Hyperactivation of MAPK Induces Loss of ER
Expression in Breast Cancer Cells
Annabell S. Oh1,
Lori A. Lorant1,
Jamie N. Holloway,
David L. Miller2,
Francis G. Kern2 and
Dorraya El-Ashry
Lombardi Cancer Center, Department of Oncology, Georgetown
University Medical Center, Washington, DC 20007
Address all correspondence and requests for reprints to: Dorraya El-Ashry, Lombardi Cancer Center, Rm. W313, TRB, 3970 Reservoir Rd., NW, Washington, DC 20007. E-mail: elashryd{at}gunet georgetown.edu
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ABSTRACT
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ER
-negative breast tumors tend to overexpress growth factor
receptors such as epidermal growth factor receptor or c-erbB-2. Raf-1
is a key intermediate in the signal transduction pathways of these
receptors. High levels of constitutive Raf kinase (
raf) activity
imparts ER
- positive MCF-7 breast cancer cells with the ability to
grow in the absence of estrogen.
raf transfectants maintained in
estrogen-depleted media showed greatly diminished responses to
17ß-estradiol or the pure antiestrogen ICI 182,780. Western blotting,
ligand binding, and immunohistochemistry assays revealed a loss of
ER
protein expression, and ribonuclease protection assays indicated
that this correlated with loss of ER
message. In examining the
basal expression of estrogen-induced genes in the stable
transfectants or in transient cotransfection assays with an
estrogen-response element- reporter construct and
raf or
constitutively active MAPK kinase (
MEK), no ligand- independent
activation of ER
was observed. Transient expression of
raf
and double-label immunostaining showed ER
was lost in those cells
that transiently expressed
raf. Abrogation of Raf signaling via
treatment with the MEK inhibitors PD 098059 or U0126 resulted in
reexpression of ER
. Similar studies performed with MCF-7 cells
overexpressing epidermal growth factor receptor or c-erbB-2 confirmed
that hyperactivation of MAPK resulted in down-regulation of ER
that
was reversible by MEK inhibition or transfection with dominant negative
ERK1 and ERK2 constructs. These data suggest that the hyperactivation
of MAPK in epidermal growth factor receptor- or
c-erbB-2-overexpressing breast cancer cells is directly responsible for
generation of an ER
-negative phenotype and, more importantly, that
this process may be abrogated by inhibiting these pathways, thus
restoring ER
expression.
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INTRODUCTION
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CLINICALLY, BREAST CANCER presents as
either ER
positive or as ER
negative. The presence of ER
is
correlated with a better prognosis both in terms of increased
disease-free survival and overall survival and predicts for response to
hormonal therapies such as tamoxifen (1, 2, 3, 4). Tamoxifen as
an adjuvant therapy is effective in both pre- and postmenopausal
patients with ER
+ tumors (5); however, 2535% of all
ER
+ tumors do not respond to tamoxifen (de novo
resistance), and even those that do initially respond ultimately
develop resistance (acquired resistance) (6). Fifty
percent of patients with ER
+ primary tumors that relapse after
adjuvant tamoxifen therapy have recurrent tumors in which ER
expression is lost (7, 8). In the metastatic setting,
tamoxifen has also proven effective, with about 30% of ER
+ patients
demonstrating response. In this case, 30% of the initially
tamoxifen-responsive metastatic tumors that subsequently develop
resistance to tamoxifen have lost ER
expression. It may also be
possible that ER
-negative tumors arise de novo.
Regardless of whether the ER
-negative phenotype is acquired or
de novo, the lack of ER
expression precludes the use of
tamoxifen, as tamoxifen has not been demonstrated to have a therapeutic
benefit in ER
-negative patients (9, 10). The
reexpression of ER
, therefore, in tumors in which ER
expression
has been lost or is not expressed could allow for restoration of
tamoxifen sensitivity, and maintenance of ER
expression may provide
a means of prolonging response to this well tolerated drug.
ER
-negative tumors frequently overexpress growth factor
receptors, such as the epidermal growth factor receptor [EGFR
(11)] or c-erbB-2, as do many ER
-negative breast
cancer cell lines, suggesting that up-regulated growth factor signaling
may provide an alternative growth stimulus. The presence of high levels
of these receptors is also an important prognostic indicator. For
example, in breast tumors, the overexpression of EGFR is inversely
correlated with ER
: in the majority of patients breast tumors
are either ER
+/EGFR- or ER
-/EGFR+ (11), and EGFR+
tumors have a poor prognosis independent of ER
status
(12, 13, 14, 15, 16). Similarly, tumors that overexpress c-erbB-2 have
a poorer prognosis and tend to be ER
- (17, 18, 19). The protooncogene
Ras, the downstream mediator of growth factor receptor activation, is
overexpressed in approximately 70% of breast cancer (20, 21), further implicating growth factor signaling mechanisms in
breast cancer. Double-label immunohistochemical detection of ER
and
EGFR in breast tumor specimens and breast cancer cell lines confirms
the inverse correlation of expression (22, 23, 24).
Furthermore, in ER
+/EGFR+ tumors, individual tumor cells express
high levels of only ER
or EGFR, but never both (22, 23). The EGFR+ cells in these tumors are also associated with a
higher growth rate than the ER
+/low EGFR cells (25, 26). Interestingly, ER
and EGFR expression in the same cell
is observed in normal and benign breast specimens (23),
suggesting that the interaction between these two signaling pathways is
altered in breast cancer cells.
Experimentally, the induction of estrogen-independent growth in the
ER
+, estrogen-dependent MCF-7 human breast cancer cell line via
stable transfection of a variety of growth signaling factors also
frequently results in tamoxifen resistance and decreased levels of
ER
(27, 28, 29, 30, 31, 32). In addition, MCF-7 cells selected for
their ability to grow in the absence of estrogen (33, 34, 35),
as well as selected for resistance to adriamycin (36),
frequently up-regulate signaling molecules such as EGFR, Raf, and
TGF
(36, 37). Up-regulation of these pathways may
therefore be an early event in progression to ER
negativity,
resulting in an intermediate ER
+/estrogen-independent phenotype.
Understanding the mechanisms underlying the role of up-regulated growth
factor signaling in estrogen-independent growth might lead to methods
of reversing this in the earlier stages when ER
is still expressed
and might provide clues on the underlying mechanism of loss of ER
expression.
We previously established a model of up-regulated growth factor
signaling in the ER
-positive, estrogen-dependent MCF-7 human breast
cancer cell line by stably expressing a constitutively active Raf
kinase (designated
raf), an important downstream effector of
tyrosine kinase receptor signaling.
raf-expressing MCF-7 cells
exhibited increased anchorage-dependent and anchorage-independent
growth in the absence of estrogen (38). In the current
study, we demonstrate that estrogen-independent growth of
raf clones
results in the loss of ER
expression. This loss in expression occurs
at both the protein and message levels. Transient expression of
raf
or constitutively active MAPK kinase (
MEK) in MCF-7 cells also leads
to down-regulation of ER
expression. It is MAPK (or ERK) activity
that is responsible for this down-regulation since abrogation of MAPK
activity via direct inhibition of MEK or dominant negative ERKs results
in rapid reexpression of ER
. Constitutive activation of stably
transfected c-erbB-2 or ligand-induced activation of stably transfected
EGFR also leads to a MAPK- induced down-regulation of ER
that is
reversible via abrogation of MAPK activity. These data suggest that
up-regulated growth factor signaling via MAPK is directly linked to
loss of ER
expression and generation of the ER
-negative phenotype
and that at some stage in the progression pathway, the ER
-negative
phenotype may not be permanent.
