Suppression of proliferation of two independent NF1 malignant peripheral nerve sheath tumor cell lines by the pan-ErbB inhibitor CI-1033.

Abstract

Neurofibromatosis Type 1 (NF1) is characterized by the abnormal proliferation of neuroectodermal tissues and the development of certain tumors, particularly neurofibromas, which may progress into malignant peripheral nerve sheath tumors (MPNSTs). Effective pharmacological therapy for the treatment of NF1 tumors is currently unavailable and the prognosis for patients with MPNSTs is poor. Loss of neurofibromin correlates with increased expression of the epidermal growth factor receptor (EGFR) and ErbB2 tyrosine kinases and these kinases have been shown to promote NF1 tumor-associated pathologies in vivo. We show here that while NF1 MPNST cells have higher EGFR expression levels and are more sensitive to EGF when compared to a non-NF1 MPNST cell line, the ability of the EGFR inhibitor gefitinib to selectively inhibit NF1 MPNST cell proliferation is marginal. We also show that NF1 MPNST proliferation correlates with activated ErbB2 and can be suppressed by nanomolar concentrations of the pan-ErbB inhibitor CI-1033 (canertinib). Consequently, targeting both EGFR and ErbB2 may prove an effective strategy for suppressing NF1 MPNST tumor growth in vivo.

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1938Cancer Biology & Therapy2008; Vol. 7 Issue 12
[Cancer Biology & Therapy 7:12, 1938-1946; December 2008]; ©2008 Landes Bioscience
© 2009 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
Neurofibromatosis Type 1 (NF1) is characterized by the
abnormal proliferation of neuroectodermal tissues and the devel-
opment of certain tumors, particularly neurofibromas, which may
progress into malignant peripheral nerve sheath tumors (MPNSTs).
Effective pharmacological therapy for the treatment of NF1
tumors is currently unavailable and the prognosis for patients with
MPNSTs is poor. Loss of neurofibromin correlates with increased
expression of the epidermal growth factor receptor (EGFR) and
ErbB2 tyrosine kinases and these kinases have been shown to
promote NF1 tumor-associated pathologies in vivo. We show here
that while NF1 MPNST cells have higher EGFR expression levels
and are more sensitive to EGF when compared to a non-NF1
MPNST cell line, the ability of the EGFR inhibitor gefitinib to
selectively inhibit NF1 MPNST cell proliferation is marginal. We
also show that NF1 MPNST proliferation correlates with acti-
vated ErbB2 and can be suppressed by nanomolar concentrations
of the pan-ErbB inhibitor CI-1033 (canertinib). Consequently,
targeting both EGFR and ErbB2 may prove an effective strategy for
suppressing NF1 MPNST tumor growth in vivo.
Introduction
Neurofibromatosis type 1 (NF1) is the most common inherited
cancer predisposition syndrome and is associated with the aberrant
proliferation of tissues derived from the neural crest.1,2 The overall
incidence of approximately 1 in 3,000 includes about 50% of cases
that arise due to new mutations in the NF1 gene,3 which encodes a
tumor suppressor protein called neurofibromin. Patients with NF1
present numerous clinical manifestations and are at an increased
risk of developing certain tumors, most commonly neurofibromas.2
The majority of cells in neurofibromas are derived from Schwann
cells and these tumors can affect any peripheral nerve. In approxi-
mately 10% of cases, plexiform neurofibromas progress to malignant
peripheral nerve sheath tumors (MPNSTs).4 There is currently no
pharmacological treatment for MPNSTs. Prognosis for NF1 patients
with MPNSTs is poor, with only 21% of patients surviving 5 years
from time of diagnosis.5
While haploinsufficiency for functional neurofibromin protein
drives some aspects of the disease and provides a favorable microen-
vironment for tumor development,6-8 loss of heterozygosity is
found in the transformed Schwann cell component of MPNSTs.9
Neurofibromin downregulates the activity of the small GTPase
Ras.10 Ras has pivotal roles in cell survival, proliferation and
differentiation by transducing responses initiated at the cell surface
to several intracellular signaling molecules, including those that
constitute the Raf-MEK-ERK and PI3K-Akt axes.11,12 We have
previously demonstrated that basal N- and K-Ras and ERK1/2
activities are higher in NF1 MPNST cells and that activation of
the ERK1/2 signaling pathway is critical for their proliferation.13 A
rational approach to NF1 MPNST therapy may derive from such
characterization of the critical signal transduction pathways that
drive MPNST proliferation.14,15
In addition to loss of functional neurofibromin, neurofibromas
and MPNSTs show significant alterations in the expression of one
or more of the epidermal growth factor receptor (ErbB) family of
receptor tyrosine kinases.16,17 These changes significantly affect the
responsiveness of Schwann cells to ErbB ligands.16 While quiescent
Schwann cells do not express the EGFR, loss of neurofibromin has
been shown to correlate with aberrant expression of this receptor
in Schwann cells.16,18 Increased EGFR abundance and activity
are associated with the development and progression of multiple
human solid tumors19,20 and play important roles in NF1-associated
malignancy. For example, transgenic murine Schwann cells that
have targeted overexpression of the EGFR exhibit features of
