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Targeted Molecular Therapy for Oral Cancer With Epidermal Growth Factor Receptor Blockade
A Preliminary Report
Jeffrey N. Myers, MD, PhD;
F. Christopher Holsinger, MD;
B. Nebiyou Bekele, PhD;
Emily Li, DDS;
Samar A. Jasser, BA;
Jerald J. Killion, PhD;
Isaiah J. Fidler, DVM, PhD
Arch Otolaryngol Head Neck Surg. 2002;128:875-879.
ABSTRACT
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Background Overexpression of epidermal growth factor receptor (EGF-R) is associated
with increased malignant potential and correlates with poor clinical outcome
in head and neck cancer. Therefore, inhibition of the EGF-R pathway provides
an ideal target for molecular therapy. We examined in vitro and in vivo effects
of PKI166, an orally administered EGF-R inhibitor, on 2 human squamous cell
carcinoma of the oral cavity cell lines, Tu159 and MDA1986.
Study Design Basic science, laboratory investigation.
Results For Western blotting, Tu159 and MDA1986 cells were pretreated for 1
hour and then stimulated with EGF. The EGF-R–specific tyrosine kinase
autophosphorylation was inhibited completely by PKI166 at all doses tested
(1-10 µg/mL). By means of a tetrazolium-based viable cell assay, PKI166
was shown to arrest the growth of Tu159 and MDA1986 cells. The inhibitory
concentration (50%), calculated from regression lines on the linear portion
of the growth inhibition graphs, was 0.18µM (R
= 0.98) for Tu159 cells and 0.23µM (R = 0.97)
for MDA1986 cells. Nude mice were inoculated subcutaneously with 1 x
106 Tu159 tumor cells and observed for 7 days. Next, daily doses
of PKI166 (0, 10, or 50 mg/kg) were delivered by orogastric lavage for 28
days and the animals were observed for tumor growth. PKI166 significantly
reduced tumor growth in mice treated for 1 month with oral PKI166 in a dose-dependent
fashion.
Conclusions Targeted molecular therapy with EGF-R blockade arrests the growth of
oral cancer in vitro and reduces its proliferation in an experimental xenograft
animal model.
INTRODUCTION
IN 2002, SQUAMOUS cell carcinoma of the head and neck (HNSCC) is predicted
to account for nearly 40 000 new cancers in the United States, equal
in incidence to leukemia and greater than all endocrine tumors.1
Worldwide, cancers of the oral cavity and pharynx represent an even greater
public health problem, responsible for almost 200 000 deaths annually.2 Squamous cell carcinoma of the oral cavity (SCCOC)
accounts for nearly 50% of all newly diagnosed cancers in India and is a leading
cause of cancer death in France.2 Despite improvements
in locoregional control, morbidity and mortality rates have improved little
during the past 30 years.3
Targeted molecular therapy offers an exciting new approach to treat
human malignancy.4 The tyrosine kinase inhibitor
STI-571 has shown promise in early clinical trials for the treatment of chronic
myeloid leukemia.5 Its success highlights the
potential for anticancer drugs based on the specific molecular abnormality
present in a human cancer.
The epidermal growth factor receptor (EGF-R) pathway provides an attractive
target for molecular therapy for HNSCC. Overexpression of the EGF-R correlates
with a poor outcome in patients with HNSCC6
and other human epithelial tumors.7 The EGF-R
is a 170-kd transmembrane glycoprotein consisting of an extracellular ligand-binding
domain, a transmembrane domain, and an intracellular domain with intrinsic
tyrosine kinase activity.8-9 Once
activated, the EGF-R intracellular domain phosphorylates both the receptor
itself and several crucial second messenger effector molecules.10
Activation of this signaling pathway triggers DNA synthesis and a mitogenic
cascade, resulting in cell proliferation.11-12
Therefore, inhibition of the EGF-R pathway and its tyrosine kinase signaling
activity may provide an ideal target for the molecular treatment of HNSCC.
