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1 Departments of Head and Neck Surgery, 2 Cancer Biology, and 3 Biostatistics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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ABSTRACT |
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INTRODUCTION |
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50% for the past 30 years (3)
. To improve the outcome for patients with SCCHN, novel therapeutic approaches are needed. The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor of the ErbB family that is expressed or highly expressed in a range of solid tumors, including head and neck cancers (4) . EGFR activation induces activation of several downstream intracellular substrates, leading to mitogenic signaling and other tumor-promoting cellular activities (5) . In human tumors, overexpression of this receptor correlates with a more aggressive clinical course (4) .
Nevertheless, recent experimental evidence suggests that the cancer cells can escape from growth inhibition by using alternative growth pathways or by constitutive activation of downstream signaling effectors. For example, human A431 cancer cells xenografts can acquire resistance to anti-EGFR monoclonal antibodies (mAbs), such as C225 and hR3, by increased tumor-induced angiogenesis due to the constitutive overexpression of proangiogenic growth factors, such as vascular endothelial growth factor (VEGF; ref. 6 ).
VEGF, a mitogen specific for vascular endothelial cells, is considered to play a key role in angiogenic processes (7) . VEGF binds to two distinct receptors on endothelial cells, flt-1 (VEGFR-1) and flk-1/KDR receptor (VEGFR-2), the latter of which is considered the dominant signaling receptor governing endothelial cell permeability, proliferation, and differentiation (8) . Expression of these receptors in normal tissues is low and is up-regulated during the development of pathological conditions associated with neovascularization (9 , 10) . Inhibition of VEGF-induced angiogenic signals would be expected to selectively target tumor-associated vessels because division of endothelial cells in the normal vasculature is infrequent (8) . Hence, inhibition of VEGF-mediated effects likely is an attractive alternative or complement to cytotoxic therapies.
Blockade of EGFR with specific mAbs or small molecule chemical inhibitors has been demonstrated to decrease tumor cell production of proangiogenic molecules (such as VEGF) and to inhibit tumor-associated angiogenesis (10 , 11) . A recent report documented that treatment combining an anti-EGFR antibody with an anti-KDR (VEGFR-2) antibody in a murine model of human colon cancer decreased tumor vascularity and increased tumor and endothelial cell apoptosis (12) . Therefore, although EGFR/ErbB2 inhibitors can inhibit production of VEGF by the tumor cells, a more potent antitumor response can be achieved through the concomitant inhibition of VEGFR. Dual inhibitors, combining both activities in the same molecule, would therefore be attractive for the treatment of numerous solid tumors.
NVP-AEE788 (AEE788; molecular weight, Mr 440,600) is an orally administered dual specific-kinase inhibitor targeting both the ErbB and VEGF receptors. It belongs to the class of the 7H-pyrrolo[2,3-d] pyrimidines. The efficacy of AEE788 in vitro against lung, bladder, and breast cancer cell lines has been demonstrated, and AEE788 has proved to be an effective antitumor compound in human tumor xenograft models (data on file at Novartis Pharma AG, Basel, Switzerland).
In the present study, we evaluated the therapeutic effect of the AEE788 administered alone or in combination with paclitaxel against established squamous cell carcinomas growing orthotopically within the tongue of athymic nude mice.
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MATERIALS AND METHODS |
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Measurement of Cell Proliferation.
The antiproliferative effects of AEE788 against JMAR and MDA1986 cells were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay that measures cell proliferation based on the ability of live cells to use MTT and convert it to a dark blue formazan (14)
. One thousand cells were plated into each well of a 96-well tissue culture plate. The cells were grown in DMEM supplemented with sodium pyruvate, essential amino acids, and 10% FBS. After a 24-hour attachment period, the cells were refed with medium (negative control with DMEM alone) or medium containing paclitaxel. After a 3-day incubation, the number of metabolically active cells was determined by MTT assay as measured by a MR-5000 96-well microtiter plate reader (Dynatech, Inc., Chantilly, VA) at an absorbance at 570 nm. Growth inhibition was calculated using 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.
Measurement of Cell Death.
