Unliganded and Liganded Estrogen Receptors Protect against Cancer Invasion via Different Mechanisms
Nadine Platet,
Séverine Cunat,
Dany Chalbos,
Henri Rochefort and
Marcel Garcia
Institut National de la Santé et de la Recherche
Médicale Unité Hormones et Cancer (U148) and
Université de Montpellier I Montpellier, France 34090
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ABSTRACT
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While estrogens are mitogenic in breast cancer
cells, the presence of estrogen receptor
(ER
) clinically
indicates a favorable prognosis in breast carcinoma. To improve our
understanding of ER
action in breast cancer, we used an original
in vitro method, which combines transient transfection and
Matrigel invasion assays to examine its effects on cell invasiveness.
ER
expression in MDA-MB-231 breast cancer cells reduced their
invasiveness by 3-fold in the absence of hormone and by 7-fold in its
presence. Integrity of hormone and DNA-binding domains and activating
function 2 were required for estradiol-induced inhibition, suggesting
that transcriptional activation of estrogen target genes was involved.
In contrast, these domains were dispensable for hormone-independent
inhibition. Analysis of deletion mutants of ER
indicated that amino
acids 179215, containing the N-terminal zinc finger of the
DNA-binding domain, were required for ligand-independent receptor
action. Among different members of the nuclear receptor family, only
unliganded ER
and ERß reduced invasion. Calreticulin, a
Ca2+-binding protein that could interact with
amino acids 206211 of ER
, reversed hormone-independent ER
inhibition of invasion. However, since calreticulin alone also
inhibited invasion, we propose that this protein probably prevents
ER
interaction with another unidentified invasion-regulating factor.
The inhibitor role of the unliganded ER was also suggested in three
ER
-positive cell lines, where ER
content was inversely correlated
with cell migration. We conclude that ER
protects against cancer
invasion in its unliganded form, probably by protein-protein
interactions with the N-terminal zinc finger region, and after hormone
binding by activation of specific gene transcription.
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INTRODUCTION
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Estrogen receptor
(ER
) is a ligand-inducible
transcription factor that belongs to the superfamily of nuclear
receptors (reviewed in Refs. 1 2 ). The more recently isolated ERß
isoform has structural similarities to ER
, but a different tissue
distribution (3 4 ). Estrogen signal transduction involved
high-affinity binding to ERs, conformational changes of ERs and
recruitment of transcriptional auxiliary factors, binding to estrogen
response elements (EREs) in gene promoters, and regulation of
transcriptional activity in conjunction with other transcription
factors bound to their cognate sites in the promoter. Molecular
analysis has shown that ER
, like other nuclear receptors, consists
of separable domains responsible for DNA binding [DNA-binding domain
(DBD), hormone binding domain (HBD)], and transcriptional activation.
The N-terminal activation function (AF-1) of the purified receptor is
constitutively active, whereas the activation function located within
the C-terminal part (AF-2) requires hormone for its activity. In
addition to this well known activation of target promoters containing
EREs, other liganded ER actions involving recruitment of ER
to gene
promoters lacking EREs via protein-protein interactions with
promoter-bound transcription factors (5 6 7 8 9 ), or activation of the
mitogen-activated protein kinase pathway (10 ), have been proposed.
Estrogens and their receptors are implicated in the etiology of
breast cancer (reviewed in Ref. 11 ). In large clinical studies, ER
has been associated with a more favorable prognostic outcome in breast
cancer patients (12 ). While the mitogenic action of estrogens in breast
cancer cells is well established (13 ), several studies have correlated
ER
expression to lower Matrigel invasiveness and reduced metastatic
potential of breast cancer cell lines (14 15 ). This paradox suggests
that ER expression could be associated with or involved in pathways
that hinder cancer progression. A working hypothesis is that ER
protects against invasion of the basement membrane, an important step
of the metastatic process required for cancer dissemination. Estrogen
effects on cell invasiveness have been studied in vitro
using Matrigel, a reconstituted basement membrane as host. Initial
studies indicated that invasiveness of MCF7 breast cancer cells was
increased by estradiol and also, paradoxically, by antiestrogens (16 17 ). More recent studies have confirmed the stimulatory effect of
antiestrogens, such as 4-OH-tamoxifen and ICI 164,384 but not the
estradiol-induced stimulation (15 18 19 20 21 22 ). By contrast, estradiol
appeared to significantly reduce invasiveness, and this inhibition was
reversed by antiestrogens. This conclusion was noted in several
ER
-positive cancer cell lines established from breast (15 ) or ovary
(20 ), and in different ER
-negative cancer cells constitutively
expressing ER
after stable transfection (18 19 22 ). Similar
results were also obtained on the migration of normal cells from
vascular smooth muscle (21 ).
In the present study, after deletion of different ER domains, we
demonstrate that ER
, expressed in ER-negative MDA-MB-231 breast
cancer cells, prevents invasion in vitro via two distinct
mechanisms, according to its unliganded or liganded status.
Estrogen-induced inhibition requires ER
domains normally involved in
the transcriptional activation of ERE-containing promoters. In
contrast, the unliganded receptor action appears to depend on a
discrete ER
region that includes the N-terminal zinc finger of DBD
and probably involves protein-protein interaction.