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RESULTS
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raf clones maintained in charcoal-stripped serum (CCS) have
significantly diminished ER
expression. We have previously shown
that the initial clonal isolates of
raf transfected MCF-7 cells had
decreased doubling times in estrogen-depleted media compared with a
pooled population of parental vector-control transfected cells, the
HCopool cell line (38). But these
raf transfectants
still responded to estrogen by further increasing their growth rate.
Each of these clones has been maintained in the absence of estrogen
[phenol red-free IMEM (PRF-IMEM) supplemented with 10% CCS].
The HCopool cell line was adapted long term for growth in the absence
of estrogen to serve as a CCS control (HCopoolc). When these
CCS
raf transfected clones (or
rafc clones) were
analyzed for their growth properties in the presence of estrogen or
antiestrogens, they exhibited significantly blunted or no responses to
estrogen and antiestrogens (data not shown). This nonresponsiveness of
the
rafc clones suggested that the cells might no longer
be expressing functional ER
. To determine whether this was indeed
the case,
rafc clones were assessed for ER
levels,
both at the protein and mRNA level. ER
protein was analyzed both by
steroid-binding assay, by Western blotting, and by
immunohistochemistry. Results from these assays are shown in Fig. 1A
and B. MCNC4 cells are a clonal
population of parental vector-control transfected cells adapted
long-term for growth in CCS and thus grow continuously in CCS like the
rafc clones. While the MCNC4 line exhibits approximately
206 fmol ER/mg protein, each of the
rafc clones has
significantly reduced levels of ER
expression, with Raf
14c, the clone exhibiting the highest
raf expression,
having only approximately 10 fmol/mg protein. Thus, all of the
rafc clones had significantly reduced ER
protein
expression. Immunohistochemistry with the H222 anti-ER monoclonal
antibody (MAb) also demonstrates loss of ER
expression in
raf-expressing cells. Nuclear immunoreactivity for ER
is clearly
observed in the HCopool cell line, increased immunoreactivity is seen
in HCopoolc cells, and no nuclear immunoreactivity is
observed in Raf 14c cells.
RNase protection assays were performed to determine ER
mRNA
levels in the clones. Compared with either the HCopool (grown in FBS
and stripped of estrogens for 5 d) or the MCNC4 (grown
continuously in CCS) control transfected cells or to the initial
raf
clonal isolates, the
rafc clones exhibited drastically
reduced ER
mRNA levels (Fig. 1C
). Again, the effect was most readily
detectable in Raf 14c where very little ER
mRNA was
detected. Furthermore, the significant decrease in ER
message
occurred relatively early upon switching to growth in CCS (CCS
e, four passages in CCS) since further significant reduction
was not seen after prolonged growth in CCS (CCS l, 16
passages in CCS).
raf-induced down-regulation of ER
Does Not Involve
Ligand-Independent Activation of ER
Transcriptional Activity
Because estrogen-induced activation of ER
results in subsequent
down-regulation of ER
(39, 40, 41, 42, 43, 44, 45), and because it has been
demonstrated in other systems that growth factor signaling via MAPK can
induce ligand-independent activation of ER
(46), we
assessed whether
raf signaling could activate ER
in the absence
of estrogen. We examined the basal expression of three
estrogen-regulated genes, pS2, cathepsin D, and PR, in our
rafc clones. If ligand-independent activation were
occurring, one would expect higher basal levels of estrogen-induced
genes in the absence of estrogen than that observed in control
transfected cells in the absence of estrogen. None of the
rafc clones displayed significantly increased basal
levels of pS2 or cathepsin D mRNA compared with the control
vector-transfected MCNC4 cell line which, like the
rafc
transfectants, grows continuously in the absence of estrogen (Fig. 2A
). Like other MCF-7 cell lines adapted
for growth in estrogen-depleted conditions, MCNC4 cells exhibit
increased basal expression over control MCF-7 cells as a result of the
adaptation (data not shown). Except for Raf 14c cells, which
express less than 10 fmol/mg protein ER
and behave more like
ER
-negative cells in that they express very low levels of pS2 mRNA,
all of the clones expressed similar levels of pS2 and cathepsin D mRNA
as MCNC4 cells. Analysis of PR mRNA expression in the
rafc clones revealed that like the control MCNC4 cells,
none of the raf clones expressed PR in the absence of estrogen.
Furthermore, while MCNC4 cells were induced by estrogen to express PR,
none of the clones expressed PR in response to estrogen (Fig. 2B
).
Collectively, these data suggest that constitutive activation of the
Raf/MEK/MAPK pathway in MCF-7 cells does not result in
ligand-independent expression of estrogen- target genes.

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Figure 2. rafc Clones Do Not Exhibit
Ligand-Independent Induction of Estrogen-Induced Genes pS2, Cathepsin
D, and PR
A, RNA was prepared from clones and analyzed for expression of pS2 and
cathepsin D by Northern blotting. GAPDH was used as a loading control.
B, RNA was prepared from clones treated (+) or not (-, vehicle) with
10-8 M estradiol for 72 h and analyzed
for PR expression by RNase Protection Assay. GAPDH was used as a
loading control.
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Because the estrogen target genes we examined contain complex promoters
with multiple regulatory factors, it is possible that an effect on
ER
activity could be masked by effects on other factors as well, or
that small variations in expression levels were due to other growth
factor-stimulated effectors such as activator protein 1.
Therefore, we next examined whether
raf or a constitutively active
MEK-1 construct (
MEK) (47) was capable of inducing
ligand-independent activation of ER
through an estrogen response
element (ERE)-luciferase construct. The ERE-luciferase construct is an
mouse mammary tumor virus (MMTV)- driven plasmid in which the
endogenous glucocorticoid response elements (GREs) have been deleted
and a double ERE consensus sequence has been inserted in their place
(48, 49). A control luciferase construct was also used
where the double ERE consensus sequence has been scrambled to a
nonsense sequence (NON-luciferase). The experiment shown in Fig. 3A
depicts a transient cotransfection
assay of MCF-7 cells with either
raf,
MEK, or the parental
control vector pCHC6 and an ERE-luciferase vector or the control
NON-luciferase vector. As can be seen, cotransfection of pCHC6 with
ERE-luc results in very little luciferase activity in the absence of
estrogen (Co) while estrogen is able to induce significant luciferase
activity. The antiestrogens 4-hydroxytamoxifen and ICI 182,780 do not
have a significant effect on activity. When
raf is cotransfected
with ERE-luc, there is no more luciferase activity in untreated cells
(Co) than when the control pCHC6 plasmid was used. These data indicate
that
raf is not inducing ligand-independent activation of ER
. Of
interest, however,
raf in conjunction with estrogen treatment
represses the estrogen induction compared with that with pCHC6. While
the absolute values vary from experiment to experiment, the
raf-induced repression in the experiment depicted in Fig. 3
is
approximately 47%, and this is statistically significant
(P < 0.005). When a constitutively active MEK is used
in the same assay, we again observe no ligand-independent activation of
ER
(Co with
MEK vs. with pCHC6), and
MEK almost
completely represses the estrogen inductionthe repression in this
case is approximately 97% (P < 0.001). These
experiments have also been performed in T47D cells with similar
results: neither
raf nor
MEK induced ligand-independent
activation, and both inhibited estrogen-induced activity (data not
shown). These data further support the finding that strong signaling
via the Raf/MEK/MAPK pathway does not induce ligand-independent
activation of ER
in breast cancer cells. Instead, signaling through
this pathway appears to decrease ER
activity.