Cells were plated at a density of 2 x 105 cells per well in 6-well plates (Costar, Cambridge, MA) and maintained for 24 hours before treatment with AEE788 and paclitaxel. Seventy-two hours later, the extent of cell death was determined by propidium iodide (PI) staining of hypodiploid DNA (15)
. For the PI staining, cells were resuspended in Nicoletti buffer 50 µg/mL PI (Sigma, St. Louis, MO) and 0.1% sodium citrate for 20 minutes at 4°C. Cells were then analyzed by flow cytometry, and the sub-G0-G1 fraction was measured with the Lysis program (Biosciences Clontech [BD], Franklin Lakes, NJ). The percentage of cells undergoing specific apoptosis was calculated by subtracting the percentage of cells that had undergone spontaneous apoptosis in the relevant controls from the total percentage of apoptotic cells in the study cultures (16)
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Western Blotting.
To determine whether AEE788 acted through its purported mechanism in vitro, we evaluated the effect of this agent on inhibition of EGF-stimulated tyrosine phosphorylation of EGFR, VEGFR, mitogen-activated protein kinase (MAPK), and AKT in human JMAR and MDA1986 oral cancer cells. Under basal conditions in both serum-containing and serum-free media, both cells demonstrated a low level of autophosphorylation, which was enhanced after exposure to EGF for 15 minutes. These cell lines were plated onto 6-well plates at a concentration of 2 x 105 cells/well and then incubated for 24 hours. On the next day, cells were incubated with serum-free media for 24 hours. The study cultures were treated with AEE788 at concentrations ranging from 0.1 to 10 µmol/L and controls with DMSO alone for 1 hour. The cells were then activated with EGF recombinant (40 ng/mL) for 15 minutes, washed with PBS, scraped with lysis buffer [1% Triton X-100, 20 mmol/L Tris (pH 8.0), 137 mmol/L NaCl, 10% glycerol (v/v), 2 mmol/L EDTA, 1-mmol/L phenylmethylsulfonyl fluoride, 20 µmol/L aprotinin-leupeptin-trypsin inhibitor, and 2 mmol/L sodium orthovanadate], and centrifuged to remove insoluble protein. The samples were diluted in sample buffer [0.5 mmol/L Tris-HCl (pH 6.8), 10% SDS, 1 mol/L DTT, 10% (v/v) glycerol, and 1% bromphenol blue] and boiled. The proteins (100 µg per lane) were resolved on 10% SDS- PAGE and transferred onto 0.45-mm polyvinylidene difluoride membranes, which then were blocked with 5% (w/v) nonfat milk in 0.1% Tween 20 (v/v) in TBS (TBS-T) for 1 hour. The membrane was probed with anti-EGFR (1:3000 dilution) and anti-phospho-EGFR (Tyr1068; 1:2000 dilution) antibodies, respectively, incubated overnight in 1% milk TBST, washed three times with 1x TBS-1, and incubated for 1 hour at room temperature with horseradish peroxidase-conjugated sheep antirabbit immunoglobulin (1:2000; Amersham, Inc., Arlington Heights, IL) in 1% nonfat milk. The blots were also probed with anti-phosphorylated AKT-Ser473 (Cell Signaling Technology, Beverly, MA), diluted 1:2000 in 1% nonfat milk TBST, phospho-MAPK (Tyr42/44), and phospho-VEGFR-2 (pY1045), and incubated with peroxidase-conjugated donkey antirabbit IgG (1:3000; Sigma) in 1% nonfat milk. Finally, all blots were probed with anti-ß-actin (1:3000; Sigma) in 1% nonfat milk, followed by horseradish peroxidase-conjugated donkey antirabbit IgG (1:4000; Amersham, Inc.) in 1% nonfat milk. Protein bands were visualized by the enhanced chemiluminescence detection system (Amersham, Inc.; ref. 16
).
Reagents.