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RESULTS
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Regulation of Invasiveness of Breast Cancer Cells by Unliganded
ER
and Estrogen/Antiestrogen Treatment
The effect of ER
on invasiveness was assayed using a new
method based on transient cotransfection of the ER
-expression vector
(HEGO), or the empty pSG1 vector, with the pGL3 vector constitutively
expressing luciferase and used as a marker to assess only transfected
cells, as described in Materials and Methods. The
percentage of luciferase-positive cells migrating through Matrigel was
compared in HEGO-transfected or control pSG1-transfected cells. The
validity and reproducibility of this cotransfection method have been
demonstrated in the highly invasive ER
-negative MDA-MB-231 cell line
(23). As shown in Fig. 1A
, estradiol or antiestrogen treatments did not significantly affect
invasion of control pSG1-transfected cells. ER
expression induced a
3.3-fold decrease in the invasiveness of transfected cells in a
ligand-independent manner. Estradiol treatment reinforced this effect
by an additional 2-fold reduction in invasiveness. Among antiestrogens,
the partial agonist/antagonist OH-tamoxifen did not significantly
affect invasion whereas the pure antiestrogen ICI 164,384 almost
totally reversed the strong inhibition due to the unliganded ER
. In
the presence of estradiol, both antiestrogens reversed the hormone
action, but ICI 164,384 also reversed the effect due to the unliganded
receptor. Immunofluorescence analysis after transient HEGO transfection
revealed that treatment by 100 nM ICI 164,384
decreased the number of ER-positive nuclei by 6-fold as compared with
control or estradiol-treated cells (data not shown). This indicated
that reversal of the unliganded receptor effect by ICI 164,384 was
probably due to this drastic decrease in receptor content.

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Figure 1. Effect of ER Transient Transfection on
MDA-MB-231 Cell Invasion and Chemotaxis
A, ER -negative MDA-MB-231 cells were transiently cotransfected with
ER -expressing vector (HEGO) or control vector (pSG1), and the
luciferase-expressing vector (pGL3) used as a marker of transfected
cells, as described in Materials and Methods. The percentage
of Matrigel-invading cells was estimated in triplicates 24 h after
invasion in the presence of 20 nM estradiol
(E2), 100 nM
4-hydroxy-tamoxifen (OHT), 100 nM ICI 164,384
(ICI), or ethanol alone (C). Values represent mean ±
SD of three experiments. B, Comparison of the
effects of ER -expressing vector or control vector on cell invasion
(with Matrigel) and motility (without Matrigel). Data represent
mean ± SD of three separate experiments. *,
P < 0.01 vs. pSG1 control; ,
P < 0.05 vs. HEGO control.
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We used the same assay in the absence of Matrigel to analyze the effect
of ER
on one component of the invasion process, i.e. cell
motility (Fig. 1B
). In the absence of ligand, ER
expression reduced
cell motility to 69% of the control value, indicating that decreased
invasion (30% of control) could be partly due to a motility
effect.
Different ER
Domains Are Implicated in the Inhibition
of Invasion Mediated by Unliganded and Hormone-Activated Receptors
To determine the ER
domains involved in the two
inhibitions obtained in the absence and presence of estradiol, ER
deletion constructs were transfected in MDA-MB-231 cells, and their
effects on cell invasion were compared (Fig. 2
). The expression and nuclear
localization of all mutated proteins were also verified by
immunofluorescence as described in Materials and Methods. In
most experiments, transfection efficiency reached about 6 ± 2%
of total cells (data not shown). In the absence of ligand, the
G400V-mutated ER
(HEO, 46% of pSG1) was slightly less inhibitory
than the native ER
(HEGO, 29% of pSG1). Mutant HE15 (1282)
lacking the hormone binding domain and AF-2 function or HE91 containing
two amino acid changes in the DBD (E203G, A207V), which prevented
transactivation via an ERE (24 ), was as effective as the wild-type
ER
, indicating that these functions are not involved in
ligand-independent inhibition. Inversely, mutants HE19 (179595) and
HE11 (
185251) truncated in A/B and C domains, respectively, were
ineffective, suggesting that these domains are important. On the other
hand, E2-induced inhibition was totally abolished
when mutants deleted in the HBD (HE15) or in the DBD (HE11) or mutated
in the ERE binding domain (HE91) or in the core region of AF2
(HEmutAF2) were transfected (Fig. 2
). In contrast, deletion of the A/B
region (HE19) did not influence the hormonal effect.
Taken together, these data indicated that inhibition due to unliganded
ER
and to the E2-activated receptor required
different receptor domains. The hormone action required ERE binding and
AF2, two functions classically involved in transcriptional activation
of target genes. In contrast, these functions were dispensable for the
unliganded receptor action, which appeared to be due only to its 1282
amino-terminal region.
Inhibition of Invasiveness by Unliganded ER Involved the First Zinc
Finger of the C Region
To further specify which part of the 1282 amino acid region is
involved in unliganded receptor action, A/B deletion mutants were
analyzed. Deletions of any different part of the A/B region did not
influence the inhibitory effect of the unliganded receptor (Fig. 3A
). Moreover, a point mutation of serine
118, previously described as essential for steroid-independent receptor
activation (25 26 ), to a nonphosphorylatable alanine (HE457 or
HE15/457) did not alter ER
and ER
1282 (HE15) efficiencies for
inhibiting invasion (data not shown). The finding that the ER100
(
3178) mutant was active while HE19 (
1178) was inefficient
indicated that the two N-terminal amino acids are required (Fig. 3A
).

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Figure 3. ER Domains Involved in the Ligand-Independent
Inhibition of Cancer Cell Invasion
Vectors expressing wild-type (HEGO) or mutated ER were
transiently transfected in MDA-MB-231 cells in estradiol-withdrawn
culture conditions, as described in Fig. 1 . For each vector, Matrigel
invasion was estimated after 24 h in triplicate wells. Values
represent mean ± SD of three experiments. A,
Deletion mutants of A/B domains. B, Deletion mutants of C/D domains.
NLS and hemagglutinin epitope tag (HA) are described in
Materials and Methods. C, Immunofluorescence detection
of ER mutants. Twenty four hours after transfection with the
indicated vectors, the cells were fixed and immunostained with
anti-ER antibody directed against the A/B domain. The percentage of
ER-positive cells was evaluated by counting immunofluorescent nuclei in
five areas, as described in Materials and Methods.
Typical staining after transfection with vectors HE15-NLS (a), ER108
(b), HE384 (c), and ER103 (d). Bar, 10 µm.
