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Synergy between inhibitors of androgen receptor
and MEK has therapeutic implications in estrogen
receptor-negative breast cancer
Naderi et al.
Naderi et al.Breast Cancer Research 2011, 13:R36
http://breast-cancer-research.com/content/13/2/R36 (1 April 2011)
RESEARCH ARTICLE Open Access
Synergy between inhibitors of androgen receptor
and MEK has therapeutic implications in estrogen
receptor-negative breast cancer
Ali Naderi
*
, Kee Ming Chia and Ji Liu
Abstract
Introduction: Estrogen receptor-negative (ER-) breast cancer is a heterogeneous disease with limited therapeutic
options. The molecular apocrine subtype constitutes 50% of ER-tumors and is characterized by overexpression of
steroid response genes including androgen receptor (AR). We have recently identified a positive feedback loop
between the AR and extracellular signal-regulated kinase (ERK) signaling pathways in the molecular apocrine
subtype. In this feedback loop, AR regulates ERK phosphorylation through the mediation of ErbB2 and, in turn, ERK-
CREB1 signaling regulates the transcription of AR in molecular apocrine cells. In this study, we investigated the
therapeutic implications of the AR-ERK feedback loop in molecular apocrine breast cancer.
Methods: We examined a synergy between the AR inhibitor flutamide and the MEK inhibitor CI-1040 in the
molecular apocrine cell lines MDA-MB-453, HCC-1954 and HCC-202 using MTT cell viability and annexin V apoptosis
assays. Synergy was measured using the combination index (CI) method. Furthermore, we examined in vivo synergy
between flutamide and the MEK inhibitor PD0325901 in a xenograft model of the molecular apocrine subtype. The
effects of in vivo therapies on tumor growth, cell proliferation and angiogenesis were assessed.
Results: We demonstrate synergistic CI values for combination therapy with flutamide and CI-1040 across three
molecular apocrine cell lines at four dose combinations using both cell viability and apoptosis assays. Furthermore,
we show in vivo that combination therapy with flutamide and MEK inhibitor PD0325901 has a significantly higher
therapeutic efficacy in reducing tumor growth, cellular proliferation and angiogenesis than monotherapy with
these agents. Moreover, our data suggested that flutamide and CI-1040 have synergy in trastuzumab resistance
models of the molecular apocrine subtype. Notably, the therapeutic effect of combination therapy in trastuzumab-
resistant cells was associated with the abrogation of an increased level of ERK phosphorylation that was developed
in the process of trastuzumab resistance.
Conclusions: In this study, we demonstrate in vitro and in vivo synergies between AR and MEK inhibitors in
molecular apocrine breast cancer. Furthermore, we show that combination therapy with these inhibitors can
overcome trastuzumab resistance in molecular apocrine cells. Therefore, a combination therapy strategy with AR
and MEK inhibitors may provide an attractive therapeutic option for the ER-/AR+ subtype of breast cancer.
Introduction
Estrogen receptor-negative (ER-) breast cancer constitu-
tes around 30% of all cases with limited therapeutic tar-
gets available for this heterogeneous disease [1]. In
contrast to ER+ breast cancer, in which anti-estrogen
therapy is an effective treatment strategy, current
therapeutic options for advanced ER-breast cancer
mostly rely on chemotherapeutic agents.
Molecular profiling of ER-breast cancer broadly classi-
fies this disease into basal and molecular apocrine sub-
types [2]. Molecular apocrine breast cancer constitutes
approximately 50% of ER-tumors and is characterized
by a steroid response gene signature that includes
androgen receptor (AR) and a high frequency of ErbB2
overexpression [2-8]. For pathological classification, this
subtype can easily be characterized as ER-/AR+ breast
* Correspondence: a.naderi@uq.edu.au
The University of Queensland Diamantina Institute, Princess Alexandra
Hospital, Ipswich Road, Brisbane, Queensland 4102, Australia
Naderi et al.Breast Cancer Research 2011, 13:R36
http://breast-cancer-research.com/content/13/2/R36
© 2011 Naderi et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creative commons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, pro vided the original work is properly cited.
cancer [6-8]. In a recent study by Park et al.[7],AR
expression was observed in 50% of ER-breast tumors
and in 35% of triple-negative cancers. In addition, ErbB2
overexpression was present in 54% of ER-/AR+ tumors
compared to 18% of the ER-/AR-group, which suggests
a significant correlation between AR expression and
ErbB2 overexpression in ER-tumors [7]. Importantly, a
growingbodyofevidencesuggeststhatARisathera-
peutic target in molecular apocrine breast cancer [4,5,9].
In this regard, AR inhibition reduces cell viability and
proliferation in molecular apocrine models [4,5,9]. In
addition, an ongoing clinical trial has demonstrated that
AR inhibition can stabilize disease progression in meta-
static ER-/AR+ breast cancer [10].
ARsignalinghasasignificantroleinthebiologyof
molecular apocrine tumors. Notably, we have identified
a functional cross-talk between the AR and ErbB2 sig-
naling pathways in molecular apocrine cells that modu-
lates cell proliferation and expression of steroid
response genes [5]. In addition, this cross-talk has been
confirmed by a genome-wide meta-analysis study [11].
Moreover, we have recently discovered a positive feed-
back loop between the AR and extracellular signal-
regulated kinase (ERK) signaling pathways in molecular
apocrine breast cancer [12]. In this feedback loop, AR
regulates ERK phosphorylation through the mediation of
ErbB2, and, in turn, ERK-CREB1 signaling regulates the
transcription of AR in molecular apocrine cells [12].
The AR-ERK feedback loop has potential therapeutic
implications in molecular apocrine breast cancer. In par-
ticular, due to the availability of effective AR and mito-
gen-activated protein kinase kinase (MEK) inhibitors,
exploiting this feedback loop would provide a practical
therapeutic approach. A number of AR inhibitors are
currently used for prostate cancer, and their safety in a
female patient population has been demonstrated in stu-
dies of breast and ovarian cancers [10,13,14]. Further-
more, several classes of MEK inhibitors have been
developed and are now being examined in various clini-
cal trials [15,16]. Therefore, a potential positive outcome
for the preclinical studies can readily be tested in future
clinical trials.
Here we carried out a preclinical study of combination
therapy with AR and MEK inhibitors using in vitro and
in vivo molecular apocrine models. Our results suggest
that this combination therapy provides a promising
therapeutic strategy in ER-/AR+ breast cancer.