Several strategies have been developed to block the EGF-R. These include
antisense technology, ligand-linked toxins, monoclonal antibodies, and small-molecule
tyrosine kinase inhibitors. A phase 1 clinical trial recently demonstrated
the safety and preliminary efficacy of the monoclonal antibody preparation
C225.13 PKI166 is a newly identified, low-molecular-weight
EGF-R tyrosine kinase inhibitor that is administered orally.14
PKI166 not only inhibited tumor growth in an orthotopic model of human pancreatic
adenocarcinoma but also induced apoptosis in tumor-associated endothelial
cells.15 Although a handful of in vitro studies
have examined the role of EGF-R tyrosine kinase inhibitors in HNSCC,16-17 to our knowledge, no confirmation
in an experimental animal model has been reported.
We present a preclinical evaluation of an EGF-R tyrosine kinase inhibitor
and its antitumoral effects on in vitro and in vivo proliferation of human
SCCOC.
MATERIALS AND METHODS
ANIMALS
Male athymic nude mice (NCR-nu) were purchased from the Animal Production
Area of the National Cancer Institute–Frederick Cancer Research and
Development Center (Frederick, Md). The mice were housed and maintained in
laminar flow cabinets under specific pathogen-free conditions in facilities
approved by the American Association for Accreditation of Laboratory Animal
Care and in accordance with current regulations and standards of the US Department
of Agriculture, the US Department of Health and Human Services, and the National
Institutes of Health. The mice were used in accordance with Animal Care and
Use Guidelines of The University of Texas M. D. Anderson Cancer Center, Houston.
They were 8 to 12 weeks old when they were used for this study.
CELL LINES AND CULTURE CONDITIONS
Tu159 and MDA1986 are human SCCOC cell lines derived from individual
patients undergoing surgery, primarily at M. D. Anderson Cancer Center.18-19 The cells were grown in vitro in
Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and
L-glutamine. Adherent monolayer cultures were maintained on plastic and incubated
at 37°C in 5% carbon dioxide and 95% air. The cultures were free of Mycoplasma species and the following pathogenic murine
viruses: reovirus type 3, pneumonia virus, K virus, Theiler encephalitis virus,
Sendai virus, minute virus, ectromelia virus, and lactate dehydrogenase virus
(assayed by MA Bioproducts, Walkersville, Md). The cultures were maintained
for no longer than 12 weeks after recovery from frozen stocks.
REAGENTS
PKI166 (4-[R]-phenethylamino-6-[hydroxyl]phenyl-7H-pyrrolo[2.3-d]-pyrimidine)
was synthesized and obtained (Novartis International AG, Basel, Switzerland).
For in vivo administration, PKI166 was dissolved in dimethyl sulfoxide (0.5%)
and then diluted 1:20 in Hanks balanced saline solution.14
Tetrazolium (MTT) was purchased (Sigma-Aldrich Corp, St Louis, Mo), and a
stock solution was prepared by dissolving 5 mg of MTT in 1 mL of phosphate-buffered
saline and filtering the solution to remove particles. The solution was protected
from light, stored at 4°C, and used within 1 month.
WESTERN BLOTTING
The Tu159 and MDA1986 cells were plated into 6-well (38 mm2)
plates at a concentration of 4 x 105 cells per well and then
incubated in serum-free medium for 24 hours. Treated cells were preincubated
with inhibitor (1-10 µg/mL) (controls were preincubated with dimethyl
sulfoxide alone) for 1 hour, then epidermal growth factor (EGF) (40 ng/mL)
was added for 15 minutes; the cells were then washed with phosphate-buffered
saline containing 5mM edetic acid and 1mM sodium orthovanadate. Cells were
scraped into lysis buffer (1% Triton X-100; 20mM Tris, pH 8.0; 137mM sodium
chloride; 10% glycerol (vol/vol); 2mM edetic acid; 1mM phenylmethylsulfonyl
fluoride; 20µM aprotinin-leupeptin-trypsin inhibitor; 2mM sodium orthovanadate)
and centrifuged to remove insoluble protein. Samples were diluted in sample
buffer (0.5mM Tris hydrochloride, pH 6.8; 10% sodium dodecyl sulfate; 1M dithiothreitol;
10% [vol/vol] glycerol; and 1% bromphenol blue) and boiled. The proteins (30
µg/mL) were resolved on 7.5% sodium dodecyl sulfate–polyacrylamide
gel electrophoresis and transferred onto 0.45-µg nitrocellulose membranes.
