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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

1.
Jemal  AThomas  AMurray  TThun  M Cancer statistics, 2002. CA Cancer J Clin.2002;52:23-47.
2.
Parkin  DMPisani  PFerlay  J Global cancer statistics. CA Cancer J Clin.1999;49:33-64, 31.
3.
Goepfert  H Squamous cell carcinoma of the head and neck: past progress and future promise. CA Cancer J Clin.1998;48:195-198.
4.
Clark  JW Targeted therapy.  In: Chabner  BA, Longo  DL, eds. Cancer Chemotherapy and Biotherapy: Principles and Practice. 3rd ed. Philadelphia, Pa: Lippincott Williams & Wilkins; 2001:891-910.
5.
Druker  BJTalpaz  MResta  DJ  et al Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med.2001;344:1031-1037.
6.
Grandis  JRMelhem  MFGooding  WE  et al Levels of TGF-α and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Natl Cancer Inst.1998;90:824-832.
7.
Nicholson  RIGee  JMHarper  ME EGFR and cancer prognosis. Eur J Cancer.2001;37(suppl 4):9-15.
8.
Ullrich  ACoussens  LHayflick  JS  et al Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature.1984;309:418-425.
9.
Pazin  MJWilliams  LT Triggering signaling cascades by receptor tyrosine kinases. Trends Biochem Sci.1992;17:374-378.
10.
Ullrich  ASchlessinger  J Signal transduction by receptors with tyrosine kinase activity. Cell.1990;61:203-212.
11.
Aaronson  SA Growth factors and cancer. Science.1991;254:1146-1153.
12.
Cantley  LCAuger  KRCarpenter  C  et al Oncogenes and signal transduction. Cell.1991;64:281-302.
13.
Baselga  JPfister  DCooper  MR  et al Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin. J Clin Oncol.2000;18:904-914.
14.
Traxler  PBuchdunger  EFuret  P  et al Preclinical profile of PKI166—a novel and potent EGF-R tyrosine kinase inhibitor for clinical development [abstract]. Clin Cancer Res.1999;5(suppl):3750.
15.
Bruns  CJSolorzano  CCHarbison  MT  et al Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma. Cancer Res.2000;60:2926-2935.
16.
Bos  MMendelsohn  JKim  YMAlbanell  JFry  DWBaselga  J PD153035, a tyrosine kinase inhibitor, prevents epidermal growth factor receptor activation and inhibits growth of cancer cells in a receptor number–dependent manner. Clin Cancer Res.1997;3:2099-2106.
17.
Faust  RATawfic  SDavis  ATAhmed  K Apoptosis and growth inhibition of head and neck tumor cell line induced by epidermal growth factor receptor tyrosine kinase inhibitor. Oral Oncol.1999;35:290-295.
18.
Lansford  CDGrenman  RBier  H  et al Head and neck cancers.  In: Palsson  B, Masters  JRW, eds. Cancer Cell Lines Part 2.Vol 2. Norwell, Mass: Kluwer Academic Publishers; 1999:185-255.
19.
Beckhardt  RNKiyokawa  NXi  L  et al HER-2/neu oncogene characterization in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg.1995;121:1265-1270.
20.
Denizot  FLang  R Rapid colorimetric assay for cell growth and survival: modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods.1986;89:271-277.
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Gullick  WJ Prevalence of aberrant expression of epidermal growth factor receptor in human cancers. Br Med Bull.1991;47:87-93.
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Grandis  JRTweardy  DJ Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer. Cancer Res.1993;53:3579-3584.
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Maurizi  MAlmadori  GFerrandina  G  et al Prognostic significance of epidermal growth factor receptor in laryngeal squamous cell carcinoma. Br J Cancer.1996;74:1253-1257.
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Sturgis  EMSacks  PGMasui  HMendelsohn  JSchantz  SP Effects of antiepidermal growth factor receptor antibody 528 on the proliferation and differentiation of head and neck cancer. Otolaryngol Head Neck Surg.1994;111:633-643.
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Goldstein  NIPrewett  MZuklys  KRockwell  PMendelsohn  J Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin Cancer Res.1995;1:1311-1318.
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Fan  ZBaselga  JMasui  HMendelsohn  J Antitumor effect of anti-epidermal growth factor receptor monoclonal antibodies plus cis-diamminedichloroplatinum on well established A431 cell xenografts. Cancer Res.1993;53:4637-4642.
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Papadimitrakopoulou  VAMurren  JRFidler  IJ  et al A phase I dose-escalating study to evaluate the biological activity and pharmacokinetics of PKI166, a novel tyrosine kinase inhibitor, in patients with advanced cancers [abstract]. Clin Cancer Res.2001;7(11 suppl):3708s.
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Traxler  PBold  GBuchdunger  E  et al Tyrosine kinase inhibitors: from rational design to clinical trials. Med Res Rev.2001;21:499-512.
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Kim  ESKhuri  FRHerbst  RS Epidermal growth factor receptor biology (IMC-C225). Curr Opin Oncol.2001;13:506-513.
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Original Article
August 2002