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Since these results excluded involvement of a specific A/B subregion,
we then constructed mutants deleted in the C and D regions. The SV40
large T antigen nuclear localization signal (NLS) was added at the C
terminus of HE15 to maintain nuclear targeting of mutants deleted in
the three nuclear localization sequences in the 256303 region of
ER
(Fig. 3B
). Analysis of mutants ER106, ER107, and ER108
progressively deleted from amino acids 281 to 216 indicated that the D
region and the second zinc finger of the C region are not necessary for
inhibition. Taken together, these data indicated that the 3178 (Fig. 3A
) and 216281 (Fig. 3B
) regions were dispensable for the inhibitory
effect, and that the 179215 first (or N-terminal) zinc finger region
was essential. Finally, this was demonstrated using mutant ER103
(
150215) derived from HE384 by additional deletion of this region,
which was totally inactive. The relevance of the 179215 region in the
inhibitory effect of the unliganded receptor was also demonstrated
using a short receptor mutant (ER110) containing only amino acids 1 and
2 and 178215 of the receptor followed by NLS and a
hemagglutinin epitope to detect its expression. This minimal sequence
significantly reduced invasion to 52 ± 14% of control.
Immunofluorescence analysis demonstrated that the marked differences in
the efficiency of these mutants were a direct consequence of the
mutations and not due to differences in protein expression (Fig. 3C
).
These data clearly identify an ER
region between amino acids 179 and
215, containing the first zinc finger, which is essential for
inhibition of cell invasiveness in the absence of hormone.
The reversibility of the inhibitory effect of unliganded ER by ICI
164,384 was tested using receptor mutants either mutated in the ERE
binding region (HE91) or deleted in HBD (HE15 and ER108). As shown in
Fig. 4
, the effect of ICI 164,384 was not
impaired when the ERE binding domain was mutated. By contrast, deletion
of E/F domains totally prevented antiestrogen action. These data
strongly suggest that ICI 164,384 inhibits receptor action after
binding to the HBD, but independently of ERE-mediated
transcription.

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Figure 4. Reversal of ER Inhibition of Invasion by
ICI 164,384 Requires HBD but not ERE Binding
After transfection with the indicated vectors, Matrigel-invading
cells were estimated in triplicate after 24 h in the presence of
100 nM ICI 164,384 (ICI) or ethanol alone (control) as
described in Fig. 3 . Values expressed as percentage of pSG control
transfection are mean ± SD of three experiments.
*, Significantly different from corresponding ethanol-treated cells,
P < 0.05.
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Unliganded Receptor Action Is Specific to ER
and ERß: Analysis
of Common Amino Acids
Since the DBD is well conserved within the nuclear receptor
superfamily, we analyzed the effects of several receptors on MDA-MB-231
cell invasiveness in steroid-deprived culture conditions. As shown in
Fig. 5A
, receptors specific to androgens
or glucocorticoids were totally ineffective on invasion in the absence
of hormone. Among the estrogen/thyroid/retinoic acid receptor
subfamily, the thyroid hormone receptor
1 (TR
1), vitamin D
receptor (VDR), and retinoid acid receptor
(RAR
) were also
inactive. Invasion was specifically decreased 3-fold and 2-fold by the
expression of ER
or ERß, respectively. The lower efficiency of the
ERß could be due to its endogenous presence in these cells, as
previously suggested by mRNA studies (27 ). These data indicated that
inhibition of invasiveness in the absence of hormone was ER
- and
ERß-specific and pointed to amino acids common to these receptors.
The 179215 amino-acid sequence of ER
was compared with the
corresponding regions of the other receptors tested (TR
1, VDR, and
RAR
) (Fig. 5B
). ER
showed higher homology with ERß than with
any other receptor in this region. Seven amino acids (A186, Y191, W200,
S201, A207, K210, G215) were found to be common to only ER
and ERß
and might be essential for the action of the unliganded ER.
To further characterize the active region, we used two mutants,
i.e. HE 73 and HE74 derived from HEO, each containing six
amino acid changes from the ER sequence to the GR sequence (Fig. 6
). The HE73 plasmid containing mutations
of four critical amino acids (A186, Y191, W200, and S201) common to
only ER
and ERß inhibited invasion as efficiently as the wild-type
receptor. By contrast, the HE74 plasmid mutated at six positions (203,
204, 207, 212, 213, 214) including only the ER common A207 had no
significant effect on invasion (88 ± 11%) even by transfecting a
3-fold higher plasmid concentration (data not shown). This inactivation
confirms the importance of the whole 203215 region but is not only
dependent upon mutation of the common A207 since this amino acid
was also mutated in the active mutant HE91 (see Fig. 2
).
Modulation of the Hormone-Independent Antiinvasive Effect of ER by
Calreticulin Expression
Calreticulin, a Ca2+-binding protein, is known to bind
integrins at the cell surface and to modulate gene expression by
interacting with the consensus motif KxFF[K/R]R present in the DBD of
all nuclear receptors (reviewed in Ref. 28 ). In ERs, the KAFFKR motif
(amino acids 206211) is located in the first zinc finger domain (28 )
and contains an ER-specific alanine that is essential for ERE binding
specificity (29 ). Calreticulin thus appears as a potential
ER-interacting protein possibly involved in hormone-independent
ER-induced inhibition of invasion. When calreticulin was coexpressed
with HE15 by transient transfection in MDA-MB-231 cells, the inhibitory
effect of this HBD-deleted mutant was totally repressed, and invasion
was unaltered as compared with control cells (Fig. 7
). These data confirmed the implication
of the first zinc finger domain in the antiinvasive effect. However,
when tested alone, calreticulin expression reduced Matrigel invasion to
31%, i.e. as efficiently as ER
expression. Similar
inhibition was observed after transfection of 110 µg of
calreticulin expression vector (data not shown). It is not surprising
that calreticulin was a negative modulator of invasion since this
protein is known to increase integrin-mediated cell adhesion and
spreading (28 ). Taken together, these data indicate that calreticulin
prevents the hormone-independent inhibition of invasion by ER
,
probably through interaction with its first zinc finger region, and
suggests that calreticulin prevents ER
interaction with another
unknown nuclear factor that activates invasion.