neurofibromas, such as hyperplasia, collagen deposition, mast cell
Research Paper
Suppression of proliferation of two independent NF1 malignant
peripheral nerve sheath tumor cell lines by the pan-ErbB inhibitor CI-1033
Joshua T. Dilworth,1 Jonathan W. Wojtkowiak,1 Patricia Mathieu,5 Michael A. Tainsky,2,3,6 John J. Reiners Jr,1,4-6 Raymond
R. Mattingly1,3,6,* and Chad N. Hancock1
1Department of Pharmacology; and 2Center for Molecular Medicine and Genetics; Wayne State University School of Medicine; Detroit, Michigan USA; Programs in 3Molecular
Biology and Genetics and in 4Proteases; Barbara Ann Karmanos Cancer Institute; Detroit, Michigan USA; and 5Institute of Environmental Health Sciences and 6Environmental
Health Sciences Center for Molecular & Cellular Toxicology with Human Applications; Wayne State University; Detroit, Michigan USA
Abbreviations: NF1, neurofibromatosis Type 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MAP, mitogen-
activated protein; ERK, extracellular-signal regulated protein kinase; MPNST, malignant peripheral nerve sheath tumor; CI-1033,
N-[4-(3-chloro-4-fluoro-phenylamino)-7-(3-morpholin4-yl-propoxy)-quinazol-in-6-yl]-acrylamide
Key words: EGF receptor, ErbB2, NF1, tyrosine kinase inhibitor, MPNST
*Correspondence to: Raymond R. Mattingly; Department of Pharmacology
Wayne State University School of Medicine; 540 East Canfield Ave.;
Detroit, Michigan 48201 USA; Tel.: 313.577.6022; Fax: 313.577.6739;
Email: r.mattingly@wayne.edu
Submitted: 07/30/08; Accepted: 09/07/08
Previously published online as a Cancer Biology & Therapy E-publication:
http://www.landesbioscience.com/journals/cbt/article/6942

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Suppression of NF1 MPNST proliferation with CI-1033
set of antibodies, and which also showed an absence of ErbB3 or
ErbB4 receptors.16
Loss of the NF1 tumor suppressor is believed to result in exag-
gerated Ras-mediated signaling.22 One of the primary Ras effector
pathways is the ERK MAP kinase pathway, whose activation is
required for proliferation of MPNST lines.13 To determine whether
loss of neurofibromin correlated with prolonged EGF-mediated ERK
signaling, we treated the MPNST lines with 10 ng/ml of EGF and
harvested cells over a 4 h time period. Both of the NF1 MPNST lines
show sustained ERK activation in response to EGF stimulation (Fig.
1B and C). The NF1 lines, particularly the NF90-8 cells, also show
prolonged EGFR phosphorylation subsequent to EGF treatment,
as determined by immunoblotting for phosphorylation of Tyr1068.
infiltration and dissociation from axons.21 The EGFR confers a
mechanism by which Schwann cells respond to mitogenic factors and
activate Ras-mediated proliferative and pro-survival signaling path-
ways, which are greatly potentiated in the context of neurofibromin
deficiency.22
Another ErbB family member that has been connected to
NF1 tumor pathologies is the ErbB2 tyrosine kinase. ErbB2 is
expressed in normal Schwann cells and heterodimerization of ErbB2
with ErbB3 is believed to direct neuregulin-mediated signaling in
Schwann cells.23 While there is no known ligand for ErbB2, this
protein readily heterodimerizes with the other ErbB family members
and greatly enhances their signaling.24 Overexpressed ErbB2 can
also homodimerize, resulting in ligand-independent signaling and
contributing to tumorigenesis.25,26 Previous reports have found an
inverse relationship between neurofibromin expression and ErbB2
levels,27 and constitutive activation of ErbB2 in Schwann cells results
in formation of invasive Schwann cell tumors.27,28
In this study, we found that two independent NF1 MPNST lines
expressed higher levels of EGFR and have a prolonged and more
potent ERK activation in response to EGF. We also found strong
coupling between EGFR and ErbB2 in these NF1 MPNST cells.
Despite the increase in EGFR signaling in NF1 MPNST cells, there
was little inhibition of cell growth in response to the EGFR-selective
inhibitor gefitinib unless exogenous ligand was present. In contrast,
the pan-ErbB inhibitor CI-1033 (canertinib) strongly suppressed
proliferation of the NF1 MPNST cells. Considering the roles of
EGFR and ErbB2 in NF1-related pathology, inhibition of both of
these ErbB kinases may prove an effective therapy for treatment of
MPNSTs in vivo.
Results
NF1 MPNSTs express higher levels of EGFR and ErbB2 and
have a more potent response to EGF. Previous studies have found
that MPNST cell lines express significantly different levels of the
ErbB receptors.17 We analyzed a panel of MPNST cell lines: two
(ST88-14 and NF90-8) were isolated from separate NF1 patients,
and one (STS-26T) was isolated from a non-NF1 patient. STS-26T
cells express neurofibromin and maintain lower levels of basal Ras
activity.13 Both NF1 lines expressed substantially higher levels of
EGFR and ErbB2 receptor than the STS-26T line (Fig. 1A). These
results are consistent with previous reports that characterized the
expression of ErbB family members in these lines using an alternate
www.landesbioscience.com Cancer Biology & Therapy1939
Figure 1. Characterization of protein expression and MPNST cell line
response to EGF. (A) Total cell lysates from the ST88-14 (“88”), NF90-8
(“90”) and STS-26T (“26”) MPNST lines were analyzed by immunoblot-
ting for antibodies versus total protein levels of EGFR and ErbB2, and
active and total ERK. Expression of β-tubulin is shown for a loading control.