For in vivo administration, AEE788 was dissolved and administered to mice at a concentration of 50 mg/kg three times a week. Paclitaxel was obtained from Sigma and administered at 200 µg/week. The following antibodies were used: polyclonal rabbit anti-EGF, anti-VEGF, anti-EGFR, anti-VEGFR-2, and anti-activated EGFR (Santa Cruz Biotechnology, Santa Cruz, CA); anti-activated VEGFR-2 (Oncogene, Cambridge, MA); anti-activated AKT and anti-activated MAPK (Cell Signaling Technology); mouse anti-proliferating cell nuclear antigen clone PC-10 (DAKO A/S, Copenhagen, Denmark); rat antimouse CD31/PECAM-1 and rat antimouse CD31 peroxidase-conjugated rat antimouse IgG1 (PharMingen, San Diego, CA); peroxidase-conjugated F(ab')2 goat antirabbit IgG F(ab')2, peroxidase-conjugated rat antimouse IgG F(ab')2 fragment, Affinipure Fab-fragment goat antimouse IgG, peroxidase-conjugated goat antirat IgG, and Texas Red-conjugated goat antirat IgG (Jackson Research Laboratories, West Grove, CA); Alexa Fluor 594-conjugated goat antimouse IgG, Alexa Fluor 594-conjugated goat antirabbit IgG, and Alexa Fluor 488-conjugated goat antirabbit IgG (Molecular Probes, Eugene, OR); peroxidase-conjugated rat antimouse IgG2a (Serotec; Harlan Bioproducts for Science, Inc., Indianapolis, IN); horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham, Inc.); and sheep antimouse and human IgG (Sigma). Other reagents were Hoechst Dye 3342 MW 615.9 (Hoechst, Warrington, PA), stable 3,3'-diaminobenzidine (Research Genetics, Huntsville, AL), 3-amino-9-ethylcarbazole AEC (Biogenex Laboratories, San Ramon, CA), and Gill’s hematoxylin (Sigma). Prolong solution was purchased from Molecular Probes and pepsin from Biomeda (Foster City, CA).
PI and MTT were both purchased from Sigma. Stock solutions were prepared by dissolving 1 mg of each compound in 1 mL of PBS and filtering the solution to remove particles. The solution was protected from light, stored at 4°C, and used within 1 month. The enhanced chemiluminescence detection system was purchased from Amersham, and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay was done with a commercial apoptosis detection kit (Promega, Madison, WI) with modifications as described below.
Animals and Maintenance.
Male athymic nude mice (NCI-nu), ages 8 to 12 weeks, 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 United States Department of Agriculture, United States Department of Health and Human Services, and the NIH. The mice were used in accordance with Animal Care and Use Guidelines of The University of Texas M. D. Anderson Cancer Center under a protocol approved by the Institutional Animal Care Use Committee.
Orthotopic Injections of Oral Tumors.
To produce tongue tumors, JMAR and MDA1986 cells were harvested from subconfluent cultures. The cells were resuspended in HBSS. Only suspensions consisting of single cells with >90% viability were used for the injections. A total number of 2 x 105 cells were resuspended in 50 µL of HBSS and injected into the tongue with a 30-gauge hypodermic needle and a tuberculin syringe as described previously (17)
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Once per week, the mice were weighed, and tongue tumors were measured with microcalipers. Tumor volume was calculated using the formula V = (A)(B2)
/6, where A is the length of the longest dimension of the tumor, and B is the length of the tumor perpendicular to A. Mice were monitored for 42 days. Mice were killed after >25% loss of the recorded initial body weight in this period, and the others were killed on day 42, after which, the body weight was determined and recorded. The tongues with tumors were resected; one half was fixed in formalin and embedded in paraffin, and the other half was embedded in OCT compound (Miles, Inc., Elkhart, IN), rapidly frozen in liquid nitrogen, and stored at –80°C. H&E staining confirmed the presence of tumor in each sample included in the analysis. Frozen and paraffin tissues were submitted for immunohistochemical analysis. Necropsy was performed in the mice to determine the extent of disease.
Therapy for Established Squamous Cell Carcinoma Tumors in Athymic Nude Mice.
Mice were examined 7 days following injection of cells into the tongue. Mice with similarly sized tumors were randomized into one of four groups (n = 10 mice/group): group 1, the control group, received an oral diluent for AEE788 (90% polyethylene glycol 300 + 10% 1-methyl-2-pyrrolidinone) and i.p. HBSS; group 2, the AEE788 group, received AEE788 (50 mg/kg) by oral administration three times a week; group 3, the paclitaxel group, received paclitaxel by i.p. injection once per week (200 µg/week); and group 4, the combination therapy group, received both the oral AEE788 regimen of group 2 and the i.p. paclitaxel regimen of group 3 concomitantly.