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Figure 7. Calreticulin Expression Reversed Ligand-Independent
ER Inhibition of Invasion
MDA-MB-231 cells were transfected with the indicated concentrations
(µg) of HE15 or calreticulin (CRT) expression vectors as described in
Materials and Methods. In the absence of these vectors,
identical concentrations of the corresponding empty vectors (pSG1 for
HE15, pcDNA3 for CRT) were used as controls. The percentages of
migrating cells were compared with those obtained with the
corresponding control vectors alone. HE15 and calreticulin expression
was verified in parallel by immunocytochemistry. Values represent
mean ± SD of three independent experiments.
*, Significantly different from control, P < 0.05.
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Inverse Correlation between ER
Expression and Cell Motility in
ER-Positive Cell Lines
ER-positive breast cancer cell lines are less invasive in
vitro and less metastatic in vivo than their
ER-negative counterparts (14 15 ). In all breast cancer cell lines
studied by immunocytochemistry, we observed heterogeneous expression of
ER
, with a wide range of intense to negative nuclear staining (Fig. 8A
). We used this heterogeneity to test
whether the ER
content in individual cells was correlated with their
motility capacities. A motility test was used instead of the invasion
test because of the low invasiveness of ER
-positive cell lines and
the low seeding cell density required for immunostaining of individual
cells. The percentage of immunostained cells was determined in the
upper and lower side of the filter containing nonmigrating and
migrating cells, respectively (Fig. 8A
). The percentage of
ER
-positive cells was lower in the migrating cell population than in
the nonmigrating one. This difference was significant in three
different cell lines, MCF7, ZR75.1, and T47D (Fig. 8B
). In control
experiments, we verified that this decrease in ER
positivity was not
due to differences in cell densities between the two sides of the
filter by immunostaining of serial dilutions of MCF7 cells (data not
shown).

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Figure 8. ER Immunostaining of Three Cell Lines before and
after Migration through Transwells
A, Immunostaining of MCF7 cells on the upper (nonmigrating cells)
(left panel) and lower (migrating cells) (right
panel) sides of the filter after 6 h migration in phenol
red-free medium containing 10% DCC-treated FCS was performed using
antihuman ER , 1D5 monoclonal antibody. Photographs at x20 showed
strong nuclear immunoperoxidase staining of ER-expressing cells, light
nuclear counterstaining with hematoxylin of ER-negative cells, and
colorless circles corresponding to filter pores. Bar, 30
µm. B, Cells were immunostained with antihuman ER antibody as in
panel A. The percentage of cells with nuclear staining was evaluated
from 400800 cells in five different areas on the upper and lower
sides of filters. *, P < 0.05 vs.
nonmigrating cells.
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The fact that unliganded ER
expression was correlated with decreased
motility of the three breast cancer cell lines was consistent with
direct evidence obtained by transfection of ER
-negative cells. Taken
together, these in vitro data indicated a protective role of
ER
against invasion by breast cancer cells.
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DISCUSSION
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Several studies indicated that estrogen decreased in
vitro invasiveness and motility of breast (15 18 22 ) and ovarian
(20 ) cancer cells. In addition, evidence of estrogen-induced inhibition
of cancer aggressiveness was obtained in an experimental murine
metastasis model using ER
-expressing MDA-MB-231 cells (18 ).
Moreover, epidemiological studies of breast cancer risk in women using
hormone replacement therapy (HRT) are in accordance with a decrease of
tumor invasion mediated by estrogen (30 ). Among women using HRT (80%
received preparations containing estrogen alone), the risk of breast
cancer increased, but these tumors were stage 1 with more favorable
prognosis. Compared with tumors in never-users, those in HRT-users are
less invasive to axillary lymph nodes and to distant sites. These data
suggest that, in addition to their initial promoter role in breast
cancer, estrogens could prevent spreading of cancer cells.
In this study, we used an original method that combines transient gene
expression with Matrigel invasion assay to specify the receptor domains
involved in estradiol inhibition of invasion. The estradiol effect is
probably due to the classical activation of target gene transcription
since it required ERE binding and AF2 domain integrity, two
transactivation prerequisites. This strongly suggests that some
estrogen-regulated genes negatively controlled invasion. Among
estrogen-regulated proteins, those increasing cell-cell adhesion, such
as E-cadherin, or decreasing matrix degradation, such as
1-antichymotrypsin are possible candidates (reviewed in Ref.
31 ).
The major finding of this study is the strong inhibitory effect of
unliganded ER
on cancer cell invasion. In previous experiments using
stable transfection, analysis of two transfectants was not sufficient
to render a conclusive decision on the inhibitory effect of
unliganded ER
on invasion (18 ). Possible artefacts related to the
cell cloning procedure were eliminated by this new transient
transfection approach. We could reproducibly inhibit cell invasion by
full-length ER
expression in the absence of hormone. Evidence that
ER
acted through an E2-independent mechanism
was demonstrated by the action of different mutants lacking the hormone
binding domain. Moreover, the strong inhibitory effect of the
unliganded receptor was reversed by addition of the pure antiestrogen
ICI 164,384 but not by the partial agonist/antagonist OH-tamoxifen. We
have also shown that ICI 164,384 activity requires the E/F region, but
not ERE-mediated transcription, and is associated with a 6-fold
decrease in the number of ER-stained nuclei. This suggests that ICI
164,384 prevents unliganded receptor action by ER degradation after its
binding to the HBD. These data are in agreement with previous studies
in native ER-positive cells showing that ER
stability was decreased
in the presence of ICI 164,384 (32 ) but not in the presence of
OH-tamoxifen (33 ).