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Figure 3. raf and MEK Do Not Induce Ligand-Independent
Activation of an ERE-Reporter Construct
A, MCF-7 cells were quick-stripped of estrogens as described in
Materials and Methods and transiently cotransfected with
2.5 µg of either parental vector pCHC6, raf, or MEK and 2.5
µg of either ERE-luciferase or NON-luciferase. Post-transfection
treatments were vehicle (Co), 10-8 M estradiol
(E2), 10-7 M 4-OH-tamoxifen (Tam), or
10-7 M ICI 182,780 (ICI). B, MCF-7 cells
cotransfected with pCHC6 or MEKK1. C, MCF-7 cells transfected with
ERE-luc or NON-luc were treated with vehicle (Co), 10-8
M estradiol (E2), 12 ng/ml EGF (EGF), or 10-8
M estradiol plus 12 ng/ml EGF (E2+EGF).
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Constitutive MEKK1 Activity Does Not Repress ER
Activity
Since growth factor activation of Ras can activate other signaling
pathways in addition to Raf, one question arising from these data is
the role of other growth factor signaling pathways initiated by growth
factor binding or activation of Ras in inducing ligand-independent
activation or down-regulation of ER
. Transient cotransfection of a
constitutively active MEKK1 with either ERE-luciferase or
NON-luciferase indicates that MEKK1 signaling does not repress ER
activity (Fig. 3B
). The activity of both the ERE-luciferase and the
NON-luciferase constructs increased when cotransfected with MEKK1, most
likely a reflection of the two imperfect activator protein 1 or
serum response element sites that lie in the MMTV promoter driving
these two reporter plasmids. These data suggest that the effect of
raf and
MEK on ER
activity and expression is specific to
signaling via MAPK.
We then moved upstream of Raf or MEKK1. MCF-7 cells transiently
transfected with either the ERE- or NON-luciferase vector were treated
with EGF, a ligand whose tyrosine kinase receptor signals through Raf-1
but also activates other pathways such as MEKK1. Again, no
ligand-independent activation of ER
was observed (Fig. 3C
). Instead,
EGF treatment with estrogen resulted in a repression of estrogens
ability to activate ER
. These data indicate that EGF signaling in
MCF-7 cells does not result in ligand-independent activation of
ER
.
Transient Expression of
raf Results in Down-Regulation of ER
Expression
To examine more closely the temporal component of
raf-mediated
loss of ER
expression,
raf was transiently transfected into MCF-7
cells or HCopoolc cells and followed by double-label
immunohistochemistry for both ER
with the 1D5 anti-ER MAb and the
transfected
raf. Those cells that stain for the transfected
raf
no longer display nuclear immunoreactivity for ER
(Fig. 4
). We have repeated this using
MEK
and obtain the same results. Interestingly, in all cases, we always
observe that some cells immediately adjacent to the transfected cells
also exhibit decreased nuclear staining, suggesting that perhaps over
the 2448 h post transfection, cell-cell signaling is occurring. To
quantitate the effect of this transient expression of
raf on ER
expression, 11 low-power fields with a total of 4,459 cells were
assessed.
raf expression was observed in 369 of these cells. Of
these
raf-expressing cells, 94.1% displayed no nuclear
immunoreactivity for ER
. Only 3% of the non-
raf-expressing cells
displayed this lack of ER
immunoreactivity, indicating a specific
effect of
raf expression on ER
expression.
Abrogation of Raf Signaling by a MEK Inhibitor Results in
Reexpression of ER
To assess the effect of transient shut down of
raf signaling on
ER
expression in Raf 14c cells, we used the MEK-specific
inhibitor, PD 098059, to suppress the activity of this Raf effector as
monitored by expression of activated, phosphorylated MAPK (Fig. 5A
). PD 098059-treated and untreated Raf
14c cells were then analyzed for ER
expression by
immunohistochemistry with the H222 anti-ER MAb. Untreated cells
are ER
negative. However, ER
is reexpressed in approximately 60%
of the cells after 12 h of continuous MEK inhibition (Fig. 5B
).
The staining intensity indicates that ER
is expressed at a
relatively high level in some cells, but not every cell returns to
ER
positivity during this time frame, suggesting a cell cycle
component to the ER
reexpression mediated by MEK inhibition in this
cell line, which has a doubling time of approximately 22 h. We
have also assessed the time course in which ER
is reexpressed in Raf
14c cells in response to MEK inhibition, this time using the
newer, more potent MEK inhibitor U0126. Raf 14c cells were
treated with U0126 for 1 h, and treated cells were analyzed for
ER
expression at various times post U0126 treatment. A modest
increase in ER
levels is observed at 1 or 3 h post U0126
treatment, but a significant increase in ER
expression to levels
comparable to that of MCF-7 cells is observed at 7 h post U0126
treatment. Thus, the abrogation of Raf signaling via MEK and MAPK is
sufficient to allow for reexpression of ER
.
Overexpression and Hyperactivation of Growth Factor Signaling Does
Not Result in Ligand-Independent Activation of ER
, but Rather
MAPK-Induced Loss of ER
Expression
MCF-7 cells express relatively low levels of growth factor
receptors such as EGFR or c-erbB-2, and the above studies were
performed where only one arm of signaling was hyperactivated
(i.e.
raf or
MEKK1). Therefore, we were interested in
determining whether overexpression of these receptors and the
subsequent concomitant hyperactivation of growth factor signaling
pathways that is found in some breast tumors would induce either
ligand-independent activation of ER
or, alternatively,
down-regulation of ER
protein and activity. To do this, we have used
two other MCF-7 stably transfected cell lines, MCE5 cells (stably
overexpressing EGFR) and MB3 cells (a clone stably overexpressing
c-erbB-2 with high levels of constitutive autophosphorylation)
(29, 30). Like our
rafc clones,
overexpression of EGFR and c-erbB-2 facilitates the continuous growth
of both cell lines in the absence of estrogen. The original ER assays
performed with the initial isolates of these cell lines indicated that
the EGFR overexpressing cell line (MCE5) displayed a modest reduction
in ER
levels compared with control transfected cells adapted for
growth in estrogen-free conditions (29), while the
c-erbB-2 overexpressing cell line with high levels of constitutive
autophosphorylation (MB3) displayed a significant reduction in ER
levels compared both to control transfected cells and to two c-erbB-2
overexpressing clones not exhibiting high constitutive activity
(30). MCE5s and MB3s were transiently transfected with the
ERE- or NON-luciferase vectors and analyzed for luciferase activity in
the absence or presence of estrogen. Neither of these cell lines
exhibits significant activation of ERE-luciferase in the absence of
estrogen (Fig. 6A
) indicating there is no
ligand-independent activation. The estrogen-induced activation in MB3 s
was blunted, corresponding to the significantly lower levels of ER
expressed by these cells. Since there is little EGF in the media for
these cells, it was possible that the majority of the overexpressed
EGFR in MCE5 cells was not activated. To address this, the same
transient transfections were repeated with MCE5 cells in the absence
and presence of EGF. Again, no ligand-independent activation is seen
with EGF treatment (Fig. 6B
), and furthermore, the addition of EGF
actually repressed the estrogen-induced activation as we had seen with
raf and
MEK.

