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... Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research ABT-510 Is an Effective Chemopreventive Agent in the Mouse 4-Nitroquinoline 1-Oxide Model of Oral Carcinogenesis Rifat Hasina,1 Leslie E. Martin,1 Kristen Kasza,2 Colleen L. Jones,1 Asif Jalil1 and Mark W. Lingen1 Abstract Despite numerous advances, the 5-year survival rate for head and neck squamous cell cancer (HNSCC) has remained largely unchanged. This poor outcome is due to several variables, including the development of multiple primary tumors. Therefore, it is essential to supplement early detection with preventive strategies. Using the 4-nitroquinoline 1-oxide (4-NQO) mouse model, we sought to define an appropriate dose and duration of administration that would predict the histologic timeline of HNSCC progression. Additionally, we sought to determine the timing of the onset of the angiogenic phenotype. Finally, using ABT-510 as a proof-of-principle drug, we tested the hypothesis that inhibitors of angiogenesis can slow/delay the development of HNSCC. We determined that 8 weeks of 100 g/mL 4-NQO in the drinking water was the optimal dosage and duration to cause a sufficient incidence of hyperkeratoses, dysplasias, and HNSCC over a period of 32 weeks with minimal morbidity and mortality. Increased microvessel density and vascular endothelial growth factor expression in hyperkeratotic lesions provided evidence that the initiation of the angiogenic phenotype occurred before the development of dysplasia. Importantly, ABT-510 significantly decreased the overall incidence of HNSCC from 37.3% to 20.3% (P = 0.021) as well as the combined incidence of dysplasia and HNSCC from 82.7% to 50.6% (P < 0.001). These findings suggest that our refinement of the 4-NQO model allows for the investigation of the histologic, molecular, and biological alterations that occur during the premalignant phase of HNSCC. In addition, these data support the hypothesis that inhibitors of angiogenesis may be promising chemopreventive agents. At current rates, approximately 400,000 cases of head and led Slaughter et al. (4) to propose the concept of field cancerization. This theory suggests that multiple individual primary tumors may develop independently in the upper aerodigestive tract as a result of years of chronic exposure to carcinogens. The occurrence of these new primary tumors can be particularly devastating for individuals whose initial lesions are small. Their 5-year survival rate for the first primary tumor is considerably better than patients with late-stage disease. However, second primary tumors are the most common cause of treatment failure and death among early-stage HNSCC patients (5). Therefore, it is insufficient treatment to address only the initial lesion. To improve the outcome of such patients, some form of chemopreventive treatment is essential. Chemoprevention can be defined as the systemic use of natural or synthetic agents to reverse or halt the progression of premalignant lesions. Chemopreventive agents are being tested for their efficacy in the preclinical and clinical settings for several malignancies, including HNSCC (6). However, the initial promising responses have not been consistently reproduced and toxicity was often a significant issue. Therefore, more effective and better tolerated therapy is needed for premalignant oral disease. Angiogenesis, the growth of new blood vessels from preexisting ones, is an essential phenotype in several physiologic and pathologic processes, including growth and development, wound healing, reproduction, arthritis, and tumor formation neck squamous cell cancer (HNSCC) will be diagnosed worldwide this year (1). Despite numerous advances in therapy, the long-term survival for these patients has remained largely unchanged. Several factors contribute to this poor outcome. First, oral cancer is often diagnosed in an advanced stage. The 5-year survival rate of early-stage oral cancer is approximately 80%, whereas the survival drops to 19% for late-stage disease (2). Second, the development of multiple primary tumors has a major effect on survival. The rate of second primary tumors in these patients has been reported to be 3% to 7% per year, higher than for any other malignancy (3). The observation of frequent second primary tumors in oral cancer Authors' Affiliations: Departments of 1Pathology, Medicine, and Radiation and Cellular Oncology and 2Health Studies, The University of Chicago, Chicago, Illinois Received 11/13/08; revised 2/7/09; accepted 3/4/09; published OnlineFirst 3/31/09. Grant support: Abbott Laboratories and NIH grant DE012322. 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: Mark W. Lingen, Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, MC 6101, Chicago, IL 60637. Phone: 773-702-5548; Fax: 773-702-9903; E-mail: mark.lingen@uchospitals. edu. 2009 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0211 www.aacrjournals.org 385 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research (7). Whether active neovascularization occurs is dependent on the relative concentrations of inducers and inhibitors of angiogenesis present in a given tissue microenvironment. Therefore, the inhibition of tumor-associated angiogenesis, using natural or synthetic inhibitors of angiogenesis, is an attractive target for therapy that has been gaining traction in the field of oncology. Like all solid tumors, HNSCCs must develop multiple direct and indirect ways to induce angiogenesis. Importantly, the expression of the angiogenic phenotype is one of the first recognizable phenotypic changes observed in both experimental models as well as in human HNSCC (811), suggesting that inhibitors of angiogenesis may also hold promise as chemopreventive agents. In addition to their main biological/molecular effects, some of the drugs currently under investigation in the chemopreventive setting have potential antiangiogenic activity. However, to date, no pure inhibitors of angiogenesis have been tested for their ability to act as chemopreventive agents in HNSCC. Animal models that faithfully recapitulate the human condition are critical to further our understanding of the molecular, biological, and clinical aspects of various diseases, including cancer. The hamster buccal pouch and the rat tongue models of oral carcinogenesis are well-established surrogate models for the human condition. However, although the hamster buccal pouch and rat tongue models have been extensively investigated, they have several limitations, resulting in the recent development of mouse oral cancer models that have several advantages (1218). In particular, mouse models enable the development and testing of new approaches to prevention and treatment, identification of early diagnostic markers, and an understanding of the biology and genetics of tumor initiation, promotion, and progression in an animal model whose genome is most similar to humans (1922). Although the 4-nitroquinoline 1-oxide (4-NQO) mouse model of oral carcinogenesis has gained increased attention as an alternative model, several parameters require further investigation. For example, although various treatment protocols have been described, the optimal administration, timing, and dosage of 4-NQO required to develop lesions that clinically mimic the human condition (singular or synchronous neoplasms) have not been fully established. In addition, a detailed and systematic investigation of the histologic changes during progression in the 4-NQO mouse model before the development of HNSCC has not been done. Finally, the timing and mechanisms of the development of various tumor-related phenotypes have not been determined in this model. Each of these issues is of critical importance and must be addressed to determine how closely this model system mimics the human condition such that it can be used effectively for future preclinical studies. Therefore, the purpose of this work was 4-fold. First, we sought to establish a dosage and treatment schedule that resulted in the development of singular or occasionally synchronous HNSCC rather than innumerable lesions throughout the oral cavity over a protracted period of time. Second, we established a predictable timeline for the histologic progression of mucosal lesions in mice treated with 4-NQO. Third, we sought to clarify when the expression of the angiogenic phenotype can be first observed in this model. Finally, ABT510, a mimetic peptide of thrombospondin-1 (Fig. 1; refs. 23, 24), was administered to 4-NQOtreated mice as a proof of principle to test the hypothesis that inhibitors of angiogenesis can be successfully used as chemopreventive agents for HNSCC. Materials and Methods Administration of 4-NQO Two hundred thirty male CBA mice, 6 to 8 wk of age, were purchased from The Jackson Laboratory and housed in the Animal Resource Facility under controlled conditions and fed normal diet and autoclaved water. All animal procedures were carried out in accordance with Institutional Animal Care and Use Committee approved protocols. Mice were administered 4-NQO in their drinking water on a continuous basis at the required dose for the required duration. 