(B) The MPNST cell lines were stimulated as shown with 10 ng/ml EGF. EGFR
and ERK activation were assessed by immunoblotting with phospho-specific
antibodies against each. Expression of β-tubulin is shown for a loading
control. (C) Densitometry of the amount of active ERK1,2 in each sample
in (B). Background was calculated from equivalent areas in each lane and
subtracted from the value of protein in that lane. Active ERK1,2 levels were
recorded as percent maximum signal and are shown as mean ± S.E.M. from
three independent experiments. (D) MPNST cells were treated for 10 min
with the indicated pg/ml concentrations of EGF or vehicle (RPMI media, “V”)
and the activation of EGFR and ERK determined by immunoblotting. β-tubulin
is included as a protein loading control. (A, B and D) are representative of
at least three independent experiments.
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Suppression of NF1 MPNST proliferation with CI-1033
alternative method to detect endogenous EGFR activity,
we examined whether the EGFR showed detectable
levels of receptor turnover. We treated these MPNST
lines with either 10 ng/ml EGF, 50 μg/ml of the protein
synthesis inhibitor cycloheximide, or both. As shown in
Figure 2A and B, treatment of cultures with EGF alone,
without pretreatment with cycloheximide, resulted in
negligible time-dependent decreases of EGFR levels
over time. A much more pronounced EGF-induced
decrease in total EGFR was detected in cycloheximide-
pretreated cultures. These results are consistent with
reports that EGF treatment triggers transport of the
EGFR to lysosomes and subsequent receptor degrada-
tion.33 In addition, these data indicate that each of the
MPNST lines was synthesizing EGFR protein to replace
receptor lost due to EGF-induced receptor internaliza-
tion and degradation. There was no detectable change
in EGFR levels in cells treated with cycloheximide
alone, indicating that protein synthesis was not required
to maintain basal EGFR levels in the MPNST lines over
the 3 h time frame of the experiment.
EGF treatment has previously been shown to result
in cyclin-D1 induction via Ras and ERK MAP kinase
pathway activation.34 To confirm inhibition of protein
synthesis by cycloheximide, we immunoblotted our
samples for cyclin-D1. While cyclin D1 was detected
in untreated cells, a 4 h pretreatment with cyclohex-
imide resulted in a complete loss of immunoreactivity
(Fig. 2, lanes “S” versus “CHX + VEH”). In addition,
cycloheximide treatment prevented EGF-stimulated
induction of cyclin D1 (Fig. 2, lanes “CHX + EGF”).
The ST88-14 cell line showed similar results to the
other two lines tested (data not shown). These results
indicate that the concentration and duration of cyclo-
heximide treatment were sufficient to inhibit protein synthesis in
these MPNST cell lines.
Gefitinib suppresses EGF-mediated EGFR and ERK activation
in MPNSTs. Gefitinib (Iressa or ZD1893) is an EGFR-selective
inhibitor that competes for the ATP-binding site within the EGFR
and inhibits ligand-induced tyrosine phosphorylation and receptor
activation.19 Gefitinib has previously been shown to inhibit the
EGFR-mediated proliferation of the A431 cell line at nanomolar
concentrations.35 We thus hypothesized that gefitinib would inhibit
EGFR-mediated proliferation of these cell lines. We first determined
the concentrations of gefitinib required to inhibit EGF-mediated
phosphorylation of the EGFR and the subsequent phosphorylation
of ERK. We pretreated the three MPNST cell lines with increasing
concentrations of gefitinib for 2 h and then treated them with 10
ng/ml of EGF for 10 min. We found that 1 μM of gefitinib was
sufficient to inhibit phosphorylation of the EGFR at Tyr1068, as
well as EGF-induced activation of ERK, in each of these MPNST
cell lines (Fig. 3A). Gefitinib was also able to inhibit EGF-stimulated
proliferation of MPNST cells grown in very low (0.1%) serum
(supplementary data).
Gefitinib does not significantly inhibit proliferation of MPNST
cell lines in 5% serum at EGFR-selective concentrations. We next
tested the ability of gefitinib to reduce the proliferation of cells grown
Phosphorylation of the EGFR on Tyr1068 results in recruitment
of the Grb2 adaptor protein and activation of the Ras-MEK-ERK
pathway.31
To begin investigation of whether loss of neurofibromin in the
ST88-14 and NF90-8 lines correlates with an increased sensitivity of
the EGFR and the ERK pathway to EGF stimulation, we compared
the ability of decreasing concentrations of EGF to stimulate EGFR
and ERK activation in these NF1 lines versus the non-NF1 STS-26T
line. Cells from each MPNST line were treated with 1 pg/ml–10
ng/ml EGF for 10 min. We detected phosphorylation of the EGFR
on Tyr1068 at concentrations as low as 1 ng/ml in the NF1 lines,
whereas Tyr1068 phosphorylation was detected in the non-NF1
STS-26T line only after treatment with 10 ng/ml EGF (Fig. 1D).