Immunohistochemical-Immunofluorescent Determination of Proliferating Cell Nuclear Antigen (PCNA), CD31/PECAM-1, EGFR, Activated EGFR, VEGFR, Activated VEGFR, Activated MAPK, and Activated AKT.
Specimens of the resected tongue tumors were processed for routine histologic and immunohistochemical analyses for markers of vascularization, survival, proliferation, and cell death. In vivo cell proliferation and apoptosis were evaluated using anti-PCNA antibodies and the TUNEL method, respectively. Paraffin-embedded tissues were used for identification of PCNA, EGF, VEGF, EGFR, and VEGFR-2. Sections (4 to 6-µm thick) were mounted on positively charged Superfrost slides (Fisher Scientific Co., Houston, TX) and dried overnight. Sections were deparaffinized in xylene, followed by treatment with a graded series of alcohol (100, 95, and 80% etomidate/double distilled H2O v/v) and rehydrated in PBS (pH 7.5). Sections analyzed for PCNA and VEGFR-2 were heated in a microwave oven for 5 minutes; sections analyzed for VEGF and EGFR were incubated for 20 minutes with pepsin at 37°C for antigen retrieval as described previously (18
, 19) .
Frozen tissues were used for identification of CD31/PECAM-1, activated EGFR, activated VEGFR-2, activated MAPK, and activated AKT; TUNEL and double staining (CD31/TUNEL, CD31/ActEGFR, and CD31/ActVEGFR-2) were also performed. The tissues were sectioned (8 to 10 µm), mounted on positively charged Plus slides (Fisher Scientific Co.), and air-dried for 30 minutes. Frozen sections were fixed in cold acetone (5 minutes), 1:1 acetone/chloroform (v/v; 5 minutes), and acetone (5 minutes) and washed with PBS. Immunohistochemical procedures were performed as described previously (20) . A positive reaction was visualized by incubating the slides with stable 3,3'-diaminobenzidine for 10 to 20 minutes for CD31/PECAM-1 and all paraffin slides. The sections were rinsed with distilled water, counterstained with Gill’s hematoxylin for 1 minute, and mounted with Universal Mount (Research Genetics). A positive reaction for activated EGFR and activated VEGFR was visualized by incubating the slides for 1 hour with a 1:400 dilution of Alexa Fluor 488-conjugated goat antirabbit IgG at room temperature for 1 hour in the dark. For activated MAPK and activated AKT, a 1:600 dilution of Alexa Fluor 594-conjugated goat antirabbit IgG was used. Fluorescent bleaching was minimized by covering the slides with a mixture of 90% glycerol and 10% PBS. Control samples exposed to the secondary antibody alone showed no specific staining.
TUNEL assay was performed using an apoptosis detection kit (Promega) with the following modifications: samples were fixed with 4% paraformaldehyde (methanol-free) for 10 minutes at room temperature, washed twice with PBS for 5 minutes, and then incubated with 0.2% Triton X-100 for 15 minutes at room temperature. After two 5-minute washes with PBS, the samples were incubated with equilibration buffer for 10 minutes at room temperature. The equilibration buffer was drained, and the reaction buffer containing 44 µL of equilibration buffer, 5 µL of nucleotide mix, and 1 µL of terminal deoxynucleotidyl transferase (Promega kit) was added to the tissue sections and incubated in a humid atmosphere at 37°C for 1 hour, avoiding exposure to light. The reaction was terminated by immersing the samples in 2x SSC for 15 minutes. Samples were then washed three times for 5 minutes to remove unincorporated fluorescein-dUTP.