4-NQO powder (Sigma) was first dissolved in DMSO at 50 mg/mL as a stock solution and stored at 20C until used. On the days of 4-NQO administration, the stock solution was dissolved in propylene glycol (Sigma) and added to the drinking water bottles containing autoclaved tap water to obtain a final concentration of either 50 or 100 g/mL. A fresh batch of water was prepared every week for each of the 8 or 16 wk of carcinogenic treatment. Normal autoclaved drinking water was resumed at the end of this period. Control mice not receiving 4-NQO were given water containing vehicle only. Treatment with ABT-510 ABT-510, a synthetic peptide that mimics the antiangiogenic activity of the naturally occurring protein thrombospondin-1, was provided by Abbott Laboratories. The peptide was dissolved in sterile Fig. 1. Chemical structure of ABT-510. ABT-510 is a nonapeptide derived from the antiangiogenic fragment of the second type 1 repeat of thrombospondin-1. The key structures in the synthesis and the chemical structure from which this figure was derived can be found in the original publication by Haviv et al. (23). Cancer Prev Res 2009;2(4) April 2009 386 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer Fig. 2. Histopathology of 4-NQOinduced oral lesions in the mouse tongue. Photomicrographs show the histopathologic progression in this model system: histologically normal (control; A), hyperkeratosis (B), epithelial dysplasia (C), and squamous cell carcinoma (D). 5% dextrose, immediately filter sterilized, and stored at 4C. Mice receiving ABT-510 treatment were given a daily i.p. injection of 50 mg/kg body weight for the required duration of 4, 8, 12, 16, 20, or 24 wk. The route of administration and dosage given was determined based on previously published studies (2429). polymer-labeled horseradish peroxidasebound secondary reagent (EnVision+, DAKO). CD31 antigen retrieval was done using the Dako Target Retrieval System (pH 9.0) in a decloaking chamber. The primary antibody PECAM (Santa Cruz Biotechnology) was applied at 1:200 dilution in PBS for 1 h at room temperature. Antibody binding was visualized using the LSAB kit (DAKO). For determination of cell proliferation, sections were treated in ET buffer using decloaking chamber, incubating at 1:300 dilution using a Ki-67 antibody (NeoMarkers) for 1 h at room temperature. This was followed by anti-rabbit polymer-labeled horseradish peroxidasebound secondary reagent (EnVision+). All three immunohistochemistry stains were developed with 3,3-diaminobenzidine chromogen and counterstained with hematoxylin. Corresponding negative control experiments were done by omitting the incubation step with the primary antibody. Histologic examination Mice were sacrificed in accordance with Institutional Animal Care and Use Committee recommendations. Specifically, cervical dislocation was done after anesthesia by i.p. injection of xylazine and ketamine. Immediately following death, the tongues were excised, longitudinally bisected, and processed in 10% buffered formalin and embedded in paraffin. Fifty 5-m sections from each specimen were then cut and the 1st, 10th, 20th, 30th, 40th, and 50th slides were stained with H&E for histopathologic analysis. Histologic diagnoses were rendered using established criteria. Hyperkeratoses were characterized by a thickened keratinized layer, with or without a thickened spinous layer (acanthosis), and an absence of nuclear or cellular atypia. Dysplasias were characterized as lesions that showed various histopathologic alterations, including enlarged nuclei and cells, large and/or prominent nucleoli, increased nuclear to cytoplasmic ratio, hyperchromatic nuclei, dyskeratosis, increased and/or abnormal mitotic figures, bulbous or teardrop-shaped rete ridges, loss of polarity, and loss of typical epithelial cell cohesiveness. Because of the subjective nature of grading of epithelial dysplasia and its limited ability to predict biological progression (30, 31), we chose to not assign descriptive adjectives of severity to the dysplastic lesions. Rather, we grouped all lesions showing cytologic atypia but lacking evidence of invasion into the single category of dysplasia. HNSCCs were characterized by lesions that showed frank invasion into the underlying connective tissue stroma. Scoring of immunohistochemistry A combined scoring method that accounts for intensity of staining as well as percentage of cells stained was used for the evaluation of VEGF as previously described (32). Strong, moderate, weak, and negative staining intensities were scored as 3, 2, 1, and 0, respectively. For each of the intensity scores, the percentage of cells that stained at that level was estimated visually. The resulting combined score consisted of the sum of the percentage of stained cells multiplied by the intensity scores. For example, a case with 10% weak staining, 10% moderate staining, and 80% strong staining would be assigned a score of 270 (10 1 + 10 2 + 80 3 = 270) out of a possible score of 300. The determination of microvessel density (MVD) using CD31 as a marker was done as previously described (33). Briefly, using low-power magnification, the region containing the most intense area of tumor neovascularization was chosen for counting in each of the tumors. For the normal control tissue, MVD was determined by finding the most intense area of neovascularization directly below the overlying mucosa. Individual microvessels were counted using a 100 field (10 objective lens and 10 ocular lens). Any brown staining endothelial cells that were clearly separate in appearance were counted as individual vessels. Ten random fields within this hotspot area were viewed and counted at 100. Results were expressed as the total number of microvessels observed in the hotspot region of each individual tumor. For Ki-67, the labeling indices were determined by randomly analyzing at least 500 nuclei in 10 high-powered fields (400 magnification) for Immunohistochemistry Antigen retrieval was achieved on deparaffinized sections and endogenous peroxidase activity was quenched in 3% hydrogen peroxide and blocked in milk peroxidase. For vascular endothelial growth factor (VEGF) detection, slides were treated in ET buffer in a decloaking chamber and mouse primary antibody (Santa Cruz Biotechnology) was applied at 1:50 dilution in PBS for 1 h at room temperature. Antibody binding was visualized with anti-mouse www.aacrjournals.org 387 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research each tissue section. Labeling indices were expressed as a percentage of the total number of cells. quent deaths were observed in study A. At week 4 (n = 4), 75% of the tongues showed hyperkeratotic lesions, whereas 25% contained dysplasia. In the week 8 mice (n = 4), 25% had hyperkeratosis, 25% had dysplasia, and 50% had carcinoma. At 12 weeks (n = 4), 75% contained dysplasia and 25% had squamous cell carcinoma. Finally, by 24 weeks (n = 4), 25% showed dysplasia and 75% contained squamous cell carcinoma. Pilot study B (50 g/mL for 16 weeks) was terminated early because of an excessive number of deaths. Of the initial mice, 40% (8 of 20) died either during carcinogen treatment or within 6 weeks after the completion of the carcinogen treatment. In pilot studies C and D, mice were treated with 100 g/mL 4-NQO for either 8 weeks (pilot study C) or 16 weeks (pilot study D). In pilot study C (100 g/mL for 8 weeks), there were no deaths during the study. At week 4 (n = 5), 100% of animals showed hyperkeratotic lesions (Fig. 2B). At week 8 (n = 5), 60% showed hyperkeratosis and 40% contained epithelial dysplasia (Fig. 2C). By 12 weeks (n = 5), 100% of animals had dysplasia. Finally, at week 24 (n = 5), 40% had dysplasia and 60% showed squamous cell carcinoma (Fig. 2D). Similar to pilot study B, unacceptable mortality rates were observed in pilot study D after animals were treated with 100 g/mL for 16 weeks, resulting in the premature termination of this arm of the study as well. Although this was a relatively small sample size, these results showed that a sequential histologic progression could be observed using this type of carcinogenic induction protocol. It further suggested that the majority of the dysplastic and cancerous lesion were likely to be found starting at 12 weeks after carcinogen treatment. Therefore, to expand on the pilot studies, a cohort of 55 additional mice was treated with 100 g/mL 4-NQO for 8 weeks and tongues were subsequently harvested at weeks 16, 20, and 24. At week 16 (n = 20), 15% contained hyperkeratosis, 55% had dysplasia, and 30% showed squamous cell carcinoma. At 20 weeks (n = 15), 13% had hyperkeratosis, 60% contained dysplasia, and 23% showed squamous cell carcinoma. By 24 weeks after carcinogen treatment (n = 20), 25% of specimens contained dysplasia and 75% of the tissues showed squamous cell carcinoma. Collectively, the