In addition, both of the NF1 MPNST lines show ERK pathway
activation in response to much lower concentrations of EGF than
the STS-26T line (Fig. 1D).
MPNST cells show little turnover of the EGFR in the absence
of exogenous EGF. Previous studies have identified the EGFR
as partially driving the proliferation of NF1 MPNST cells.16
In addition, phosphorylation of the EGFR has been observed
in histological sections of NF1 MPNSTs.32 However, we were
not able to detect basal phosphorylation of the EGFR on Tyr1068
in non-stimulated MPNST cultures (Fig. 1B and D). As an
1940Cancer Biology & Therapy2008; Vol. 7 Issue 12
Figure 2. Assessment of EGFR turnover in the MPNST cell lines. NF90-8 NF1 MPNST
cells (A) and the non-NF1 MPNST line STS-26T (B) were pretreated with 50 μg/ml cyclo-
heximide (or vehicle) for 2 h. Cells were then treated with 10 ng/ml EGF (or vehicle)
for the indicated times. Lanes are indicated: “S”, untreated control; “CHX + VEH”, 2 h
cycloheximide treatment followed by treatment with vehicle over the indicated time-course;
“VEH + EGF”, pretreatment with ddH20 vehicle followed by treatment with 10 ng/ml EGF
over the indicated time-course; “CHX + EGF”, 2 h treatment followed by treatment with
10 ng/ml EGF over the indicated time-course. Total EGFR protein levels were compared
using immunoblotting. Cyclin-D1 is included to verify inhibition of EGF-stimulated protein
synthesis by cycloheximide. β-tubulin is included as a protein loading control. These data
are representative of at least three experiments per cell line.
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Suppression of NF1 MPNST proliferation with CI-1033
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Figure 3. Gefitinib inhibits EGF-stimulated activation of the
EGFR-ERK pathway but does not inhibit MPNST proliferation. (A)
Serum-starved cultures were treated with the indicated concentra-
tion of gefitinib (or vehicle) for 1 h, and subsequently treated
with 10 ng/ml EGF for 10 min. EGFR and ERK activation were
compared by immunoblotting with phospho-specific antibodies.
β-tubulin was used as a total protein loading control. (B) Gefitinib
does not inhibit the proliferation of MPNST lines at EGFR-selective
concentrations. On day 0, plates were treated with either vehicle
(DMSO) or the indicated concentration of gefitinib. Plates were
harvested and cells counted at the indicated time-points and, the
ability to exclude Trypan Blue was used to assess viability. These
data show the effects of gefitinib on both total cell number (top)
and percent viability (lower). Data represent the mean ± S.D. of
three independent cultures for each line. (C) High concentrations
of gefitinib have EGFR-independent effects. HepG2 cells were
treated with either DMSO vehicle or the indicated concentration
of gefitinib. Cells were harvested at the indicated time-points and
counted using the Trypan Blue exclusion assay. Data represent the
mean ± S.D. of three independent cultures.
Thus, our data indicate that the EGFR does not drive proliferation of
NF1 MPNST cells in vitro in the absence of exogenous EGF.
NF1 MPNST lines show a strong transactivation of ErbB2 in
response to activation of EGFR by EGF. Differential expression of
the various ErbB receptors has been demonstrated in both MPNST
cell lines and primary tumor specimens.17 We have determined
that the ST88-14 and NF90-8 NF1 lines expressed significantly
in 5% serum. Neurofibromas and MPNSTs are highly
vascular tumors and are likely to be exposed to numerous
cytokines and growth factors.36,37 In addition, a number
of growth factors besides EGFR ligands are believed to
drive MPNST proliferation.38,39 Therefore, we believe that
testing the ability of gefitinib to inhibit the proliferation of
these MPNST lines in the presence of serum more accu-
rately modeled the environment these tumor cells would
encounter in vivo. We cultured the ST88-14 NF1 line and
the STS-26T non-NF1 line in 5% serum supplemented
with 1–10 μM of gefitinib and counted the number of
cells. Gefitinib had an IC50 between 3 and 10 μM in
the NF1 ST88-14 line, and did not effectively suppress
proliferation of the non-NF1 STS-26T line at concentra-
tions below 10 μM (Fig. 3B). Cell proliferation decreased
significantly at 10 μM of gefitinib in both lines and this
was accompanied by a significant decrease of viable cells
in the STS-26T MPNST line. The NF90-8 NF1 MPNST
line responded similarly to gefitinib, with no inhibition of
proliferation seen until treatment with 10 μM gefitinib
(data not shown).