Materials and methods
Cell culture and treatments
Breast cancer cell lines MDA-MB-453, HCC-202, and
HCC-1954 were obtained from the American Type Cul-
ture Collection (Manassas, VA, USA). All the culture
media were obtained from Invitrogen (Melbourne, VIC,
Australia). MDA-MB-453 cell line was cultured in L15
media/10% fetal bovine serum (FBS). HCC-202 and
HCC-1954 cells were cultured in RPMI 1640 media
with 10% FBS. Cell cultures were carried out in a humi-
dified 37°C incubator supplied with 5% CO
2
.Thefol-
lowing treatments were applied for the cell culture
experiments: (1) AR inhibitor flutamide (Sigma-Aldrich,
Sydney, NSW, Australia) at 5 to 200 μM concentrations;
(2) MEK inhibitor CI-1040 (PD184352) (Selleck Chemi-
cals,Houston,TX,USA)at2to30μMconcentrations;
and (3) ErbB2 inhibitor trastuzumab (Roche, Sydney,
NSW, Australia) at 10 to 80 μg/ml concentrations.
Treatments with the inhibitors were performed in media
containing FBS.
Cell viability assay
MDA-MB-453, HCC-202 and HCC-1954 cells were
grown in 96-well plates to 50% confluence followed by
inhibitor treatments for 48 hours in full media. A sol-
vent-only-treated group was used as a control. Cell via-
bility was assessed using the Vybrant MTT Proliferation
Assay Kit (Invitrogen) as previously described [5,17].
Absorbance at 570 nm was measured for the experi-
mental groups using a plate reader. MTT experiments
were performed in eight biological replicates.
Apoptosis assay
Apoptosis measurement with flow cytometry was carried
out using Annexin V-FITC Apoptosis Detection Kit I
(BD Biosciences, Sydney, NSW, Australia). All experi-
ments were performed in four biological replicates.
Combination indices
Drug synergy was assessed using a combination index
(CI) method as described before [9,18]. We first mea-
sured cell viability and apoptosis for the combination
therapies with flutamide and CI-1040 using MTT and
annexin V assays, respectively. We next identified the
concentrations of flutamide and CI-1040 monotherapies,
which resulted in a level of reduction in cell viability
and apoptosis similar to that observed with each of the
combination therapy conditions. Subsequently, CI for
the combined treatments were calculated as follows:
CI = [Ca,x/ICx,a] + [Cb,x/ICx,b], Ca,xand Cb,xare the
concentrations of drug A and drug B used in combina-
tion to achieve x% drug effect [18]. ICx,a and ICx,b are
the concentrations for single agents to achieve the same
effect. A CI less than 1 indicates synergy with the com-
bination therapy.
Tumor xenograft studies
Animal ethics approval was obtained for the project, and
mice were maintained in accordance with the Institu-
tional Animal Care guidelines. Six-week-old female
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nonobese diabetic/severe combined immunodeficient
mice were purchased from Animal Resource Center
(Perth, WA, Australia). The methodology for generating
the tumors in mice was performed as previously
described [9,12]. A total of 5 × 10
6
MDA-MB-453 cells
were injected into the flank of each mouse to generate
the xenograft tumors [9]. Drug treatments were initiated
7 days after the cell injections.
Flutamide treatment was carried out with 25 mg/60-
day slow-release flutamide pellets (Innovative Research
of America, Sarasota, FL, USA), and the control group
received placebo pellets (Innovative Research of Amer-
ica). MEK inhibitor treatment was carried out with daily
oral gavage of PD0325901 (Selleck Chemicals) at 5 to
20 mg/kg/day as described before [19]. PD0325901 was
prepared at a stock concentration of 50 mg/ml in
dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and made
up to the daily working concentration in 0.05% methyl-
cellulose/0.02% Tween 80 (Sigma-Aldrich). The control
group received daily gavage of a volume of DMSO equal
to that of the treatment group in the same carrier
solution.
The tumor volumes were assessed every 3 days by
measuring the length (l)andwidth(w) and then calcu-
lating the volume as π/6 × l×w×(l+w)/2 as
described before [20]. Xenograft tumors were harvested
30 days following the start of treatments. Fold change in
tumor volume was calculated as [volume on treatment
day 30/volume on treatment day 1]. Harvested tumors
werefixedinformalinandembeddedinparaffinfor
immunohistochemistry (IHC) staining.
Toxicity studies in mice
We assessed toxicity to MEK inhibitor in mouse xeno-
graft model by measuring body weight change during
30 days of treatment with PD0325901 at 5 to 20 mg/kg/
day. The control group received daily gavage of carrier
solution. Xenograft experiments were carried out as
explained before, and two mice were treated per each
treatment group. Mice were weighed daily during the
course of treatment. In the event of weight reduction
for two consecutive days, drug was withheld until weight
stabilized before therapy reinitiation. Toxicity was evalu-
ated by the measurement of (1) weight change pre- and
post-treatment in each group and (2) number of treat-
ment days lost due to weight reduction or mortality.
Immunohistochemistry
IHC staining was performed using EnVision+ System-
HRP (AEC, DakoCytomation, Melbourne, VIC, Austra-
lia) following the manufacturers’instruction. Antigen
retrieval was carried out using Target Retrieval Solution
(DakoCytomation). Rabbit polyclonal Ki-67 and rabbit
polyclonal CD31 antibodies were obtained from Abcam
(Cambridge, UK). Primary antibody incubation was car-
ried out at 1:50 dilution for each antibody. Slides were
counterstained with hematoxylin (Sigma-Aldrich) and
mounted using Glycergel Mounting Medium (DakoCy-
tomation). For IHC scoring, slides were examined using
a light microscope at ×60 magnification (Nikon Instru-
ments Inc., Tokyo, Japan).
The percentage of cells showing Ki-67 nuclear staining
in a total of 600 cells was calculated as the proliferation
index for each tumor. The total number of CD31-
positive blood vessels in a tumor cross-section was
counted to measure angiogenesis in each sample. Scor-
ing was carried out separately by two investigators, and
the average scores were used for the final analysis.
Generation of trastuzumab-resistant line
To generate a trastuzumab-resistant line, MDA-MB-453
cells were continuously cultured with increasing doses
of trastuzumab at 10 to 20 μg/ml concentrations for
90 days. The MDA-MB-453 control line was treated
with solvent only and grown for the same duration. Cell
viability of resistant and control lines were assessed
using MTT assay.
