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Tumor Biology |
Departments of Medicine [H. K., A. E. G. L., J. T. F., C. L. A.], Cell Biology [C. L. A.], and Pathology [J. F. S., J. T. F.], Vanderbilt University School of Medicine, Department of Veteran Affairs Medical Center [C. L. A.] and Vanderbilt-Ingram Cancer Center [J. F. S., C. L. A.], Nashville, Tennessee 37232, and Genentech, Inc. South San Francisco, California 94080 [P. I. P., M. X. S.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Multiple lines of experimental evidence suggest that overexpression of HER2 confers antiestrogen resistance to breast tumor cells. MCF-7 human breast cancer cells transfected with either a full-length HER2 cDNA or with ectopic heregulin-ß1, the HER3/4 ligand that activates HER2, lose sensitivity to tamoxifen or estrogen dependence (6, 7, 8) . Several clinical studies have shown that tumors with high HER2 expression and/or with high circulating levels of the HER2 ectodomain exhibit a statistically lower clinical response rate and/or shorter durations of response after antiestrogen therapy (9, 10, 11, 12, 13, 14, 15, 16, 17) , further suggesting an association between high tumor levels of the proto-oncogene and resistance to endocrine therapy. Two studies, however, failed to show a reduced response to tamoxifen in HER2-overexpressing tumors (18 , 19) . These discrepancies may be explained by the variable number of patients in these studies, different antiestrogen therapies used, variable methods to assess HER2 overexpression, among others.
The mechanisms by which HER2 potentially mediates tamoxifen resistance are unclear. However, HER2 overexpression results in activation of the Ras/MAPK signaling pathway in breast tumor cell lines and carcinomas (4 , 20) . MAPK has been shown to phosphorylate Ser-118 in the ER, leading to ligand-independent ER activation with loss of the inhibitory effect of tamoxifen on ER-mediated transcription (21 , 22) , providing a viable mechanism to explain the association of HER2 with tamoxifen resistance. Finally, an antibody against the ectodomain of HER2 increased the inhibitory effect of tamoxifen against HER2-overexpressing BT-474 breast tumor cells in culture (23) . Therefore, we have studied the effect of blocking HER2 and MAPK in antiestrogen-resistant breast tumor cells. Interruption of these signaling pathways with small molecule inhibitors or dominant-negative mutants of MAPK enhanced the inhibitory effect of tamoxifen on both ER-mediated transcription in vitro and on tumor cell proliferation in vitro and in vivo. Taken together, our results support a role for MAPK in HER2-induced tamoxifen resistance in ER-positive breast tumor cells, thus providing a mechanistic target for future research and treatment approaches.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Colony-forming Assay.
Growth effects of the different inhibitors were tested in an
anchorage-independent soft agarose colony-forming assay. Tumor cells
were plated at a density of 3 x 104
cells/35-mm dish in IMEM, 10% FCS, 0.8%
agarose, 10 mM HEPES in the absence or presence of 4-OH
tamoxifen, U0126, and AG1478, either alone or in combination. Dishes
were incubated in a humidified CO2 incubator at
37°C and colonies measuring 
70 µm in diameter were counted after
7 days using an OMNICON 3800 Tumor Colony Analyzer (Biologicals,
Gainesville, VA).
Plasmids and Luciferase ER Reporter Assays.
The pGLB-MERE and pGLB-MNON plasmids (24)
were provided by
D. El-Ashry (Georgetown University, Washington, DC). Each
plasmid contains either a double consensus ERE (MERE) or the same
sequence with the ERE palindromes scrambled (MNON) into the
HindIII site of pGLB (Promega Corp., Madison, WI). The
dominant-negative MEK-1 (pREP4-K97A MEK1) and dominant-negative MEK-2
(pREP4-K101A MEK2) plasmids (25)
were provided by J. T.
Holt (Vanderbilt University, Nashville, TN). MCF-7/HER2–18 and
MCF-7/neo cells in 24-well plates were transiently transfected with
0.25 µg/well pGLB-MERE or pGLB-MNON using 2.5 µl/well Lipofectamine
reagent (Life Technologies, Inc.) in phenol red-free Opti-MEM I reduced
serum medium (Life Technologies, Inc.) for 8 h. In some cases,
0.25 µg/well pREP4-K97A MEK1, pREP4-K101A MEK2, or pREP4 plasmids
(Invitrogen, Carlsbad, CA) were cotransfected with the ERE-containing
plasmids. After an additional 24-h incubation in phenol red-free IMEM
supplemented with 10% charcoal-stripped FCS (Cocalico Biologicals,
Reamstown, PA), each well was replenished with fresh medium
containing 1 nM 17ß-estradiol with or without 1
µM 4-OH tamoxifen in the presence or absence of
kinase inhibitors for 16 h. Firefly luciferase and Renilla
reniformis luciferase activities in cell lysates were determined
using the dual-luciferase reporter assay system (Promega) according to
the manufacturer’s instructions in a Monolight 2010 luminometer
(Analytical Luminescence Laboratory, San Diego, CA). Firefly luciferase
activity was normalized to R. reniformis luciferase activity
and expressed as relative luciferase units.