Using ER
mutants, we demonstrated that the 1215 region (ER108) of
the receptor was as efficient as the full-length receptor in the
absence of ligand. Moreover, ERE-mediated transactivation and the A/B
region containing the AF1 transactivation function were dispensable for
this action. This contrasts with the previously proposed mechanism for
steroid-independent activation of ER
requiring the functional A/B
region, serine 118 phosphorylation, and ERE binding (25 26 ).
Analysis of mutants in the C and D regions revealed that deletion of
the first zinc finger of the C region totally prevented
ligand-independent inhibition, whereas other deletions flanking this
zinc finger sequence were ineffective. Moreover, the minimal 178215
sequence preceded by two terminal amino acids appeared sufficient to
inhibit invasion. The active conformation of the critical 179215
region is probably favored by the presence of a short (two or more
amino acids) terminal sequence. As inhibition of invasion is restricted
to the two ER isoforms among different members of the nuclear receptor
superfamily, we identified seven amino acids common to only ER
and
ERß by sequence comparison of the 179215 region. However, the
analysis of HE73 and HE74 mutants pointed out the importance of the
203214 sequence rather than common amino acids located in the
186207 region.
On the basis of these data, we propose that unliganded ER decreases
invasiveness via interaction of the first zinc finger region with an
unknown nuclear factor. The possibility that unliganded ER reversed
invasion via a direct genomic effect due to DNA interaction was
excluded by the activity of HE91, ER108, and ER110 mutants. Previous
published data have shown that 1) mutations in HE91 prevented
transactivation of ERE-mediated responses (24 ); and 2) deletions of the
C-terminal zinc finger or of the 222226 dimerization domain, as found
in ER108 and ER110 mutants, abolished DNA binding of ER (34 ) and DBD
dimerization (35 ). Moreover, other examples of DBD regions that have a
transcriptional role distinct from DNA binding have already been
described (36 37 ).
Among the possible candidate proteins interacting with the DBD region
of ER
(38 39 40 ), we tested calreticulin since this protein was known
to specifically interact with the first zinc finger of different
nuclear receptors (28 40 ). Calreticulin expression was able to reverse
the inhibitory effect of HE15 but also inhibited invasion when tested
alone. This indicates that calreticulin is not the ER
interacting
factor that positively regulates invasion, but its interaction could
prevent the binding of ER
to this unknown activator. The possibility
that ER
could interfere with AP1-directed gene activity (6 8 9 41 ) via a protein-protein interaction with the c-Jun protein (9 ) was
excluded since the ER
region interacting with c-Jun is probably not
the first zinc finger (Ref. 9 and C. Teyssier and D. Chalbos,
unpublished data), and we showed that c-Jun overexpression did not
influence the ER
effect on cell invasiveness (our unpublished
data).
In mammary carcinogenesis, the promoting role and mitogenic effect of
estrogens are well demonstrated, whereas the presence of ER
paradoxically seems to be associated with more differentiated and less
invasive tumors. Moreover, large clinical studies have shown that ER
is a favorable prognostic marker in primary breast tumors. This was
also confirmed in breast cancer cell lines in which ER
positivity
was associated with low Matrigel invasiveness and low metastatic
potential in mice. In this study, we present the following evidence
that ER
per se could have a protective role against
cancer progression: 1) Transient expression of the unliganded ER
and
several mutants deleted in the hormone binding domain drastically
reduced MDA-MB-231 cell invasiveness in Matrigel tests. 2) Studies in
three ER
-positive cell lines showed that in hormone-deprived
conditions, the ER
content was inversely correlated with cell
motility. These hormone-independent effects of ER
also suggest a
function for ER variants deleted in exon 3 (second zinc finger) or exon
4 (part of HBD) that have been found to occur naturally in normal and
neoplastic estrogen target tissues (42 43 ). In addition to the
antitumor properties of the transfected ER
previously shown in the
presence of estrogen (18 44 45 ), the strong antiinvasive activity
associated with expression of the unliganded receptor also suggests
potential practical applications in cancer gene therapy.
We conclude that unliganded and hormone-activated ER
decrease
in vitro cancer cell invasiveness via distinct mechanisms.
This is evidence of a protective role of ER
in cancer progression
and supports hormone-independent ER
function. This finding could
help to explain the favorable prognosis associated with the presence of
ER
in primary breast cancers and lead to new therapeutic
applications.
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MATERIALS AND METHODS
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Plasmids
Plasmids derived from the pSG5 or pSG1 vector expressing
full-length human ER
(HEGO) or mutated forms (HE series) of the
receptor were generously donated by P. Chambon, and their constructions
have been previously described (46 47 ) except for HEmut-AF2 containing
four mutations in the core activating domain of AF2 that prevent its
action (Ref. 48 and V. Vivat and P. Chambon, unpublished).
ER100 plasmid (
3178), a derivative of the HE344/384 (
350 and
150178), was created by XhoI excision of amino acids 50
to 150. The ER103 expression vector (
150216) was constructed by
replacement of the HE384 XhoI-XbaI fragment
(amino acids 179 to 379) with a PCR-generated fragment containing
residues 216379 with a XhoI site preceding amino acid 216.