Western blot analysis
Rabbit monoclonal ERK1/2 and phospho-ERK1/2
(Thr202/Tyr204) antibodies were obtained from Cell
Signaling Technology (Danvers, MA, USA). Western
blot analysis was carried out at 1:1,000 dilution of each
primary antibody using 10 μgand20μg of cell lysates
for total and phospho-ERK1/2, respectively. Protein con-
centrations from the cell isolates were measured using
BCA Protein Assay Kit (Thermo Scientific, Melbourne,
VIC, Australia). Rabbit polyclonal a-tubulin antibody
(Abcam) was used as loading control. Analysis of band
densities was performed using Bio-Profil Densitometer
Software (Vilber Lourmat, Eberhardzell, Germany). Fold
changes in band densities were measured relative to the
control groups. Western blot analysis was done in two
biological replicates, and the average fold change was
shown for each set of experiments.
Statistical analysis
Biostatistical analysis was done using the SPSS version
17.0 statistical software package (SPSS, Inc., Chicago, IL,
USA). The Mann-Whitney Utest was applied for the
comparison of nonparametric data.
Results
Synergy between AR and MEK inhibitors in reducing cell
viability
To assess a potential synergy between the AR inhibitor
flutamide and the MEK inhibitor CI-1040, we used pre-
viously characterized molecular apocrine cell lines
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MDA-MB-453, HCC-1954 and HCC-202 [5,9]. CI-1040
has been commonly used to examine the effects of MEK
inhibition on cell lines, and therefore it was chosen for
in vitro experiments in this study [21-23]. The effect of
monotherapies with flutamide at 5 to 200 μMandCI-
1040 at 2 to25 μM concentrations on cell viability of
molecular apocrine lines was assessed by MTT assay.
We observed that monotherapies with these inhibitors
reduced cell viability in a dose-dependent manner across
three cell lines (Figures 1A to 1F and 2A to 2D).
It is notable that MDA-MB-453 cells were relatively
more sensitive to flutamide treatment compared to the
HCC-1954 and HCC-202 lines. In MDA-MB-453 cells,
flutamide at 30 μM concentration reduced cell viability
by approximately 75% compared to control (Figure 1A).
However, in HCC-1954 and HCC-202 cell lines, there
was a 50% reduction in cell viability with flutamide at
100 μM concentration (Figure 1C and 1E). Furthermore,
HCC-202 cells were relatively less sensitive to CI-1040
treatment compared to the other two cell lines. In this
respect, CI-1040 at 25 μM concentration reduced cell
viability by over 75% in MDA-MB-453 and HCC-1954
cells compared to an approximately 30% reduction in
the HCC-202 line (Figure 1B, D and 1F).
Next, we calculated CI values for the combined ther-
apy with flutamide and CI-1040 at four dose combina-
tions in each cell line (Figure 2). In MDA-MB-453 cell
line, which had a high level of sensitivity to flutamide,
this drug was applied at 5 and 10 μM in combination
with CI-1040 at 5 and 10 μM concentrations (CI-1040
(5 μM)/flutamide (5 μM), CI-1040 (10 μM)/flutamide
(5 μM), CI-1040 (5 μM)/flutamide (10 μM), and CI-
1040 (10 μM)/flutamide (10 μM)). In HCC-1954 and
HCC-202 cell lines, flutamide at 20 and 40 μMconcen-
trations was assessed for synergy in combination with
CI-1040 at 5 and 10 μM concentrations (CI-1040 (5
μM)/flutamide (20 μM), CI-1040 (10 μM)/flutamide (20
μM), CI-1040 (5 μM)/flutamide (40 μM), and CI-1040
(10 μM)/flutamide (40 μM)). Importantly, we observed a
synergy at all four dose combinations across three cell
lines. In MDA-MB-453 cell line, CI values for the com-
bination therapy with flutamide and CI-1040 were 0.64
to 0.75 (Figure 2B). Furthermore, in HCC-1954 and
HCC-202 lines, CI values for the combination therapy
were 0.49 to 0.75 and 0.6 to 0.83, respectively (Figure
2C and 2D). These data suggest that AR inhibitor fluta-
mide and MEK inhibitor CI-1040 have synergy in redu-
cing cell viability of molecular apocrine cell lines.
Synergy between AR and MEK inhibitors in inducing
apoptosis
To further investigate the synergy between flutamide
and CI-1040, we assessed the effect of this combination
therapy on apoptosis in molecular apocrine cell lines.
Apoptosis was detected using annexin V assay and ana-
lyzed by flow cytometry. Using this approach, we calcu-
lated CI values for the combination therapy with
flutamide and CI-1040 at four dose combinations in
each cell line. CI-1040 was applied at 5 and 10 μMin
combination with flutamide at 20 and 30 μM concentra-
tions (CI-1040 (5 μM)/flutamide (20 μM), CI-1040
(10 μM)/flutamide (20 μM), CI-1040 (5 μM)/fluatmide
(30 μM), and CI-1040 (10 μM)/flutamide (30 μM)).
Notably, we observed synergy at all four dose combi-
nations in molecular apocrine cell lines. In HCC-1954
and MDA-MB-453 cell lines, CI values for the combina-
tion therapy were 0.7 to 0.8 and 0.65 to 0.75, respec-
tively (Figure 3A to 3H and Table 1). Furthermore, in
the HCC-202 cell line, CI values for the combination
therapy were 0.6 to 0.75 (Figure 4A to 4D and Table 1).
Therefore, we can conclude that AR inhibitor flutamide
and MEK inhibitor CI-1040 have synergy in the induc-
tion of apoptosis in molecular apocrine cell lines.
Assessment of MEK inhibitor toxicity in mice
We investigated the in vivo toxicity of PD0325901 to
identify a tolerable dose of this MEK inhibitor for xeo-
nograft studies. PD0325901 is a potent MEK inhibitor
with chemical characteristics similar to that of CI-1040;
however, a better oral bioavailability makes this agent
more suitable for in vivo studies [19,24]. Following
xenografts with MDA-MB-453 cells, mice were treated
with daily oral gavage of PD0325901 at 5, 10, 15 and
20 mg/kg/day for 30 days. Daily gavage of carrier solu-
tion was used as control. Toxicity was evaluated by the
measurement of weight change during treatment and
number of treatment days lost due to weight reduction
or mortality as described in Materials and methods.
We observed a significantly higher weight gain in mice
treated with PD0325901 at 5 and 10 mg/kg/day doses
compared to the control group (P<0.01,Figure5A).