Immunoprecipitation and Immunoblot Analysis.
After washes with ice-cold PBS, cells were lysed in EBC buffer [50
mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% NP40,
100 mM NaF, 200 µM
Na3VO4, 1 mM
PMSF, 1 µg/ml pepstatin A, 2 µg/ml aprotinin, and 0.5 µg/ml
leupeptin] for 20 min at 4°C. For analysis of ER and N-CoR, cells
were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0),
5 mM EDTA, 1% NP40, 0.2% Sarcosyl, 0.4 M
NaCl, 20 mM NaF, 100 µM
Na3VO4, 10 mM
sodium molybdate, 1 mM PMSF, 1 µg/ml pepstatin A, 2
µg/ml aprotinin, and 0.5 µg/ml leupeptin as described previously
(26)
. Cell lysates were cleared by centrifugation (14,000
rpm for 10 min at 4°C), and the protein content in the supernatants
was determined by the BCA method (Pierce, Rockford, IL). Equal amounts
of total protein were resolved by SDS-PAGE and then subjected to
immunoblot analysis using polyclonal antibodies against erbB-2
(NeoMarkers, Freemont, CA), phospho-MAPK (Promega), total MAPK (New
England BioLabs, Beverly MA), or N-CoR (Santa Cruz Biotechnology, Santa
Cruz, CA), and an ER
monoclonal antibody (Novocastra laboratories,
Burlingame, CA). In some cases, cell lysates were precipitated at 4°C
with the erbB-2 antibody (10 µg/mg protein) and protein A-Sepharose
CL-4B (Sigma). Immune complexes were then subjected to immunoblot
analysis, as described, for HER2 or phosphotyrosine (P-Tyr; monoclonal
antibody, Upstate Biotechnology, Lake Placid, NY). To detect an
ER/N-CoR association, cell lysates were first precipitated with the
N-CoR antibody (2.0 µg/mg protein) and protein G-Sepharose (Sigma),
followed by immunoblot analysis for N-CoR and ER.
Chemical Cross-Linking.
Chemical cross-linking in intact cells was performed as described
previously (27)
. In brief, after a 1-h treatment with
1–10 µM AG1478 at 37°C, MCF-7/HER2–18 cells were
washed with PBS and incubated in PBS containing 1 mM of the
nonpermeable cross-linker bis(sulfosuccinimidyl) suberate (Pierce) for
20 min at 4°C. The reaction was terminated by adding 250
mM glycine for 5 min while rocking. Cells were then lysed
in EBC buffer, and equivalent amounts of protein were resolved by
5–7% gradient SDS-PAGE, followed by immunoblot analyses for HER2 and
P-Tyr.
In Vitro MEK Activity Assay.
MEK activity was assayed in vitro as described by Favata
et al. (28)
. MEK-1 was precipitated overnight
at 4°C from MCF-7/HER2–18 cell lysates (EBC buffer) with a MEK-1
monoclonal antibody (2.5 µg/mg protein; Transduction Laboratories,
San Diego, CA) and protein A-Sepharose. The immune complexes were
washed three times with EBC buffer and resuspended in 40 µl of kinase
assay buffer [20 mM HEPES (pH 7.0), 5
mM 2-mercaptoethanol, 10 mM
MgCl2, and 0.1 mg/ml BSA] containing 5 µg of
GST-(K71A)ERK-1 (Upstate Biotechnology, eluted from agarose beads with
10 mM glutathione), 10 µM
ATP, and 10 µCi of [
-32P]ATP (specific
activity, 3000 Ci/mmol; Amersham, Piscataway, NJ) at 25°C for 30 min.
Kinase reactions were conducted in the presence or absence of exogenous
U0126 or AG1478 and terminated by adding Laemmli sample buffer.
Phosphorylated ERK-1 species were resolved by SDS-PAGE and visualized
by autoradiography.
Real-Time Quantitative PCR.
Gene expression was quantified using real-time quantitative PCR
or TaqMan technique as described previously (29, 30, 31)
.