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Figure 6. Growth Factor Signaling Represses ER
A, MCE5 and MB3 cells were transfected with 2.5 µg of either ERE-luc
or NON-luc. Post-transfection treatments were for 48 h in media
plus vehicle (Co), 10-8 M estradiol (E2),
10-7 M 4-OH tamoxifen (Tam), or
10-7 M ICI 182,780 (ICI). B, MCE5 cells were
transfected with ERE-luc and treated with vehicle (Co),
10-8 M estradiol (E2), 12 ng/ml EGF (EGF), or
10-8 M estradiol plus 12 ng/ml EGF (E2+EGF).
C, ER levels were determined for cell lines by ligand-binding assay
and are expressed as femtomoles/mg protein. D, Whole-cell lysates (2.5
µg) were electrophoresed in 10% polyacrylamide gels, transferred,
and probed with an antiphospho-MAPK antibody. MCE5 cells were treated
or not with 12 ng/ml EGF for 10 min. E, Whole-cell lysates (150 µg)
from MCE5 cells treated or not with 12 ng/ml EGF for 8 h were
electrophoresed in 10% polyacrylamide gels, transferred, and probed
with H151 anti-ER MAb.
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We were next interested in determining whether there was a correlation
between ER
status and MAPK activity in these cells. We have repeated
the ER
analysis and assessed the phospho-MAPK expression of these
cell lines alongside our Raf 14c cells. Ligand-binding assay
results confirmed the earlier ER
analysis of these cell lines. Raf
14c cells were close to ER
negative, the MB3 cells that
contain the constitutively active c-erbB-2 exhibit significantly
reduced ER
levels, and the EGFR-overexpressing MCE5s display
moderately reduced levels compared with their control-transfected cells
(Fig. 6C
). Analysis of the MAPK activity levels in MB3s and MCE5s
revealed that MB3s had significantly more activity, similar to that
observed with Raf 14c and other ER
-negative breast cancer
cells, than ER
+ control cells or MCE5s (Fig. 6D
). These data
suggested that a threshold level of hyperactivation of downstream
signaling pathways, not mere overexpression, was involved in
down-regulating ER
levels. In support of this, EGF treatment of
MCE5s to activate the EGFR resulted in a significant increase in MAPK
activity, which corresponded to a significant reduction in ER
levels
at 8 h post EGF treatment, from 223 fmol/mg protein to 5 fmol/mg
protein (Fig. 6E
).
Abrogation of MAPK Activity in MCE5 and MB3 Cells Reverses the
ER
Down-Regulation
To confirm that it was high MAPK activity that was responsible for
the negative effects on ER
expression and activity in the EGFR and
constitutively active c-erbB-2-overexpressing MCF-7s, MAPK activity was
abrogated using dominant negative ERK1 and ERK 2 constructs
(50). MCE5s were again transiently cotransfected with
ERE-luciferase or NON-luciferase and with dominant negative (dn) ERK1
and ERK2 before hormonal treatments. Cotransfection of the dnERKs had
no effect on ERE-luciferase activity in the absence of estrogen, but
did result in significant enhancement of estrogen-induced activity,
suggesting that even the modest reduction in ER
expression in MCE5s
is a result of increased MAPK activity (Fig. 7A
). We did not obtain a significant EGF
repression of estrogen- induced activity, probably due to the fact
that with continued culture, these cells produce TGF
(D. Miller,
unpublished data). In support of this, the MCE5 cells used in this
experiment exhibited significantly higher levels of MAPK activity in
the absence of EGF, and addition of EGF did not result in a further
significant induction of this MAPK activity (data not shown). Use of
the MEK inhibitor U0126 in these experiments gave similar results (data
not shown). To assess the effects of MAPK abrogation on ER
levels
directly, ligand-binding assays were performed with EGF-treated and
untreated MCE5 cells transiently transfected with control vector or
dnERK1 and dnERK2 constructs. Again, abrogation of MAPK activity
resulted in increased ER
expression in the presence or absence of
EGF (Fig. 7B
). Most impressive was the increase in ER
levels in
cells transiently transfected with the dnERKs, considering that only a
fraction of the cells are taking up the vectors. Similar data was also
obtained with the constitutively active c-erbB2-overexpressing MB3
cells (Fig. 7
, C and D). These results indicate that even with
activation of several signal transduction cascades, the resultant high
MAPK activity in these cells results in loss of ER
expression
and suggests that ER
-negative, EGFR, or c-erbB-2-overexpressing
breast cancer cells are ER
negative due to the direct
down-regulation of ER
induced by the high MAPK activity.

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Figure 7. Abrogation of EGFR- or c-erbB-2-Induced MAPK
Activity Restores ER Expression and Activity
MCE5 (panel A) and MB3 (panel C) cells were cotransfected with 2.5 µg
ERE-luc or NON-luc and 2.5 µg of control vector pCHC6 or dnERKs (1.25
µg of both dnERK1 and dnERK2 were used) as indicated.
Post-transfection treatments were as indicated. ER levels in
treated, untreated and transfected MCE5 (panel B) or MB3 (panel D)
cells were determined by ligand binding assay.
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Abrogation of MAPK Activity in Conjunction with Demethylation of
the ER
Promoter Results in Reexpression of ER
in MDA-231
Cells
To extend further our hypothesis of hyperactivation of MAPK being
responsible for down-regulation of ER
expression, we were interested
in whether the ER
-negative phenotype of established ER
-negative
breast cancer cells lines could be reversed by abrogation of MAPK
activity. However, most ER
-negative breast cancer cell lines, such
as MDA-MB-231 cells, MCF-7/Adr cells, MDA-MB-468, and SkBr3 cells,
exhibit site- specific methylation of CpG islands in the ER
promoter, thus requiring treatment with a demethylating agent such as
5-aza-cytidine to obtain reexpression of ER
in these cells
(51, 52). This reexpressed ER
should then be
down-regulated again due to the high levels of MAPK activity
present in these cells while inhibition of this high MAPK activity
would allow for the continued reexpression of ER
. MDA-231 cells
treated for 3 d with 5-aza-cytidine to allow for the reexpression
of ER were then treated with the MEK inhibitor U0126 for varying times.
As demonstrated in Fig. 8
, a 3-d
treatment with 5-aza-cytidine results in ER
being expressed. Further
treatment of these cells with U0126 results in an increased expression
of ER
at 8 h (a 1.5-fold increase), with expression then
decreasing over the next 416 h back to pre-U0126 treatment
levels. This 8-h time frame for increased ER
expression is highly
consistent with the U0126 and/or dnERK data in our Raf, EGFR, and
c-erbB-2 MCF-7 cell models. Our models, however, would have predicted
that because of the high MAPK activity in MDA-231 cells, demethylation
of the ER
promoter alone should not have resulted in detectable
ER
expression at the 3-d time point of 5-aza-cytidine treatment;
i.e. concomitant abrogation of MAPK activity would be
necessary to detect ER
expression. Interestingly, analyses of
the MAPK activity from these MDA-231 cells demonstrated that there is a
significant decrease in the MAPK activity of MDA-231 treated with
5-aza-cytidine alone, a 9.4-fold decrease compared with untreated
cells. Treatment with U0126 results in further reduction in MAPK
activity of 4.4-fold at 4 h with activity returning to pre-U0126
levels at the longer time points. The inhibition of MAPK activity over
the first 4 h of U0126 treatment is sufficient to detect an
increase in ER
expression at 8 h. The subsequent return of the
MAPK activity to pre-U0126-treated levels then results in ER
expression reduced to that of pre-U0126-treated levels at later time
points. Both the U0126-induced increase in ER
expression and
decrease in MAPK activity were specific since neither ER
expression
nor MAPK activity varied over the time course in the
5-aza-cytidine-treated cells incubated with DMSO as the vehicle control
(data not shown). These data indicate that abrogation of MAPK in an
established ER
-negative breast cancer cell line with methylation of
the ER
promoter correlates with reexpression of ER
.