Importantly, treatments with higher doses of PD0325901
at 15 and 20 mg/kg/day resulted in a significant weight
reduction compared to the lower doses of this agent (P<
0.01, Figure 5A). Furthermore, the number of treatment
days lost due to toxicity was significantly lower with
PD0325901 doses of 5 and 10 mg/kg/day compared to
that of 15 and 20 mg/kg/day (P< 0.01, Figure 5B). Nota-
bly, PD0325901 treatment at 5 mg/kg/day did not result
in any measurable toxicity using this approach (Figure
5A and 5B). These findings indicate that PD0325901
treatment at lower doses is significantly less toxic than
higher doses of this agent in a xenograft mouse model.
In vivo therapeutic efficacy of combination therapy with
AR and MEK inhibitors
To further assess the therapeutic efficacy of combined
AR and MEK inhibition in molecular apocrine breast
Naderi et al.Breast Cancer Research 2011, 13:R36
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0
0.1
0.2
0.3
0.4
0.5
0.6
CTL
FLU10
FLU20
FLU40
FLU60
FLU80
FLU100
HCC-1954 (Flutamide)
0
0.1
0.2
0.3
0.4
0.6
HCC-202 (Flutamide)
CTL
FLU10
FLU20
FLU40
FLU60
FLU80
FLU100
A) B)
N= 8
N= 8
0
0.1
0.2
0.3
0.4
Absorbance at 570 nM by MTT assay
C
)
MDA-MB-453 (Flutamide)
N= 8
0
0.1
0.2
0.3
0.4
CTL
CI-2
CI-5
CI-10
CI-15
CI-20
CI-25
N=8
MDA-MB-453 (CI-1040)
0
0.2
0.4
0.6
0.8
1
D)
CTL
CI-2
CI-5
N=8
CI-10
CI-15
CI-20
CI-25
HCC-1954 (CI-1040)
E)
0
0.1
0.2
0.3
0.4
CTL
CI-2
CI-5
CI-10
CI-15
CI-20
CI-25
N=8
HCC-202 (CI-1040)
F)
FLU30
FLU25
FLU20
FLU15
FLU10
FLU5
CTL
Absorbance at 570 nM by MTT assay
Absorbance at 570 nM by MTT assay
Absorbance at 570 nM by MTT assay
Absorbance at 570 nM by MTT assay
Absorbance at 570 nM by MTT assay
Figure 1 The effect of flutamide and CI-1040 on cell viability of molecular apocrine lines.(A) MTT assay to measure cell viability in MDA-
MB-453 cell line after treatment with flutamide (FLU) at 5 to 30 μM concentrations. CTL: control. (B) MTT assay to measure cell viability in MDA-
MB-453 cell line after treatment with CI-1040 (CI) at 2 to 25 μM concentrations. (C) MTT assay to measure cell viability in HCC-1954 cell line after
treatment with flutamide at 10 to 100 μM concentrations. (D) MTT assay to measure cell viability in HCC-1954 cell line after treatment with CI-
1040 at 2 to 25 μM concentrations. (E) MTT assay to measure cell viability in HCC-202 cell line after treatment with flutamide at 10 to 100 μM
concentrations. (F) MTT assay to measure cell viability in HCC-202 cell line after treatment with CI-1040 at 2 to 25 μM concentrations. All error
bars: ± 2 SEM.
Naderi et al.Breast Cancer Research 2011, 13:R36
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HCC-1954
CTL
CI-15
FLU-150
CI-5/FLU-20
0.2
0
0.4
0.6
0.8
1
1.2
A)
C)
0
0.1
0.2
0.3
0.4
Absorbance at 570 nM by MTT assay
CTL
CI-5
CI-10
FLU-5
FLU-10
CI-5/FLU-5
CI-10/FLU-5
CI-5/FLU-10
CI-10/FLU-10
MDA-MB-453
N=8
Absorbance at 570 nM by MTT assay
Absorbance at 570 nM by MTT assay
B)
-0.1
0
0.1
0.2
0.3
0.4
MDA-MB-453
CTL
CI-10
FLU-20
CI-5/FLU-5
N=8
Bars:+/-2SEM
CI=0.75
CI-20
FLU-35
CI-10/FLU-5
CI=0.64
CI-15
FLU-27.5
CI-5/FLU-10
CI=0.69
CI-25
FLU-40
CI-10/FLU-10
CI=0.65
0
0.1
0.2
0.3
0.4
Absorbance at 570 nM by MTT assay
D)
HCC-202
CI= 0.83
CI= 0.76
N= 8
Bars: +/-2SEM
CI-10/FLU-20
CI-5/FLU-40
CI-20
FLU-200
CI-10/FLU-40
CI= 0.49
CI= 0.75 CI= 0.6
CI= 0.7
N= 8
Bars: +/-2SEM
CTL
CI-30
FLU-80
CI-10/FLU-20
CI-10/FLU-40
CI-5/FLU-40
CI-25
FLU-50
CI-5/FLU-20
CI= 0.73
0.6
Figure 2 Synergistic effect of AR and mitogen-activated protein kinase kinase inhibitors on cell viability.(A) MTT assay to measure cell
viability in MDA-MB-453 cell line after monotherapies and combination treatments with flutamide (FLU) and CI-1040 (CI) at 5 and 10 μM
concentrations. CTL: control. Error bars: ± 2 SEM. (B) Combination indices (CI) for flutamide and CI-1040 combination therapy in MDA-MB-453 cell
line using MTT assay. Cell viability was measured after combination therapies with flutamide at 5 and 10 μM with each concentration of CI-1040 at
5 and 10 μM. The concentrations of FLU and CI-1040 monotherapies with an effect similar to that of each combination therapy are depicted. Error
bars: ± 2 SEM. (C) Combination indices for flutamide and CI-1040 combination therapy in HCC-1954 cell line using MTT assay. Cell viability was
measured after combination therapies with flutamide at 20 and 40 μM with each concentration of CI-1040 at 5 and 10 μM. (D) Combination
indices for flutamide and CI-1040 combination therapy in HCC-202 cell line using MTT assay at concentrations described in Figure 2C.