Poly(A) RNAs from MCF-7, MCF-7/neo, and MCF-7/HER2–18 were prepared by
Oligotex mRNA kit (Qiagen, Valencia, CA). The sequences of the
primer/probe sets used for each analysis are: EGFR: F,
5'-TTCCTGTGGATCCAGAGGA-3'; R, 5'-AGCGTAATCCCAAGGATGT-3'; and P,
5'-FAM-AGGACGGACCTCCATGCCTTTGAGAA-TAMRA-p-3'; HER2: F,
5'-TCTGGACGTGCCAGTGTGAA-3'; R,
5'-TGCTCCCTGAGGACACATCA-3'; and P,
5'-FAM- CAGAAGGCCAAGTCCGCAGAAGCC-TAMRA-p-3'; HER3: F,
5'-TTCTCTACTCTACCATTGCCCAAC-3'; R, 5'-CACCACTATCTCAGCATCTCGGTC-3'; and
P, 5'-FAM-ACACCAACTCCAGCCACGCTCTGC-TAMRA-p-3'; HER4: F,
5'-GAGATAACCAGCATTGAGCACAAC-3'; R, 5'-AGAGGCAGGTAACGAAACTGATTA-3'; and
P, 5'-FAM-CCTCTCCTTCCTGCGGTCTGTTCGA-TAMRA-p-3', where F and R are the
forward and reverse primers, respectively, and P is the fluorescent
labeled probe. Ribosomal protein 19 (RPL19) was used as a
housekeeping gene. Primer/probe sets for RPL19 are: F,
5'-ATGTATCACAGCCTGTACCTG-3'; R, 5'-TTCTTGGTCTCTTCCTCCTTG-3'; and P,
5'-FAM-AGGTCTAAGACCAAGGAAGCACGCAA-TAMRA-p-3'. TaqMan analysis was
performed in a standard 96-well plate format. Standard curves were
constructed using 3.9–1000 ng of total mRNA prepared from cell lysates
of T47D human breast cancer cells. Standard curves for RPL19 were
constructed using 31.25–500 ng of total mRNA. Each dilution was run in
duplicate. All samples were run in triplicate using 100 ng of mRNA for
each reaction.
Xenograft Studies in Athymic Mice.
Female athymic mice (Harlan Sprague Dawley, Madison, WI), 4–5
weeks of age, were supplemented with a 0.25-mg, 21-day release,
17ß-estradiol pellet (Innovative Research, Toledo, OH) in the dorsal
space and then injected s.c. at a distant site with 5 x 106 MCF-7/HER2–18 cells. Once tumors reached an
approximate volume of 200 mm3
, 10 mice/group were
randomly allocated to treatment with: (a) vehicle (DMSO)
alone; (b) tamoxifen delivered as a 25-mg, 60-day release
s.c. pellet (Innovative Research) plus DMSO; (c) AG1478 (50
mg/kg/day) given by i.p. injection; or (d) tamoxifen plus
AG1478. Tumor diameters were serially measured with calipers and tumor
volumes calculated by the formula: volume = width2 x length/2. On day 30 after
tumor cell inoculation (2 weeks of treatment), three mice/group were
injected with the daily dose of AG1478 or DMSO, followed by i.p.
injection with BrdUrd (50 mg/kg), and their tumors were harvested
2 h later. Tumors were homogenized using a Polytron homogenizer
(Brinkmann, Westbury, NY) in TNE lysis buffer [50
mM Tris-HCl (pH 7.6), 150
mM NaCl, 2 mM EDTA, 1
mM
Na3VO4, 1
mM PMSF, 1 µg/ml pepstatin A, 2 µg/ml
aprotinin, and 0.5 µg/ml leupeptin]. After homogenization, NP40 was
added (1%, v/v). Equivalent amounts of protein from the tumor lysates
were then subjected to HER2, P-MAPK, and total MAPK immunoblot analysis
or to precipitation with HER2 antibodies, followed by P-Tyr
immunoblotting. For histological analysis, portions of the excised
tumors were fixed overnight in Histoprep (Fisher Scientific,
Pittsburgh, PA), sectioned, and stained with H&E. BrdUrd labeling of
tumor cell nuclei was visualized by staining the sections with BrdUrd
antibody (Biogenics, San Ramon, CA), as indicated by the Histo-mouse
kit (Zymed Laboratories, Inc., South San Francisco, CA). Additional
sections were used to stain for apoptotic cells using the DNA in
situ nick end-labeling (TUNEL) method (Intergen).
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RESULTS |
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. By immunoblot analysis, ER levels in MCF-7/HER2–18 cells
and in the tamoxifen-sensitive MCF-7/neo and parental MCF-7 cell lines
were similar (Fig. 1B)
. To rule whether transfection of HER2
had modified the levels of homologous EGF receptor-type kinases, we
used a quantitative PCR (TaqMan; Refs. 29
and
30
). Relative to a housekeeping ribosomal protein L19
(RPL19) gene, only the HER2 mRNA (13-fold), but not EGFR
(HER1), HER3, or HER4, was found to be in excess in MCF-7/HER2–18
cells (Fig. 1C)
. Antiestrogen resistance was also documented
by examining transcriptional responses of luciferase reporter
constructs containing ERE. Addition of 1 nM
estradiol activated ERE-luciferase transcription 18.7-fold in
MCF-7/HER2–18 and 5.3-fold in MCF-7/neo cells above each control (Fig. 2)
. Because of different transfection efficiencies, a comparison of the
constitutive ER-mediated transcription between both lines with high and
low HER2 levels cannot be done. The difference in the level of
estrogen-induced transcription did not translate into a difference in
estrogen-stimulated cell proliferation in that both cell lines
exhibited a similar 2–3-fold increase in cell number in response to
estradiol in estrogen-free medium after 4 days (data not shown).