The 7.5% gels were used to probe with anti–EGF-R and antiphosphotyrosine
antibodies. The membranes were blocked with 5% (wt/vol) nonfat milk in 0.1%
Tween 20 (vol/vol) in Tris-buffered saline, probed with mouse monoclonal antiphosphotyrosine
(IgG2bk) (1:5000) (Upstate Biotechnology, Inc, Lake Placid, NY) in 5% nonfat
milk, and incubated with horseradish peroxidase–conjugated sheep anti–mouse
immunoglobulin (1:2000) (Amersham Life Science Inc, Arlington Heights, Ill)
in 5% nonfat milk. The blots were also probed with sheep anti–EGF-R
(UBI Inc), diluted 1:1000, in 5% nonfat milk and incubated with peroxidase-conjugated
donkey anti–sheep IgG (1:3000) (Sigma Immunochemicals, St Louis, Mo)
in 5% nonfat milk. Finally, all blots were probed with antiactin (1:1000)
in 5% nonfat milk (Sigma Immunochemicals), followed with horseradish peroxidase–conjugated
donkey anti–rabbit immunoglobulin (1:2000) (Amersham Inc) in 5% nonfat
milk. Protein bands were visualized by the Enhanced ChemiLuminescence detection
system (Amersham Inc).
MTT CELL PROLIFERATION ASSAY
PKI166 was tested against the Tu159 and MDA1986 cell lines by means
of an MTT-based assay. The MTT assay measures cell proliferation, based on
the ability of live cells to use MTT and convert it into dark-blue formazan.20 One thousand cells were plated into 38-mm2
wells of 96-well tissue culture plates. The cells were grown in Dulbecco modified
Eagle medium supplemented with sodium pyruvate, essential amino acids, and
10% fetal bovine serum. After a 24-hour attachment period, the cells were
refed with medium (negative control with dimethyl sulfoxide alone) or medium
containing PKI166. After a 5-day incubation, the number of metabolically active
cells was determined by MTT assay. The conversion of MTT to formazan by metabolically
active cells was measured by a 96-well microtiter plate reader at an optical
density at 570 nm (MR-5000; Dynatech Laboratories Inc, Chantilly, Va). Growth
inhibition was calculated from the following formula: cytostasis (%) = {[1 -
(A/B)] x 100}, where A is the absorbance of treated cells and B is the
absorbance of control cells.
IN VIVO TUMOR XENOGRAFTS
Tu159 cells were harvested from subconfluent cultures by a brief exposure
to 0.25% trypsin and 0.02% edetic acid. Trypsinization was reversed with the
addition of medium containing 10% fetal bovine serum, and the cells were washed
once in serum-free medium and resuspended in Hanks balanced saline solution.
Tumor cells were then implanted subcutaneously in the flanks of nude mice
at a concentration of 1 x 106 cells per mouse. One week elapsed,
at which time subcutaneous tumors could be palpated. The mice were then treated
for 28 days with daily oral doses of 0, 10, or 50 mg of PKI166 per kilogram.
Tumor sizes were measured by calipers and recorded weekly. Measurements were
recorded as the products of the length and width of tumors.
STATISTICS
Sigma Plot software (SPSS Science, Chicago, Ill) was used to calculate
the inhibitory concentration (50%) (IC50) by means of equations
based on an exponential rise algorithm. SPSS software (SPSS Science) was used
for statistical analysis. A repeated-measures regression analysis was used
to assess the effects of time, dose, and time x dose interaction on
growth of in vivo tumor Tu159 xenografts in mice. The primary analysis was
a repeated-measures analysis. Repeated-measures analysis was performed on
the log-transformed data to mitigate skewness observed in the tumor volume
data. Repeated-measures analysis was also performed on the original data and
on the square root–transformed data to ensure consistent inferences.
The effects in the model were assessed at an  significance level of
.05. All computations were carried out on a DELL personal computer (Dell Computer
Corp, Austin, Tex) with Windows NT operating system (Microsoft Corp, Redmond,
Wash) and using the SAS Proc Mixed procedure (SAS Institute Inc, Cary, NC).
