Published OnlineFirst July 3, 2012; DOI: 10.1158/1535-7163.

MCT-11-0730

Molecular
Cancer
Therapeutics

Preclinical Development

Dacomitinib (PF-00299804), an Irreversible Pan-HER

Inhibitor, Inhibits Proliferation of HER2-Amplied Breast
Cancer Cell Lines Resistant to Trastuzumab and Lapatinib
Ondrej Kalous1, Dylan Conklin1, Amrita J. Desai1, Neil A. O'Brien1, Charles Ginther1,
Lee Anderson1, David J. Cohen1, Carolyn D. Britten1, Ian Taylor2, James G. Christensen2,
Dennis J. Slamon1, and Richard S. Finn1

Abstract
The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other
malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a
significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro
activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of
human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with
variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast
epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to
determine IC50 values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified
lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated
with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority
of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0G1 arrest and an
induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and
acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to
lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative
effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers
resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 197887. 2012 AACR.

Introduction
The HER (ErbB) receptor family consists of 4 type I
receptor tyrosine kinases, which include the EGF receptor (EGFR, HER1, and ErbB-1), HER2 (HER2/neu,
ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4), and their
associated ligands, including EGF, neuregulins (NRG14), TGF-a, amphiregulin, betacellulin, heparin-binding
EGF-like growth factor and epiregulin. Ligand binding
to the extracellular domain leads to homodimerization
or heterodimerization of the receptors and autophosphorylation within the intracellular domain. This
results in the activation of signaling cascades involved
in mediating cell growth and differentiation. Unlike
Authors' Afliations: 1Department of Medicine, Division of Hematology/
Oncology, Geffen School of Medicine at UCLA, Los Angeles; and 2Pzer
Global Research and Development, Pzer Inc., San Diego, California
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Richard S. Finn, Department of Medicine, Division
of Hematology/Oncology, Geffen School of Medicine at UCLA, 10833 Le
Conte Ave, 11-934 Factor Bldg, Los Angeles, CA 90095. Phone: 310-5862091; Fax: 310-586-6830; E-mail: Rnn@mednet.ucla.edu
doi: 10.1158/1535-7163.MCT-11-0730
2012 American Association for Cancer Research.

1978

other family members, HER2 has no known ligand and,

under normal conditions, relies on forming heterodimers with other family members that have ligand interactions. In cancer, this tight regulation of HER family
signaling is disrupted and contributes to transformation
(1).
HER2 amplification occurs in 20% to 25% of patients
with breast cancer. It is associated with a poor prognosis
and is a validated target for therapy (2, 3). Trastuzumab
(Herceptin; Genentech), a humanized monoclonal antibody that binds to the extracellular domain of HER2,
has been shown to improve survival in the metastatic
and adjuvant settings (411), and lapatinib (Tykerb;
GlaxoSmithKline), a small-molecule selective inhibitor of
the HER2 and EGFR tyrosine kinases, is approved for
HER2-positive advanced breast cancer that progressed
after trastuzumab-based therapy (12).
Pan-HER inhibitors were developed with the goal of
improving therapeutic response and overcoming drug
resistance that is seen with trastuzumab and lapatinib.
Unlike lapatinib, which is a reversible inhibitor that competes with ATP at the binding sites (13), most pan-HER
inhibitors bind to the kinases in a covalent and irreversible
form (14, 15). It is hypothesized that the prolonged suppression of multiple targets within the HER receptor

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Published OnlineFirst July 3, 2012; DOI: 10.1158/1535-7163.MCT-11-0730

Dacomitinib (PF-00299804) and Breast Cancer

Lapatinib

Dacomitinib

Figure 1. Chemical structures of

investigated molecules in this article.

N
N

H3CO

N
N

HN
O

HN

CI
F

H2O

HN

HN
O

O
O

CI
F

family may overcome the problems with adaptability and

redundancy in signaling pathways.
Dacomitinib (PF-00299804; chemical structure in Fig. 1)
is a second-generation irreversible pan-HER receptor
tyrosine kinase inhibitor (selectively inhibiting EGFR,
HER2, and HER4) under clinical development (16, 17).
Dacomitinib showed marked activity in several xenograft
models with variable levels of HER family receptors,
including lung tumors resistant to EGFR small molecule
tyrosine kinase inhibitors (16, 18). The primary aim of this
study was to describe the in vitro activity of dacomitinib in
a panel of 47 human breast cancer and immortalized
breast cell lines, to identify potential biomarkers of
response and/or resistance to guide clinical development.

Materials and Methods

Cell lines, cell culture, and reagents
The cell line panel included 44 breast cancer lines and 3
immortalized breast epithelial cell lines representing the
known molecular subgroups of breast cancer and has
been described in detail previously (19). The panel included MDA-MB-415, MDA-MB-134, HCC-1500, ZR-75-30,
HCC-202, HCC-1419, HCC-38, HCC-70, HCC-1187,
HCC-1806, HCC-1937, HCC-1954, MDA-MB-436, HCC1569, Hs578t, HCC-1143, MDA-MB-175, BT-474, SK-BR-3,
MDA-MB-361, UACC-893, UACC-812, UACC-732, T47D, MDA-MB-453, MDA-MB-468, CAMA-1, MDAMB-157, MCF-7, MDA-MB-435, ZR-75-1, BT-20, MDAMB-231, BT-549, DU4475, HCC-1395, HCC-2218, 184A1,
184B5, and MCF-10A that were purchased from American
Type Culture Collection. The cell lines EFM-192A, KPL-1,
EFM-19, COLO-824, and CAL-51 were obtained from the
German Tissue Repository DSMZ. Both cell line banks
carry out the cell lines authentication by short tandem
repeat analysis. The cell lines SUM-190 and SUM-225 were
obtained from the University of Michigan (Ann Arbor,
MI). Upon receipt, all cell lines were assessed for Mycoplasma contamination using a multiplex PCR method (20),
and mitochondrial DNA from the cells was sequenced to
confirm their correct identity (21). Cell lines were then
expanded and these procedures were repeated for all cell
lines before cryopreservation. All cell lines were passaged
for less than 6 months before use in this study. Trastuzumab-resistant BT-474 (BT-474-TR) and SKBR3 (SK-BR-3TR) cell lines (pools of resistant cells) were established
after serial passage in the continued presence of trastu-