data for the 100 g/mL 4-NQO administered for 8 weeks show a reproducible timeline of histologic progression. Specifically, the data show that hyperkeratotic Data analysis For comparison of immunohistochemical scores across groups, ANOVA was done. A transformation of the data (square root or natural log) was used, as needed, to stabilize the variance across groups. If a significant overall difference was found by ANOVA, then pairwise comparisons were done with a Bonferroni adjustment for multiple comparisons. A test for trend was also done using ANOVA with the appropriate linear contrasts, and these results were confirmed using a nonparametric trend test as described by Cuzick (34). For comparison of ABT-510 treatment groups, Fisher's exact test was done by collapsing data across the six sacrifice times. All analyses were done using Stata version 10 (StataCorp). Results Histopathologic progression of 4-NQOtreated mice Articles describing the mouse 4-NQO model have typically reported the incidence of dysplasia and/or cancer at the end of the prescribed treatment protocols. However, these articles have not systematically characterized the sequential timing of histologic atypia development after carcinogen treatment over a protracted period of time. This is an important aspect of the model because a more thorough understanding of the histologic and molecular progression in this system is required if it is to be used to model oral premalignancy as well as preclinical chemoprevention studies. To carefully characterize the development of the histopathologic changes in this model, a series of pilot studies were done to identify the optimal carcinogen dosage and duration of exposure required to develop a predictable time line of progression. The animals were sacrificed at planned intervals after completion of carcinogen treatment, and their tongues were excised and examined histologically. No histopathologic changes were noted in the tongue mucosa from the control mice (Fig. 2A). Mice were treated with 50 g/mL 4-NQO for 8 weeks (pilot study A) and 16 weeks (pilot study B). They were subsequently randomly assigned to groups and sacrificed at 4, 8, 12, and 24 weeks after 4-NQO treatment. From the initial 20 animals in pilot study A (50 g/mL for 8 weeks), 4 mice died during treatment from undetermined causes. However, no subse- Fig. 3. Incidence of each histologic diagnosis of control and ABT-510 treatment groups at each sacrifice time. The number located at the top of each bar indicates the total sample size of each group. ABT-510 significantly decreased the incidence of HNSCC from 37.3% to 20.3% (P = 0.021) as well as the combined incidence of dysplasia and HNSCC from 82.7% to 50.6% (P < 0.001). Cancer Prev Res 2009;2(4) April 2009 388 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer Fig. 4. Immunohistochemical staining of Ki-67 in the 4-NQO mouse model of HNSCC. A, tissue sections containing areas of normal, hyperkeratosis, dysplasia, and squamous cell carcinoma immunohistochemically stained for Ki-67 and labeling indices were quantified (B and C). Ki-67 labeling indices in the specimens containing hyperkeratosis, dysplasia, and HNSCC were all significantly greater when compared with the normal mucosa (P < 0.001). to the mouse tongue epithelium from untreated control mice (n = 5) contained only occasional capillaries that were relatively evenly dispersed throughout the connective tissue stroma (Fig. 5A). However, the number and distribution of microvessels at the mucosal/connective tissue interface increased in the hyperplastic, dysplastic, and malignant mucosa (Fig. 5A). With increasing histologic atypia, the vessels were more densely packed directly adjacent to the basal cell layer. Quantification of MVD during histologic progression revealed a statistically significant difference (P < 0.001 by ANOVA) in CD31 scores when normal epithelium (mean SD: 23.2 4.3) was compared with hyperplasia (90.2 6.7), dysplasia (191.6 10.6), and squamous cell carcinoma (309.3 17.9). Pairwise comparisons indicated that each group was significantly different from all other groups (Bonferroni adjusted P < 0.001 in all cases). Furthermore, there was a significant linear trend in CD31 levels across the four naturally ordered groups (i.e., increasing CD31 levels with increasing disease severity; P for trend < 0.001; Fig. 5B and C). lesions predominate at weeks 4 and 8, dysplasias are the most common diagnosis at weeks 12, 16, and 20, and carcinoma is the predominant histologic finding at week 24 (Fig. 3). Ki-67 Expression during histologic progression in 4NQOtreated mice Expression of Ki-67, a nuclear proliferation-associated antigen that is specific for cells in the active phases of the cell cycle, was determined via immunohistochemistry to quantify the relative proliferative rates of normal, hyperkeratotic, dysplastic, and malignant mouse tongue mucosa. Cells positive for Ki-67 expression showed distinct nuclear staining. In normal mucosa, Ki-67 expression was limited to basilar and occasional parabasilar cells (Fig. 4A), whereas greater suprabasilar labeling was observed with increasing histologic atypia (Fig. 4A). There was a statistically significant difference (P < 0.001 by ANOVA) in labeling indices when normal epithelium (mean SD: 16.4 3.0) was compared with hyperplasia (23.5 2.6), dysplasia (32.3 3.5), and squamous cell carcinoma (47.8 3.6). Subsequent pairwise comparisons indicated that each group was significantly different from all other groups (Bonferroni adjusted P < 0.001 in all cases). Additionally, there was evidence for an increasing linear trend in Ki-67 levels across the four ordered groups (P for trend < 0.001; Fig. 4B and C). Expression of VEGF during the histologic progression in 4-NQOtreated mice Like all solid tumors, HNSCC must develop direct and indirect mechanisms to induce the production of new blood vessels. Several dozen candidate angiogenic molecules are produced by oral keratinocytes, and VEGF is an important angiogenic factor in both physiologic and pathologic settings, including HNSCC (35). Therefore, we did immunohistochemistry for VEGF to quantify its expression at various stages of histologic progression in the mouse 4-NQO model. Expression of VEGF by tongue keratinocytes from untreated control mice (n = 5) was rare and limited to the basilar and parabasilar cells (Fig. 6A). In addition, occasional stromal cells as well as endothelial lined vascular channels stained positively. The intensity of VEGF expression as well as the overall expression Increased MVD occurs before histologic atypia in 4NQOtreated mice The induction of blood vessel growth is an early phenotypic change in both human HNSCC as well as in the hamster buccal pouch and rat tongue models of oral carcinogenesis (811). To determine the timing of the expression of the angiogenic phenotype in the 4-NQO mouse model, we did immunohistochemistry for CD31 to quantify MVD as a surrogate for in vivo angiogenesis activity. The connective tissue stroma adjacent www.aacrjournals.org 389 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research of the cytokine by various keratinocytes present in the different layers of the epithelium increased in the hyperplastic, dysplastic, and malignant mucosa (Fig. 6A). Quantification of VEGF expression among histologic stages revealed a statistically significant difference (P < 0.001 by ANOVA) when normal epithelium (mean SD: 17.0 5.7) was compared with hyperplasia (70.6 14.0), dysplasia (144.9 35.4), and squamous cell carcinoma (237.6 41.4). Each group was significantly different from all other groups (Bonferroni adjusted P < 0.001 in all cases). Most importantly, there also was evidence for an increasing linear trend in VEGF levels with disease progression (P for trend < 0.001; Fig. 6B and C). normal, 40% contained hyperkeratosis, and 10% had dysplasia. At week 8 (n = 10), 10% were normal, 70% contained hyperkeratosis, and 20% showed dysplasia. By week 12 (n = 10), 10% were normal, 30% contained hyperkeratosis, and 60% had dysplasia. At week 16 (n = 12), 58% had hyperkeratosis, 25% had dysplasia, and 17% showed carcinoma. Progressing to week 20 (n = 16), 25% had hyperkeratosis, 25% had dysplasia, and 50% showed carcinoma. Finally, at week 24 (n = 21), 33% had hyperkeratosis, 38% had dysplasia, and 29% showed carcinoma. With respect to overall incidence of cancer, a 46% reduction in HNSCC was observed, with the 4-NQO group having an incidence rate of 37.3% and the ABT-510 treatment group having an incidence of 20.3% (P = 0.021). In addition, the combined incidence of dysplasia and HNSCC was 82.7% in the 4-NQO control group and 50.6% in the ABT-510 treatment group. This difference was highly statistically significant (P < 0.001). Effects of ABT-510 administration in 4-NQOtreated mice The expression of the angiogenic phenotype is one of the first recognizable phenotypic changes observed in both experimental models as well as in human HNSCC (811), suggesting that inhibitors of angiogenesis may