 |
DISCUSSION
|
---|
Raf is a downstream effector of growth factor receptors,
such as EGFR or c-erbB-2, the overexpression of which is either
inversely correlated with ER
expression or directly correlated with
decreased sensitivity to antiestrogens (8, 12, 13, 53). We
have used the expression of constitutively activated Raf as a model of
up-regulated growth factor signaling to study the interaction of the
growth factor and ER- mediated signaling systems in the progression of
breast cancer from estrogen-dependent growth to estrogen-independent
growth. We have previously found that expression of a constitutively
active form of the Raf-1 kinase in MCF-7 ER
+, estrogen-dependent
human breast cancer cells results in two seemingly linked phenomena.
First, it induced estrogen-independent growth of these cells, both
anchorage dependent and anchorage independent (38).
Second, the expression of
raf was not tolerated by the cells when
grown in the presence of estrogen. In dissecting the mechanisms
underlying both of these phenomena, we were interested in determining
the direct effect of Raf signaling on ER
that imparts estrogen
independence.
raf transfected clones growing continuously in the absence of
estrogen (
rafc) were characterized for their growth
capabilities in the presence and absence of estrogen or antiestrogens.
Interestingly, in addition to the fact that these cells grew in the
absence of estrogen, they no longer responded positively to the
addition of estrogen nor did they respond to antiestrogens as the
control cells did. These data raised the possibility that the
rafc cells no longer expressed functional ER. Analysis of
the cells for ER
expression at the levels of steroid binding,
overall protein by Western blotting and immunohistochemistry, and RNA
all demonstrated significant reduction in ER
expression (Fig. 1
).
Furthermore, double-label immunohistochemistry demonstrated that high
expression of both ER
and stably transfected
raf did not occur in
the same cell (data not shown). These data were quite exciting because
they recapitulate results of published studies in which double-label
immunohistochemical detection of ER
and EGFR in breast tumor
specimens from ER
+/EGFR+ tumors revealed that individual tumor cells
express high levels of only ER
or EGFR, but not both (22, 23). The EGFR+ cells in these tumors are also associated with a
higher growth rate than the ER
+ cells (25, 26),
suggesting that active signaling is occurring perhaps through MAPK.
Stable transfections of growth factor signaling components into ER
+
MCF-7 breast cancer cells, i.e. EGFR, erbB-2, heregulin
(HRG), or Ras, lead to significantly decreased levels of ER
in most
cases, in addition to estrogen-independent growth
(27, 28, 29, 30, 31, 32). For example, decreased ER
resulting from
overexpression of c-erbB-2 in MCF-7 cells has been described by two
different groups. In one case, an approximate 50% decrease in ER
levels was observed (31). In the second study, an
approximate 4-fold reduction in ER
was found in one clone where the
transfected erbB-2 exhibited a high level of constitutive
autophosphorylation (30). In this latter study, clones not
exhibiting constitutive activation of the transfected c-erbB-2
displayed no alteration in ER
expression, suggesting that
up-regulated signaling, not merely overexpression, was linked to ER
down-regulation. In support of this, ER
levels in fibroblast growth
factor-1-overexpressing MCF-7 cells were unaffected (49)
and displayed a much more modest increase in MAPK activity (data not
shown) while EGF treatment of EGFR-overexpressing cells resulted in
both significantly increased MAPK activity and decreased ER
expression (Fig. 6
, D and E). Thus, our system of chronic activation of
a specific downstream growth signaling pathway resulting in both
estrogen-independent growth and loss of ER
implicates the activation
of MAPK as the mediator of signal transduction-induced down-regulation
of ER
. This is further supported by the reversal of ER
down-regulation that occurs upon abrogation of MAPK activity either by
MEK inhibition or dominant negative ERKs in our different cell line
models (Figs. 5
and 7
).
This loss of ER
expression could occur by one of three mechanisms.
First,
raf induction of estrogen-independent growth could be
occurring through an ER
-independent mechanism, and the loss of ER
in our cells could merely be due to the fact that since the cells no
longer need ER
for growth, they have down-regulated its expression.
However, this would be in contrast to cells naturally selected for
estrogen independence by long-term growth and selection in the absence
of estrogen where these cells remain responsive to estrogen and/or
antiestrogens and actually up-regulate expression of ER
(Refs.33, 34, 35, 54 and our own control cells).
Second, if Raf activation of the MAPK kinase cascade activates ER
in
the absence of estrogen (ligand-independent activation), then it is
likely that the constitutive activation of MAPK that occurs in our
cells would result in the constitutive activation of ER
. Dopamine,
which activates adenylate cyclase, has been shown to activate ER
in
the absence of estrogen (55), also termed
ligand-independent activation, and is able to potentiate the partial
agonist activity of tamoxifen (56). In the rat uterus,
where ER
and EGFR are expressed in the same cell, EGF can result in
the same effects on uterine tract growth and differentiation as
estrogen (57), and this effect can be decreased by
pretreatment with the pure antiestrogen ICI 164,384 (58),
suggesting that these effects are ER
mediated (57). The
uteri of ER
knockout mice do not exhibit EGF-induced DNA synthesis
or induction of PR expression (59), indicating that these
EGF effects are mediated through this form of ER
. EGF or TGF
can
also activate ER
transcriptional activity in endometrial
adenocarcinoma cells and ovarian adenocarcinoma cells
(60). MAPK, a downstream effector of growth factor
signaling, has been shown to phosphorylate and activate ER
in
in vitro cotransfection experiments of MAPK and ER
into
HeLa or Cos cells. EGF treatment of ER-negative cells transiently
transfected with ER
also activates ER
(46). Because
an end result of ER
activation by estrogen is its eventual
down-regulation, a constitutive and continual activation of ER
could
ultimately result in the chronic and total down-regulation of
ER
.
Experiments performed to examine ligand-independent activation in
ER
+ breast cancer cells have given conflicting results. Agents that
increase intracellular cAMP, such as IGF-I, cause increased rat uterine
and MCF-7 breast cancer cell PR levels (61, 62), as well
as activation of transiently transfected reporter constructs containing
EREs (63, 64), in a manner similar to that of estrogen. In
addition to increasing transcriptional activity of ER
, ER
phosphorylation was increased in these studies (63). This
type of interaction between IGF-I and ER
signaling corresponds to
the physiological relationship between these two in MCF-7 cells in
which IGF-I synergizes with estrogen in inducing growth
(65, 66, 67, 68, 69). The transfection of HRG or erbB-2 into MCF-7
cells has also been shown to result in ligand-independent activation of
ER
in one study (31). However, other HRG transfection
studies resulted in down-regulation of ER
expression without
transcriptional activation (32, 70, 71). In addition,
treatment of MCF-7 cells with EGF results in down-regulation of ER
without induction of PR (72), as does treatment with a
novel 52-kDa form of HRG (73). These data suggest that
signaling via these pathways in ER
+ breast cancer cells can induce
the down-regulation of ER
expression without ligand-independent
activation, which corresponds to the clinical observation that ER
expression is inversely correlated with EGFR or erbB-2
overexpression.