www.aacrjournals.org

zumab 105 mg/mL. Lapatinib-resistant BT-474 (BT-474LR) and SK-BR-3 (SK-BR-3-LR) cell lines (pools of resistant cells) were established after serial passage in the
presence of gradually increasing concentrations of lapatinib (0.17 mmol/L). These lines were established in our
laboratories, authenticated, and checked for Mycoplasma
contamination as described above.
HER2 amplification was defined as greater than 2
HER2/neu FISH signals per chromosome 17 centromere
FISH signal (22). A SpectrumOrange labeled HER2/neu
probe (ABBOTT Molecular) and SpectrumGreen labeled
chromosome 17 alpha-satellite centromere probe were
used (ABBOTT Molecular).
Proliferation assays
Cells were seeded in duplicate at 5
103 to 5
104 cells
per well in 24-well plates, and growth inhibition data was
calculated as described previously (19). Briefly, day after
plating, dacomitinib was added at 10 mmol/L and 2-fold
dilutions over 12 concentrations were carried out to generate a doseresponse curve. Control wells without the
drug were also seeded. The cells were counted on day 1
when the drug was added, as well as after 6 days when the
experiment ended. After the trypsinization cells were
placed in an Isotone solution and immediately counted
using a Coulter Z1 particle counter (Beckman Coulter,
Inc.). The suspension cultures were counted using a Coulter Vi-Cell counter (Beckman Coulter, Inc.).
Microarray analysis of cell lines
Agilent microarray analyses were developed for each
cell line as described previously (23, 24). These data are
available with GEO accession number GSE18496.
Western blots and protein quantification
To determine the effect of dacomitinib on the expression
of analyzed proteins, cells in log-phase growth were
treated with 0.1 or 1 mmol/L dacomitinib and lysates
were taken as described (19). Details about antibodies
and immunoprecipitation techniques can be found in
Supplementary Material.
Cell-cycle analysis and apoptosis studies
The effects of dacomitinib on the cell cycle were
assessed using Nim-DAPI (40 , 6-diamidino-2-phenylindole) staining (NPE Systems). The cells were plated

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1979

Published OnlineFirst July 3, 2012; DOI: 10.1158/1535-7163.MCT-11-0730

Kalous et al.

evenly in control and experimental wells, allowed to

grow to log phase, and then treated with 100 nmol/L or
1 mmol/L dacomitinib for 48 hours or 5 days and analyzed
as described previously (19).
For apoptosis, cells were plated evenly in control and
experimental wells, allowed to grow to log phase, and
then treated with 100 nmol/L or 1 mmol/L dacomitinib for
48 hours or 5 days and analyzed using Annexin-Vfluorescein isothiocyanate (FITC) as described (19).
Statistical analysis
CochranMantelHaenszel (CMH) c2 analysis was carried out using the PROC FREQ function in SAS for
Windows version 9.2 (SAS Institute, Inc.). Simple linear
regression was also carried out in SAS using PROC REG
function.
Pearson correlation coefficients and their corresponding P values were calculated using the PROC CORR
function in SAS. Graphs were created in Microsoft Excel.

Results
Dacomitinib preferentially inhibits growth of HER2amplified breast cancer cell lines in vitro
A panel of 44 human breast cancer cell lines, representing
luminal, nonluminal subtypes, and 3 immortalized breast
epithelial lines (19), was used to test the antiproliferative
effect of dacomitinib (PF-00299804). The calculated IC50
values for each cell line and its molecular classification, as
well as HER2 amplification status (by FISH) and estrogen
receptor (ER) status, were determined (Table 1 and Fig. 2).
Sensitivity was defined as IC50 < 1 mmol/L (the rationale for
selecting this cut-off is discussed below).
As a group, the HER2-amplified cell lines were most
sensitive to growth inhibition by dacomitinib (IC50 < 1
mmol/L in 14 of 16 lines; 87.5%) as compared with 5 of 28
(17.9%) of HER2-nonamplified lines (excluding immortalized lines). MDA-MB-453 and UACC-732 were the only
HER2-amplified lines resistant to the antiproliferative
effect of the compound (IC50 > 1 mmol/L). The nonluminal
lines were the most resistant to this compound. c2 (CMH)
analysis was carried out to compare the response classification (response defined as IC50 < 1 mmol/L) in the
HER2-amplified versus nonamplified groups of cell lines.
HER2-amplified lines were far more likely to be classified
as responders to dacomitinib [RR, 3.39; 95% confidence
interval (CI), 1.826.33; P < 0.0001].
ER status correlated with response when treated as a
separate, independent variable (P 0.015). However, ER
status was highly cross-correlated with HER2 status. After
controlling for HER2 status by stratified analysis, ER
status was no longer a statistically significant predictor
of response (P 0.17).
Quantitative HER2 expression associates with
response to dacomitinib
We also aimed to explore the quantitative effect of
HER2 expression on response to dacomitinib, in compar-