Because 1 μM of gefitinib is sufficient to inhibit EGFR-
dependent proliferation in other cancer cell lines,40 and
concentrations higher than 1 μM have been shown to have
EGFR-independent effects,41 we hypothesized that the
effects of high concentrations of gefitinib observed in our
studies were due to anti-proliferative effects independent
of this drug’s action at the EGFR. To test this hypothesis,
we assessed whether gefitinib influences the proliferation
of cells that do not rely on the EGFR for proliferation. We
used two human cancer cell lines: the human hepatocellular
carcinoma line HepG2, which expresses very low levels of
EGFR,42 and a doxorubicin-resistant SKNSH neuroblastoma line,
which does not express detectable levels of EGFR.43 Concentrations
of 5 and 10 μM gefitinib significantly reduced HepG2 proliferation
(Fig. 3C). In addition, 10 μM gefitinib also inhibited proliferation
of the SKNSH line by approximately 25% (data not shown). These
data support the conclusion that high concentrations of gefitinib
result in anti-proliferative effects that are not EGFR-dependent.
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Suppression of NF1 MPNST proliferation with CI-1033
1942 Cancer Biology & Therapy2008; Vol. 7 Issue 12
phosphorylation were detected with both a rabbit monoclonal and
polyclonal antibody against these phosphorylation sites of ErbB2.
The rabbit monoclonal antibody does not immunoreact with
several other phosphorylated receptor tyrosine kinases, including
the EGFR and ErbB4. In addition, the phospho-ErbB2 immu-
noreactive band overlaid with ErbB2 when blots were reprobed
with a mouse monoclonal antibody detecting total ErbB2 protein
(Fig. 4B). We are therefore confident that this band represents basal
and EGF-stimulated phosphorylation of ErbB2 on Tyr1221/1222.
The studies presented in Figure 2 indicated that the MPNST
lines utilize protein synthesis to maintain EGFR levels after EGF
stimulation. Previous studies have determined that EGFR/ErbB2
heterodimers are resistant to ligand-induced endocytosis as well as
ubiquitination of the EGFR through c-cbl activation.24 To deter-
mine whether ErbB2 was downregulated similarly to the EGFR after
EGF stimulation, we examined ErbB2 phosphorylation status and
expression levels in the NF1 lines following a 3 h treatment with 50
μg/ml cycloheximide, 10 ng/ml EGF, or both. We found that ErbB2
expression levels did not decline in response to treatment with EGF
alone, or in combination with cycloheximide in either the ST88-14
(Fig. 4A) or NF90-8 (Fig. 4B) cells. In addition, EGF-induced
phosphorylation of Tyr1221/1222 of ErbB2 was detected 3 h after
addition of 10 ng/ml of EGF to the media (Fig. 4B). In contrast,
both EGFR phosphorylation and total protein levels were decreased
significantly at this time (Figs. 4B, and 2A and B, respectively).
These results indicate that activation of the EGFR is associated with
prolonged transactivation of ErbB2, and that EGFR downregula-
tion does not correlate with downregulation of ErbB2 in the NF1
MPNST cell lines.
CI-1033 inhibits phosphorylation of Tyr1221/1222 of ErbB2
and Tyr1068 of EGFR. Our data indicated that NF1 MPNST lines
express higher levels of both the EGFR and ErbB2 versus a non-NF1
line and that prolonged transactivation of ErbB2 occurs after activa-
tion of the EGFR with exogenous EGF. CI-1033 is an irreversible
inhibitor of all ErbB family member kinase activities.45 Treatment
of both NF1 MPNST cell lines with 1 μM CI-1033 effectively
suppressed EGF-induced phosphorylation of EGFR at Tyr1068 and
ErbB2 at Tyr1221/1222 (Fig. 4C). In addition, CI-1033 also inhib-
ited the EGF-stimulated activation of ERK in these lines (Fig. 4C).
CI-1033 inhibits the proliferation of NF1 but not the non-NF1
MPNST line in 5% serum. The in vivo proliferation of MPNSTs
can be driven through ErbB-mediated neuregulin signaling as well
as EGFR-mediated signaling.16,17 We hypothesized that inhibition
of ErbB signaling through a selective pan-ErbB inhibitor would be
effective at suppressing multiple ErbB-mediated proliferation path-
ways in MPNST cells. Concentrations of CI-1033 as low as 250 nM
and 500 nM suppressed the proliferation of ST88-14 and NF90-8
cultures, respectively (Fig. 5). In contrast, concentrations of CI-1033
up to 1 μM did not markedly affect the proliferation of the non-NF1
STS-26T line (Fig. 5). At the concentrations examined, CI-1033 had
no effect on NF90-8 or STS-26T viability. A modest loss of viability
in the ST88-14 cell line was only seen at the highest concentration
tested (1 μM) (Fig. 5).
We next determined the effects of CI-1033 on MPNST cell cycle
distribution (Table 1). Concentrations of CI-1033 having graduated
effects on NF1 ST88-14 proliferation caused parallel gains in the
percentage of cells in G1, coupled with parallel losses of S-phase cells.
higher levels of the EGFR and ErbB2 receptors than the non-NF1
STS-26T MPNST line (Fig. 1A). EGFR:ErbB2 heterodimers are
significantly more active than EGFR homodimers24 and could
further amplify EGFR-mediated NF1 tumor pathologies. To deter-
mine whether ErbB2 was involved in EGFR-mediated signaling,
we treated the MPNST lines with 10 ng/ml EGF for 10 min.