Using ERE-luciferase reporter constructs and analysis of the basal
level of transcription of estrogen-induced genes, we observe no
ligand-independent activation of ER
by
raf. Similarly, when we
move further downstream of Raf and analyze the effect of a
constitutively active MEK or upstream and analyze the effects of EGF
treatment, we also find no increase in ER
activity in ERE-luciferase
reporter assays. Finally, in two cell line models exhibiting
up-regulated growth factor signaling due to activation of overexpressed
EGFR or to overexpression of constitutively active c-erbB-2, we do not
observe any ligand-independent activation of ER
, but rather
decreased ER
expression and activity. Thus, a third possibility is
supported by our data, which indicate that the Ras/Raf/MEK/MAPK
signaling pathway can induce the down-regulation of ER
expression
without ligand-independent activation.
An important aspect of this loss of ER
expression involves its
reversibility. It has been demonstrated that most ER
-negative breast
cancer cell lines such as MDA-MB-231 cells, MCF-7/Adr cells, and
MDA-MB-468 cells exhibit site-specific methylation of CpG islands in
the ER
promoter, and that reversion of this ER
negativity
requires treatment with a demethylating agent such as 5-aza-cytidine
(51, 52). In addition, about 25% of ER
-negative breast
tumors were found to contain methylated ER
(74). More
recent data using a highly sensitive methylation-specific PCR assay
determined that 100% of these same ER
-negative tumors displayed
some degree of methylation; however, a number of ER
-positive tumors
also showed similar degrees of methylation (75).
Methylation, by modifying the DNA structure assisting in the
recruitment of histone deacetylases, ensures that the gene is in an
inactive conformation. If methylation occurs as a step subsequent to
another mechanism of ER
repression, then one would predict that some
ER
- negative breast tumors at an earlier point in a progression
pathway do not express ER
because one of these other mechanisms is
still operative. In such tumors, it then may be possible to reverse
this phenotype and restore antiestrogen sensitivity.
Therefore, we were extremely interested in determining whether the
Raf-induced loss of ER
expression observed in our transfectants
could be reversed. It was first determined that the cells did not
contain methylated ER
(76). The resultant shut off of
raf signaling through MEK and MAPK via treatment with PD 098059 or
U0126 allowed for the reexpression of ER
, indicating that the
down-regulation of ER
in our system is MEK-dependent (Fig. 5
). This
time frame is not inconsistent with a purely transcriptional mechanism.
Alternatively, it may be that accelerated protein degradation is also
involved, thus accounting for the rapid reexpression or that the return
of ER
expression is cell cycle dependent.
It is important to note here that, unlike other transfections of
constitutively active Raf, namely an estrogen-regulated
Raf:ER
ligand-binding domain version, where aberrant activation of other
signaling pathways such as the c-Jun N-terminal kinase (JNK) pathway
via establishment of autocrine growth factor loops has been
demonstrated (77, 78, 79, 80), our
raf transfectants do not
exhibit increased JNK, NF
B, or MEKK1 activity (our
unpublished data), indicating that the effects on ER
expression in
these transfectants is through Raf/MEK/MAPK. This conclusion is further
supported by results of transient expression experiments with
raf
and
MEK vectors in which effects in the short term are similar to
those seen with the stable transfectants. Finally, the experiments
using dnERKs to reverse the ER
down-regulation in Raf 14c cells and
in the EGFR and c-erbB-2-overexpressing cells (Fig. 7
) clearly
establish MAPK as the signal transduction mediator of growth
factor-induced down-regulation of ER
. Furthermore, even in an
established ER
-negative breast cancer cell line with methylation of
the ER
promoter, the demethylation-induced increase in ER
expression correlated with significant reduction in MAPK activity, and
further specific inhibition of MAPK resulted in further increases in
ER
expression (Fig. 8
).
These data raise the possibility that potentially reversible mechanisms
can be responsible for the lack of ER
expression in some breast
cancer cells, and thus there may exist two subpopulations of
ER
-negative tumors: those in which the ER
gene is methylated, and
thus permanently off, and those in which the lack of ER
expression
can be reversed by blocking the mechanism responsible for its
down-regulation.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
HCopool cells (pooled, hygromycin-resistant, control parental
vector transfectants) were maintained in IMEM (Life Technologies, Inc., Gaithersburg, MD) with phenol red supplemented with 10%
FBS Intergen, Purchase, NY). For growth in the absence of
estrogen, media were switched to PRF-IMEM supplemented with 10% CCS
(Life Technologies, Inc.). For certain experiments, cells
growing in FBS-containing media were quick-stripped of estrogen by
repeated rinsing of cells in PRF-IMEM and growth in PRF-IMEM + 10% CCS
(three times per day for 2 d). All other cells lines
(HCopoolcHCopool cells long-term adapted for growth in the
absence of estrogen, MCNC4a G418-resistant clone of control
vector-transfected cells long-term adapted for growth in the absence of
estrogen, and Raf 14c, Raf 27c, Raf
30c, and Raf 35cdifferent
raf transfected
clones growing continuously in the absence of estrogen) were maintained
in PRF-IMEM supplemented with 10% CCS. Hormone treatments, when
performed, were with 17ß-estradiol (Sigma, St. Louis,
MO) at 10-8 M, the partial
estrogen antagonist 4-hydroxy-tamoxifen at 10-7
M, or the pure antiestrogen, ICI 182,780
(obtained from Alan Wakeling, AstraZeneca Pharmaceuticals,
Macclesfield, UK) at 10-7 M. Cells were
plated in 75-cm2 T-flasks (Costar
Cambridge, MA) and grown in a forced-air humidified incubator at an
atmosphere of 5% CO2 and 37 C.
Gel Electrophoresis and Western Blotting
Whole cell lysates for ER
detection were prepared from cells
grown to approximately 80% confluence. Cells were harvested by
incubation in trypsin-EDTA (Life Technologies, Inc.),
washed once in PRF-IMEM plus 10% CCS, once in PRF-IMEM, and then in
TEG buffer (10 mM Tris-OH, pH 7.4, 1 mM EDTA,
10% glycerol). Cell pellets were homogenized at 0 C in TEDG (10
mM Tris-OH, pH 7.4, 1 mM EDTA, 1
mM, 10% glycerol) plus 0.5 M NaCl and a
cocktail of proteolysis inhibitors (as described in Ref.
81 except that leupeptin was at 1 mg/ml). Homogenates were
centrifuged at 105,000 x g at 4 C for 30 min in a
Ti70.1 rotor (Beckman Coulter, Inc., Fullerton, CA) to
yield a whole-cell extract. One hundred fifty micrograms of cellular
lysate were electrophoresed through 10% SDS-polyacrylamide gels with
0.1% SDS included in the gel and running buffers. Rainbow mol wt
markers were from Amersham Pharmacia Biotech (Arlington
Heights, IL). Electrophoresed gels were transferred to 0.45 µm
nitrocellulose (Bio-Blot NC, Costar, Cambridge, MA) for
4 h at 0.4 Amps in phosphate transfer buffer [20
mM sodium phosphate, pH 6.8, 20% methanol,
0.05% SDS (82)], and the blots were blocked in TBST (10
mM Tris, pH 7.5, 150 mM
NaCl, and 0.1% Tween-20) with 3% BSA and an additional 0.15%
Tween-20 added for 60 min at room temperature. Blots were then
incubated with H151, an anti-ER
MAb (made against a hinge-region
peptide, kindly provided by Dean Edwards, University of Colorado Health
Sciences Center, Denver, CO), diluted to 1 µg/ml in TBST plus 1% BSA
overnight at 4 C. After washing the blots 3 x 5 min with TBST,
they were incubated with goat antimouse antiserum linked to horseradish
peroxidase (Amersham Pharmacia Biotech) diluted 1:2,000 in
TBST/1% BSA for 60 min at room temperature. The blots were again
washed in TBST, once for 20 min and then 3 x 5 min, and the bound
secondary antibody was visualized using enhanced chemiluminescence
(Amersham Pharmacia Biotech) according to the
manufacturers instructions.