1980

Mol Cancer Ther; 11(9) September 2012

ison with the qualitative amplified/nonamplified approach discussed above. Specifically, we wanted to determine whether a higher relative expression of HER2
mRNA is associated with a stronger response to dacomitinib, even within the HER2-amplified and nonamplified
groups. For this purpose, we ran a linear regression
analysis using log(IC50) as the response variable and
log-ratio of expression by microarray (compared with
mixed breast cancer RNA reference pool) as the predictor
variable. This analysis was carried out first on the entire
panel of lines, then separately on each group of HER2amplified and nonamplified lines. The association was
significant in all 3 analyses. (Full panel: b 1.07, SE
0.15, P < 0.0001, r 0.73; HER2 amplified: b 1.6, SE
0.57, P 0.014, r 0.6; HER2-nonamplified: b 1.57,
SE 0.6, P 0.014, r 0.44; Supplementary Fig. S1).
However, the significance of this relationship in cell
lines with higher IC50 values (resistant) is unknown. In
a separate analysis for HER2-amplified lines, we found
that the total HER2 protein levels (quantified by Western
blot) correlated with HER2 mRNA levels by microarray
(r 0.67, P 0.004). Finally, we found an association
between the HER2 protein levels and response to dacomitinib in HER2-amplified lines (b 1.99, SE 0.55, P
0.003, r 0.69; Supplementary Fig. S2).
All 4 HER family receptors were analyzed for their
relationship between mRNA expression and IC50. The
association between HER2 expression and response to
dacomitinib was by far the strongest. In addition, there
was a marginal inverse correlation between HER3 mRNA
levels and log(IC50) values (r 0.25, P 0.09). No
correlation was observed between the response to dacomitinib and EGFR mRNA (r 0.07, P 0.62) and HER4
mRNA (r 0.05, P 0.75) levels, respectively (Supplementary Fig. S3).
Biochemical effects of dacomitinib on signal
transduction
The effects of dacomitinib on HER2, EGFR, HER4, AKT,
and ERK activation were determined using Western blot
analysis of a subset of lines with variable levels of HER
receptors and sensitivities to dacomitinib.
After a 10-minute exposure to 100 nmol/L or 1 mmol/L
of dacomitinib, we observed no effect on total HER2, AKT,
and ERK in a majority of lines and a minor decrease in total
EGFR in several lines. Most of the selected lines expressed
very low baseline levels of total HER4 that mostly did not
change after the treatment with dacomitinib. Dacomitinib
caused an inhibition of HER2 phosphorylation in all lines
with detectable baseline phosphorylation (Fig. 3, densitometry in Supplementary Table S1). Dacomitinib caused
an inhibition of EGFR phosphorylation in sensitive lines,
but only 2 resistant lines (MDA-MB-453 and MDA-MB231). Dacomitinib decreased the phosphorylation of
HER4 in sensitive lines with detectable baseline phosphorylation. Dacomitinib inhibited phosphorylation of
AKT in all tested sensitive lines, but only 2 resistant lines
(HCC-70 and MDA-MB-453). Similarly, dacomitinib

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Published OnlineFirst July 3, 2012; DOI: 10.1158/1535-7163.MCT-11-0730

Dacomitinib (PF-00299804) and Breast Cancer

Table 1. The calculated IC50 for each cell line and its molecular classication
Cell line

IC50 value (mmol/L)

IC50 SE

Breast cancer subtype

HER2 status

ER status

HCC-202
ZR-75-30
MDA-MB-175
HCC-2218
SUM-225
HCC-1419
184A1
MCF-10A
SK-BR-3
EFM-192A
UACC-893
BT-474
MDA-MB-361
UACC-812
HCC-1954
184B5
SUM-190
ZR-75-1
HCC-1500
MDA-MB-415
HCC-1143
HCC-1569
EFM-19
COLO-824
MDA-MB-157
T-47D
MDA-MB-468
HCC-70
HCC-1187
UACC-732
MDA-MB-134
HCC-1395
MDA-MB-453
HCC-1937
HCC-1806
BT-549
HCC-38
MDA-MB-436
BT-20
CAMA-1
CAL-51
MDA-MB-435
Hs578T
DU-4475
MCF-7
MDA-MB-231
KPL-1

<0.005
<0.005
<0.005
<0.005
0.006
0.006
0.007
0.008
0.015
0.016
0.017
0.018
0.03
0.04
0.06
0.08
0.11
0.24
0.53
0.67
0.73
0.77
1.07
1.20
1.36
1.41
1.42
1.44
1.46
1.69
1.74
1.77
2.00
2.04
2.05
2.22
2.40
2.56
2.58
2.68
2.76
2.92
2.92
3.55
3.67
4.23
4.88

n/a
n/a
n/a
n/a
0.002
0.002
0.0003
0.001
0.003
0.002
0.006
0.011
0.01
0.00
0.01
0.00
0.01
0.12
0.30
0.07
0.05
0.17
0.16
0.00
0.08
0.69
0.03
0.06
0.16
0.09
0.17
0.64
0.11
0.02
0.18
0.03
0.15
0.00
0.29
0.46
0.46
0.00
0.28
0.99
0.35
0.11
0.57

Luminal
Luminal
Luminal
Luminal
Luminal
Luminal
Immortalized
Immortalized
Luminal
Luminal
Luminal
Luminal
Luminal
Luminal
Basal
Immortalized
Luminal
Luminal
Luminal
Luminal
Basal
Post-EMT
Luminal
Basal
Post-EMT
Luminal
Basal
Basal
Basal
Luminal
Luminal
Post-EMT
Luminal
Post-EMT
Basal
Post-EMT
Basal
Post-EMT
Basal
Luminal
Post-EMT
Post-EMT
Post-EMT
Basal
Luminal
Post-EMT
Luminal

Amplied
Amplied
Normal
Amplied
Amplied
Amplied
n/a
n/a
Amplied
Amplied
Amplied
Amplied
Amplied
Amplied
Amplied
n/a
Amplied
Normal
Normal
Normal
Normal
Amplied
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Amplied
Normal
Normal
Amplied
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal

Positive
Positive
Positive
Positive
Negative
Positive
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Positive
Positive
Positive
Positive
Negative
Negative
Positive
Negative
Negative
Positive
Negative
Negative
Negative
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
Positive
Negative
Positive

NOTE: Included are molecular subtype and HER2 and ER status. HER2-amplied cell lines were most sensitive to the growth inhibition
effects of dacomitinib. Post-EMT, cell lines classied as representing breast cancers that had undergone an epithelial-to-mesenchymal
transition.
Abbreviation: n/a, not applicable.

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Kalous et al.

10

IC50 (mol/L)

0.1

0.01

* * * *

HC
C
M ZR -2 0
DA - 7 2
-M 5-3
HC B- 0
1
C 75
SU -2 2
1
HC M-2 8
C- 2 5
14
1 1
M 84 9
CF A1
SK -1 0
EF - B A
M R
UA - 19 -3
CC 2 A
M B -89
DA T 3
-M -47
UA B- 4
C 36
HC C - 1
C- 81 2
19
1 54
SU 8 4
M B5
Z -19
HC R- 7 0
M C 5
DA - -1
-M 1 50
HC B- 0
4
C 1
HC -1 5
C- 143
1
E 56
C FM 9
M O L -1
DA O 9
-M -82
B- 4
1
M
DA T 5 7
-M -4 7
B- D
H 46
HC C C 8
-7
U C- 0
M AC 1 18
DA C 7
-M -73
H B 2
M C C -13
DA - 4
-M 1 39
HC B- 5
4
C 5
HC -1 3
C- 937
1
BT 806
M H -54
DA C 9
-M C-3
B- 8
4
B 36
CA T-2
M 0
M C ADA A 1
-M L- 5
B- 1
Hs 43 5
DU 578
-4 T
4
M
DA MC 75
-M FB- 7
2
KP 3 1
L1