Naderi et al.Breast Cancer Research 2011, 13:R36
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A)
Annexin V-FITC Annexin V-FITC
B)
C) D)
Annexin V-FITC Annexin V-FITC
Count
Count
Count
Count
P1= 7% P1=19.1%
P1=19.1%
P1=20.4% CI= 0.7
HCC-1954 (control) HCC-1954 (CI-20 μM)
HCC-1954 (FLU-100 μM) HCC-1954 (CI-10/FLU-20 μM)
Count
Annexin V-FITC
MDA-MB-453 (control)
E)
P1= 8%
0100 200 300 400
Count
Annexin V-FITC
MDA-MB-453 (CI-20 μM)
F)
= 85.1%
Count
0100 200 300 400
G)
Annexin V-FITC
MDA-MB-453 (FLU-80 μM)
= 88.6%
0100 200 300 400
Count
Annexin V-FITC
MDA-MB-453 (CI-10/FLU-20 μM)
= 86.3%
CI= 0.75
H)
Figure 3 Synergistic induction of apoptosis by AR and mitogen-activated protein kinase kinase inhibitors in HCC-1954 and MDA-MB-
453 cell lines.(A) Histogram showing the percentage of apoptosis (P1) in control (solvent-only treated) HCC-1954 cell line using annexin V-FITC
flow cytometry. (B) Histogram showing the percentage of apoptosis following CI-1040 treatment at 20 μM (CI 20 μM) in HCC-1954 cell line.
(C) Histogram showing the percentage of apoptosis following flutamide (FLU) treatment at 100 μM in HCC-1954 cell line. (D) Histogram
showing the percentage of apoptosis following combination therapy with CI-1040 at 10 μM and flutamide at 20 μM in HCC-1954 cell line.
Combination index (CI) is calculated using the concentrations of monotherapies with these agents as shown in Figures 3B and 3C that induced
a level of apoptosis similar to that of combination therapy. (E) Histogram showing the percentage of apoptosis in control MDA-MB-453 cell line.
(F) Histogram showing the percentage of apoptosis following CI-1040 treatment at 20 μM in MDA-MB-453 cell line. (G) Histogram showing the
percentage of apoptosis following flutamide treatment at 80 μM in MDA-MB-453 cell line. (H) Histogram showing the percentage of apoptosis
and CI following combination therapy with CI-1040 at 10 μM and flutamide at 20 μM in MDA-MB-453 cell line.
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cancer, we generated xenograft tumors using MDA-MB-
453 cell line. This cell line was chosen for the xenograft
studies because it is a prototype of molecular apocrine
subtype and has been previously employed for in vivo
studies of the AR-ERK feedback loop [4,5,9,12].
PD0325901 treatment was carried out at 5 mg/kg/day
based on the results of our toxicity studies. Mouse treat-
ments were carried out in the following four groups: (1)
placebo pellet and daily oral gavage of carrier solution
(control group), (2) flutamide 25 mg/60 days pellet +
gavage of carrier solution (flutamide monotherapy),
(3) daily oral gavage of PD0325901 at 5 mg/kg/day +
placebo pellet (PD0325901 monotherapy) and (4) fluta-
mide pellet + PD0325901 (combination therapy). Six
mice were treated in each experimental group for 30
days, and fold change in tumor volume was calculated
as described in Materials and methods. We observed a
threefold lower tumor volume change in the combina-
tion therapy group compared to that of control (P<
0.01, Figure 6A). Importantly, mice treated with combi-
nation therapy had approximately 2.5-fold lower tumor
growth compared to that of monotherapy groups (P<
0.01, Figure 6A and 6B).
We next investigated the effect of different in vivo
treatments on cellular proliferation and angiogenesis
using harvested xenograft tumors. Proliferation index
and angiogenesis were assessed with IHC using Ki-67
and CD31 antibodies, respectively. The results were
then compared between different in vivo therapy groups.
Notably, we observed a proliferation index of 22% ± 2
Table 1 Combination indices for apoptosis induced by
flutamide and CI-1040 treatments
Cell line
Treatment HCC-1954 MDA-MB-453 HCC-202
CI-1040, μM 5 10 5 10 5 10
CI values FLU 20, μM 0.7 0.7 0.7 0.75 0.6 0.7
CI values FLU 30, μM 0.8 0.75 0.65 0.7 0.75 0.7
CI: combination index, FLU: flutamide. CI-1040 and flutamide concentrations
are shown in μM.
P1
A) B
)
C) D)
HCC-202 (control) HCC-202 (CI-30 μM)
HCC-202 (FLU-80 μM)
P1 P1
P1
C
ount
Count
Count
Count
Annexin V-FITC
Annexin V-FITC
Annexin V-FITC
Annexin V-FITC
HCC-202 (CI-10/FLU-30 μM)
: 5% : 37%
: 43% : 40%
CI= 0.7
Figure 4 Synergistic induction of apoptosis by AR and MEK inhibitors in HCC-202 cell line.(A) Histogram showing the percentage of
apoptosis (P1) in control (solvent-only treated) HCC-202 cell line using annexin V-FITC flow cytometry. (B) Histogram showing the percentage of
apoptosis following CI-1040 treatment at 30 μM (CI-30 μM) in HCC-202 cell line. (C) Histogram showing the percentage of apoptosis following
flutamide (FLU) treatment at 80 μM in HCC-202 cell line. (D) Histogram showing the percentage of apoptosis following combination therapy
with CI-1040 at 10 μM and flutamide at 30 μM in HCC-202 cell line. CI is calculated using the concentrations of monotherapies with these
agents as shown in Figures 4B and 4C that induced a level of apoptosis similar to that of combination therapy.
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in tumors treated with the combination therapy, which
was significantly lower than that of control (56% ± 2)
and monotherapy groups (flutamide: 39% ± 3,
PD0325901: 31% ± 4), (P< 0.05, Figure 6C to 6E).
Furthermore, angiogenesis was significantly lower in the
combination therapy group with a CD31-positive blood
vessel count of 5.3 ± 3 compared to that of control (44
± 6) and monotherapy groups (flutamide: 43 ± 7,
PD0325901: 24 ± 7) (P< 0.03, Figure 7A to 7D). More-
over, CD-31-positive blood vessels in the combination
therapy group were smaller and less distinct than those
in other groups (Figure 7B to 7D).
These findings indicate that the combination therapy
with fluatmide and PD0325901 has a significantly higher
level of in vivo activity in the reduction of xenograft tumor
growth, cellular proliferation and angiogenesis compared
to that of monotherapies with these agents. It is also nota-
ble that flutamide and PD0325901 monotherapies did not
significantly reduce tumor growth compared to the con-
trol group (Figure 6A and 7A). Therefore, a significantly
higher efficacy in the combination therapy group com-
pared to that of monotherapies suggests an in vivo synergy
between fluatmide and PD0325901.