For quantification of endothelial cells, the samples were incubated with 1 µg/mL Hoechst stain for 2 minutes at room temperature. Fluorescent bleaching was minimized by treating slides with glycerol/PBS mounting medium containing 0.1 mol/L propyl gallate. Immunofluorescence microscopy was done with a x100 objective on an epifluorescence microscope equipped with narrow band pass excitation filters mounted on a filter wheel (Ludl Electronic Products, Hawthorne, NY) to individually select for green, red, and blue fluorescence. Images were captured with a three-chip camera (Sony Corporation of America, Montvale, NJ) mounted on a universal microscope (Carl Zeiss, Tohnwood, NY) and Optimas image analysis software (Bioscan, Edmond, WA) installed on a Compaq computer with a Pentium chip, a frame grabber, an optical disk storage system, and a Mavigraph UP-D7000 digital color printer (Sony, Tokyo, Japan). To produce prints, images were additionally processed with Adobe PhotoShop software (Adobe Systems, Mountain View, CA). Endothelial cells were identified by red fluorescence, and DNA fragmentation was detected by localized green and yellow fluorescence within the nuclei of apoptotic cells.
For the quantification analysis, five slides were prepared for each group, and two areas were selected in each slide. The percentage of stained cells among the total number of cells in each area and the average proportion of stained cells in each group were calculated and compared. For total TUNEL and PCNA expression, the cells were counted in 10 random 0.159-mm2 fields at x100 magnification. Quantification of apoptotic endothelial cells was expressed as an average of the ratio of apoptotic endothelial cells to the total number of endothelial cells in 10 random 0.011-mm2 fields at x400 magnification. To quantify microvessel density (MVD), 10 random 0.159-mm2 fields at x100 magnification were captured for each tumor, and microvessels were quantified as described previously (21 , 22) .
Statistical Analysis.
The Wilcoxon nonparametric test was used to test for differences in mice tumor volume and mice weight between each treatment group and control group on each day. To statistically model treatment, time, and treatment-by-time interactions on mouse tumor volume and mouse weight over the treatment period, a generalized linear mixed model was used. Because half of the mice in the control group had to be killed and the missing values did not appear at random, only the data before 21 days were used in the analysis in all experimental models. Survival was analyzed with the Kaplan-Meier method. Differences between treatment and control groups were compared with the log-rank test. To test for differences of quantitative immunohistochemical analyses between treatment groups, a mixed model was used. Student’s t test from the mixed model was used to assess between-treatment differences. The linear mixed model with a compound symmetric correlation matrix was used to adjust for within-slide correlation (23)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 0.8 µmol/L for the JMAR cells (data not shown) in medium supplemented with 2% serum. Moreover, treatment of the MDA1986 cells (Fig. 1B)
and JMAR cells (data not shown) with AEE788 increased their sensitivity to paclitaxel but did not increase the level of inhibition achieved with paclitaxel at a high concentration.
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and JMAR cell line (data not shown) was 50%. The addition of 5 µmol/L AEE788 significantly increased tumor cell death. This concentration of AEE788 decreased the concentration of paclitaxel needed to induce 50% apoptosis from 2.2 to 1.0 nmol/L in the JMAR cell line (data not shown). At 5 nmol/L concentration, paclitaxel alone induced 45% cell death. At the same paclitaxel concentration (5 nmol/L), when 0.5 µmol/L AEE788 was added, the percentage cells undergoing apoptosis increased to 78% (Fig. 1D)
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AEE788 Inhibits EGF-induced EGFR, VEGFR, MAPK, and AKT Phosphorylation in SCCHN Cells.
To determine whether AEE788 could inhibit EGFR-stimulated growth and survival signaling pathways of MDA1986 and JMAR cells, the cells were stimulated in vitro with EGF and then treated for 1 hour with AEE788 at various concentrations. Cell lysis and Western blotting for phosphorylated forms of EGFR, VEGFR-2, AKT, and MAPK revealed inhibition of phosphorylation of all four of these kinases at 0.1 µmol/L and complete inhibition of the activity of these kinases at 2 µmol/L. Total levels of EGFR and AKT remained unchanged by AEE788 treatment (Fig. 2)
. Results were similar in the JMAR cells using slightly higher concentrations of AEE788 (data not shown).
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As shown in Table 1
, in the MDA1986 orthotopic model, the tumor volume of mice treated with AEE788 alone or in combination with paclitaxel was significantly lower than that of the mice in the control group (P = 0.0048 for the AEE788 group and P = 0.0022 for the AEE788 plus paclitaxel group at day 21). Overall, the tumor volume was significantly lower in the groups treated with AEE788 alone, paclitaxel alone, and the combination of AEE788 and paclitaxel than in the control group (P < 0.0001, P = 0.0313, and P < 0.0001, respectively). Mouse tumor volume increased over time.