0.001

Figure 2. Inhibitory concentration and cell type. Bar graph of IC50 values (mmol/L) and cell type. Cell lines are color coded by subtype: dark blue bars (stripes),
HER2 amplied; light blue, luminal; yellow, nonluminal/undergone an epithelial-to-mesenchymal transition; red, nonluminal; turquoise, immortalized.  , cell
lines with IC50 < 0.005 mmol/L.

caused a decrease in phosphorylation of ERK in all lines,

except for resistant MDA-MB-231.
Effects of dacomitinib on cell cycle and apoptosis
The effects of dacomitinib on the cell cycle were
analyzed in a subset of both sensitive and resistant cell
lines. Cells were exposed to dacomitinib at 1 mmol/L for
48 hours, and then flow cytometry was carried out using
Nim-DAPI staining. We observed a significant G0G1
cell-cycle arrest in the very sensitive HER2-amplified
BT-474 and SK-BR-3 lines and less pronounced yet
statistically significant G0G1 cell-cycle arrest in the
resistant HER2-normal MCF-7 line. Dacomitinib caused
no changes in cell cycle in the less sensitive ZR-75-1 and
HCC-1143 lines and in the resistant MDA-MB-231 line
(Fig. 4).
Similarly, the effect of dacomitinib on apoptosis was
determined in the same subset of lines. For this assay, cells
were exposed to 1 mmol/L of dacomitinib for 5 days and
then analyzed with a dual stain flow cytometry protocol
using Annexin-VFITC and propidium iodide. A significant increase in apoptosis was seen in the very sensitive
BT-474 and SK-BR-3 lines and in the less sensitive HCC1143. The changes in ZR-75-1 (P 0.08) did not reach a
statistical significance. No changes were observed in
resistant lines (Fig. 5).
In a separate experiment, we analyzed the differences in the cell cycle and apoptosis between the 48hour and 5-day treatment with dacomitinib in the
sensitive BT-474 and SK-BR-3 lines. There was no
difference in the cell cycle between these 2 time points.
We observed no induction in apoptosis for SK-BR-3 at
48 hours, which indicates that dacomitinib needs
more than 48 hours to induce apoptosis in this line. In
the BT-474 line, we observed an induction of apoptosis
at 48 hours, albeit at lesser degree than at 5 days
(Supplementary Fig. S4).

1982

Mol Cancer Ther; 11(9) September 2012

Together, these data suggested that the antiproliferative

effect of dacomitinib is mediated by both inhibition of the
cell cycle and the induction of apoptosis.
Dacomitinib overcomes acquired resistance to
trastuzumab and lapatinib in vitro
BT-474-trastuzumab- (BT-474-TR) and SK-BR-3-trastuzumabresistant (SK-BR-3-TR) cell lines were established
in our laboratory after prolonged exposure of the parental
cell lines in medium with 105 mg/mL trastuzumab over
several months (25). Despite resistance to trastuzumab,
dacomitinib inhibited the proliferation of both these cell
lines with a similar potency as their parental cell lines
(Table 2). Although the exact mechanism of trastuzumab
resistance in these lines is unknown, they clearly maintain
dependence on HER signaling.
BT-474-lapatinib- (BT-474-LR) and SK-BR-3-lapatinib
resistant (SK-BR-3-LR) cell lines were developed in our
laboratory after prolonged exposure of the parental cell
lines to progressively higher concentrations of lapatinib
over several months. Unlike the trastuzumab-resistant
cell lines, the sensitivity of these lines to dacomitinib was
less than their parental counterparts. For the BT-474-LR,
there was a 1.7-fold increase in IC50, and for the SK-BR-3LR, there was a 23-fold increase in IC50 (Table 2). However, the decrease in sensitivity to dacomitinib in the
lapatinib-resistant lines compared with their parental
lines was much less pronounced than their decrease in
sensitivity to lapatinib. Specifically, there is an 89-fold and
119-fold increase in lapatinib IC50 for BT-474-LR and SKBR-3-LR compared with respective parental cell lines, and
both lapatinib-resistant cell lines would still be considered
sensitive to dacomitinib with low IC50 values of 0.031 and
0.339 mmol/L, respectively.
These data indicated that, in the HER2-amplified cell
lines, dacomitinib overcomes the acquired resistance to

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Dacomitinib (PF-00299804) and Breast Cancer

1 mol/L

MDA-MB-231
(4.23 mol/L)
100 nmol/L

MDA-MB-453
(2 mol/L)
1 mol/L

1 mol/L

HCC-70
(1.44 mol/L)
100 nmol/L

1 mol/L

100 nmol/L

MDA-MB-468
(1.42 mol/L)

1 mol/L

HCC-1143
(0.73 mol/L)
100 nmol/L

1 mol/L

HCC-1954
(0.06 mol/L)
100 nmol/L

100 nmol/L

BT-474
(0.02 mol/L)
1 mol/L

1 mol/L

EFM-192A
(0.02 mol/L)
100 nmol/L

1 mol/L

SK-BR-3
(0.02 mol/L)
100 nmol/L

1 mol/L

HCC-202
(<0.005 mol/L)
100 nmol/L

Cell line
IC50

Resistant

100 nmol/L

Sensitive

pHER2

Total HER2

pEGFR

Total EGFR

pHER4
Total HER4

pAKT

Total AKT

pERK

Total ERK
-Tubulin

Figure 3. The effects of dacomitinib on total and phosphorylated HER2, EGFR, HER4, AKT, and ERK. The effects of dacomitinib on total and phosphorylated
HER2, EGFR, HER4, AKT, and ERK were measured by Western blot as described in Materials and Methods. All cell lines were treated with 100 nmol/L
or 1 mmol/L dacomitinib for 10 minutes. Cell lines are arranged from most sensitive (low IC50; left) to least sensitive (high IC50; right). Dacomitinib had no effect
on total HER2, AKT, and ERK in a majority of lines and caused a minor decrease in total EGFR in several lines. Most of the selected lines expressed low levels of
total HER4, which remained mostly unchanged posttreatment. Dacomitinib inhibited HER2 phosphorylation in all lines with detectable baseline
phosphorylation. Dacomitinib inhibited EGFR phosphorylation in sensitive lines, but only 2 resistant lines (MDA-MB-453 and MDA-MB-231). Dacomitinib
decreased HER4 phosphorylation in sensitive lines with detectable baseline levels. Dacomitinib inhibited phosphorylation of AKT in all sensitive lines, but only
2 resistant lines (HCC-70 and MDA-MB-453). Dacomitinib caused a decrease in phosphorylation of ERK in all lines, except for resistant MDA-MB-231.
Densitometry data are available in Supplementary Table S1.