also hold promise as chemopreventive agents. However, to date, no pure inhibitors of angiogenesis have been tested for their ability to act as chemopreventive agents in HNSCC. Therefore, using ABT-510 as a proof-of-principle drug, we tested the hypothesis that inhibitors of angiogenesis can be used as chemopreventive agents in the realm of HNSCC. Based on our findings described above, mice were administered 4-NQO (100 g/mL) in their drinking water for 8 weeks. At the completion of this initiation phase, the mice were returned to normal water and given daily i.p. injections of ABT-510 (50 mg/kg/d) and sacrificed at regular intervals over the next 24 weeks. During the 24-week chemopreventive period, no significant differences in food or fluid consumption among the groups were observed. Similarly, there was no difference in body weight or activity between the control and the treatment group mice (data not shown). Data for the incidence of tongue lesions are shown in Fig. 3. At week 4 (n = 10), 50% of the tongues were histologically Discussion Animal models of HNSCC have traditionally used either 7,12-dimethylbenz(a)anthracene or 4-NQO as the carcinogenic agent. The induction of HNSCC using 4-NQO has been achieved in several different rodent species, including mice, hamsters, and rats, and has been generally found to be preferable for several reasons. First, in contrast to the topical application of 7,12-dimethylbenz(a)anthracene, 4-NQO can be delivered via the drinking water, thereby making the outcomes more predictable. Second, the molecular alterations induced in mouse mucosa by 4-NQO closely mimic the human disease. For example, similar to the human condition, epidermal growth factor receptors are overexpressed in this model (36). Similar to humans, altered expression of p53 as well as mutation of p53 have been shown in 4-NQO models (37). In addition, 4-NQO induces activating point mutations in the H-ras oncogenes (38). Although H-ras mutations are infrequent events in U.S. HNSCC (39), they are common in the Fig. 5. Expression of the angiogenic phenotype in the 4-NQO mouse model of HNSCC. A, tissue sections containing areas of normal, hyperkeratosis, dysplasia, and squamous cell carcinoma immunohistochemically stained for CD31 and MVD was quantified (B and C). MVD in the specimens containing hyperkeratosis, dysplasia, and HNSCC was all significantly greater when compared with the normal mucosa (P < 0.001). Cancer Prev Res 2009;2(4) April 2009 390 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer Fig. 6. Expression of VEGF in the 4-NQO mouse model of HNSCC. A, tissue sections containing representative areas of normal, hyperkeratosis, dysplasia, and squamous cell carcinoma immunohistochemically stained for VEGF and expression levels were quantified (B and C). Expression of VEGF in the specimens containing hyperkeratosis, dysplasia, and HNSCC was significantly greater when compared with normal mucosa (P < 0.001). opment of singular or occasionally synchronous oral lesions. This aspect was critical because we wanted to refine the model to ensure that the mice developed lesions in a fashion that was most similar to the human condition rather than forming innumerable ones. In addition, the prescribed dosage and timing of treatment resulted in a predictable histologic progression over the 24-week period of time. Specifically, the data show that after the completion of carcinogen treatment, the predominant histopathologic diagnoses are hyperkeratosis at weeks 4 and 8, dysplasia at weeks 12, 14, and 16, and HNSCC at week 24 (Fig. 3). Importantly, the establishment of the progression timeline in this model will allow for future studies aimed at investigating the molecular and biological alterations that occur during the premalignant phase of HNSCC as well as for the validation of potential diagnostic biomarkers. The induction of new blood vessel growth is a critical tumor phenotype in all malignancies, including HNSCC. There is considerable interest in determining how cells, progressing from normal to tumorigenic, make this switch. In some animal models, a distinct switch to the angiogenic phenotype is seen (44). In other cases, the cells developing into tumors sequentially become more angiogenic in a stepwise fashion (8, 45). Although the phenotype has been studied using animal models as well as human cells and tissue, the mechanisms about how this occurs in HNSCC are unknown. However, although the phenotypic changes in these models are similar to the human condition, the specific mechanisms involved in the induction of new blood vessel growth can be quite different. For example, although the major angiogenic factor in the hamster buccal pouch model is transforming growth factor (8), this growth factor has not been found to be a significant participant in the induction of angiogenesis in human HNSCC. Rather, a different subset of growth factors, including interleukin-8 and VEGF, seems to play predominant roles (35). As there were no data about the angiogenic phenotype in the 4NQO mouse model, we sought to determine the timing of the rest of the world (40). Therefore, because the majority of the 400,000 cases of HNSCC are outside of the United States/Europe and may therefore harbor H-ras mutations (in conjunction with epidermal growth factor receptor and p53 alterations), we believe that this is an excellent model from an experimental, histologic, and molecular perspective. Finally, 4-NQOinduced lesions develop in the absence of nonspecific inflammatory changes. This is a critical point because substances such as 7,12-dimethylbenz(a)anthracene can be significant irritants, resulting in chronic inflammation, necrosis, sloughing of tissue, and the formation of organizing granulation tissue (41). The etiology, type of inflammatory cell infiltrate, and therefore perhaps mechanisms in this type of injurious situation are likely to be different from what one sees in the human condition. Furthermore, these factors make it difficult to study premalignant lesions because inflammation itself can cause cytologic and/or morphologic changes that can be confused with dysplasia. Because 4-NQO treatment does not result in this type of injurious nonspecific inflammation, it is more likely to reflect the events that occur during human HNSCC. Although 4-NQO has been used as a carcinogenic agent to induce tumors in animal models since the 1950s, and in the oral cavity since the 1970s (42, 43), a thorough review of the literature failed to reveal studies that were specifically designed to characterize the histopathologic changes that develop over time in the 4-NQO mouse model. Furthermore, the route of administration, the amount of carcinogen used, and the duration of treatment schedules have been highly variable. Therefore, we sought to characterize the optimal dosage and timing of 4-NQO treatment and to establish a timeline for the reproducible development of lesions showing hyperkeratosis, dysplasia, and HNSCC. Using two different doses and two different durations of treatment, we determined that 100 g/mL in the drinking water for 8 weeks provided the most preferable results. This decision was based on the fact that this treatment protocol resulted in the devel- www.aacrjournals.org 391 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research cumulative decrease of 39% in the incidence of both dysplasia and HNSCC between the control and ABT-510 treatment groups (82.7% versus 50.6%; P < 0.001), the rates being higher in the control groups at each time point except for week 4 where there was only one case of dysplasia. Overall, these cumulative data strongly support the contention that ABT-510 was effective at decreasing the incidence of dysplasia and HNSCC in the mouse 4-NQO model of oral carcinogenesis. The integration of inhibitors of angiogenesis into the treatment regimens of various diseases is increasing in frequency (47, 48). However, although there has been considerable discussion about the potential role of antiangiogenic agents in the area of chemoprevention, there are limited data to support the role of this class of drugs in this clinical setting (49, 50). ABT-510 is a mimetic peptide of thrombospondin-1 and acts as an inhibitor of angiogenesis by modulating the ability of endothelial cells to respond to various angiogenic factors. Specifically, ABT-510 binds to CD36, thereby inducing caspase-8 mediated endothelial cell apoptosis. This mechanism has been shown to block angiogenesis in vitro and in vivo as well as slow tumor growth in preclinical studies (2429). Further, it has been tested clinically for the treatment of inflammatory bowel disease and in cancer therapy as a single agent or in combination with chemotherapeutic agents (5155). Because it directly abrogates the ability of endothelial cells to respond to angiogenic factors, rather than altering the expression of these factors by tumor and/or stromal cells, one does not typically observe the direct modulation of various angiogenic factors in tumor cells. In