For detection of phosphorylated MAPK, cell lysates were prepared as
described previously (38). Briefly, cells were rinsed in
PBS and then lysed in a modified gold lysis buffer [20 mM
Tris, pH 7.9, 137 mM NaCl, 5 mM EDTA, 10%
glycerol, 1% Triton X-100, 1 mM EGTA, 1 mM
Pefabloc A (instead of phenylmethylsulfonyl fluoride), 1
mM aprotinin, 1 mM leupeptin, 1
µM pepstatin A, 1 mM
Na3VO4, 1 mM
sodium pyrophosphate, 10 mM sodium fluoride] on ice,
scraped into a microfuge tube, and centrifuged at 12,000 x
g to pellet nuclear debris. Supernatants were analyzed for
protein content using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL) and stored at -20 C. Fifty micrograms of
cellular lysate were electrophoresed through 10% SDS-polyacrylamide
gels as above. Electrophoresed gels were transferred for 2 h at
0.4 Amps in Towbins buffer (20 mM Tris, 150
mM glycine, pH 8.3, 20% methanol, 0.1% SDS),
and the blots were blocked as above. Blots were then incubated with an
antiphospho-MAPK polyclonal antibody that specifically recognizes only
the phosphorylated form of MAPK or with an anti-MAPK polyclonal
antibody that recognizes all forms of MAPK (New England Biolabs, Inc., Beverly, MA) diluted 1:1,000 in TBST plus 1% BSA
overnight at 4 C. Blots were processed as above except the secondary
antibody was donkey antirabbit antiserum linked to horseradish
peroxidase (Amersham Pharmacia Biotech) and diluted
1:4,000 in TBST/1% BSA for 60 min at room temperature.
ER Steroid-Binding Assay
Whole-cell extracts were prepared as described above for ER
Western blotting. Extracts were incubated with 10 nM
[3H]-17ß-estradiol ± a 100-fold excess
of unlabeled estradiol for 16 h at 4 C. Binding was assayed using
the dextran-coated charcoal assay as described previously
(83). In short, the dextran-coated charcoal was added to
adsorb free hormone and was then pelleted by centrifugation. Aliquots
of supernatant were removed and counted in 10 ml of liquid
scintillation fluid in a Beckman Coulter, Inc. liquid
scintillation counter. Values are expressed as femtomoles/mg protein.
Steroid-binding assays have been performed on all cell lines a minimum
of three times, with comparable results shown as a representative
experiment.
Immunohistochemistry Assay
Cells were plated in two-well chamber slides (Falcon, Becton
Dickinson, Franklin Lakes, NJ), allowed to attach, and grown as
a monolayer. For ER
expression, cells in FBS were quick stripped of
estrogen. Cells were fixed by incubation for 10 min at room temperature
with 10% formaldehyde-PBS, followed by ice-cold acetone for 15 sec.
Fixed cells were then blocked by incubation for 60 min at room
temperature in PBS with 1% BSA. For ER
detection, cells required
permeabilization by incubation in PBS with 0.1% Triton X-100 for 5 min
at room temperature. Primary antibodies were incubated overnight at
room temperature in a humidified chamber and were at 2.5 µg/ml for
anti-ER
(H222 kindly provided by Geoffrey Greene) or at 1:400
dilution (1D5 from Zymed Laboratories, Inc., South San
Francisco, CA), and 0.5 µg/ml for anti-Raf (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted in PBS/1% BSA.
Both ER
antibodies gave comparable immunostaining results. After
three PBS washes, secondary antibody incubations were for 60 min at
room temperature and were a 1:200 dilution of biotinylated antirat (for
ER
) and a 1:300 dilution of horseradish peroxidase-linked antirabbit
(for
raf) in PBS/1% BSA. Detection of ER
required a further
incubation of 30 min at room temperature with streptavidin-alkaline
phosphatase (AP) and then visualization with Vector Red (to give a red
color). Detection of
raf required just visualization with Vector SG
(to give a blue-gray color, Vector Laboratories, Inc.).
For double-label immunohistochemistry, both primary antibodies
were incubated together, followed by both secondaries together,
visualization of
raf, and then streptavidin-AP and visualization of
ER
. Stained cells were then dehydrated through a graded series of
ethanol, followed by xylene, and mounted in Permount. All incubations
were followed by three washes of 30 sec each, and no counterstain was
used.
RNA Extraction, Northern Analysis, and RNase Protection
Assays
RNA was extracted using a one-step acid-guanidinium method as
described in Ref. 84 . For Northern analysis, 10 µg of
total RNA were electrophoresed through a 1% agarose/formaldehyde gel
as described previously (29). After capillary transfer to
nitrocellulose, the membrane was baked at 80 C for 2 h. The blot
was first probed for Cathepsin D. The Cathepsin D riboprobe was
transcribed from a 319-bp KpnI- EcoRI fragment
consisting of nucleotides 90 to 409. After cloning the fragment into
pBluescriptKS+ and linearizing with XbaI, the fragment was
transcribed with T3 polymerase. After probing for Cathepsin D, the blot
was stripped and reprobed for expression of pS2 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as a loading
control. The pS2 antisense riboprobe was transcribed from a 305-bp
PstI fragment, inserted into pBluescriptKS+, linearized with
HindIII, and transcribed with T7 polymerase. Construction
and synthesis of GAPDH antisense riboprobe has been previously
described (29). Prehybridization, hybridization, and
membrane stripping were carried out as previously described
(29). Briefly, the blot was prehybridized in 50%
formamide, 5x SSC, 5x Denhardts, 25 mM
NaHPO4 for 4 h at 42 C. Hybridization was
carried out in prehybridization buffer plus 10% (wt/vol) dextran
sulfate overnight at 42 C. The blot was washed twice at room
temperature in 0.2x SSC-0.1% SDS and twice in 0.1x SSC-0.1% SDS at
65 C for 20 min each. Blots were exposed at -70 C to x-ray film.
RNase Protection assays were performed as previously described
(29). The ER
riboprobe, corresponding to Exon 5, was
transcribed with T7 polymerase from pORB-300 (kindly provided by
MaryBeth Martin, Georgetown University Medical Center) linearized with
EcoRI as previously described in Ref. 85 . It
generates a protected fragment of 305 bases. The PR riboprobe was
transcribed from an AvaI fragment spanning nucleotides
283545 inserted into pGEM4. After linearization with
EcoRI, it was transcribed using SP6 polymerase. It generates
a protected fragment of 262 bases. The GAPDH riboprobe generates a
protected fragment of 104 bases.