trastuzumab and that it maintains a considerable antiproliferative activity in the cell lines with acquired resistance to lapatinib.
Effects of dacomitinib on cell cycle, apoptosis, and
signal transduction in cell lines conditioned for the
acquired resistance to trastuzumab and lapatinib
We analyzed the effect of dacomitinib on cell cycle and
apoptosis in the cell lines conditioned for acquired resistance to trastuzumab (BT-474-TR, SK-BR-3-TR) and lapatinib (BT-474-LR, SK-BR-3-LR). Cells were exposed to
100 nmol/L or 1 mmol/L of dacomitinib for 48 hours (cell
cycle) and 5 days (apoptosis).
In the trastuzumab-resistant lines, dacomitinib caused a
G0G1 arrest comparable with the parental lines. In lapatinib-resistant lines, the G0G1 arrest caused by 1 mmol/L
of dacomitinib was similar to the parental lines. The effect
was less pronounced at 100 nmol/L of dacomitinib (Supplementary Fig. S5).

www.aacrjournals.org

The effect of dacomitinib on apoptosis in trastuzumabresistant lines was stronger than in the parental lines. In
lapatinib-resistant lines, dacomitinib at 100 nmol/L did
not induce apoptosis, and the effect at 1 mmol/L was less
pronounced than in the parental lines (Supplementary
Fig. S6).
In addition, the effect of dacomitinib on the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the
acquired resistant lines was analyzed by Western blots
(Supplementary Fig. S7, densitometry in Supplementary
Table S2). Dacomitinib decreased the phosphorylation of
HER2, EGFR, AKT, and ERK in trastuzumab- and lapatinib-resistant lines. The expression of baseline total and
phosphorylated HER4 was very low, making assessment
of dacomitinib effects difficult.

Discussion
Using a broad panel of 44 human breast cancer cell lines
and 3 immortalized breast epithelial lines, we have shown

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Published OnlineFirst July 3, 2012; DOI: 10.1158/1535-7163.MCT-11-0730

Kalous et al.

A100
90
80
70
60
% 50
40
30
20
10
0

100
90
80
70
60
50
% 40
30
20
10
0

BT-474 (IC50 = 0.02 mol/L)

G0/G1

G0/G1

B100
90
80
70
60
% 50
40
30
20
10
0

G2

ZR-75-1 (IC50 = 0.24 mol/L)

G2

MCF-7 (IC50 = 3.67 mol/L)

G0/G1

G2

100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0

100
90
80
70
60
50
40
30
20
10
0

SK-BR-3 (IC50 = 0.02 mol/L)

G0/G1

G0/G1

Mol Cancer Ther; 11(9) September 2012

G2

G2

MDA-MB-231 (IC50 = 4.23 mol/L)

G0/G1

that HER2 amplification status was a strong predictor of

response to dacomitinib, with IC50 values below 1 mmol/L
in the vast majority of HER2-amplified lines. MDA-MB453 and UACC-732 were the only HER2-amplified lines
that were resistant to dacomitinib. Of note, these 2 lines
have some of the lowest levels of HER2 DNA copy
number by comparative genomic hybridization (data not
shown) and HER2 mRNA among the HER2-amplified
lines. In addition, these 2 lines express low baseline levels
of total and phosphorylated HER2 (25) and total and
phosphorylated EGFR (data not shown). These 2 cell lines
were also previously shown to be resistant to trastuzumab
(25) and lapatinib (25, 26). These findings may have
potential clinical implications for selecting the patients
for clinical trials with dacomitinib.
Dacomitinib showed an antiproliferative activity superior to trastuzumab in vitro. The HER2-amplified lines
SUM-225, HCC-1419, HCC-1954, UACC-893, and HCC1569 were resistant to trastuzumab (25) but sensitive to
dacomitinib (IC50 < 1 mmol/L). These data suggest that
dacomitinib can potentially overcome de novo trastuzumab resistance. Lapatinib and dacomitinib generated
comparable IC50 values in HER2-amplified lines; only
HCC-1569 was resistant to lapatinib (IC50 > 1 mmol/L)
but sensitive to dacomitinib (25). Similarly to trastuzumab
and lapatinib, the nonluminal, HER2-nonamplified cell
lines were the most resistant to dacomitinib.
We have confirmed that dacomitinib acts by blocking
the phosphorylation of HER2, EGFR, and HER4 in breast
cancer cell lines. The inhibition of HER2 phosphorylation
was dose dependent and specific to HER2-amplified lines.

1984

HCC-1143 (IC50 = 0.73 mol/L)

Figure 4. Effects of dacomitinib

on cell cycle. A, sensitive cell
lines BT-474 and SK-BR-3
(IC50 0.02 mmol/L) show a
signicant G0G1 arrest and a
decrease in the S-phase and G2
-phase fractions as compared with
the less sensitive ZR-75-1 and
HCC-1143 (IC50 0.24 and 0.73,
respectively). B, resistant cell lines
MCF-7 (the effect is less
pronounced yet statistically
signicant) and MDA-MB-231 after
incubation with 1 mmol/L
dacomitinib for 2 days. Solid bars,
control samples; striped bars,
treated samples. Error bars
represent SE for 2 separate
experiments.  , P < 0.05 compared
with control.