Synergy between AR and MEK inhibitors overcomes
trastuzumab resistance
It is known that at least 50% of ER-/AR+ breast tumors
have ErbB2 overexpression, and anti-ErbB2 treatment is
an established part of management for this subgroup
[7,8,25]. Importantly, trastuzumab resistance is a major
clinical problem in this patient population [26]. There-
fore, we investigated the activity of combination therapy
with flutamide and CI-1040 in overcoming trastuzumab
resistance using molecular apocrine cell lines MDA-MB-
453 and HCC-1954 with known ErbB2 overexpression
[5,9]. We first examined the effect of trastuzumab treat-
ment at 10 to 80 μg/ml concentrations for 48 hours on
cell viability of MDA-MB-453 and HCC-1954 lines
using MTT assay. A solvent-only-treated group was
used as control. We observed a significant reduction in
cell viability by approximately 40% following trastuzu-
mab treatments in MDA-MB-453 cell line (P<0.01,
Figure 8A). In addition, trastuzumab activity reached a
plateau at 10 μg/ml concentration without any addi-
tional reduction in cell viability at higher concentrations
of this agent (Figure 8A). Furthermore, HCC-1954 cell
line showed an intrinsic resistance to trastuzumab treat-
ment with no significant reduction in cell viability at
any of the tested concentrations (Figure 8B).
Next, we generated a trastuzumab-resistant MDA-
MB-453 line (MDA-MB-453-R) as described in Materi-
als and methods. We confirmed that MDA-MB-453-R
cells are resistant to trastuzumab at 20 μg/ml concentra-
tion using MTT assay. MDA-MB-453-R line showed a
level of cell viability in the presence of trastuzumab
similar to that observed in untreated control line (Figure
-4
-2
0
2
4
Weight change in grams during study
CTL PD-5 PD-10 PD-15 PD-20
N= 2
* p<0.01
N= 2
PD-5 PD-10 PD-15 PD-20
0
1
2
3
4
5
6
Number of treatment days lost due to toxicity
* p<0.01
A) B)
Figure 5 Assessment of in vivo toxicity to MEK inhibitor PD0325901.(A) Weight change in grams is shown for each PD0325901 (PD)
treatment group in the MDA-MB-453 xenograft model. Weight change is the difference between pre- and post-treatment weight in each group.
PD0325901 treatments were carried out at 5, 10, 15 and 20 mg/kg/day for 30 days, and daily gavage of carrier solution was used as control. *P<
0.01 for PD-5/PD-10 vs. control groups and PD-5/PD-10 vs. PD-15/PD-20 groups using Mann-Whitney Utest. Error bars: ± 2 SEM. (B) Number of
days lost due to toxicity is shown for each PD0325901 treatment group in mouse xenograft model explained in Figure 5A. *P< 0.01 for PD-5/
PD-10 vs. PD-15/PD-20 groups.
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B
)
FLU FLU + PD
D)
Control Ki-67 (60X) 20 μMFLU + PD Ki-67 (60X)
CTL FLU PD FLU + PD
0
1
2
3
4
5
6
7
Fold-change in tumor volume
p< 0.01
N= 6
*
A)
Bars: +/- 2SEM
C)
E)
p< 0.05
N= 6
*
*
**
0
10
20
30
40
50
60
Percentage of Ki-67 staining
C
TL FL
U
PD FL
U
+ PD
Figure 6 The therapeutic effects of AR and MEK inhibitors on in vivo tumor growth and cellular proliferation.(A) Fold change in tumor
volume is shown for each in vivo treatment group using MDA-MB-453 xenograft model. CTL: control group; FLU: flutamide; PD: PD0325901. *P<
0.01 for the combination therapy group vs. control or monotherapy groups using Mann-Whitney Utest. Error bars: ± 2 SEM. (B) Representative
image of xenograft tumors in flutamide monotherapy and combination therapy groups. (C) Immunohistochemistry (IHC) was used to measure
the proliferation index in a control xenograft tumor. Staining was carried out using a Ki-67 rabbit polyclonal antibody. Original magnification,
×60. (D) IHC was used to measure Ki-67 proliferation index in a xenograft tumor treated with the combination therapy. Original magnification,
×60. (E) Tumor proliferation indices using Ki-67 nuclear staining for the xenograft experiments. *P< 0.05 for monotherapy groups vs. control and
**P< 0.05 for combination therapy vs. monotherapy groups. Error bars: ± 2 SEM.
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8C). In contrast, the control line demonstrated a signifi-
cant reduction in cell viability following trastuzumab
treatment at 20 μg/ml concentration for 48 hours (P<
0.01, Figure 8C). Subsequently, we calculated CI values
to assess synergy between flutamide and CI-1040 in
MDA-MB-453-R line. Flutamide and CI-1040 treat-
ments were carried out at the same four dose combina-
tions applied before in the nonresistant line (CI-1040 (5
μM)/flutamide (5 μM), CI-1040 (10 μM)/flutamide (5
μM), CI-1040 (5 μM)/flutamide (10 μM), and CI-1040
(10 μM)/flutamide (10 μM)). Importantly, we observed a
synergy at all four dose combinations in MDA-MB-453-
R line with CI values of 0.68 to 0.76 (Figure 8D).
The synergy between flutamide and CI-1040 in MDA-
MB-453-R line raises the possibility of a functional role
for ERK phosphorylation in the process of trastuzumab
resistance in molecular apocrine cells. To investigate
this possibility, we assessed the level of phosphorylated
B
)
C) D)
Control CD31 (40X) 40 μM
FLU + PD CD31 (40X)
PD CD31 (40X)
A)
0
10
20
30
40
50
60
Number of CD31-positive blood vessels
CTL FLU PD FLU + PD
*
**
p< 0.03
N= 6
Figure 7 The therapeutic effect of AR and MEK inhibitors on in vivo angiogenesis.(A) Angiogenesis index for each in vivo treatment
group. Angiogenesis was measured as the number of CD-31-positive blood vessels in a cross-section of each xenograft tumor. CTL: control
group; FLU: flutamide; and PD: PD0325901. *P< 0.03 for PD0325901 monotherapy vs. control and **P< 0.03 for combination therapy vs.
monotherapy groups using Mann-Whitney Utest. Error bars: ± 2 SEM. (B) Immunohistochemistry (IHC) was used to measure angiogenesis in a
control xenograft tumor. Staining was performed using a CD31 rabbit polyclonal antibody. Original magnification, × 40. (C) IHC was used to
measure angiogenesis in a PD0325901 monotherapy tumor. Original magnification, × 40. (D) IHC was used to measure angiogenesis in a
xenograft tumor treated with combination therapy. Original magnification, × 40.