Transient Transfections and Luciferase Assays
MCF-7 cells were plated in Falcon six-well plates, allowed to
attach overnight, and were then quick stripped of estrogens by repeated
washing and replacing of the media with PRF-IMEM supplemented
with 10% CCS three times per day for 2 d. At the end of the
second day, cells were transfected by
N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic
acid-buffered saline (BBS), calcium phosphate, 2%
CO2 protocol as described above (86) except
each well was transfected with 2.5 µg of ERE or NON luciferase
plasmid and 2.5 µg of
raf,
MEK, or
MEKK1 plasmid suspended
in CaCl2 and mixed with BBS. The MCE5 and MB3
cells were transfected with 2.5 µg of ERE or NON luciferase. The
luciferase plasmids were either pGLB-MERE or pGLB-MNON
(48), obtained by inserting an altered MMTV promoter where
the endogenous glucocorticoid response element is replaced with a
tandem repeat of a consensus ERE (MERE) or the same sequence with the
ERE palindromes scrambled (MNON) (48)] into the
HindIII site of pGLB basic luciferase plasmid (Promega Corp., Madison, WI) . The
raf plasmid is the same vector we
used for generation of the stable transfectants (38). The
MEK construct was obtained from Natalie Ahn; it is a cytomegalovirus
(CMV)-driven MEK containing two substitutions (Ser-218 and Ser-222 have
been replaced with acidic residues) and a truncation from residues
3251 (a suspected kinase regulation domain) that result in
approximately 400 times the activity of wild-type MEK
(47). The
MEKK1 plasmid is a CMV-driven constitutively
active MEKK1 obtained from Stratagene (La Jolla, CA).
Additional transfections included dominant negative ERK1 and ERK2
constructs (kindly provided by Peter Shaw, Max Planck Institute for
Immunology). These were transfected at 1.25 µg each to give a total
of 2.5 µg, and pCHC6 was again used as the vector control. The cells
were incubated for 1618 h at 2% CO2 and 35 C,
washed two times with PBS, and then incubated for 48 h in media
containing vehicle (0.01% ethanol), 10-8
M estradiol, 10-7
M 4-OH-tamoxifen, 10-7
M ICI 182,780, 12 ng/ml EGF, or a combination of
EGF and estrogen. Cells were assayed for luciferase activity (expressed
as relative light units or RLU) using the Luciferase Assay System of
Promega Corp. according to the manufacturers
instructions. The luciferase values were normalized for protein to
obtain RLU/mg, and the RLU/mg values were adjusted to specific RLU/mg
by subtracting out the value obtained with lysate prepared from
mock-transfected cells. The triplicates were then averaged, and the
values were plotted as specific RLU/mg protein with error bars
depicting the standard error. A set of triplicate wells transfected
with pCMV-luciferase was included in all transfection experiments as a
measure of transfection efficiency. When transfection experiments were
performed with multiple cell lines, the CMV luciferase activity was
used to normalize the data for transfection efficiencies.
Transient Transfection of
raf and Double-Label
Immunohistochemistry
MCF-7 cells were plated in two-well chamber slides, allowed to
attach overnight, and were then quick stripped of estrogens by repeated
washing and replacing of the media with PRF-IMEM supplemented with 10%
CCS 3 times per day for two days. At the end of the second day, cells
were transfected by the BBS-calcium phosphate, 2%
CO2 protocol (86). Briefly, each
well was transfected with 1.0 µg of
raf suspended in
CaCl2 and mixed with BBS. The cells were
incubated for 18 h at 2% CO2 and 35 C, were
washed two times with PBS, and then incubated for 48 h in fresh
media. Cells were then processed for immunohistochemistry.
MEK Inhibition
Inhibition of MEK activity was accomplished using PD 098059 [a
Parke-Davis (Morris Plains, NJ) compound obtained from
New England Biolabs, Inc. (Beverly, MA)]. PD 098059 was
resuspended in DMSO according to the manufacturers instructions. A 1-h
treatment with 50 µM has been shown to effectively
suppress the MEK induction of MAPK activity in response to various
growth factors in other cell systems, but much higher doses are
required to inhibit a strong or sustained activator (as described in
Product Data Sheet from NEB). Since
raf results in high and
sustained MEK activity and because it was necessary to inhibit
raf
signaling for several hours to allow for ER
protein to be
reexpressed, the conditions required for abrogating
raf signaling
for 12 h in Raf 14c cells using PD 098059 were first
determined by analysis of the effects of varying doses and times on the
phosphorylation of MAPK. It was determined that while 100
µM was sufficient to abrogate
raf activation
of MAPK for 1 h, a 200 µM dose was
required to abrogate activation of MAPK in Raf 14c cells for
3 h. After 4 h, some return of phospho-MAPK was still
observed with the 200 µM dose. Therefore, cells
were treated four times consecutively with the 200
µM dose for 3 h to a total of 12 h of
complete MEK inhibition. Cells were then washed in PBS and processed
for immunohistochemistry. For other experiments, a newer and more
effective MEK inhibitor, U0126 (Promega Corp.), was used.
Experiments were performed to determine the appropriate dose and time
of treatment. For all cell lines, a 30-min pretreatment with U0126 at
10 µM was sufficient to abrogate MAPK activity.
In either case, control treatments were vehicle alone (DMSO).
Demethylation of the ER Promoter
MDA-MB-231 cells were plated in PRF-IMEM plus 10% CCS and grown
to approximately 60% confluency. Cells were treated or not with 0.5
µM 5-aza-2'-deoxycytidine (Sigma) to return
ER
expression, as described previously (52). Cells were
then treated with 10 µM UO126 (Promega Corp.) and harvested at 0, 4, 8, 12, and 24 h. In parallel,
cells were treated with DMSO as the vehicle control for U0126. After
harvesting, whole-cell extracts were prepared and analyzed for ER
,
phospho-MAPK, and MAPK expression by gel electrophoresis and Western
blotting as described as above. Densitometric scanning was performed
using Image Quant software.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to acknowledge Dr. Susan Chrysogelos and Dr.
Sandy McLesky for critical review of this manuscript and helpful
discussions.
 |
FOOTNOTES
|
---|
This work was supported in part by NIH Grants CA-71465 (to D.E.A.) and
CA-50376 (to F.G.K.), and by the Lombardi Cancer Center Tissue Culture
and Microscopy Core Facilities (Cancer Center Support Grant
2P30-CA-51008, and SPORE Grant 2P50-CA-58185). D.E.A. was supported by
a Career Development Award from the Department of Defense (USAMRDC,
DAMD1794-J-4172).
1 Equal contributions were made by these authors. 
2 Current addresses: Department of Microbiology and Kaplan Cancer
Center, New York University School of Medicine, New York, New York
10016 [D.L.M.] and Southern Research Institute, Birmingham, Alabama
35255 [F.G.K.]. 
Abbreviations: BBS,
N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic
acid-buffered saline; CCS, charcoal-stripped serum; DMSO,
dimethylsulfoxide; dn, dominant negative; EGF, epidermal growth factor;
EGFR, EGF receptor; ERE, estrogen response element; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; HRG, heregulin; IMEM,
improved MEM; MAb, monoclonal antibody;
MEK, constitutively active
MAPK kinase; MMTV, mouse mammary tumor virus; PRF-IMEM, phenol red-free
IMEM;
raf, constitutive Raf kinase; TBST, 10 mM Tris, pH
7.5, 150 mM NaCl, and 0.1% Tween-20.
Received for publication June 9, 2000.
Accepted for publication April 26, 2001.
 |
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