G2

The blockage of EGFR phosphorylation was more pronounced in HER2-amplified lines but was also present in
some resistant lines. The inhibition of HER4 phosphorylation was observed in several sensitive HER2-amplified
lines. These effects lead to the inhibition of the PI3K/AKT
signaling pathway in sensitive lines and 2 resistant lines,
as evidenced by a reduction of phosphorylated AKT. We
have also observed a loss of phosphorylated ERK in most
lines, suggesting that dacomitinib also acts by inhibiting
the RAS/MAPK pathway. These effects of dacomitinib on
HER receptors and downstream signaling pathways ultimately resulted in cell-cycle inhibition and apoptosis.
In this study, 1 mmol/L was chosen based on the distribution of IC50 values in our dataset, which has biologic
significance (i.e., there seemed to be a natural increase in
the IC50 distribution around the 1 mmol/L mark; cell lines
below this cutoff point were mostly HER2 amplified;
dacomitinib induced changes in cell cycle and apoptosis
in cell lines with IC50 values below 1 mmol/L but not
above). In addition, phase I data was recently reported
(17). In general, the maximum plasma levels of dacomitinib
in the phase I study were between 200 to 300 nmol/L and
are within the range of the 1,000 nmol/L cut-off used here.
Trastuzumab and lapatinib have an important role in
the current clinical therapy of HER2-amplified breast
cancer. Unfortunately, less than half of patients respond
to monotherapy, and though the response rates increase
when combined with chemotherapy, the responses are
often only temporary in a significant number of patients
(411, 2730). For these reasons, it is important to keep
searching for new therapies for this subset of patients.

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Dacomitinib (PF-00299804) and Breast Cancer

% Annexin-Vpositive cells

% Annexin-Vpositive cells

70
60

60
50
40

30

30

20

20

10

10

ZR-75-1 (IC50 = 0.24 mol/L)

70

60

60

50

50

40

40

30

30

20

20

10

10

MCF-7 (IC50 = 3.67 mol/L)

HCC-1143 (IC50 = 0.73 mol/L)

80

70

MDA-MB-231 (IC50 = 4.23 mol/L)

80

70

70

60

60

50

50

40

40

30

30

20

20

10

10

Our findings suggest different, largely nonoverlapping

mechanisms of resistance to dacomitinib, trastuzumab,
and lapatinib. We found that 2 HER2-amplified cell lines
that we had conditioned for acquired resistance to trastuzumab (BT-474-TR and SK-BR-3-TR) were very sensitive
to the antiproliferative effect of dacomitinib. This sensitivity to dacomitinib was comparable with the sensitivity
observed in their parental cell lines. To examine the effect
of dacomitinib in the models of acquired resistance to
lapatinib, we have generated 2 lines with acquired lapatinib resistance (BT-474-LR and SK-BR-3-LR). We found
these 2 lines to be sensitive to dacomitinib, though their
sensitivity was less pronounced than in the parental lines.
Several mechanisms of resistance to trastuzumab have
been proposed; however, their significance is not yet

www.aacrjournals.org

70

40

80

SK-BR-3 (IC 50= 0.02 mol/L)

80

50

80

% Annexin-Vpositive cells

Figure 5. Effects of dacomitinib on

apoptosis. A, sensitive cell lines BT474 and SK-BR-3 show signicant
increase in the percentage of
Annexin-Vpositive cells. Less
sensitive cell lines ZR-75-1 and
HCC-1143 show less pronounced
increase in Annexin-Vpositive cells
(statistically signicant in HCC1143). B, resistant cell lines MCF-7
and MDA-MB-231 show no changes.
Cells were incubated with 1 mmol/L
dacomitinib for 5 days. Solid bars,
control samples; striped bars,
treated samples. Error bars represent
SE for 2 separate experiments.

, P < 0.05 compared with control.

BT-474 (IC50 = 0.02 mol/L)

80

clearly defined. Several studies have implicated an

increased activation of EGFR, HER3, and other receptor
tyrosine kinases (e.g., IGF-1R) and their ligands in resistance to trastuzumab (25, 3135). Alterations in the PI3K/
AKT pathway through PIK3CA mutation (36, 37) and/or a
loss of expression of the PTEN tumor suppressor (38, 39)
have also been associated with resistance to trastuzumab.
We have shown previously that, contrary to dacomitinib,
the response to trastuzumab was not significantly associated with total HER2 protein levels (vs. amplification;
ref. 25). A recent publication exploring mechanisms of
trastuzumab resistance (40) also supports the use of a panHER inhibitor in overcoming resistance to trastuzumab.
The mechanisms of lapatinib resistance are not well
understood either. Among the proposed mechanisms of

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Kalous et al.

Table 2. Comparison of dacomitinib and lapatinib responses (IC50) in parental and trastuzumab- and
lapatinib-resistant cell lines

Cell line

Dacomitinib
IC50 (mmol/L)

Lapatinib
IC50 (mmol/L)

Dacomitinib
IC50 fold change
compared with
parental line

BT-474
BT-474-TR
BT-474-LR
SK-BR-3
SK-BR-3-TR
SK-BR-3-LR

0.018  0.011
0.010a
0.031  0.020
0.015  0.003
0.005a
0.339  0.137

0.016  0.011
0.077  0.039
1.424  0.120
0.054  0.008
0.039  0.010
6.400  1.119

0.56a
1.72

0.33a
22.60

Lapatinib
IC50 fold change
compared with
parental line

4.81
89

0.72
118.52

NOTE: Growth rate decrease (fold change) in the presence or absence of 15 mg/mL of trastuzumab: BT-474 (5.00  1.05), BT-474-TR
(1.16  0.21), SK-BR-3 (1.45  0.09), SK-BR-3-TR (1.19  0.05); both resistant lines t the criteria for trastuzumab resistance (<1.2-fold
decrease in growth rate; data in part published by O'Brien and colleagues; ref. 25).
Abbreviations: TR, trastuzumab-resistant cell lines; LR, lapatinib-resistant cell lines.
a
Approximate average value from a minimum of 2 experiments.

lapatinib resistance are HER2 mutations (41), activation of

prosurvival pathways through ER signaling (42), and
overexpression of membrane-bound receptor tyrosine
kinase AXL (43). There are several possible explanations
for the effect of dacomitinib in overcoming the acquired
resistance to lapatinib. Unlike lapatinib, dacomitinib
binds to the active site of kinases in a covalent and
irreversible form, permanently blocking the kinase activity. In addition to blocking HER2 and EGFR, dacomitinib
also inhibits HER4 kinase activity; however, the clinical
significance of HER4 inhibition remains to be determined.
We confirmed that dacomitinib blocks the phosphorylation of HER4 in several sensitive lines. However, we did
not find a correlation between the HER4 mRNA levels and
the response to dacomitinib.
In summary, this study shows that dacomitinib has a
strong antiproliferative activity in HER2-amplified breast
cancer cell lines and maintains this activity in HER2amplified cell lines with de novo and acquired resistance
to trastuzumab and acquired resistance to lapatinib.
Given the importance of finding new therapies for
drug-resistant breast cancer, these findings are a strong
rationale for clinical development of this compound.