Addition of 1 µM 4-OH tamoxifen suppressed
estradiol-induced ERE-luciferase transcription 26%
(P = 0.28) and 58% (P < 0.001) in MCF-7/HER2–18 and MCF-7/neo cells, respectively
(Fig. 2)
. Of note, tamoxifen alone increased ERE-luciferase
transcription 1.5-fold above basal in MCF-7/HER2–18 but not in control
cells. These data indicate that both HER2-overexpressing and control
MCF-7 cells exhibit wild-type ER function in response to exogenous
ligand but a clear difference in sensitivity to tamoxifen in both
growth and transcription assays.
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. To explain the inhibitory effect of AG1478, a relatively
specific EGFR kinase inhibitor, on HER2 phosphorylation, we examined
whether AG1478 would interact with HER2 in MCF-7/HER2–18 cells. A
brief 1-h incubation with 1–10 µM AG1478
induced the formation of HER2 dimers. At the higher dose of AG1478,
these HER2 dimers lacked a P-Tyr signal (Fig. 3B)
,
potentially explaining the inhibitory effect of AG1478 on HER2
phosphorylation shown in Fig. 3A
. Finally, we examined the
target specificity of the kinase inhibitors in an in vitro
kinase assay using exogenous ERK-1 (p44/MAPK) as a substrate for MEK-1
immunoprecipitated from MCF-7/HER2–18 cells. U0126 but not AG1478
inhibited MEK-1 induced phosphorylation of ERK-1 in vitro
(Fig. 3C)
, suggesting that the inhibitory effect of AG1478
on MAPK activity in intact cells (Fig. 3A)
is not
attributable to a nonspecific interaction of AG1478 with MEK-1.
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. To support that this enhancement of tamoxifen action was
attributable to the interruption of MAPK activity, we used constructs
encoding dominant-negative MEK-1 and MEK-2 (25)
. Relative
to control vector, transiently transfected dominant-negative MEK-2 or
the combination of dominant-negative MEK-2 and MEK-1 mutants
significantly enhanced tamoxifen-mediated inhibition of ER-induced
luciferase expression (P < 0.05; Fig. 4B
), whereas the effect of dominant-negative MEK-1
approached statistical significance (P = 0.1;
Fig. 4B
). It has been proposed that tamoxifen blocks
estrogen action by recruiting transcriptional corepressors to the
ligand-binding domain in the ER (32)
. Hence, we examined
the association of ER with the N-CoR in both MCF-7/neo and
MCF-7/HER2–18 cells. ER and N-CoR content in these cells was similar.
Tamoxifen induced a strong association of ER with N-CoR in the
antiestrogen-sensitive MCF-7/neo cells, whereas this effect was
markedly reduced in MCF-7/HER2–18 cells (Fig. 5)
. However, treatment of the tamoxifen-resistant cells with AG1478 or
U0126 enhanced tamoxifen-mediated association of N-CoR with ER without
any alterations in ER and N-CoR levels. These results are
consistent with the transcriptional responses shown in Fig. 4
.
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. Similar data were obtained with ER-positive BT-474 cells
that exhibit gene amplification and constitutive phosphorylation of the
HER2 receptor (33)
with an IC50 to
tamoxifen comparable with that in MCF-7/HER2–18 cells. In both cell
lines, the synergistic inhibitory effect of AG1478 with tamoxifen
appeared to be dose dependent. In BT-474 cells, however, the higher
concentration of U0126 was markedly inhibitory per se (Fig. 6B)
. By immunoblot, neither AG1478 nor U1026 altered ER
levels in BT-474 cells (not shown). Thus, in two HER2-overexpressing
systems, blockade of HER2 and MAPK partially abrogates antiestrogen
resistance.