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Overall, the groups receiving AEE788 alone or in combination with paclitaxel had significantly lower tumor volumes than did the control group (P < 0.001 for both study groups).
Two of the 10 mice in each of the combination treatment groups for both the JMAR and the MDA1986 orthotopic tongue models had no histologic evidence of tumors at the end of the study.
In the JMAR orthotopic model, the survival rate differed significantly between the treatment groups and the control group (all P < 0.05). Compared with the control group, mice treated with AEE788, paclitaxel, or the combination of AEE788 and paclitaxel had significantly increased survival rates, with P values of 0.0029, 0.0403, and 0.0009, respectively.
In the MDA1986 orthotopic model, the survival rate of the mice treated with AEE788 alone or in combination with paclitaxel was significantly greater than that of the mice in the control group (P < 0.0001 for both study groups; Fig. 3
). There was no significant difference in survival rates between the group treated with paclitaxel alone and the control group.
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. Therefore, AEE788 reduced MVD and increased apoptosis in vivo.
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DISCUSSION |
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EGFR elicits multiple biological effects in cells that overexpress the receptor, including proliferation, inhibition of apoptosis, invasion, and transformation (24) . Comprehensive data over the past 20 years strongly support the role of EGFR and its ligands in the development and progression of SCCHN (25) .
Several mAbs demonstrate antitumor efficacy against SCCHN cell lines and squamous cell carcinoma xenografts in nude mice (25) . Of the EGFR-specific mAbs that have been developed to date, the human-murine chimeric mAb C225 has been studied most extensively. C225 dramatically enhances the in vitro and in vivo radiosensitivity of human SCCHN tumor cells and xenografts (26, 27, 28) and may also increase the efficacy of standard chemotherapy (29) . The results of phase I clinical trials of C225 (30 , 31) , EMD 72000 (32) , and h-R3 (33) in patients with SCCHN are encouraging. A phase II trial of h-R3 and phase II and III trials of C225 in patients with SCCHN are currently under way (34) .
ZD1839 (Iressa), the most widely studied of the current EGFR tyrosine kinase inhibitors, greatly inhibits the growth of squamous carcinoma cell lines and human tumor xenografts in athymic mice (35) . The combination of ZD1839 with a chemotherapeutic agent such as cisplatin or paclitaxel produced remarkable antitumor efficacy both in vitro and in vivo (36) . Synergistic effects were also observed when ZD1839 was combined with radiotherapy in xenograft models (37) . Another widely studied EGFR tyrosine kinase inhibitor, OSI-774, also inhibited the growth of head and neck carcinoma xenografts in athymic mice (38) . A promising new protein tyrosine kinase inhibitor, PKI-166, arrests the growth of oral cancer in vitro and reduced tumor cell proliferation in a xenograft animal model (39) . CI-1033, which targets not only EGFR but also all four members of the ErbB family of growth factor receptors, inhibited growth in several human cancer cell lines and suppressed tumor growth in athymic nude mice with human squamous cell carcinoma xenografts (40) .
Several of these tyrosine kinase inhibitors are currently being assessed in phase I trials. On the basis of a successful phase I trial of OSI-774 (41) , this inhibitor recently was studied in a phase II trial involving patients with advanced SCCHN (42) . ZD1839 demonstrated clinically significant antitumor activity in a phase II trial against head and neck cancer (43) . However, two recently completed large randomized phase III studies of ZD1839 failed to demonstrate that adding ZD1839 to a standard chemotherapeutic regimen increased survival rates in patients with advanced non–small-cell lung cancer (44 , 45) .
Because multiple growth-controlling pathways may be altered in cancer cells, the combination of biological therapeutics targeting two or more such pathways should be tested in clinical settings to allow development of multitargeted therapies. Indeed, studies of preclinical models demonstrated that significant sustained antitumor activity can be obtained by combining anti-EGFR agents with other targeted therapy, such as inhibitors of VEGFR activity (46) .