RESULTS
INHIBITION OF EGF-R AUTOPHOSPHORYLATION IN HUMAN SCCOC
In our first experiment, we determined whether treatment of Tu159 and
MDA1986 cells with PKI166 could inhibit EGF-stimulated tyrosine phosphorylation
of the EGF-R. Tu159 and MDA1986 cells, incubated 15 minutes with serum-free
medium but containing EGF, exhibited high levels of autophosphorylated EGF-R
as detected by antiphosphotyrosine antiserum on Western blots of anti–EGF-R–immunoprecipitated
cell lysates. Next, pretreatment of cells with PKI166 for 60 minutes, followed
by a 15-minute treatment with EGF, inhibited the autophosphorylation in a
dose-dependent manner (0-10 µg/mL). Expression of the 170-kd EGF-R protein
was found to be down-modulated by the addition of EGF in the absence of PKI166.
Receptor autophosphorylation was found to be maximal under these conditions,
and the EGF-R–specific tyrosine autophosphorylation was inhibited completely
by PKI166 at all doses tested (1-10 µg/mL) in both cell lines (Figure 1 and Figure 2).
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Figure 1. Effect of PKI166 on tyrosine phosphorylation
in Tu159 cells of squamous cell carcinoma of the oral cavity by Western blotting.
EGF indicates epithelial growth factor; EGF-R, EGF receptor; and P, phosphorylated.
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Figure 2. Effect of PKI166 on tyrosine phosphorylation
in MDA1986 cells of squamous cell carcinoma of the oral cavity by Western
blotting. EGF indicates epithelial growth factor; EGF-R, EGF receptor; and
P, phosphorylated.
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MEDIATION OF IN VITRO CYTOTOXICITY OF SCCOC
Tu159 and MDA1986 cells were incubated for 5 days in medium, either
with or without PKI166. As seen in Figure
3 and Figure 4, a dose-dependent
cytotoxicity was seen with the addition of PKI166. Both cell lines had similar
patterns of growth inhibition by PKI166. The IC50 was calculated
from regression lines on the linear portion of the growth inhibition graphs.
The IC50 was 0.18µM (R = 0.98) for
Tu159 cells and 0.23µM (R = 0.97) for MDA1986
cells.
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Figure 3. PKI166-mediated cytotoxicity of
Tu159 oral cancer cells as measured by tetrazolium-based viable cell assay.
Tu159 cells were sensitive to the effects of PKI166 in a dose-dependent manner.
The inhibitory concentration (50%) was 0.18µM (R = 0.98) for Tu159 cells.
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Figure 4. PKI166-mediated cytotoxicity of
MDA1986 oral cancer cells as measured by tetrazolium-based viable cell assay.
The MDA1096 cells were sensitive to the effects of PKI166 in a dose-dependent
manner. The inhibitory concentration (50%) was 0.23µM
(R = 0.97) for MDA1986 cells.
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REDUCTION OF IN VIVO GROWTH OF Tu159 SCCOC XENOGRAFTS
On the basis of these in vitro data, a pilot study was performed to
determine the effect of PKI166 on the growth of SCCOC in nude mice. Groups
of 5 mice were inoculated subcutaneously with 1 x 106 Tu159
cells each and observed for 7 days. Then, the animals were treated daily with
oral PKI166 (0, 10, or 50 mg/kg) for 28 days. As shown in Figure 5, the growth of Tu159 xenografts was reduced in a dose-dependent
manner. With the repeated-measures analysis (log-transformed data), differences
in the growth in tumor size over time (as measured by the time x dose
interaction) were statistically significant (P =
.008). Differences between the control animals and the 50-mg dose group (P = .004) and between the 10-mg and 50-mg groups (P = .02) were statistically significant. Differences between
control animals and the 10-mg dose group were not statistically significant.
Inferences drawn from the untransformed tumor volume data and the square root–transformed
data were similar to those observed for the log-transformed data.
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Figure 5. Therapeutic effect of epidermal
growth factor receptor blockade by means of systemic treatment with PKI166
on the growth of Tu159 xenograft tumors in nude mice. The growth of Tu159
tumor xenografts was inhibited in a dose-dependent manner in animals treated
for 1 month with oral PKI166. Differences in the growth of tumors were statistically
significant (P = .008). Differences between the control
animals and the 50-mg dose group (P = .004) and between
the 10-mg and 50-mg groups (P = .02) were statistically
significant. Differences between control animals and the 10-mg dose group
were not statistically significant.