keeping with these previous observations, we did not observe an alteration in the expression of VEGF by the oral keratinocytes in the treatment group animals (data not shown). However, the nearly 2-fold decrease in the incidence of HNSCC in the ABT-510 group at 24 weeks shows that the drug has a potent effect in vivo in this animal model. One of the long-term goals of chemoprevention must be the development of treatments that can be easily taken by at-risk individuals for prolonged periods of time with minimal side affects to achieve widespread acceptance and long-term compliance. This would be particularly important in the case of high-risk patients who have not yet developed their first malignancy. As such, because ABT-510 is not an orally administered agent, it is unlikely that it would be found acceptable in its current form. Nonetheless, our proof-of-principle findings support the hypothesis that inhibitors of angiogenesis may have activity as chemopreventive agents. Furthermore, many of the chemopreventive agents currently under investigation, such as epidermal growth factor receptor tyrosine kinase inhibitors and cyclooxygenase-2 inhibitors, have multiple activities, including the inhibition of angiogenesis. However, the toxicities observed at the current prescribed dosages may preclude them from being used widely as chemopreventive agents. Therefore, the combination of one or both of these agents at lower doses in concert with other antiangiogenic agents may reduce toxicities and improve efficacy. The data presented here show proof of principle that the induction of new blood vessel growth by premalignant cells may be one such phenotype that could be targeted in a chemoprevention cocktail. appearance of this phenotype as well as identify the angiogenic factors that might be involved. The determination of MVD in tissue sections, using endothelial cell markers such as factor VIII, CD31, and/or CD34, is an accepted surrogate marker for in vivo angiogenesis. In the 4-NQOtreated animals, we observed a statistically significant increase in MVD as early as the hyperkeratotic stage, thereby showing that, much like other models as well as the human condition, the expression of the angiogenic phenotype is an early phenotypic change (Fig. 6). Furthermore, we observed sequentially increasing levels of VEGF expression at the stages of hyperkeratosis, dysplasia, and HNSCC (Fig. 6B and C). The mechanisms for this sequential increase are not known at this time. However, one could hypothesize that the progressive increase in VEGF expression might in part be the result of potential tumor-stroma paracrine interactions (46). The design of the study did not allow us to functionally validate whether VEGF was the key angiogenic factor produced in this animal model. However, the concordant increased expression of both MVD and VEGF supports the hypothesis that VEGF plays a central role in the expression of the angiogenic phenotype in the mouse 4-NQO model. We have shown that daily treatment with ABT-510 for 24 weeks resulted in limited toxicity and significantly decreased the incidence of dysplasias and carcinomas in the mouse 4-NQO model of HNSCC (Fig. 3). The rationale for the 24-week treatment schedule was based on the observed cancer incidence and histologic diagnosis in our preliminary studies. As a result of these findings, animals were subsequently sacrificed at the same regular intervals over the 24-week time course to synchronize the treatment arm results with the initial histologic observations. Specifically, we observed most HNSCC (72%) at week 24, whereas dysplasia was the predominant histologic diagnosis at weeks 16 (55%) and 20 (60%). Because we were testing the hypothesis that ABT-510 would reduce the incidence of HNSCC, we believe it was most appropriate to carry out the prevention study to a time point where the predominant histologic diagnosis in the control group would be expected to be HNSCC. Overall, we observed a 46% cumulative reduction in the incidence of HNSCC between the control and treatment groups (37.3% versus 20.3%; P = 0.021). We did observe a small increase of HNSCC in the treatment group at week 20 when compared with the 20-week control group. However, this difference was not statistically significant (P = 0.273). The reasons for this observation are unknown but may be the result of uneven cohort sizes between the control and treatment groups within and at different sacrifice points. For example, the week 20 treatment group contained fewer animals (n = 16) than some of the other time points, such as week 24 (n = 21). The differences in cohort sizes at the given time points were a reflection of the fact that our initial experiments determined that week 24 should be the major cancer end point of this study. As such, although we were interested in synchronizing the treatment arm results with the histologic studies, we also felt it was most important to have the largest n at the major end point of the study. Further, it should be noted that the 16- and 24-week time points showed significant differences in the incidence of HNSCC between the control and treatment groups. For example, at 24 weeks, the difference in HNSCC incidence between the control and treatment groups was 72% versus 29%, respectively (P = 0.007). Finally, we also observed a Cancer Prev Res 2009;2(4) April 2009 Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. 392 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer References 1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:7196. 2. Murphy GP, Lenhhardt LW. American Cancer Society textbook of clinical oncology. 2nd ed. Atlanta: American Cancer Society; 1995. 3. Day GL, Blot WJ. Second primary tumors in patients with oral cancer. Cancer 1992;70:149. 4. Slaughter DP, Southwick HW, Smejkal W. 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... Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Cancer Prevention Research Research Article Dual Inhibition of Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor is an Effective Chemopreventive Strategy in the Mouse 4-NQO Model of Oral Carcinogenesis Guolin Zhou1, Rifat Hasina1, Kristen Wroblewski2, Tanmayi P. Mankame1, Colleen L. Doi1, and Mark W. Lingen1 Abstract Despite recent therapeutic advances, several factors, including field cancerization, have limited improvements in long-term survival for oral squamous cell carcinoma (OSCC). Therefore, comprehensive treatment plans must include improved chemopreventive strategies. Using the 4-nitroquinoline 1-oxide (4-NQO) mouse model, we tested the hypothesis that ZD6474 (Vandetanib, ZACTIMA) is an effective chemopreventive agent. CBA mice were fed 4-NQO (100 g/mL) in their drinking water for 8 weeks and then randomized to no treatment or oral ZD6474 (25 mg/kg/d) for 24 weeks. The percentage of animals with OSCC was significantly different between the two groups (71% in control and 12% in the ZD6474 group; P 0.001). The percentage of mice with dysplasia or OSCC was significantly different (96% in the control and 28% in the ZD6474 group; P 0.001). Proliferation and microvessel density scores were significantly decreased in the ZD6474 group (P 0.001 for both). Although proliferation and microvessel density increased with histologic progression in control and treatment cohorts, epidermal growth factor receptor and vascular endothelial growth factor receptor-2 phosphorylation was decreased in the treatment group for each histologic diagnosis, including mice harboring tumors. OSCC from ZD6474-treated mice exhibited features of epithelial to mesenchymal transition, as shown by loss E-cadherin and gain of vimentin protein expression. These data suggest that ZD6474 holds promise as an OSCC chemopreventive agent. They further suggest that acquired resistance to ZD6474 may be mediated by the expression of an epithelial to mesenchymal transition phenotype. Finally, the data suggests that this model is a useful preclinical platform to investigate the mechanisms of acquired resistance in the chemopreventive setting. Cancer Prev Res; 3(11); 1493502. 