Addition of EGF resulted in a pronounced phosphorylation of
ErbB2 at the Tyr1221/1222 autophosphorylation sites in the NF1
MPNST lines (Fig. 4A). Phosphorylation of these residues couples
ErbB2 activation with the Ras-ERK pathway and Schwann cell
mitotic activity.24,44 Basal and EGF-induced Tyr1221/1222
Figure 4. Coupling of EGFR and ErbB2 in NF1 MPNST cell lines. (A) EGF
induces phosphorylation of ErbB2 in NF1 MPNST cell lines. Serum-starved
cultures of ST88-14 (“88”); NF90-8 (“90”); and STS-26T (“26”) cells were
treated with vehicle or 10 ng/ml EGF for 10 min. Phosphorylation of
Tyr1221/1222 of ErbB2 was assessed using a phospho-specific antibody
raised against these sites (indicated as “pErbB2”). These data are repre-
sentative of four independent experiments. (B) ErbB2 protein levels and
activation remain elevated following EGF stimulation. ST88-14 (left) and
NF90-8 (right) cells were pretreated with cycloheximide or vehicle for 2 h
and subsequently treated with EGF or vehicle for 3 h. Lanes are indicated
with “C”, cycloheximide treated only; “E”, EGF treated only; “C + E”,
treated first with cycloheximide and then with EGF. (C) CI-1033 inhibits EGF-
mediated phosphorylation of EGFR and ErbB2 in NF1 MPNSTs. ST88-14
(left) and NF90-8 (right) cells were treated with vehicle or 1 μM of CI-1033
for 1 h and subsequently treated with 10 ng/ml EGF for 10 min. Lanes are
“V”, vehicle treated only; “E”, treated with EGF alone; “CI”, treated first
with CI-1033 for 1 h and then with EGF. Results are representative of three
separate experiments in both lines. In all panels, β-tubulin is included as a
protein loading control.
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Suppression of NF1 MPNST proliferation with CI-1033
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and inhibition of ErbB3/ErbB2 signaling by pan-ErbB inhibitors
may be significant to the effectiveness of this treatment approach
in MPNSTs.17 Aberrant expression of the EGFR in NF1 MPNSTs
introduces an additional dimerization partner for ErbB2.47 These
EGFR:ErbB2 heterodimers are significantly more active than EGFR
homodimers.24
The two NF1 MPNST lines used in our studies express greater
levels of ErbB2 than the non-NF1 MPNST line (Fig. 1A). Others
have demonstrated that the NF1 MPNST cell lines used here do not
express detectable ErbB3 and ErbB4 receptors.16 Thus, these lines
are an excellent model to test the relationship between the EGFR and
ErbB2 receptors in the absence of the other ErbB family members.
Activation of EGFR with exogenous EGF resulted in phosphoryla-
tion of the Tyr1221/1222 phosphorylation sites and transactivation
of ErbB2 in the NF1 MPNST lines (Fig. 4A). A previous study did
In contrast, but in agreement with proliferation analyses, CI-1033
treatment did not alter cell cycle phase distributions in STS-26T
cells (Table 1).
Discussion
Targeted expression of human EGFR in Schwann cells of newborn
mice results in characteristics of the initial stages of NF1 tumor
formation, including mast cell accumulation, collagen deposition,
and disruption of axon-glial interactions.32 These EGFR-dependent
developmental pathologies are inhibited by cetuximab administra-
tion, but only if administered for 2 weeks beginning immediately
after birth. Treatments started later during development are much
less effective, even if administered over a greater period of time.32
These results suggest that there may be a critical period during which
aberrantly expressed EGFR mediates remodeling of the normal
Schwann cell environment into a tumor-promoting environment.
In the present study, we examined the effectiveness of EGFR inhibi-
tion in suppressing the proliferation of MPNST cell lines. While
our data suggest that inhibition of the EGFR with gefitinib does not
effectively suppress proliferation of MPNSTs in vitro, inhibition of
aberrantly expressed EGFR may suppress development of the tumor-
supporting microenvironment in vivo.
In addition to evaluating the EGFR as an inhibitor of MPNST
proliferation, we examined the ErbB2 receptor as a potential
pharmacological target. While there is no known ligand for ErbB2,
this protein heterodimerizes with other members of the ErbB family
and potentiates their ligand-mediated signaling.24 Overexpressed
ErbB2 can undergo ligand-independent activation and contribute
to tumorigenesis.25,26 In normal Schwann cells, the only available
heterodimerization partner for ErbB2 is thought to be ErbB3,46
Figure 5. CI-1033 selectively inhibits the proliferation of NF1 MPNST cell lines. On day 0, plates were treated with either vehicle or the indicated concentra-
tion of CI-1033. Cells were harvested and counted using the Trypan Blue exclusion assay. These data show the effects of CI-1033 on both total cell number
(top) and percent viability (lower). Data are mean ± S.D. and are representative of three separate experiments for each line.
Table 1 CI-1033 selectively decreases the proportion of
ST88-14 NF1 MPNST cells in the S-phase of the
cell cycle
Treatment:
[CI-1033]
for 48 h
0
100 nM
250 nM
1000 nM


ST88-14
% of cells in:
S
28
25
22
16




STS-26T
% of cells in:
S
21
21
21
24
G1
63
66
70
80
G2/M
9
9
8
4
G1
70
70
70
66
G2/M
9
9
9
10
ST88-14 and STS-26T MPNST cells were treated as indicated with a range of CI-1033 concentrations that
dose-dependently inhibit proliferation of NF1 MPNST cells (Fig. 5). The cells were processed for analysis of
cell cycle distribution by flow cytometry.