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COMMENT
Blockade of the EGF-R signaling pathway by the novel tyrosine kinase
inhibitor PKI166 suppressed the growth of human SCCOC. PKI166 specifically
inhibited the autophosphorylation mediated by the EGF-R tyrosine kinase pathway,
as measured by Western blotting. Furthermore, the in vitro cytotoxicity of
PKI166 on 2 SCCOC cell lines was confirmed by MTT assay. Finally, we present
the first report, to our knowledge, of in vivo suppression of SCCOC tumor
growth in an experimental animal model using oral tyrosine kinase inhibitor–EGF-R
blockade.
The proliferation of HNSCC has been correlated with increased expression
of EGF-R and its ligands, EGF and transforming growth factor (TGF-).
The HNSCCs express higher levels of EGF-R and TGF- than corresponding
normal tissues.21 Furthermore, the concomitant
expression of both EGF-R and its ligand TGF- suggests that an autocrine
control mechanism may be important in the development of these tumors.22-23 In head and neck cancer, overexpression
of EGF-R and TGF- has been shown to correlate with aggressive malignant
progression and poor clinical outcome.6, 24
Targeted molecular therapy for HNSCC has focused on the use of anti–EGF-R
antibody preparations. Tumor proliferation in cell culture and tumor xenografts
in athymic mice have been inhibited by these antibodies, which block EGF binding
to EGF-R.25-26 When injected into
mice bearing tumor xenografts, mouse anti–EFG-R antibody preparations
can cause partial tumor regression. The addition of concomitant chemotherapeutic
agents (cisplatin or doxorubicin) is necessary for a more complete tumor response.27 A chimeric version of the 225 monoclonal antibody
(C225) in which the mouse antibody variable regions are linked to human constant
regions exhibited an improved in vivo therapeutic effect at high doses. These
promising results with C225 led to phase 1 and 2 clinical trials that are
now under way.13
The use of an orally administered compound that inhibits the proliferation
of HNSCC has several advantages over treatment with receptor-specific antibodies.
These advantages include drug availability, no immunologic reactivity, and
direct intracellular effects on EGF-R. Phase 1 trials are currently under
way to assess the pharmocokinetic bioavailability and toxicity of PKI166.28 Early reports14, 28-29
suggest that this class of compounds has low levels of systemic toxic effects,
limited to fatigue, nausea, and rash. The most common complications from monoclonal
antibody therapy targeted at EGF-R signaling were fever, asthenia, elevation
of aminotransferase levels, nausea, and rash.30
The crucial biological activity of EGF-R pathway inhibition may be derived
from its ability to suspend cell proliferation. Blockade of the EGF-R signaling
pathway results in cellular arrest at the G1 restriction point, which has
been shown to increase sensitivity to cytotoxicity mediated by radiation or
chemotherapeutic agents.31 While halted by
EGF-R inhibition, cancer cells may be more susceptible to concomitant cytotoxic
agents (paclitaxel or cisplatin) and adjuvant radiotherapy. Studies are under
way in our laboratory to evaluate the efficacy of PKI166 when given in combination
with paclitaxel, with the use of an orthotopic nude mouse model of oral cancer.32
In summary, the blockade of the EGF-R signaling pathway with a tyrosine
kinase inhibitor arrests the growth of oral cancer in vitro and reduces its
proliferation in an experimental xenograft animal model. These preliminary
results require further confirmation with an orthotopic model of oral cancer
as well as preclinical studies to determine the safety of tyrosine kinase
inhibitors in humans. These studies are now under way in our laboratory.
AUTHOR INFORMATION
Accepted for publication January 18, 2002.
This study was presented at the Fifth International Conference on Head
and Neck Cancer, San Francisco, Calif, July 31, 2000.
Corresponding author and reprints: Jeffrey N. Myers, MD, PhD, Department
of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center,
1515 Holcombe Blvd, Box 441, Houston, TX 77030-4009 (e-mail: jmyers{at}mdanderson.org).
From the Departments of Head and Neck Surgery (Drs Myers, Holsinger,
and Li and Mr Jasser), Biostatistics (Dr Bekele), and Cancer Biology (Drs
Killion and Fidler), The University of Texas M. D. Anderson Cancer Center,
and the Bobby R. Alford Department of Otorhinolaryngology and Communicative
Sciences, Baylor College of Medicine (Dr Holsinger), Houston.
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