Although EGFR inhibitors can inhibit the production VEGF by tumor cells, a more potent antitumor response can be achieved through the concomitant inhibition of VEGFR. In the present study, we determined whether the simultaneous blockade of the EGFR and VEGFR signaling pathways by the novel EGFR and VEGFR tyrosine kinase inhibitor, AEE788, alone or in combination with paclitaxel could inhibit the growth of JMAR and MDA1986 cells injected into the tongues of nude mice. Our results showed that therapy with AEE788 inhibited cell growth, induced apoptosis, and reduced the phosphorylation of EGFR, VEGFR-2, AKT, and MAPK in both the JMAR and MDA1986 cell lines. Treatment with AEE788 inhibited tumor growth and prolonged survival, and immunohistochemical study revealed that treatment with AEE788 alone and in combination with paclitaxel was associated with decreased MVD, a decreased proliferative index, and increased apoptosis of tumor and endothelial cells.
Enhanced expression of VEGF is generally associated with increased neovascularization, as measured by MVD within the tumor (47) . In this study, oral administration of AEE788 alone or in combination with paclitaxel significantly decreased MVD. Furthermore, only the tumors from mice treated with AEE788 alone or in combination with paclitaxel had decreased double staining for CD31/Act-EGFR and CD31/Act-VEGFR. These decreases may be attributable to inhibition of EGFR blockade given that endothelial cells within many neoplasms have been shown to express EGFR (48) . Nevertheless, our immunohistochemical findings showed an inhibition of VEGFR activity in lesions from mice treated with AEE788 alone. These findings suggest that AEE788 can also directly inhibit the VEGFR.
In summary, we report that simultaneous blockade of the EGFR and VEGFR signaling pathway by the novel PTK inhibitor AEE788 alone and in combination with paclitaxel significantly inhibits oral carcinoma in nude mice. This inhibition of tumor growth is mediated by both direct antitumor effects and antiangiogenic effects. These data support the development of AEE788 for clinical use in the treatment of patients with SCCHN.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Jeffrey N. Myers, Department of Head and Neck Surgery, Unit 441, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: (713) 792-6920; Fax: (713) 794-4662; E-mail: jmyers{at}mdanderson.org
Received 4/26/04. Revised 7/29/04. Accepted 8/27/04.
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Z. Wang, G. Chakravarty, S. Kim, Y. D. Yazici, M. N. Younes, S. A. Jasser, A. A. Santillan, C. D. Bucana, A. K. El-Naggar, and J. N. Myers Growth-Inhibitory Effects of Human Anti-Insulin-Like Growth Factor-I Receptor Antibody (A12) in an Orthotopic Nude Mouse Model of Anaplastic Thyroid Carcinoma Clin. Cancer Res., August 1, 2006; 12(15): 4755 - 4765. [Abstract] [Full Text] [PDF]
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M. N. Younes, Y. D. Yazici, S. Kim, S. A. Jasser, A. K. El-Naggar, and J. N. Myers Dual Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Inhibition with NVP-AEE788 for the Treatment of Aggressive Follicular Thyroid Cancer. Clin. Cancer Res., June 1, 2006; 12(11): 3425 - 3434. [Abstract] [Full Text] [PDF]
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J. M. Summy and G. E. Gallick Treatment for Advanced Tumors: Src Reclaims Center Stage Clin. Cancer Res., March 1, 2006; 12(5): 1398 - 1401. [Full Text] [PDF]
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A. Bozec, P. Formento, J. Ciccolini, R. Fanciullino, L. Padovani, X. Murraciole, J.-L. Fischel, and G. Milano Response of endothelial cells to a dual tyrosine kinase receptor inhibition combined with irradiation Mol. Cancer Ther., December 1, 2005; 4(12): 1962 - 1971. [Abstract] [Full Text] [PDF]
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 M. N. Younes, O. G. Yigitbasi, Y. W. Park, S.-J. Kim, S. A. Jasser, V. S. Hawthorne, Y. D. Yazici, M. Mandal, B. N. Bekele, C. D. Bucana, et al. Antivascular Therapy of Human Follicular Thyroid Cancer Experimental Bone Metastasis by Blockade of Epidermal Growth Factor Receptor and Vascular Growth Factor Receptor Phosphorylation Cancer Res., June 1, 2005; 65(11): 4716 - 4727. [Abstract] [Full Text] [PDF]
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