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Suppression of NF1 MPNST proliferation with CI-1033
1944Cancer Biology & Therapy 2008; Vol. 7 Issue 12
Materials and Methods
Cell culture and treatments. Human MPNST cell lines ST88-14
and NF90-8 (generous gifts from T. Glover, University of Michigan,
Ann Arbor, MI, USA), and STS-26T (a generous gift from D. Scoles,
Cedars-Sinai Medical Center, Los Angeles, CA, USA) were cultured
in RPMI (Cellgro, Herndon, VA, USA) supplemented with 5% fetal
bovine serum, L-glutamine, phenol red, 50 units/mL penicillin and
50 μg/mL streptomycin in a humidified 37°C chamber, supple-
mented with 5% CO2. For EGF stimulation experiments, cells were
grown to ~80% confluency, serum-starved for 1 h, and stimulated
with human, recombinant EGF (Invitrogen, Carlsbad, CA, USA) as
indicated in the figures. Gefitinib (AstraZeneca, Alderly Park, UK)
and CI-1033 (Pfizer, Groton, CT) were prepared as stock solutions
in dimethyl sulfoxide (DMSO) and double distilled H2O, respec-
tively, and stored at -80°C.
Western blotting. After the indicated treatments, cells were lysed
with 2x SDS sample buffer and boiled immediately for 5 min.29
Proteins were separated using SDS-PAGE and transferred to nitrocel-
lulose or PVDF membranes. The membranes were blocked with 5%
milk in Tris-buffered saline with 0.5% Tween-20 (TBS-T) for 1 h to
overnight. Primary antibodies were rabbit polyclonal IgG anti-EGFR
at 1:1,000 (Cell Signaling Technology, Danvers, MA); rabbit immu-
noaffinity purified IgG anti-phospho-Tyr1068 of EGFR at 1:1,000
(Cell Signaling Technology); anti-MAP Kinase at 1:2,000 (Upstate
Cell Signaling Solutions, Charlottesville, VA); monoclonal IgG1
isotype anti-MAP Kinase activated, clone MAPK-YT at 1:2,000
(Sigma-Aldrich, St. Louis, MO); mouse monoclonal anti-ErbB2
(LabVision, Freemont, CA) at 1:1,000; rabbit monoclonal and poly-
clonal anti-pTyr1221/1222 of ErbB2 (Cell Signaling Technology) at
1:500; mouse monoclonal anti-Cyclin D1 (Santa Cruz Biotechnology,
Santa Cruz, CA) at 1:1000; mouse monoclonal IgG1 anti-β-tubulin
“E7” at 1:2,000 (developed by Michael Klymkowsky and obtained
from the Developmental Studies Hybridoma Bank, University of
Iowa, Iowa City, IA). Secondary antibodies were HRP-conjugated
goat anti-rabbit or goat anti-mouse IgG at 1:10,000 (Jackson
Immunoresearch Laboratories, Inc., West Grove, PA). Immunoblots
were incubated with Western Blotting Detection Reagents (Amersham
Bioscience, Buckinghamshire, United Kingdom) or DuraSignal
(Pierce Biotechnology, Inc., Rockford, IL) and recorded on X-ray
film (KODAK Biomax Light; Fisher Scientific, Pittsburgh, PA).
When necessary, brightness and contrast levels of scanned immu-
noblot images were uniformly adjusted using ADOBE Photoshop
software (Adobe Systems, San Jose, CA). ImageQuant software
(GE Healthcare, Piscataway, NJ) was used for densitometry and
quantification of immunoblot signals.
Cell proliferation assays. Approximately equal numbers of cells
(20,000–30,000) were plated onto either 30 mm or 60 mm dishes
and allowed to attach overnight. For proliferation assays using gefi-
tinib, cells were treated with the indicated concentration of gefitinib
dissolved in DMSO or an equal volume of DMSO alone and the
cells harvested at the indicated time-points. For proliferation assays
using CI-1033, cells were treated with the indicated concentration
of CI-1033 dissolved in ddH2O, or ddH2O alone and the cells
harvested at the indicated time-points. Media and floating cells
were collected and dishes rinsed with ice-cold PBS. Attached cells
were incubated with 0.5 ml trypsin for 5 min to allow complete
not find heterodimerization between EGFR and ErbB2 in normal
Schwann cells.21 In that case, however, the result could have been
affected by the system in which human EGFR was transgenically
expressed and tested for interaction with endogenous murine ErbB2
in mouse Schwann cells.21 EGFR:ErbB2 heterodimers have previ-
ously been shown to avoid degradation through ubiquitination, as
well as endocytosis-mediated transport to the lysosome, two mecha-
nisms that efficiently downregulate EGFR homodimer activity.33 Our
results indicate that ErbB2 protein levels remain relatively unchanged
and that ErbB2 remains phosphorylated on Tyr1221/1222 despite
EGFR deactivation and downregulation at the same time-points.