The deletion mutants ER106, ER107, and ER108 were constructed as
follows. The expression vector HE15-NLS was first constructed using two
complementary oligonucleotides coding for the SV40 large T antigen NLS
Pro-Lys-Lys-Lys-Arg-Lys-Val (49 ), which were inserted at the 3'-end of
ER
1282 (HE15 coding sequence) between XhoI and
AocI sites. These oligonucleotides contained a new
KpnI site, in front of the NLS. Mutants ER106 (
270281),
ER107 (
251281), and ER108 (
216281) were constructed by
replacement of the HE15-NLS NotI-XhoI fragment
(amino acids 65 to 282) with PCR-generated fragments containing
residues 65269 (plasmid ER106), 65250 (plasmid ER107), and 65215
(plasmid ER108) in front of a new XhoI site. Mutant ER110
was constructed from ER108 by replacement of EcoRI fragment
by PCR-generated fragments containing human influenza hemagglutinin tag
sequence YPYDVPDYA between the NLS and stop codon. Other human receptor
cDNAs corresponding to ERß (provided by S. Mosselman) vitamin D
(VDR), thyroid hormone (TR
1), retinoic acid (RAR
), androgen (AR),
and glucocorticoid (GR) receptors (provided by P. Chambon) were used in
pSG5 expression vector. Calreticulin cDNA inserted in pcDNA3 expression
vector (Invitrogen, San Diego, CA) was provided by M.
Michalak.
Cell Culture and Transfection
Human breast cancer cell lines MDA-MB-231, MCF7, T47D, and ZR
75.1 were maintained in monolayer cultures in DMEM supplemented with
10% FCS and 50 µg ml-1 gentamycin. In all
experiments, steroids were withdrawn from cells by 6 days of culture in
phenol red-free DMEM supplemented with 10% dextran-coated
charcoal-treated FCS (FCS-DCC).
Transient transfections of MDA-MB-231 cells were performed using the
calcium phosphate DNA coprecipitation method. Near confluent cells were
cotransfected in a 75-cm2 flask with 3.75 µg of
steroid receptor expression vector or pSG control vector and 33.75 µg
of pGL3 vector (Promega Corp., Madison WI) coding for
luciferase. Cells were exposed to the precipitate for 8 h, washed
three times with phenol red-free medium, and incubated for an
additional 16 h in fresh medium before the invasion assay.
Matrigel Invasion Assay, Recovery of Luciferase Activity, and
Calculation of the Percentage of Matrigel-Invading Cells
For the invasion assay, a suspension of
3.105 transiently transfected cells was layered
in the upper compartment of a Transwell (Costar,
Cambridge, MA) on a polycarbonate filter (8 µm pore size) previously
coated with 30 µg of Matrigel basement membrane (Becton Dickinson and Co., Le Pont de Claix, France), vs. 30
µg/ml fibronectin (Sigma, St. Louis, MO) as attractant
in the lower compartment. Cells were incubated for 24 h at 37 C in
DMEM + 10% FCS-DCC in the presence of the indicated hormone or
antihormone concentration. In parallel, a suspension of
3.105 transfected cells was layered on a 24-well
plate to determine the total luciferase activity. After 24 h,
cells were rinsed and lysed for 30 min with 100 µl (for migrating
cells on the lower side of the filter) or 300 µl (for cells plated on
24-well plate) of lysis buffer containing 10% glycerol and 1% Triton
X100 (Promega Corp.). Luciferase activities were
determined on 100 µl samples by measuring the luminescence (15-sec
integration time) in a LKB luminometer (LKB,
Rockville, MD) after the injection of 100 µl of luciferase assay
reagent (Promega Corp.). The percentage of migrating cells
is given by the ratio of luciferase activity of invasive
cells to luciferase activity of total cells x
100. Values are means of three independent experiments
performed in triplicate. The interexperiment coefficient of variation
determined from 30 values was 4%. The validity and reproducibility of
the transfection/invasion technique was described in greater detail by
Platet and Garcia (23 ).
For chemotaxis studies, cells were seeded on the Transwell filter as
described for the invasion assay, but in the absence of Matrigel.
Immunocytochemistry
ER
immunostaining was performed 24 h after transient
transfection of vectors expressing full-length and mutated ER
. Cells
were fixed and permeabilized with 3.7% formaldehyde for 12 min, cold
methanol for 4 min, and cold acetone for 2 min and then saturated with
2.5% goat serum in phosphate saline buffer containing 4% BSA
overnight at 4 C. Immunostaining was performed using mouse monoclonal
ER
antibodies directed against either the amino-terminal A/B region
(50 ) (2.5 µg ml-1 of 1D5 antibody, from
DAKO Corp., Carpinteria, CA) or carboxy-terminal
amino-acids 495594 (1 µg ml-1 C311 antibody,
from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), with
goat antimouse rhodamine-conjugated antibody (Immunotech,
Marseille, France) as second antibody. For chemotaxis studies in
ER-positive cell lines, ER
was detected by immunoperoxidase staining
using 1D5 antibody, antimouse biotinylated antibody, and the
Vectastain kit (Vector Laboratories, Inc.,
Burlingame CA). Calreticulin and hemagglutinin expression was detected
by double immunofluorescence using C-17 goat antihuman calreticulin
antibody from Santa Cruz Biotechnology, Inc., and mouse
monoclonal antihemagglutinin antibody from Roche Molecular Biochemicals (Mannheim, Germany).
 |
ACKNOWLEDGMENTS
|
---|
We thank P. Chambon (IGMBC, Strasbourg, France) for provision of
and assistance with many of the ER expression vectors used in this
study; we also thank S. Mosselman (N. V. Organon,
Oss, Netherlands) and M. Michalak (University of Alberta, Edmonton,
Canada) for providing the ERß and calreticulin expression vectors,
respectively. We thank D. Derocq for technical suggestions and J.
Y. Cance for artwork.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Marcel Garcia, Unité 540 INSERM, 60, rue de Navacelles, 34090 Montpellier, France.
N.P. is a recipient of MRES fellowship. This work was supported
by the Institut National de la Santé et de la Recherche
Médicale and the Association pour la Recherche sur le Cancer.
Received for publication August 30, 1999.
Revision received March 27, 2000.