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200 mm3
,
tumor-bearing mice were randomly allocated to no treatment or treatment
with tamoxifen, AG-1478, or both. Tamoxifen minimally delayed tumor
growth (P = 0.23), whereas AG1478 had no
effect. However, the combination of tamoxifen and AG1478 almost
completely arrested tumor growth relative to untreated controls
(P < 0.01; Fig. 7A
). To determine whether the effect of this combination was
antiproliferative versus cytotoxic, three mice/group were
pulsed with BrdUrd prior to tumor harvesting. Compared with control
tumors, there was a significant reduction in the number of
BrdUrd-labeled nuclei in the tumors treated with tamoxifen plus AG1478
(P < 0.01) but not in the other two
experimental groups (Fig. 7B)
. Histologically, tumors from
all groups were identical. To evaluate whether programmed cell death
was occurring as a result of treatment, tumor sections were evaluated
for the presence of apoptotic cells in situ as measured by
TUNEL assay. A modest increase in TUNEL-positive tumor cells was only
observed in the sections from xenografts treated with the combination
compared with control tumor cells (P = 0.06;
Fig. 7B
). Finally, to validate our molecular targets
in vivo, we examined whether HER2 signaling had been
inhibited in control and treated tumors. A representative cohort is
shown in Fig. 7C
. Levels of HER2 and total MAPK were similar
in tumors from all four experimental groups. However, phosphorylated
HER2 and active MAPK were markedly reduced in the tumors treated with
AG1478, suggesting that HER2 signaling had been disabled by AG1478
in vivo. Overall, these data suggest that interference of
the HER2 signaling pathway enhances tamoxifen action against
antiestrogen-resistant cells both in vitro and in
vivo.
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DISCUSSION |
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100-fold higher IC50 to tamoxifen compared
with MCF-7 cells with a single copy of the HER2 gene. In
this model, ER content and function, as measured by estradiol-induced
responses of ER reporters and cell proliferation, are maintained. These
data suggest that the differences in tamoxifen response in this model
are not attributable to differences in ER levels but to the potential
interaction of signal transduction elements downstream from HER2 with
the ER. To disengage HER2 function, we used the small molecule kinase
inhibitor AG1478, which displays an IC50 against
EGF-stimulated phosphorylation of an EGFR/HER2 chimeric receptor in NIH
3T3 cells of 1.4 µM.4
AG1478 has been shown to induce the formation of inactive,
unphosphorylated EGFR/HER2 heterodimers in tumor cells that naturally
overexpress HER2 (27)
. In MCF-7/HER2–18 cells, AG1478
also induces inactive HER2 dimers (Fig. 3B)
, suggesting that
it may interact directly with the HER2 kinase. By inducing the
formation of inactive, unphosphorylated HER2 dimers, it can sequester
the receptor from functional interactions with other HER receptor
partners and, therefore, disrupt signaling. Treatment of intact cells
with AG1478 eliminated active MAPK (Fig. 3A)
but had no
effect on MEK-1 activity in vitro (Fig. 3C)
,
suggesting that the effect of the quinazoline on MAPK occurred as a
result of the interruption of HER2 signaling upstream.
Several issues suggested the possibility that the EGFR kinase may have
been involved in the antiestrogen-resistant phenotype of MCF-7/HER2–18
cells: (a) AG1478 has a predominant effect against the EGFR
kinase (IC50, 3 nM; Ref.
27
); and (b) transfection
of EGFR into hormone-dependent breast cancer cells also induces
tamoxifen resistance (34
, 35)
. Moreover, HER2 can prolong
EGFR stability and both receptors contribute to transformation in a
cooperative manner (4
, 5
, 36)
. However, we were unable to
detect EGFR by immunoblot analysis of EGFR precipitates from
MCF-7/HER2–18 cell lysates. In addition, the C225 antibody against the
EGFR ectodomain (37)
had no effect on colony survival of
the HER2-transfected MCF-7 cells (data not shown). Moreover, EGFR mRNA
levels, as measured by real-time quantitative PCR, were low in
HER2-transfected and control cells (Fig. 1C)
. Hence,
although we cannot rule out some contribution of the EGFR to tamoxifen
resistance in our experimental system, we do not feel that this kinase
is playing a major causal role. There are no published reports
implicating the homologous family members HER3 and/or HER4 with loss of
estrogen dependence or antiestrogen resistance in breast cancer cells.
Nonetheless, the levels of these mRNAs were not altered by HER2
transfection into MCF-7 cells (Fig. 1C)
.
The following results implicate MAPK signaling as the mediator of antiestrogen resistance in HER2-overexpressing tumor cells: (a) overexpression of HER2 in MCF-7 cells results in both activation of MAPK and antiestrogen resistance; (b) inhibition of MAPK with U0126 enhances the ability of tamoxifen to inhibit both ER-mediated transcription and cell growth; (c) dominant-negative mutants of MEK-1 and MEK-2 also enhance the inhibitory effect of tamoxifen on ER-mediated transcription; and (d) AG1478 markedly reduces active MAPK in MCF-7/HER2–18 xenografts, and this reduction is temporally associated with tamoxifen-induced growth restraint of tumors in vivo. Neither HER2 overexpression (in MCF-7/HER2–18 cells) nor inhibition of HER2 and MAPK altered ER levels or reporter activity, suggesting that the restoration of tamoxifen action was independent of ER levels and function. In a more recent report, however, another HER2-transfected MCF-7 cell line (8) exhibited significant down-regulation of ER protein. Inhibition of MEK or MAPK restored ER expression and, presumably, sensitivity to antiestrogens (38) . This result suggests that HER2 hyperactivity may subvert the antiestrogen response by multiple potential mechanisms.