Thus, in addition to prolonged EGFR-Ras mediated signaling due
to loss of functional neurofibromin, our results suggest that coupling
between EGFR and ErbB2 may result in further amplification of
aberrant in vivo EGFR signaling in MPNSTs. We demonstrate here
that the pan-ErbB kinase inhibitor CI-1033 was effective at inhib-
iting EGF-mediated phosphorylation of Tyr1221 of ErbB2, Tyr1068
of EGFR and EGF-mediated activation of ERK. While efforts to
selectively inhibit EGFR-mediated NF1 pathologies have met with
mixed success,16,17,48,49 our results suggest that inhibition of both
EGFR and ErbB2 may provide an effective alternative.
The potency of CI-1033 correlates with suppression of ErbB2
transactivation in the NF1 MPNST cell lines. In agreement with
a previous report that showed that micromolar levels of CI-1033
(PD158780) were required to block DNA synthesis in a chemically-
induced MPNST cell line,50 we found that the non-NF1 STS-26T
MPNST cell line was significantly less sensitive to inhibition of
proliferation by CI-1033. Notably, CI-1033 effectively suppressed
the proliferation of NF1 MPNST lines in the presence of 5% serum.
Thus, we predict that ErbB inhibition may be effective in vivo,
despite the presence of growth factors and cytokines that promote
NF1 MPNST proliferation. This idea is supported by reports in
which inhibition of ErbB2 in benign vestibular schwannomas
significantly suppressed their proliferation.51,52 Fortunately, inhibi-
tion of ErbB-mediated neuregulin signaling does not reduce the
survival of mature Schwann cells in culture.53 Hence, ErbB inhibi-
tion may have the advantage of inhibiting NF1 tumor-promoting
ErbB signaling without affecting normal Schwann cell survival.
In summary, we show that two independent NF1 MPNST cell
lines demonstrate enhanced sensitivity and prolonged ERK activa-
tion in response to EGF stimulation. While EGFR expression has
been shown to correlate with NF1 tumor pathologies, the EGFR
per se does not appear to play a primary role in the proliferation
of MPNST cells in culture. We show that EGFR activation by
exogenously added EGF results in prolonged transactivation of
ErbB2 in these NF1 MPNST lines and that ErbB2 transactivation
provides an additional potential mechanism for prolonged EGFR-
Ras-ERK signaling in MPNSTs in vivo. Further, inhibition of both
EGFR and ErbB2 with the inhibitor CI-1033 produces a profound
and selective inhibition of NF1 MPNST cell proliferation. CI-1033
has reached Phase II clinical trials and so its ultimate clinical status
is not yet clear.54 Our results suggest that targeted inhibition of the
EGFR and ErbB2 may be an effective approach for inhibiting ErbB-
mediated NF1 tumor pathologies and MPNST growth. The recent
success of another combined EGFR/ErbB2 inhibitor, lapatinib, in
advanced breast cancer demonstrates that such an approach can be
effective in the treatment of human cancer.55
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Suppression of NF1 MPNST proliferation with CI-1033
www.landesbioscience.com Cancer Biology & Therapy1945
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detachment. Trypsinized cells and floating cells were combined and
centrifuged at 1000x g for 5 min to pellet. Media and trypsin were
removed by aspiration and the cells resuspended in an equal volume
of serum-free media. Numbers of live and dead cells were determined
by the Trypan blue exclusion assay.
Flow cytometry. MPNST cell lines were harvested and processed
for FACS analyses of DNA content as described previously.30 DNA
analyses were made with a FACScalibur instrument (BD Biosciences,
San Jose, CA). Percentages of cells in the G0/G1, S and G2/M stages
of the cell cycle were determined with a DNA histogram-fitting
program (MODFIT; Verity Software, Topsham, ME). A minimum
of 104 events/sample was collected for subsequent analyses.
Receptor turnover assays. Cells were plated in 5% serum and
grown to 80% confluency. Cells were serum-starved overnight, and
then treated with either 50 μg/ml cycloheximide dissolved in ddH2O
(Sigma-Aldrich) or ddH2O alone as indicated for 2 h. Cells were
then treated with either 10 ng/mL of EGF in serum-free media, or
serum-free media alone and harvested at the indicated time-points.
For assays involving a single time-point, EGF treatments were
for 3 h. Cells were harvested in 2x SDS sample buffer and boiled
immediately for 5 min. Proteins were separated using SDS-PAGE,
transferred to nitrocellulose, and immunoblotted for EGFR, phos-
phorylated and total ErbB2, cyclin D1, and β-tubulin as described
above.
Acknowledgements
We thank AstraZeneca for providing us with gefitinib and Pfizer
for providing us with CI-1033.
This study was supported by grants DAMD17-03-1-0182 and
W81XWH-05-1-0193 from the Department of the Army [The
content of the information does not necessarily reflect the position
or policy of the U.S. government and no official endorsements
should be inferred] and a Strategic Research Initiative award from the
Karmanos Cancer Institute. J.W.W. was supported by the National
Institutes of Health grant T32 ES012163. This project was aided
by Imaging and Cytometry Core facilities that were supported
by National Institutes of Health grants P30 ES06639 and P30
CA22453.
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