Accepted for publication April 3, 2000.
 |
REFERENCES
|
---|
-
Beato M, Herrlich P, Schütz G 1995 Steroid hormone
receptors: many actors in search of a plot. Cell 83:851857[Medline]
-
Mangeldorf DJ, Thummel C, Beato M, Herrlich P, Schütz
G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson
J-A 1996 Cloning of a novel estrogen receptor expressed in rat prostate
and ovary. Proc Natl Acad Sci USA 93:59255930[Abstract/Free Full Text]
-
Mosselman S, Polman J, Dijkema R 1996 ERß:
identification and characterization of a novel human estrogen receptor.
FEBS Lett 392:4953[CrossRef][Medline]
-
Blobel GA, Sieff CA, Orkin SH 1995 Ligand-dependent
repression of the erythroid transcription factor GATA-1 by the estrogen
receptor. Mol Cell Biol 15:31473153[Abstract]
-
Philips A, Chalbos D, Rochefort H 1993 Estradiol increases
and antiestrogens antagonize the growth factor-induced activator
protein-1 activity in MCF7 breast cancer cells without affecting
c-fos and c-jun synthesis. J Biol Chem 268:1410314108[Abstract/Free Full Text]
-
Stein B, Yang MX 1995 Repression of the interleukin-6
promoter by estrogen receptor is mediated by NF-
B and C/EBPß.
Mol Cell Biol 15:49714979[Abstract]
-
Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N,
Morishima T, Yamasaki Y, Kajimoto Y, Kamada T 1994 Estrogen regulation
of the insulin-like growth factor I gene transcription involves an AP-1
enhancer. J Biol Chem 269:1643316442[Abstract/Free Full Text]
-
Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen
activation of the estrogen receptor/AP-1 pathway: potential origin for
the cell-specific estrogen-like effects of antiestrogens. Mol
Endocrinol 9:443456[Abstract]
-
Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo
P, Nola E, Auricchio F 1996 Tyrosine kinase/p21 ras/MAP-kinase pathway
activation by estradiol-receptor complex in MCF7 cells. EMBO J 15:12921300[Abstract]
-
Pike MC, Krailo MD, Henderson BE, Casagrande JT, Hoel DG 1983 Hormonal risk factors, breast tissue age and the age-incidence
of breast cancer. Nature 303:767770[Medline]
-
McGuire WL 1978 Hormone receptors: their role in predicting
prognosis and response to endocrine therapy. Semin Oncol 5:24282433
-
Lippman ME, Bolan G, Huff K 1976 The effects of estrogens and
antiestrogens on hormone-responsive human breast cancer in long term
culture. Cancer Res 36:45954601[Abstract]
-
Thompson EW, Paik S, Brunner N, Sommers CL, Zugmaier G, Clarke
R, Shima TB, Torri J, Donahue S, Lippman ME, Martin GR, Dixon RB 1992 Association of increased basement membrane invasiveness with absence of
estrogen receptor and expression of vimentin in human breast cancer
cell lines. J Cell Physiol 150:534544[Medline]
-
Rochefort H, Platet N, Hayashido Y, Derocq D, Lucas A, Cunat
S, Garcia M 1998 Estrogen receptor mediated inhibition of cancer cell
invasion and motility: an overview. J Steroid Biochem Mol Biol 65:163168[CrossRef][Medline]
-
Albini A, Graf J, Kitten GT, Kleinman HK, Martin GR, Veillette
A, Lippman ME 1986 17ß-Estradiol regulates and v-Ha-ras
transfection constitutively enhances MCF7 breast cancer cell
interactions with basement membrane. Proc Natl Acad Sci USA 83:81828186[Abstract]
-
Thompson EW, Reich R, Shima TB, Albini A, Graf J, Martin GR,
Dickson RB, Lippman ME 1988 Differential regulation of growth and
invasiveness of MCF-7 breast cancer cells by antiestrogens. Cancer Res 48:67646768[Abstract]
-
Garcia M, Derocq D, Freiss G, Rochefort H 1992 Activation of
estrogen receptor into a receptor-negative breast cancer cell line
decreases the metastatic and invasive potential of the cells. Proc Natl
Acad Sci USA 89:153811542
-
Garcia M, Derocq D, Platet N, Bonnet S, Brouillet JP, Touitou
I, Rochefort H 1997 Both estradiol and tamoxifen decrease proliferation
and invasiveness of cancer cells transfected with a mutated estrogen
receptor. J Steroid Biochem Mol Biol 61:1117[CrossRef][Medline]
-
Hayashido Y, Lucas A, Rougeot C, Godyna S, Argraves WS,
Rochefort H 1998 Estradiol and fibulin-1 inhibit motility of human
ovarian- and breast-cancer cells induced by fibronectin. Int J Cancer 75:654658[CrossRef][Medline]
-
Kolodgie FD, Jacob A, Wilson PS, Carlson GC, Farb A, Verma A,
Virmani R 1996 Estradiol attenuates directed migration of vascular
smooth muscle cells in vitro. Am J Pathol 148:969976[Abstract]
-
Long BJ, Rose DP 1996 Invasive capacity and regulation of
urokinase-type plasminogen activator in estrogen receptor (ER)-negative
MDA-MB-231 human breast cancer cells, and a transfectant (S30) stably
expressing ER. Cancer Lett 99:209215[CrossRef][Medline]
-
Platet N, Garcia M 1999 A new bioassay using transient
transfection for invasion-related gene analysis. Invest Metastasis 18:198208
-
Mader S, Kumar V, de Verneuil H, Chambon P 1989 Three amino
acids of the oestrogen receptor are essential to its ability to
distinguish an oestrogen from a glucocorticoid-responsive element.