Disclosure of Potential Conicts of Interest

C.D. Britten received a commercial research grant from Pfizer Inc.
(major) and was paid for travel to a scientific meeting (minor, Pfizer). I.
Taylor is an employee of Pfizer Inc. as Senior Director, has received
compensation (major) and has ownership interest in Pfizer Inc. (major).
D.J. Slamon has received honoraria from Speakers Bureau of Genentech
(minor), Sanofi-Aventis (minor), and GlaxoSmithKline (minor), has ownership interest in Amgen (major), and is a consultant and an advisory
board member of Novartis Pharmaceuticals (minor). No potential conflicts
of interest were disclosed by the other authors.

Acknowledgments
The authors thank Veerauo Konkankit and Teodora Kolarova for their
excellent technical assistance and Dr. Habib Hamidi for reviewing the
manuscript.

Grant Support
D.J. Slamon received Department of Defense Innovator Award
W81XWH-05-1-0395. The work is also funded by a gift to D.J. Slamon by
The Wittich Family Project for Emerging Therapies in Breast Cancer at
UCLAs Jonsson Comprehensive Cancer Center.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received September 26, 2011; revised May 11, 2012; accepted June 1,
2012; published OnlineFirst July 3, 2012.

References
1.
2.

3.

4.

1986

Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat

Rev Mol Cell Biol 2001;2:12737.
Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al.
Studies of the HER-2/neu proto-oncogene in human breast and
ovarian cancer. Science 1989;244:70712.
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL.
Human breast cancer: correlation of relapse and survival with amplication of the HER-2/neu oncogene. Science 1987;235:17782.
Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, et al. Multinational study of the efcacy and safety of
humanized anti-HER2 monoclonal antibody in women who have
HER2-overexpressing metastatic breast cancer that has pro-

Mol Cancer Ther; 11(9) September 2012

5.

6.

gressed after chemotherapy for metastatic disease. J Clin Oncol

1999;17:263948.
Marty M, Cognetti F, Maraninchi D, Snyder R, Mauriac L, Tubiana-Hulin
M, et al. Randomized phase II trial of the efcacy and safety of
trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as rst-line treatment: the M77001 study group. J Clin Oncol
2005;23:426574.
Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, et al. Efcacy and safety of trastuzumab as a single agent in
rst-line treatment of HER2-overexpressing metastatic breast cancer.
J Clin Oncol 2002;20:71926.

Molecular Cancer Therapeutics

Downloaded from mct.aacrjournals.org on November 29, 2015. 2012 American Association for Cancer Research.

Published OnlineFirst July 3, 2012; DOI: 10.1158/1535-7163.MCT-11-0730

Dacomitinib (PF-00299804) and Breast Cancer

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.
21.
22.

23.

24.

25.

Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE Jr, Davidson NE,
et al. Trastuzumab plus adjuvant chemotherapy for operable HER2positive breast cancer. N Engl J Med 2005;353:167384.
Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A,
et al. Use of chemotherapy plus a monoclonal antibody against HER2
for metastatic breast cancer that overexpresses HER2. N Engl J Med
2001;344:78392.
Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch
M, Smith I, et al. Trastuzumab after adjuvant chemotherapy in HER2positive breast cancer. N Engl J Med 2005;353:165972.
Perez EA, Romond EH, Suman VJ, Jeong JH, Davidson NE, Geyer CE
Jr, et al. Four-year follow-up of trastuzumab plus adjuvant chemotherapy for operable human epidermal growth factor receptor 2-positive breast cancer: Joint Analysis of Data from NCCTG N9831 and
NSABP B-31. J Clin Oncol 2011;29:336673.
Slamon DJ, Eiermann W, Robert N, et al. Phase III randomized trial
comparing doxorubicin and cyclophosphamide followed by docetaxel
(AC->T) with doxorubicin and cyclophosphamide followed by docetaxel and trastuzumab (AC->TH) with docetaxel, carboplatin and
trastuzumab (TCH) in HER2/neu positive early breast cancer patients:
BCIRG 006 study (abstract 62). Cancer Res 2009;69.
Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T,
et al. Lapatinib plus capecitabine for HER2-positive advanced breast
cancer. N Engl J Med 2006;355:273343.
Cockerill S, Stubbereld C, Stables J, Carter M, Guntrip S, Smith K,
et al. Indazolylamino quinazolines and pyridopyrimidines as inhibitors
of the EGFr and C-erbB-2. Bioorg Med Chem Lett 2001;11:14015.
Ocana A, Amir E. Irreversible pan-ErbB tyrosine kinase inhibitors and
breast cancer: current status and future directions. Cancer Treat Rev
2009;35:68591.
Bose P, Ozer H. Neratinib: an oral, irreversible dual EGFR/HER2
inhibitor for breast and non-small cell lung cancer. Expert Opin Investig
Drugs 2009;18:173551.
Engelman JA, Zejnullahu K, Gale CM, Lifshits E, Gonzales AJ, Shimamura T, et al. PF00299804, an irreversible pan-ERBB inhibitor, is
effective in lung cancer models with EGFR and ERBB2 mutations that
are resistant to getinib. Cancer Res 2007;67:1192432.
Janne PA, Boss DS, Camidge DR, Britten CD, Engelman JA, Garon EB,
et al. Phase I dose-escalation study of the pan-HER inhibitor,
PF299804, in patients with advanced malignant solid tumors. Clin
Cancer Res 2011;17:11319.
Gonzales AJ, Hook KE, Althaus IW, Ellis PA, Trachet E, Delaney AM,
et al. Antitumor activity and pharmacokinetic properties of PF00299804, a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor. Mol Cancer Ther 2008;7:18809.
Finn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai AJ, et al. PD
0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits
proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro. Breast Cancer Res 2009;11:R77.
Uphoff CC, Drexler HG. Detection of Mycoplasma in leukemia-lymphoma
cell lines using polymerase chain reaction. Leukemia 2002;16:28993.
Ginther C, Issel-Tarver L, King MC. Identifying individuals by sequencing mitochondrial DNA from teeth. Nature genetics 1992;2:1358.
Pauletti G, Godolphin W, Press MF, Slamon DJ. Detection and quantitation of HER-2/neu gene amplication in human breast cancer
archival material using uorescence in situ hybridization. Oncogene
1996;13:6372.
Finn RS, Dering J, Ginther C, Wilson CA, Glaspy P, Tchekmedyian N,
et al. Dasatinib, an orally active small molecule inhibitor of both the src
and abl kinases, selectively inhibits growth of basal-type/"triple-negative" breast cancer cell lines growing in vitro. Breast Cancer Res Treat
2007;105:31926.
Wilson CA DJ, Bernardo G, Rong HM, Ginther C, Ferdman R, Cook AM,
et al. Cell differentiation and dominant signaling pathway signatures in
the molecular classication of human breast cancer cell lines. Breast
Cancer Res 2005;7.
O'Brien NA, Browne BC, Chow L, Wang Y, Ginther C, Arboleda J, et al.
Activated phosphoinositide 3-kinase/AKT signaling confers resistance
to trastuzumab but not lapatinib. Mol Cancer Ther 2010;9:1489502.