2010 AACR. Introduction With an annual incidence of nearly 600,000 cases, oral and pharyngeal squamous cell carcinoma is the sixth most common malignancy in the world today (1). There will be over 35,000 new cases in the United States in 2010 with nearly 8,000 deaths from the disease (2). When focusing specifically on the oral cavity squamous cell carcinoma (OSCC), it is estimated that there will be over 23,000 new cases and more than 5,300 deaths (3). Despite advances in diagnosis and treatment, improved long-term survival for OSCC patients has remained modest. Several factors contribute to this relatively poor outcome. First, OSCC is often diagnosed at an advanced stage. The 5-year Authors' Affiliations: Departments of 1 Pathology, Medicine, and Radiation and Cellular Oncology, and 2Health Studies, The University of Chicago, Chicago, Illinois Corresponding Author: Mark W. Lingen, Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, MC 6101, Chicago, IL 60637. Phone: 773-702-5548; Fax: 773-702-9903; E-mail: mark. lingen@uchospitals.edu. doi: 10.1158/1940-6207.CAPR-10-0135 2010 American Association for Cancer Research. survival rate of early stage disease is approximately 80%, although the survival drops to approximately 20% for late stage disease (2). Second, as a result of field cancerization, the development of multiple primary tumors has a major effect on survival. For patients with early stage disease, second primary tumors are their most common cause of treatment failure and death (4, 5). Therefore, to improve outcomes, a comprehensive treatment plan must include both improved early detection and secondary prevention. Chemoprevention can be defined as the use of natural or synthetic agents to reverse or halt the progression of premalignant lesions. Chemopreventive agents are currently being tested for their efficacy in preclinical and clinical settings for many malignancies including OSCC (6, 7). However, initial promising results for OSCC chemoprevention have not been consistently reproduced and toxicity has often been a significant complication. The issue of toxicity is particularly important in the realm of chemoprevention as it is conceivable that patients may require therapy for prolonged periods of time. Angiogenesis is an essential phenotype in both physiologic and pathologic settings including growth and development, www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. 1493 Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Zhou et al. wound healing, reproduction, arthritis, and tumor formation (8). Because of its critical role in cancer biology, the inhibition of tumor angiogenesis is an attractive target for cancer therapy. The induction of the angiogenic phenotype in OSCC is mediated by the direct and indirect production of various factors capable of inducing blood vessel growth (9). Among these, the vascular endothelial growth factor (VEGF) family is thought to play an important role. The biological effects of the VEGF ligands are mediated through their binding to members of the VEGF receptor family (VEGFR-1, VEGFR-2, and VEGFR-3). This interaction leads to the autophosphorylation of specific tyrosine residues and subsequent downstream activation of intracellular signaling pathways, such as the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways. Importantly, the expression of the angiogenic phenotype is one of the first recognizable phenotypic changes observed in both experimental models as well as in human OSCC (1013), suggesting that inhibitors of angiogenesis may also hold promise in the field of chemoprevention. The development, growth, and survival of OSCC are also highly dependent on the epidermal growth factor receptor (EGFR) signaling pathway. EGFR is a transmembrane glycoprotein that is a member of the ErB/HER receptor tyrosine kinase family. Upon ligand binding, EGFR signaling is mediated by the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways. Increased expression of EGFR and its ligand transforming growth factor- (TGF-) are observed in most OSCC and premalignant oral lesions, and this expression correlates with poor prognosis (14). In addition to directly influencing tumor cell growth, members of the EGFR pathway can contribute to the expression of the angiogenic phenotype. For example, the expression of either TGF- or EGFR results in increased expression of VEGF (15, 16). Because of its importance in epithelial malignancies, there is considerable interest in targeting the EGFR pathway in the realm of chemoprevention. ZD6474 (Vandetanib, ZACTIMA) is an orally available tyrosine kinase inhibitor with direct activity against multiple signal transduction pathways including VEGFR-2 and EGFR (1719). ZD6474 has an IC50 of 0.04 mol/L for VEGFR-2 and an IC50 of 0.5 mol/L for EGFR (18, 20). In preclinical studies, ZD6474 was found to be a potent inhibitor of tumor angiogenesis and the proliferation of a number of different tumor cell types including OSCC xenografts (2130). Furthermore, it is currently under active investigation in clinical trials for the treatment of various malignant neoplasms (31). To date, it has been found to have greatest activity in nonsmall cell lung cancer and recurrent medullary thyroid cancer (3234). However, the clinical utility of this agent in the realm of chemoprevention, particularly for OSCC, is unknown. Because it has the potential to inhibit two pathways that are essential for the development of OSCC, we tested the hypothesis that ZD6474 is an effective chemopreventive agent in the 4-NQO model. 1494 Cancer Prev Res; 3(11) November 2010 Materials and Methods Administration of 4-NQO and treatment with ZD6474 CBA mice, 6 to 8 weeks of age, were purchased from The Jackson Laboratory and housed in the Animal Resource Facility under controlled conditions and fed normal diet and autoclaved water. All animal procedures were carried out in accordance with Institutional Animal Care and Use Committeeapproved protocols. Mice were given 4-NQO in their drinking water on a continuous basis at the required dose for the required duration as previously described (35). Briefly, 4-NQO powder (Sigma) was first dissolved in DMSO at 50 mg/mL as a stock solution and stored at 20C until used. On the days of 4-NQO administration, the stock solution was dissolved in propylene glycol (Sigma) and added to the drinking water bottles containing autoclaved tap water to obtain a final concentration of 100 g/mL. A fresh batch of water was prepared every week for each of the 8 weeks of carcinogenic treatment. Normal autoclaved drinking water was resumed at the end of this period. Control mice not receiving 4-NQO were given water containing vehicle only. ZD6474 was provided by Astra Zeneca and dissolved in Tween 80 solution (P8192-5X10ML, Sigma). Mice receiving ZD6474 treatment were given a daily dosage of 25 mg/kg/d for 24 weeks via oral gavage. Histologic examination Mice were sacrificed in accordance with Institutional Animal Care and Use Committee recommendations. Specifically, cervical dislocation was done subsequent to anesthesia by i.p. injection of xylazine and ketamine. Immediately following death, the tongues were excised, longitudinally bisected, and processed in 10% buffered formalin and embedded in paraffin. Fifty 5-m sections from each specimen were then cut and the 1st, 10th, 20th, 30th, 40th, and 50th slides were stained with H&E for histopathologic analysis. Histologic diagnoses were rendered as previously described (35). Briefly, hyperkeratoses were characterized by a thickened keratinized layer, with or without a thickened spinous layer (acanthosis), and an absence of nuclear or cellular atypia. Dysplasias were characterized as lesions that showed various histopathologic alterations including enlarged nuclei and cells, large and/or prominent nucleoli, increased nuclear to cytoplasmic ratio, hyperchromatic nuclei, dyskeratosis, increased and/or abnormal mitotic figures, bulbous or teardrop-shaped rete ridges, loss of polarity, and loss of typical epithelial cell cohesiveness. Because of the subjective nature of grading of epithelial dysplasia and its limited ability to predict biological progression (36, 37), we chose not to assign descriptive adjectives of severity to the dysplastic lesions. Rather, we grouped all lesions demonstrating cytologic atypia but lacking evidence of invasion into the single category of dysplasia. HNSCC were characterized by lesions that showed frank invasion into the underlying connective tissue stroma. Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 ZD6474 Chemoprevention of Oral Cancer Immunohistochemistry For detection of phosphorylated EGFR (pEGFR) and phosphorylated VEGFR-2 (pVEGFR-2), antigen retrieval was achieved on deparaffinized 5 m sections using Immuno/ DNA retriever with citrate (Bio SB). Endogenous peroxidase activity was quenched with mouse/rabbit ImmunoDetector Peroxidase Block Kit. Sections were incubated using primary antibody to pVEGFR-2 1:300 (Abcam) or pEGFR 1:250 (Cell Signaling) for 1 hour at room temperature. Antibody binding was visualized by using mouse/rabbit ImmunoDetector HRP/DAB Detection System (Bio SB). For detection of CD31 and vimentin, antigen retrieval was achieved by using 10 mmol/L of citrate buffer (pH 6.0) on 5 m deparaffinized sections. Endogenous peroxidase activity was quenched with 1% hydrogen/methanol. The primary antibody for vimentin (Epitomics) was applied at 1:250 dilution for a 1-hour incubation at room temperature. For CD31 (Abcam), a 1:50 dilution was applied, followed by anti-rabbit polymer-labeled horseradish peroxidase (HRP)bound secondary reagent (DAKO EnVision+ System, HRP). For detection of E-cadherin and Ki67, antigen retrieval was achieved on 5-m deparaffinized sections using 10 mmol/L of Tris-base and 1 mmol/L of EDTA (pH 9.0). Endogenous peroxidase activity was quenched with 1% hydrogen peroxide/methanol. The primary antibody for E-cadherin (Zymed) was applied at a 1:25 dilution for 1 hour