Notably, treatment with AG1478 alone inhibited tumor MAPK but not tumor
growth (Fig. 7)
. This result raises the intriguing possibility that
estrogen-primed tumor growth is not dependent on MAPK in
vivo and that, by inhibiting HER2, AG1478 is inhibiting other
HER2-dependent signaling pathways that mediate antiestrogen resistance.
This possibility will require further studies with MAPK inhibitors in
combination with tamoxifen. In addition, tumors treated with AG1478
alone and in which P-MAPK was reduced (Fig. 7C)
were
harvested 2 h after treatment with AG1478. Thus, it is conceivable
that the combination of tamoxifen plus AG1478 induced a more sustained
down-regulation of MAPK function compared with treatment with the
kinase inhibitor alone. This speculation requires further investigation
beyond the scope of this report. Our in vitro studies on the
role of MAPK would seem to be in some disagreement with a recent report
by Lobenhofer et al. (39)
in which
estrogen-dependent cell proliferation and transcription were inhibited
by 25 µM U0126 over a 24-h period. It should be
noted, however, that these concentrations were 10-fold higher than
those of U0126 that inhibited ERK-1 activity (Fig. 3C)
and
P-MAPK content (Fig. 3A)
, which enhanced the inhibitory
effect of tamoxifen on ER reporter activity (Fig. 4A)
in our
study. Therefore, these discrepancies could be explained by the
different concentrations of U0126 that were used.
MAPK has been shown to posttranslationally modify the ER by
phosphorylating Ser-118. The evidence that p42/44 MAPK directly
phosphorylates Ser-118 is based on the in vitro observation
that activated MAPK phosphorylates the AF-1 domain in the
NH2 terminus of the ER but not a mutant in which
Ser-118 was replaced by Ala (21
, 22 , 40)
. In some cell
types, this mutation causes a reduction in estradiol-dependent
transcriptional activation (21
, 41)
. The robust activation
of ERE-luciferase expression in MCF-7/HER2–18 cells (Fig. 2)
would
argue against a mutation in AF-1 in the HER2-overexpressing cells. In
addition, we have not been able to detect phosphorylated ER bands in
MCF-7/HER2–18 or BT-474 cells maintained in estrogen-containing
medium, despite the use of optimal conditions to inhibit cellular
phosphatases (42)
, implying that under conditions in which
MAPK is hyperactive (Fig. 3)
, Ser phosphorylation may not be abundant
in HER2-overexpressing tumor cells.
Inhibition of HER2 and MAPK also sensitized BT-474 human breast cancer
cells to tamoxifen (Fig. 6)
. These cells exhibit HER2 gene
amplification, a constitutively active HER2 kinase, and at best a 50%
growth inhibition in the presence of high (1
µM) concentrations of 4-OH tamoxifen.
Preliminarily, we have also observed a modest synergistic inhibitory
effect of tamoxifen and AG1478 in ER-positive MDA-361 human breast
tumor cells, which exhibit HER2 overexpression (33)
but no
HER2 gene amplification by fluorescent in situ
hybridization analysis (data not shown). Hence, we cannot rule out that
inhibition of HER2 signaling may also enhance tamoxifen action on
hormone-dependent, tamoxifen-sensitive breast tumor cells that do not
overexpress HER2, as suggested recently by Kunisue et al.
(43)
in studies using the pure antiestrogen ICI 182,780
and another humanized HER2 antibody. The exquisite sensitivity of
wild-type and MCF-7/neo (control) cells to concentrations as low as
0.01 µM tamoxifen makes these cell lines
unsuitable to address this important question. Be that as it may, the
relevance of our results to breast tumor cells with normal levels of
HER2 or more modest levels of HER2 overexpression will require further
research.
It has been proposed that tamoxifen recruits transcriptional corepressors to the AF-2 region in the hormone-binding domain of the ER to block ER-mediated transcription (3 , 32) . Mitogens such as protein kinase A, dopamine, or EGF can decrease this antiestrogen-induced receptor-corepressor interaction and limit tamoxifen action (44, 45, 46) . Thus, we examined whether HER2 signaling, which activates the same signaling pathways activated by EGF (4 , 5) , can also disrupt this interaction. Notably, tamoxifen-induced association of ER with N-CoR was reduced in HER2-overexpressing cells. Inhibition of HER2 and MAPK markedly increased the antiestrogen-mediated association of ER with N-CoR. This result implies that antiestrogen resistance in HER2-overexpressing tumor cells can, at least in part, be attributed to unrestrained MAPK signaling that alters the ER association with corepressors of transcription. The molecular mechanisms by which MAPK regulates these protein-protein associations require further study. Nonetheless, the data presented imply that: (a) MAPK may play a causal role in HER2-induced tamoxifen resistance in ER-positive breast tumor cells; and (b) that exogenous inhibitors of HER2 signal transduction can partially abrogate the antiestrogen-resistant phenotype, providing a testable therapeutic strategy in hormone-dependent human breast cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant R01 CA80195, Department
of Defense Breast Cancer Program Grant DAMD17-98-1-8262, a Clinical
Investigator Award from the Department of Veteran Affairs (all to
C. L. A.), and Vanderbilt-Ingram Cancer Center Support Grant CA68485.
A. E. G. L. is the recipient of a Susan G. Komen Breast Cancer
Foundation Fellowship Award. 