Nature 338:271274[CrossRef][Medline]
-
Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H,
Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by
mitogen-activated protein kinase. Science 270:14911494[Abstract]
-
Bunone G, Briand P-A, Miksicek RJ, Picard D 1996 Activation of
the unliganted estrogen receptor by EGF involves the MAP kinase pathway
and direct phosphorylation. EMBO J 15:21742183[Abstract]
-
Dotzlaw H, Leygue E, Watson PH, Murphy LC 1996 Expression of
estrogen receptor-ß in human breast tumors. J Clin Endocrinol
Metab 82:23712374[Abstract/Free Full Text]
-
Dedhar S 1994 Novel functions for calreticulin: interaction
with integrins and modulation of gene expression? Trends Biochem Sci 19:269271[CrossRef][Medline]
-
Green S, Kumar V, Theulaz I, Wahli W, Chambon P 1988 The
N-terminal DNA-binding zinc finger of the oestrogen and
glucocorticoid receptors determines target gene specificity. EMBO J 7:30373044[Abstract]
-
Marsden J, Backs NPM 1996 Hormone replacement therapy and
breast cancer. Endocr Relat Cancer 3:8197
-
Sheikh, MS, Garcia M, Pujol P, Fontana JA, Rochefort H 1995 Why are estrogen-receptor-negative breast cancers more aggressive than
the estrogen-receptor-positive breast cancers? Invest Met 14:329336
-
Dauvois S, Danielian PS, White R, Parker MG 1992 Antiestrogen
ICI 164,384 reduces cellular estrogen receptor content by increasing
its turnover. Proc Natl Acad Sci USA 89:40374041[Abstract]
-
Pink JJ, Jordan VC 1996 Models of estrogen receptor regulation
by estrogens and antiestrogens in breast cancer cell lines. Cancer Res 56:23212330[Abstract]
-
Chambraud B, Berry M, Redeuilh, Chambon P, Baulieu EE 1990 Several regions of human estrogen receptor are involved in the
formation of receptor-heat shock protein 90 complexes. J Biol Chem 265:2068620691[Abstract/Free Full Text]
-
Mader S, Chambon P, White JH 1993 Defining a minimal estrogen
receptor DNA binding domain. Nucleic Acids Res 21:11251132[Abstract]
-
Schena M, Freedman LP, Yamamoto KR 1989 Mutations in the
glucocorticoid receptor zinc finger region that distinguish
interdigitated DNA binding and transcriptional enhancement activities.
Genes Dev 3:15901601[Abstract]
-
Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ,
Drouin J, Karin M 1990 Transcriptional interference between c-Jun and
the glucocorticoid receptor: mutual inhibition of DNA binding due to
direct protein-protein interaction. Cell 62:12051215[Medline]
-
Budhram-Mahadeo V, Parker M, Latchman DS 1998 POU
transcription factors Brn-3a and Brn-3b interact with the estrogen
receptor and differentially regulate transcriptional activity via an
estrogen response element. Mol Cell Biol 18:10291041[Abstract/Free Full Text]
-
Powers CA, Mathur M, Raaka BM, Ron D, Samuels HH 1998 TLS
(translocated-in-liposarcoma) is a high affinity interactor for
steroid, thyroid hormone, and retinoid receptors. Mol Endocrinol 12:418[Abstract/Free Full Text]
-
Burns K, Duggan B, Atkinson EA, Famulski KS, Nemer M,
Bleackley RC, Michalak M 1994 Modulation of gene expression by
calreticulin binding to the glucocorticoid receptor. Nature 367:476480[CrossRef][Medline]
-
Gaub M-P, Bellard M, Scheuer I, Chambon P, Sassone-Corsi P 1990 Activation of the ovalbumin gene by the estrogen receptor involves
the Fos-Jun complex. Cell 63:12671276[Medline]
-
Erenburg I, Schachter B, Mira y Lopez R, Ossowski L 1997 Loss
of an estrogen receptor isoform (ER

3) in breast cancer and
consequences of its reexpression: interference with estrogen-stimulated
properties of malignant transformation. Mol Endocrinol 11:20042015[Abstract/Free Full Text]
-
Park W, Choi J-J, Hwang E-S, Lee J-H 1996 Identification of a
variant estrogen receptor lacking exon 4 and its coexpression with
wild-type estrogen receptor in ovarian carcinomas. Clin Cancer Res 2:20292035[Abstract]
-
Jiang SY, Jordan VC 1992 Growth regulation of estrogen
receptor negative breast cancer cells transfected with cDNAs for
estrogen receptor. J Natl Cancer Inst 84:580591[Abstract]
-
Levenson AS, Kwaan HC, Svoboda KM, Weiss IM, Sakurai S, Jordan
VC 1998 Oestradiol regulation of the components of the
plasminogen-plasmin system in MDA-MB-231 human breast cancer cells
stably expressing the oestrogen receptor. Br J Cancer 78:8895[Medline]
-
Ali S, Metzger D, Bornert J-M, Chambon P 1993 Modulation of
transcriptional activation by ligand-dependent phosphorylation of the
human oestrogen receptor A/B region. EMBO J 12:11531160[Abstract]
-
Metzger D, Ali S, Bornert J-M, Chambon P 1995 Characterization
of the amino-terminal transcriptional activation function of the human
estrogen receptor in animal and yeast cells. J Biol Chem 270:95359542[Abstract/Free Full Text]
-
Vom Baur E, Zechel C, Heery D, Heine MJS, Garnier JM, Vivat V,
Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential
ligand dependent interactions between the AF-2 activating domain of
nuclear recptors and the putative transcriptional intermediary factors
mSUG1 and TIF1. EMBO J 15:110124[Abstract]
-
Kalderon D, Roberts BL, Richardson WD, Smith AE 1984 A short
amino acid sequence able to specify nuclear location. Cell 39:499509[Medline]
-
Al Saati T, Clamens S, Cohen-Knafo E, Faye J-C, Prats H,
Coindre JM, Wafflart J, Caveriviere P, Bayard F, Delsol G 1993 Production of monoclonal antibodies to human estrogen-receptor protein
(ER) using recombinant ER (RER). Int J Cancer 55:651654[Medline]