at room temperature. This was followed by antirabbit polymer-labeled HRP-bound secondary reagent (DAKO, EnVision+ System, HRP). For Ki67 (NeoMarkers), sections were incubated at a 1:300 dilution at room temperature for 1 hour followed by anti-rabbit polymer-labeled HRP-bound secondary reagent (EnVision+ System, HRP). All immunohistochemistry stains were developed with DAB chromogen and counterstained with hematoxylin. Corresponding negative control experiments were done by omitting the incubation step with the primary antibody. Scoring of immunohistochemistry Scoring of immunohistochemical staining was done using the Automated Cellular Imaging System (Chroma Vision). Stained sections were scanned and acquired using Automated Cellular Imaging System. Proliferation was measured by calculating the average labeling percentage of the epithelial compartment for Ki67 for each specimen. For determination of microvessel density (MVD), the total number of CD31-stained clusters or single cells, with or without a lumen, was quantified for each specimen. For pVEGFR-2 and pEGFR, quantification was done as previously described (38, 39). Briefly, an index of staining was calculated and expressed as the percentage of staining multiplied by staining intensity after subtracting the index staining of corresponding negative controls. Data analysis Fisher's exact test was done for the comparison of cancer and cancer + dysplasia rates between groups. Two-sample t tests, assuming unequal variances, were used for com- www.aacrjournals.org parison of MVD, Ki67, pEGFR, and pVEGFR-2 levels between groups. The nonparametric Wilcoxon rank-sum test was also done to confirm the results from the t tests. For pEGFR and pVEGFR-2, the average of five measurements for each mouse was first calculated, and this summary measure was used in the analyses. P 0.05 was considered statistically significant. All analyses were done using Stata version 11 (Stata Corp.). Results Effects of ZD6474 administration on the development of dysplasia and OSCC Mice were given 4-NQO (100 g/mL) in their drinking water for a period of 8 weeks, returned to normal water, and then randomized to observation or daily oral gavage of ZD6474 (25 mg/kg/d) for 24 weeks. We have previously shown that following the 8 weeks of 4-NQO administration, mice developed histologically identifiable hyperkeratotic and/or dysplastic lesions (35). Therefore, initiation of ZD6474 treatment at this time point was chosen because it closely mimics the clinical setting in which one would consider initiating chemopreventive therapy in patients. During the 24-week chemoprevention regimen, no significant differences in food and fluid consumption or activity were observed between the groups. At the completion of the 32-week study, there was a significant difference in the incidence of dysplasia and OSCC in the ZD6474 treatment group compared with the control group (Table 1). Overall, 71% (17 of 24) of the control mice and 12% (3 of 25) of the ZD6474-treated mice showed histologic evidence of OSCC (P 0.001). Similarly, the proportion of mice with dysplasia or OSCC was significantly different between the two treatment groups. In the control group, 96% (23 of 24) of the animals showed dysplasia or OSCC, whereas 28% (7 of 25) of the ZD6474 treatment group had dysplasia or OSCC (P 0.001). In total, this represented a 71% decrease in OSCC or dysplasia and an 83% decrease in OSCC. Effects of ZD6474 administration on proliferation and MVD ZD6474 has been shown to inhibit both tumor cell proliferation and angiogenesis via its dual activity against EGFR and VEGFR-2 (1719). Therefore, we performed Table 1. Effect of ZD6474 treatment on the development of OSCC in the mouse 4-NQO model Hyperkeratosis Dysplasia OSCC Total Control ZD6474 Total 1 6 17 24 18 4 3 25 19 10 20 49 Cancer Prev Res; 3(11) November 2010 Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. 1495 Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Zhou et al. immunohistochemistry for Ki67 and CD31 as surrogate markers for cell proliferation and angiogenesis, respectively. Overall, the Ki67 proliferative index (PI) for the ZD6474treated animals was significantly decreased when compared with the control mice (Table 2). The control group had a PI of 46 10, whereas the ZD6474 treatment group had a PI of 29 10 (P 0.001). Proliferation increased with histologic progression in both control and treatment cohorts (Fig. 1). Of note, the OSCC that arose in the ZD6474 treatment group (n = 3) had a mean PI (54.3) that was similar to the PI of the control animals (n = 17) who developed OSCC (51.6), suggesting that the ZD6474-associated tumors were still actively proliferating. Overall, there was a significant decrease in MVD in the ZD6474-treated mice when compared with controls. The control group showed a MVD score of 265 60, although the ZD6474 treatment group had a MVD score of 106 73 (P 0.001). However, there was no difference in vascularity when comparing the MVD between similar histologic diagnoses (hyperkeratosis or dysplasia or OSCC) from different treatment groups (control versus ZD6474-treated; Fig. 1). The OSCC that arose in the ZD6474 treatment group had a mean MVD (253.7) that was similar to the MVD of the OSCC control group (300.2) suggesting that the tumor was still actively inducing angiogenesis. ZD6474) there was no difference in the intensity scores between the hyperkeratotic, dysplastic, or OSCC specimens (Fig. 2). Interestingly, the pEGFR intensity scores for the OSCC from the ZD6474 treatment group were much lower than the intensity scores for the control OSCC cohort (Fig. 2). Overall, tissue from control mice had a significantly higher expression of pVEGFR-2 when compared with the ZD6474-treated mice (Table 2; Fig. 3). The combined mean intensity score for the control tissue was 106 11, whereas the combined intensity score of the tissue from the ZD6474-treated animals was 32 3 (P 0.001). When comparing expression between histologic groups within the same experimental group (control or ZD6474), the intensity scores between hyperkeratotic, dysplastic or OSCC specimens were very similar (Fig. 3). Like the pEGFR findings, expression of pVEGFR-2 in the OSCC from the ZD6474-treated group was much lower in intensity when compared with the OSCC from the control group (Fig. 3). These data show that ZD6474 was pharmacologically active in the 4-NQO model. In addition, the data suggests the OSCC that arose in the ZD6474-treated group may have developed acquired drug resistance, as ZD6474 was still actively inhibiting the phosphorylation of both EGFR and VEGFR-2. Effects of ZD6474 administration on EGFR and VEGFR-2 activation In an effort to identify the potential mechanism(s) of acquired resistance to ZD6474 treatment, immunohistochemistry for pEGFR and pVEGFR-2 was done to determine if ZD6474 was still inhibiting the activation of these receptors. Overall, tissue from the control cohort of mice showed significantly stronger cytoplasmic membrane staining for pEGFR when compared with ZD6474-treated mice. The control group had a mean intensity score of 97 5, whereas the mean intensity score was 33 3 (P 0.001) for the ZD6474-treated cohort (Table 2). When comparing pEGFR expression between histologic groups within the same treatment scheme (control or ZD6474-resistant OSCC express epithelial to mesenchymal markers ZD6474 was able to significantly reduce the incidence of OSCC when compared with the control group (Table 1). Although the inhibition of tumor development was statistically significant, 12% of the animals in the ZD6474 group developed OSCC. Furthermore, our data suggests that ZD6474 was still pharmacologically active because low levels of both pEGFR and pVEGFR-2 were still observed after 24 weeks of treatment (Figs. 2 and 3). In addition, the PI and MVD data from the OSCC arising in ZD6474-treated mice were similar to the PI and MVD data in the control animals harboring OSCC (Fig. 1). Overall, these data suggest that the OSCC in the ZD6474-treated mice had developed a form of acquired drug resistance. Similar acquired resistance has been associated with the expression of an epithelial to mesenchymal (EMT) phenotype, an intricate process that can be both physiologic and pathologic in nature (3943). For example, the induction of EMT may be a novel mechanism of acquired resistance to chemotherapy and radiation in cancer therapy (40, 44). To address the possibility that the development of resistance to ZD6474 treatment in the mouse 4-NQO model of OSCC was driven by the expression of an EMT phenotype, we did immunohistochemistry for the EMT markers E-cadherin and vimentin (45). Each of the tumors from the control group expressed high levels of E-cadherin and undetectable levels of vimentin protein (Fig. 4). Conversely, the tumors from ZD6474-treated mice lost expression of E-cadherin and expressed high levels of vimentin protein (Fig. 4). Although the sample size is small (n = 3), these data show a correlation between resistance to ZD6474 Table 