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MDA-MB-453 HCC-1954
****
p<0.01
N=8
0
0.2
0.4
0.6
0.8
Absorbance at 570 nM by MTT assay
0
0.5
1
1.5
2
Absorbance at 570 nM by MTT assay
00
10 10
20 20
60 60
80 80
Trastuzumab
A) B)
p >0.1
N=8
(μg/ml) Trastuzumab
(μg/
ml)
0
0.2
0.4
0.6
0.8
1
Absorbance at 570 nM by MTT assay
MDA-MB-453
C)
CTL CTL R
+ Trastuzumab (20
μg/
ml)
*
p<0.01
N=8
E)
42 KD
Phospho-ERK
RR-ph:
Total-ERK
α−Tubulin
42 KD
CTL CTL + Tras R
MDA-MB-453
23
0
0.2
0.4
0.6
1
1.2
Absorbance at 570 nM by MTT assay
D)
N=8
Bars:+/-2SEM
CTL
CI-5/FLU-5
CI-10/FLU-5
CI-5/FLU-10
CI-10/FLU-10
CI-8
FLU-35
0.8
CI-15
FLU-50
CI-10
FLU-40
CI-18
FLU-80
CI= 0.76
CI= 0.76
CI=0.75
CI=0.68
MDA-MB-453-R
α−Tubulin
Total-ERK
42 KD
MDA-MB-453-R
F)
CTL-R CI-5/FLU-5 CI-5/FLU-10
42 KD
Phospho-ERK
< 0.05 < 0.05
RR-Total: 1111
RR-ph:
RR-Total:
Figure 8 Synergy between AR and MEK inhibitors in trastuzumab-resistant cells.(A) MTT assay was used to measure cell viability in MDA-
MB-453 cell line following trastuzumab treatment at 10 to 80 μg/ml concentrations. CTL: control. *P< 0.01 for trastuzumab groups vs. control.
Error bars: ± 2 SEM. (B) Cell viability in HCC-1954 cell line following trastuzumab treatment as described in Figure 8A. (C) Cell viability in
trastuzumab-resistant MDA-MB-453 (R) compared to the untreated control and control line treated with trastuzumab at 20 μg/ml. *P< 0.01 for R
vs. treated control cells. (D) Combination indices (CI) for flutamide (FLU) and CI-1040 combination therapy in MDA-MB-453-R line using MTT
assay. Therapies were carried out with flutamide at 5 and 10 μM with each concentration of CI-1040 at 5 and 10 μM (CI-5 and CL-10). The
concentrations of fluatmide and CI-1040 monotherapies with an effect similar to that of each combination therapy are depicted. Error bars: ± 2
SEM. (E) Western blot analysis was used to measure the phosphorylated (ph) and total ERK levels in MDA-MB-453-R, control MDA-MB-453 and
control MDA-MB-453 treated with trastuzumab at 20 μg/ml concentration (CTL + Tras). Fold changes in band densities (RR) were measured
relative to the control. (F) Western blot analysis was used to measure the phosphorylated and total ERK levels in MDA-MB-453-R line following
combination therapies with CI-1040 (5 μM)/flutamide (5 μM) and CI-1040 (5 μM)/flutamide (10 μM). RR values were measured relative to the
untreated MDA-MB-453-R line.
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and total ERK proteins in untreated MDA-MB-453 con-
trol, MDA-MB-453 control treated with trastuzumab at
20 μg/ml, and MDA-MB-453-R cell lines. Importantly,
MDA-MB-453-R line showed a threefold higher level of
ERK phosphorylation compared to that of untreated
control (Figure 8E). In addition, there was an induction
of ERK phosphorylation by twofold following trastuzu-
mab treatment for 48 hours in the control line (Figure
8E). It is notable that there was no difference between
the levels of total ERK across these experiments (Figure
8E). Moreover, combination therapies with CI-1040 (5
μM)/flutamide (5 μM) and CI-1040 (5 μM)/flutamide
(10 μM) completely abrogated ERK phosphorylation in
MDA-MB-453-R line (Figure 8F). Taken together, these
data suggest that the synergy between flutamide and CI-
1040 can overcome trastuzumab resistance in molecular
apocrine cells. In addition, this combination therapy
abrogates the induction of ERK phosphorylation
observed in trastuzumab-resistant cells.
Discussion
Management of ER-breast cancer is challenging due to
the limited therapeutic targets available in this disease.
Heterogeneity of ER-breast cancer contributes to this
challenge, and therefore identification of novel targeted
therapies requires a robust biological understanding of
different ER-subtypes. We have recently identified a
positive feedback loop between the AR and ERK signal-
ing pathways in molecular apocrine subtype of ER-
breast cancer [12]. In this process, AR regulates ERK
phosphorylation and kinase activity as well as the phos-
phorylation of ERK target proteins RSK1 and Elk-1 [12].
Notably, AR inhibition using flutamide abrogates ERK
phosphorylation in a dose-dependent manner, and AR
activation using DHT leads to an increase in ERK phos-
phorylation mediated through ErbB2 [12]. In turn, ERK
signaling regulates AR expression mediated through
transcription factor CREB1 [12].
In this study, we explored the therapeutic implications
of the AR-ERK feedback loop in molecular apocrine
breast cancer. This was investigated using the combina-
tion therapy with AR and MEK inhibitors, which are
clinically available and constitute effective targeted
therapies to block the AR and ERK signaling pathways,
respectively [14,16]. We applied CI-1040 and
PD0325901 for in vitro and in vivo inhibition of MEK,
respectively. This approach was used due to the fact
that CI-1040 has been commonly used to study the
effect of MEK inhibitors on cell lines and PD0325901 is
a derivative of CI-1040 with a better oral bioavailability,
which makes this agent more suitable for in vivo studies
[19,21-23].
Importantly, we demonstrated synergistic CI values for
the combination therapy with AR inhibitor flutamide
and MEK inhibitor CI-1040 across three molecular
apocrine cell lines (Figures 1 to 4 and Table 1). Further-
more, this synergy was present at four dose combina-
tions in each cell line using both cell viability and
apoptosis assays, suggesting a reproducible synergy
between flutamide and CI-1040 in molecular apocrine
cells. Moreover, we showed in vivo that the combination
therapy with flutamide and MEK inhibitor PD0325901
has a significantly higher therapeutic efficacy in reducing
tumor growth, cellular proliferation and angiogenesis
compared to monotherapies with these agents in a
xenograft molecular apocrine model (Figures 6 and 7).