2 To whom requests for reprints should be
addressed, at Division of Oncology/Vanderbilt University Medical
Center, 22nd Avenue South, 1956 TVC, Nashville, TN 37232-5536. Phone:
(615) 936-3524 or (615) 936-1919; Fax: (615) 936-1790; E-mail: carlos.arteaga{at}mcmail.vanderbilt.edu
3 The abbreviations used are: ER, estrogen
receptor; EGFR, epidermal growth factor receptor; MAPK,
mitogen-activated protein kinase; MEK, MAP kinase kinase; ERE, estrogen
response element; N-CoR, nuclear receptor corepressor; P-Tyr,
phosphotyrosine; BrdUrd, 8-bromodeoxyuridine; TUNEL, terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling; PMSF,
phenylmethylsulfonyl fluoride; FAM, 6-carboxy-fluorescein; TAMRA,
6-carboxy-tetramethylrhodamine.
4 L. K. Shawver, Sugen, Inc., personal
communication.
Received 5/22/00. Accepted 8/23/00.
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I. Girault, F. Lerebours, S. Amarir, S. Tozlu, M. Tubiana-Hulin, R. Lidereau, and I. Bieche Expression Analysis of Estrogen Receptor {alpha} Coregulators in Breast Carcinoma: Evidence That NCOR1 Expression Is Predictive of the Response to Tamoxifen Clin. Cancer Res., April 1, 2003; 9(4): 1259 - 1266. [Abstract] [Full Text] [PDF]
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J. M. Knowlden, I. R. Hutcheson, H. E. Jones, T. Madden, J. M. W. Gee, M. E. Harper, D. Barrow, A. E. Wakeling, and R. I. Nicholson Elevated Levels of Epidermal Growth Factor Receptor/c-erbB2 Heterodimers Mediate an Autocrine Growth Regulatory Pathway in Tamoxifen-Resistant MCF-7 Cells Endocrinology, March 1, 2003; 144(3): 1032 - 1044. [Abstract] [Full Text] [PDF]
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S. De Placido, M. De Laurentiis, C. Carlomagno, C. Gallo, F. Perrone, S. Pepe, A. Ruggiero, A. Marinelli, C. Pagliarulo, L. Panico, et al. Twenty-year Results of the Naples GUN Randomized Trial: Predictive Factors of Adjuvant Tamoxifen Efficacy in Early Breast Cancer Clin. Cancer Res., March 1, 2003; 9(3): 1039 - 1046. [Abstract] [Full Text] [PDF]
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R. Schiff, S. Massarweh, J. Shou, and C. K. Osborne Breast Cancer Endocrine Resistance: How Growth Factor Signaling and Estrogen Receptor Coregulators Modulate Response Clin. Cancer Res., January 1, 2003; 9(1): 447s - 454s. [Abstract] [Full Text]
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H. Kurokawa and C. L. Arteaga ErbB (HER) Receptors Can Abrogate Antiestrogen Action in Human Breast Cancer by Multiple Signaling Mechanisms Clin. Cancer Res., January 1, 2003; 9(1): 511s - 515s. [Abstract] [Full Text]
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S. R. D. Johnston, J. Head, S. Pancholi, S. Detre, L.-A. Martin, I. E. Smith, and M. Dowsett Integration of Signal Transduction Inhibitors with Endocrine Therapy: An Approach to Overcoming Hormone Resistance in Breast Cancer Clin. Cancer Res., January 1, 2003; 9(1): 524s - 532s. [Abstract] [Full Text]
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K. Wu, C. Wang, M. D'Amico, R. J. Lee, C. Albanese, R. G. Pestell, and S. Mani Flavopiridol and Trastuzumab Synergistically Inhibit Proliferation of Breast Cancer Cells: Association with Selective Cooperative Inhibition of Cyclin D1-dependent Kinase and Akt Signaling Pathways Mol. Cancer Ther., July 1, 2002; 1(9): 695 - 706. [Abstract] [Full Text] [PDF]
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A. Bowman, H. Gabra, S. P. Langdon, A. Lessells, M. Stewart, A. Young, and J. F. Smyth CA125 Response Is Associated with Estrogen Receptor Expression in a Phase II Trial of Letrozole in Ovarian Cancer: Identification of an Endocrine-sensitive Subgroup Clin. Cancer Res., July 1, 2002; 8(7): 2233 - 2239. [Abstract] [Full Text] [PDF]
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V. G. Keshamouni, R. R. Mattingly, and K. B. Reddy Mechanism of 17-beta -Estradiol-induced Erk1/2 Activation in Breast Cancer Cells. A ROLE FOR HER2 AND PKC-delta J. Biol. Chem., June 14, 2002; 277(25): 22558 - 22565. [Abstract] [Full Text] [PDF]