Targeted Molecular Therapy for Oral Cancer With Epidermal Growth Factor Receptor BlockadeA Preliminary Report

Author Affiliations

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.

Arch Otolaryngol Head Neck Surg. 2002;128(8):875-879. doi:10.1001/archotol.128.8.875
Abstract

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 × 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.

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 × 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)] × 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 × 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 × 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 α 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).

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.

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 × 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 × 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.

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 α (TGF-α). The HNSCCs express higher levels of EGF-R and TGF-α than corresponding normal tissues.21 Furthermore, the concomitant expression of both EGF-R and its ligand TGF-α 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-α 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.

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Article 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@mdanderson.org).

References
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Jemal  AThomas  AMurray  TThun  M Cancer statistics, 2002. CA Cancer J Clin.2002;52:23-47.
2.
Parkin  DMPisani  PFerlay  J Global cancer statistics. CA Cancer J Clin.1999;49:33-64, 31.
3.
Goepfert  H Squamous cell carcinoma of the head and neck: past progress and future promise. CA Cancer J Clin.1998;48:195-198.
4.
Clark  JW Targeted therapy.  In: Chabner  BA, Longo  DL, eds. Cancer Chemotherapy and Biotherapy: Principles and Practice. 3rd ed. Philadelphia, Pa: Lippincott Williams & Wilkins; 2001:891-910.
5.
Druker  BJTalpaz  MResta  DJ  et al Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med.2001;344:1031-1037.
6.
Grandis  JRMelhem  MFGooding  WE  et al Levels of TGF-α and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Natl Cancer Inst.1998;90:824-832.
7.
Nicholson  RIGee  JMHarper  ME EGFR and cancer prognosis. Eur J Cancer.2001;37(suppl 4):9-15.
8.
Ullrich  ACoussens  LHayflick  JS  et al Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature.1984;309:418-425.
9.
Pazin  MJWilliams  LT Triggering signaling cascades by receptor tyrosine kinases. Trends Biochem Sci.1992;17:374-378.
10.
Ullrich  ASchlessinger  J Signal transduction by receptors with tyrosine kinase activity. Cell.1990;61:203-212.
11.
Aaronson  SA Growth factors and cancer. Science.1991;254:1146-1153.
12.
Cantley  LCAuger  KRCarpenter  C  et al Oncogenes and signal transduction. Cell.1991;64:281-302.
13.
Baselga  JPfister  DCooper  MR  et al Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin. J Clin Oncol.2000;18:904-914.
14.
Traxler  PBuchdunger  EFuret  P  et al Preclinical profile of PKI166—a novel and potent EGF-R tyrosine kinase inhibitor for clinical development [abstract]. Clin Cancer Res.1999;5(suppl):3750.
15.
Bruns  CJSolorzano  CCHarbison  MT  et al Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma. Cancer Res.2000;60:2926-2935.
16.
Bos  MMendelsohn  JKim  YMAlbanell  JFry  DWBaselga  J PD153035, a tyrosine kinase inhibitor, prevents epidermal growth factor receptor activation and inhibits growth of cancer cells in a receptor number–dependent manner. Clin Cancer Res.1997;3:2099-2106.
17.
Faust  RATawfic  SDavis  ATAhmed  K Apoptosis and growth inhibition of head and neck tumor cell line induced by epidermal growth factor receptor tyrosine kinase inhibitor. Oral Oncol.1999;35:290-295.
18.
Lansford  CDGrenman  RBier  H  et al Head and neck cancers.  In: Palsson  B, Masters  JRW, eds. Cancer Cell Lines Part 2.Vol 2. Norwell, Mass: Kluwer Academic Publishers; 1999:185-255.
19.
Beckhardt  RNKiyokawa  NXi  L  et al HER-2/neu oncogene characterization in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg.1995;121:1265-1270.
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