
separate SERMs into functionally different groups will provide science
based criteria for the selection of drugs for clinical evaluation.
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Acknowledgments |
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Footnotes |
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Received May 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. L. Bowers, V. V. Tyulmenkov, S. C. Jernigan, and C. M. Klinge Resveratrol Acts as a Mixed Agonist/Antagonist for Estrogen Receptors {alpha} and {beta} Endocrinology, October 1, 2000; 141(10): 3657 - 3667. [Abstract] [Full Text] [PDF]
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I. Tcherepanova, P. Puigserver, J. D. Norris, B. M. Spiegelman, and D. P. McDonnell Modulation of Estrogen Receptor-alpha Transcriptional Activity by the Coactivator PGC-1 J. Biol. Chem., May 19, 2000; 275(21): 16302 - 16308. [Abstract] [Full Text] [PDF]
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A. L. Wijayaratne and D. P. McDonnell The Human Estrogen Receptor-alpha Is a Ubiquitinated Protein Whose Stability Is Affected Differentially by Agonists, Antagonists, and Selective Estrogen Receptor Modulators J. Biol. Chem., September 14, 2001; 276(38): 35684 - 35692. [Abstract] [Full Text] [PDF]
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