A combination therapy approach provides an attrac-
tive option in the management of ER-/AR+ breast can-
cer, since it exploits the synergy between AR and MEK
inhibitors and at the same time minimizes their poten-
tial toxicities by requiring a lower dose of each agent in
the combination setting. This is particularly relevant for
MEK inhibitors, as higher doses of these drugs have
been associated with significant toxicities in clinical
trials [27-29]. In fact, our in vivo data clearly demon-
strated that higher doses of PD0325901 have toxicity in
mice, and this was absent at the 5 mg/kg/day dose used
for the combination therapy studies (Figure 5). Another
advantage of using lower doses of PD0325901 and fluta-
mide in xenograft studies is to show an in vivo synergy
between AR and MEK inhibitors. A similar approach
has been previously applied to assess in vivo synergy for
other agents [30,31]. Notably, we observed that mono-
therapies did not significantly reduce tumor growth in
mice, and therefore a markedly lower tumor growth
with the combination therapy compared to that of con-
trol and monotherapy groups suggests an in vivo
synergy between flutamide and PD0325901 (Figures 6A
and 7A).
The AR-ERK positive feedback loop forms the mole-
cular basis for the synergy observed between AR and
MEK inhibitors [12]. This is supported by the fact that
flutamide synergistically enhances the effect of MEK
inhibitor CI-1040 in reducing the level of ERK phos-
phorylation in molecular apocrine cells [12]. In addition,
CI-1040 treatment results in a reduction of AR expres-
sion in molecular apocrine cell lines [12]. Furthermore,
we have previously shown a synergy between flutamide
and Cdc25A inhibitor PM-20 in molecular apocrine
cells that was associated with a decrease in the phos-
phorylation levels of ERK target proteins RSK1 and Elk-
1 [9]. Therefore, cross-regulation between the AR and
ERK signaling pathways provides an attractive therapeu-
tic target in molecular apocrine breast cancer. Moreover,
a number of potent second-generation AR inhibitors
such as abiraterone and MDV3100 are currently being
studied in androgen-refractory prostate cancer [32,33].
Sincethereisgrowingevidencetosupporttheroleof
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AR as a target for therapy in molecular apocrine breast
cancer, the new AR inhibitors may potentially provide
additional treatment options in the management of this
disease.
ErbB2 amplification and overexpression are present in at
least 50% of molecular apocrine tumors, and the affected
patients are usually started on trastuzumab early in the
course of their disease [7,8,25]. However, there is a high
rate of intrinsic resistance to trastuzumab monotherapy
among patients with ErbB2-positive breast cancer, ranging
from 66% to 88% [26,34]. Furthermore, patients with a pri-
mary response to trastuzumab monotherapy have a short
median time to progression of only 4.9 months [35]. As a
result, trastuzumab monotherapy is commonly combined
with chemotherapy agents to increase response rates and
time to disease progression; however, this approach is
associated with more side effects [35,36]. In this study, we
demonstrated that flutamide and CI-1040 combination
leads to a synergistic reduction of cell viability in HCC-
1954 and MDA-MB-453-R cell lines with intrinsic and
acquired resistance to trastuzumab, respectively (Figures
2Cand8Ato8D).Therefore,combinationtherapywith
AR and MEK inhibitors may provide an effective treat-
ment option in ErbB2-positive molecular apocrine patients
with trastuzumab resistance.
A number of different mechanisms have been pro-
posed for trastuzumab resistance, including compensa-
tory signaling and altered downstream signaling
[26,37,38]. We found an increased level of ERK phos-
phorylation shortly after trastuzumab treatment in mole-
cular apocrine cells (Figure 8E). This effect on ERK
phosphorylation following acute exposure to trastuzu-
mab has been reported in other ErbB2-positive cell lines
and is similar to MAPK/ERK activation in cells stimu-
lated with exogenous ErbB ligands [39,40]. Importantly,
we observed that the level of ERK phosphorylation
further increased in trastuzumab-resistant MDA-MB-
453-R cell line, which was abrogated following flutamide
and CI-1040 combination therapy (Figures 8E and 8F).
These findings are in agreement with the previous
reports that trastuzumab-resistant cells are exquisitely
sensitivetoMEKinhibition[41].Therefore,the
observed induction of ERK in trastuzumab-resistant
molecular apocrine cells may render these cells depen-
dent on MAPK/ERK signaling and sensitizes them to
the synergy between AR and MEK inhibitors.
Conclusions
In this study, we investigated the AR-ERK feedback loop
as a therapeutic target in molecular apocrine breast can-
cer and demonstrated in vitro and in vivo synergies
between AR and MEK inhibitors in this subtype.
Furthermore, we showed that the combination therapy
with these inhibitors can overcome trastuzumab
resistance in molecular apocrine cells. Therefore, a com-
bination therapy strategy with AR and MEK inhibitors
may provide an attractive therapeutic option for mole-
cular apocrine breast cancer. Future clinical trials are
required to test the application of this approach in
patient management.
Abbreviations
DHT: dihydrotestosterone; ERK: extracellular signal-regulated kinase; MAPK:
mitogen-activated protein kinase; MEK: mitogen-activated protein kinase
kinase.
Acknowledgements
We thank Ms Kim Woolley from the University of Queensland Biological
Research Facility for assistance with animal work. This study is funded by
grants from the University of Queensland Pathfinder Program; Queensland
Department of Employment, Economic Development and Innovation; and
Princess Alexandra Hospital Private Practice Trust Fund. KMC is supported by
a Cancer Council Queensland Scholarship.
Authors’contributions
AN conceived the study, designed the experiments and drafted the
manuscript. AN, KMC and JL carried out the experiments. All authors read
and approved the final manuscript.
Competing interests
AN is named on a patent application related to the content of this
manuscript. All other authors declare that they have no competing interests.
Received: 8 February 2011 Revised: 17 March 2011
Accepted: 1 April 2011 Published: 1 April 2011
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doi:10.1186/bcr2858
Cite this article as: Naderi et al.: Synergy between inhibitors of
androgen receptor and MEK has therapeutic implications in estrogen
receptor-negative breast cancer. Breast Cancer Research 2011 13:R36.
Naderi et al.Breast Cancer Research 2011, 13:R36
http://breast-cancer-research.com/content/13/2/R36
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