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A. Adeyinka, Y. Nui, T. Cherlet, L. Snell, P. H. Watson, and L. C. Murphy Activated Mitogen-activated Protein Kinase Expression during Human Breast Tumorigenesis and Breast Cancer Progression Clin. Cancer Res., June 1, 2002; 8(6): 1747 - 1753. [Abstract] [Full Text] [PDF]
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G. K. Scott, C. Marden, F. Xu, L. Kirk, and C. C. Benz Transcriptional Repression of ErbB2 by Histone Deacetylase Inhibitors Detected by a Genomically Integrated ErbB2 Promoter-reporting Cell Screen Mol. Cancer Ther., April 1, 2002; 1(6): 385 - 392. [Abstract] [Full Text] [PDF]
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 E. R. Simpson and M. Dowsett Aromatase and Its Inhibitors: Significance for Breast Cancer Therapy Recent Prog. Horm. Res., January 1, 2002; 57(1): 317 - 338. [Abstract] [Full Text] [PDF]
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M. Dowsett, C. Harper-Wynne, I. Boeddinghaus, J. Salter, M. Hills, M. Dixon, S. Ebbs, G. Gui, N. Sacks, and I. Smith HER-2 Amplification Impedes the Antiproliferative Effects of Hormone Therapy in Estrogen Receptor-positive Primary Breast Cancer Cancer Res., December 1, 2001; 61(23): 8452 - 8458. [Abstract] [Full Text] [PDF]
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 I. Hoffmann, E. Eugene, X. Nassif, P.-O. Couraud, and S. Bourdoulous Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis J. Cell Biol., October 1, 2001; 155(1): 133 - 144. [Abstract] [Full Text] [PDF]
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A. E. G. Lenferink, D. Busse, W. M. Flanagan, F. M. Yakes, and C. L. Arteaga ErbB2/neu Kinase Modulates Cellular p27Kip1 and Cyclin D1 through Multiple Signaling Pathways Cancer Res., September 1, 2001; 61(17): 6583 - 6591. [Abstract] [Full Text] [PDF]
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R. A. McClelland, D. Barrow, T.-A. Madden, C. M. Dutkowski, J. Pamment, J. M. Knowlden, J. M. W. Gee, and R. I. Nicholson Enhanced Epidermal Growth Factor Receptor Signaling in MCF7 Breast Cancer Cells after Long-Term Culture in the Presence of the Pure Antiestrogen ICI 182,780 (Faslodex) Endocrinology, July 1, 2001; 142(7): 2776 - 2788. [Abstract] [Full Text] [PDF]
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R. Bagatell, O. Khan, G. Paine-Murrieta, C. W. Taylor, S. Akinaga, and L. Whitesell Destabilization of Steroid Receptors by Heat Shock Protein 90-binding Drugs: A Ligand-independent Approach to Hormonal Therapy of Breast Cancer Clin. Cancer Res., July 1, 2001; 7(7): 2076 - 2084. [Abstract] [Full Text] [PDF]
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J.-i. Okano and A. K. Rustgi Paclitaxel Induces Prolonged Activation of the Ras/MEK/ERK Pathway Independently of Activating the Programmed Cell Death Machinery J. Biol. Chem., May 25, 2001; 276(22): 19555 - 19564. [Abstract] [Full Text] [PDF]
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J. C. H. Donovan, A. Milic, and J. M. Slingerland Constitutive MEK/MAPK Activation Leads to p27Kip1 Deregulation and Antiestrogen Resistance in Human Breast Cancer Cells J. Biol. Chem., October 26, 2001; 276(44): 40888 - 40895. [Abstract] [Full Text] [PDF]
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) cells were plated in 35-mm dishes in IMEM, 10% FCS, 0.8%
agarose, and 10 mM HEPES in the presence or absence of the
indicated concentrations of 4-OH tamoxifen. Colonies measuring 
), MCF-7/neo (
) and
MCF-7/HER2–18 (
) were analyzed for EGFR, HER2, HER3, or HER4
expression by the quantitative RT-PCR technique as described in
"Materials and Methods." Each signal was normalized with the signal
by a control gene, RPL19, and expressed as relative mRNA
expression.
, P < 0.05, compared
with 4-OH tamoxifen (-) control, Student’s unpaired t
test. These results were confirmed in two additional experiments.