www.aacrjournals.org

26. Konecny GE, Pegram MD, Venkatesan N, Finn R, Yang G, Rahmeh M,

et al. Activity of the dual kinase inhibitor lapatinib (GW572016) against
HER-2-overexpressing and trastuzumab-treated breast cancer cells.
Cancer Res 2006;66:16309.
27. Marcom PK, Isaacs C, Harris L, Wong ZW, Kommarreddy A, Novielli N,
et al. The combination of letrozole and trastuzumab as rst or secondline biological therapy produces durable responses in a subset of
HER2 positive and ER positive advanced breast cancers. Breast
Cancer Res Treat 2007;102:439.
28. Di Leo A, Gomez HL, Aziz Z, Zvirbule Z, Bines J, Arbushites MC, et al.
Phase III, double-blind, randomized study comparing lapatinib plus
paclitaxel with placebo plus paclitaxel as rst-line treatment for metastatic breast cancer. J Clin Oncol 2008;26:554452.
29. Johnston S, Pippen J Jr, Pivot X, Lichinitser M, Sadeghi S, Dieras V,
et al. Lapatinib combined with letrozole versus letrozole and placebo
as rst-line therapy for postmenopausal hormone receptor-positive
metastatic breast cancer. J Clin Oncol 2009;27:553846.
30. Press MF, Finn RS, Cameron D, Di Leo A, Geyer CE, Villalobos IE, et al.
HER-2 gene amplication, HER-2 and epidermal growth factor receptor mRNA and protein expression, and lapatinib efcacy in women with
metastatic breast cancer. Clin Cancer Res 2008;14:786170.
31. Lee-Hoeich ST, Crocker L, Yao E, Pham T, Munroe X, Hoeich KP,
et al. A central role for HER3 in HER2-amplied breast cancer: implications for targeted therapy. Cancer Res 2008;68:587887.
32. Lu Y, Zi X, Zhao Y, Mascarenhas D, Pollak M. Insulin-like growth factorI receptor signaling and resistance to trastuzumab (Herceptin). J Natl
Cancer Inst 2001;93:18527.
33. Motoyama AB, Hynes NE, Lane HA. The efcacy of ErbB receptortargeted anticancer therapeutics is inuenced by the availability of
epidermal growth factor-related peptides. Cancer Res 2002;62:
31518.
34. Nahta R, Yuan LX, Zhang B, Kobayashi R, Esteva FJ. Insulin-like
growth factor-I receptor/human epidermal growth factor receptor 2
heterodimerization contributes to trastuzumab resistance of breast
cancer cells. Cancer Res 2005;65:1111828.
35. Ritter CA, Perez-Torres M, Rinehart C, Guix M, Dugger T, Engelman JA,
et al. Human breast cancer cells selected for resistance to trastuzumab
in vivo overexpress epidermal growth factor receptor and ErbB ligands
and remain dependent on the ErbB receptor network. Clin Cancer Res
2007;13:490919.
36. Berns K, Horlings HM, Hennessy BT, Madiredjo M, Hijmans EM,
Beelen K, et al. A functional genetic approach identies the PI3K
pathway as a major determinant of trastuzumab resistance in breast
cancer. Cancer Cell 2007;12:395402.
37. Junttila TT, Akita RW, Parsons K, Fields C, Lewis Phillips GD, Friedman
LS, et al. Ligand-independent HER2/HER3/PI3K complex is disrupted
by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC0941. Cancer Cell 2009;15:42940.
38. Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, et al. PTEN
activation contributes to tumor inhibition by trastuzumab, and loss of
PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004;6:
11727.
39. Pandol PP. Breast cancerloss of PTEN predicts resistance to treatment. N Engl J Med 2004;351:23378.
40. Gijsen M, King P, Perera T, Parker PJ, Harris AL, Larijani B, et al. HER2
phosphorylation is maintained by a PKB negative feedback loop in
response to anti-HER2 herceptin in breast cancer. PLoS Biol 2010;8:
e1000563.
41. Trowe T, Boukouvala S, Calkins K, Cutler RE Jr, Fong R, Funke R, et al.
EXEL-7647 inhibits mutant forms of ErbB2 associated with lapatinib
resistance and neoplastic transformation. Clin Cancer Res 2008;14:
246575.
42. Xia W, Bacus S, Hegde P, Husain I, Strum J, Liu L, et al. A model of
acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and
a therapeutic strategy to prevent its onset in breast cancer. Proc Natl
Acad Sci U S A 2006;103:7795800.
43. Liu L, Greger J, Shi H, Liu Y, Greshock J, Annan R, et al. Novel
mechanism of lapatinib resistance in HER2-positive breast tumor cells:
activation of AXL. Cancer Res 2009;69:68718.

Mol Cancer Ther; 11(9) September 2012

Downloaded from mct.aacrjournals.org on November 29, 2015. 2012 American Association for Cancer Research.

1987

Published OnlineFirst July 3, 2012; DOI: 10.1158/1535-7163.MCT-11-0730

Dacomitinib (PF-00299804), an Irreversible Pan-HER Inhibitor,

Inhibits Proliferation of HER2-Amplified Breast Cancer Cell Lines
Resistant to Trastuzumab and Lapatinib
Ondrej Kalous, Dylan Conklin, Amrita J. Desai, et al.
Mol Cancer Ther 2012;11:1978-1987. Published OnlineFirst July 3, 2012.

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