2. Modulation of surrogate biomarkers for angiogenesis, proliferation, and activation of the EGFR and VEGFR-2 pathways by ZD6474 Control, N = 24 MVD Ki67 pEGFR pVEGFR-2 265 46 97 106 60 10 5 11 ZD6474, N = 25 106 29 33 32 73* 10* 3* 3* *P 0.001 for comparison with the control group. All numbers indicate mean SD. 1496 Cancer Prev Res; 3(11) November 2010 Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 ZD6474 Chemoprevention of Oral Cancer Fig. 1. ZD6474-treated animals show lower proliferative indices and microvessel densities compared with control animals. A, tissue sections were immunohistochemically stained for Ki67 and labeling indices were quantified. The overall labeling indices of the ZD6474 specimens were all significantly lower when compared with the control specimens (P 0.001). B, tissue sections were immunohistochemically stained for CD31 and MVD quantified. MVD of the ZD6474 specimens were significantly lower when compared with the control specimens (P 0.001). The total group was used for all statistical analysis. and the expression of EMT markers. They further suggest that the expression of an EMT phenotype may be a novel mechanism of acquired resistance to chemoprevention therapy for OSCC. Discussion The induction of cell proliferation and blood vessel growth are two critical phenotypes that are necessary for the development of malignant neoplasms. Aberrant EGFR tyrosine kinase activity plays an important role in a number of different tumor phenotypes including proliferation, apoptosis, angiogenesis, and metastasis. Furthermore, because www.aacrjournals.org EGFR has such a critical role in the development of OSCC, this signaling pathway has considerable therapeutic potential in the areas of cancer therapy and chemoprevention. Similarly, the activation of the VEGFR-2 pathway by VEGF is a critical component for the induction of angiogenesis in both physiologic and pathologic settings including OSCC. Therefore, because ZD6474 has the ability to inhibit both EGFR and VEGFR-2 activation, it has the potential to inhibit two critical signal transduction pathways and phenotypes involved in the development of OSCC. In this study, we show that ZD6474 was pharmacologically active in the 4-NQO model of OSCC. Animals treated with 25 mg/kg/d had significantly lower expression Cancer Prev Res; 3(11) November 2010 Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. 1497 Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Zhou et al. Fig. 2. ZD6474 inhibits the phosphorylation of EGFR in the 4-NQO model of OSCC. A, tissue sections from control and ZD6474-treated animals were immunohistochemically stained for pEGFR and quantified. B, expression of pEGFR was significantly lower in the ZD6474-treated specimens when compared with the control specimens (P 0.001). levels of pEGFR and pVEGFR-2 when compared with controls (Figs. 2 and 3). We also report for the first time that daily treatment with ZD6474 decreased the incidence of dysplasias and carcinomas in the mouse 4-NQO model of OSCC (Table 1). The rationale for the 24-week treatment schedule was based on our previous work, which showed that the majority of control animals harbor OSCC by week 24, while dysplasia was the predominant histologic diagnosis at weeks 16 and 20 (35). Because we were testing the hypothesis that ZD6474 would reduce the incidence of OSCC, we believe that it is most appropriate to carry out the prevention study to a time point where the predominant histologic diagnosis in the control group would be expected to be OSCC. Overall, we observed an 83% reduction in the incidence of OSCC when comparing the control and treatment groups (71% versus 12%, P 0.001). We also observed a 71% decrease in the incidence of both dysplasia and OSCC when comparing the control and ZD6474 treatment groups (96% versus 28%, P 0.001). These data strongly support the 1498 Cancer Prev Res; 3(11) November 2010 hypothesis that ZD6474 may be an effective chemopreventive agent for OSCC. In preclinical studies, ZD6474 has been shown to be a potent inhibitor of tumor angiogenesis and proliferation for several different tumor cell types (2130). It is also under active investigation in clinical trials for the treatment of various malignant neoplasms (31), with the greatest activity observed in nonsmall cell lung cancer and medullary thyroid carcinoma (3234). However, published data regarding the potential utility of ZD6474 in the realm of chemoprevention is limited. In one study, ZD6474 markedly reduced the number and the size of intestinal polyps, resulting in a 75% decrease in tumor burden in a mouse model of colon cancer (46). The data from the colonic polyp study and our current work suggests that further studies are warranted to evaluate the potential utility of ZD6474 as a chemopreventive agent. One of the long-term goals of chemoprevention must be the development of treatments that can be easily taken by at-risk individuals for prolonged periods of time with Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 ZD6474 Chemoprevention of Oral Cancer minimal toxicities to achieve widespread acceptance and long-term compliance. This would be particularly important in the case of high-risk patients who have not yet developed their first OSCC. We have previously shown that ABT-510, a mimetic peptide of thrombospondin-1, significantly decreased the incidence of dysplasia and OSCC in the 4-NQO model (35). However, because there is no oral formulation of the drug, the translation of this agent into clinical trials for prevention seems unlikely. Conversely, because ZD6474 is an orally available drug, it is potentially more feasible for prolonged use in human prevention studies. The maximum tolerated dose as well as toxicity profile of ZD6474 when used in cancer therapy is well described. ZD6474 has been well-tolerated at doses of 100 to 300 mg/d, with the most common adverse events being rash, diarrhea, fatigue, and asymptomatic QTc prolongation (31). However, because the drug may be initiated at a lower dose range in a chemoprevention setting, one might anticipate a lesser degree of side effects. Furthermore, treatment with higher doses of ZD6474 (50 and 100 mg/kg) than the current study (25 mg/kg/d) resulted in only a modest delay, but not inhibition, of cutaneous wound healing in a mouse model (47). Taken together, these data suggest that the toxicity profile of ZD6474, when used as a chemopreventive agent, might be acceptable when lower doses of the agent are used. This might be particularly true in the context of OSCC, in which the modest long-term survival is due in part to the frequent development of multiple additional primary tumors in individuals with a previous SCC. The rate of second primary tumors in these patients has been reported to be 3% to 7% per year, which is higher than for any other malignancy (48). This observation led Slaughter et al. to propose the concept of field cancerization. This theory suggests that multiple individual primary tumors develop independently in the upper aerodigestive tract as a result of years of chronic exposure of the mucosa to carcinogens (49). As a result of field cancerization, an individual who is fortunate to live 5 years after the initial primary tumor has up to a 35% chance of developing at least one new primary tumor within that time period. The occurrence of new primary tumors can be particularly devastating for individuals whose Fig. 3. ZD6474 inhibits the phosphorylation of VEGFR-2 in the 4-NQO model of OSCC. A, tissue sections from control and ZD6474-treated animals were immunohistochemically stained for pVEGFR-2 and quantified. B, expression of pVEGFR-2 was significantly lower in the ZD6474-treated specimens when compared with the control specimens (P 0.001). www.aacrjournals.org Cancer Prev Res; 3(11) November 2010 Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. 1499 Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Zhou et al. Fig. 4. OSCC arising in ZD6474-treated mice express EMT markers. Tumor samples from control mice show strong epithelial expression of E-cadherin and stromal expression vimentin. Histologically normal epithelium from the ZD6474-treated animals show strong expression of E-cadherin and no expression of vimentin (arrows). Conversely, tumor cells from ZD6474 mice show loss of expression of E-cadherin and strong expression of vimentin. initial lesions are small. Their 5-year survival rate for the first primary tumor is considerably better than late stage disease, but second primary tumors are their most common cause of treatment failure and death (4, 5). Resistance to cytotoxic chemotherapy and radiation therapy is well appreciated in the context of cancer therapy. In addition, mechanisms of resistance in response to targeted therapies have also been described. For example, several types of intrinsic and acquired resistance to inhibitors of angiogenesis have been postulated (50, 51). Similarly, several mechanisms of resistance related to antiEGFR therapy have been reported for nonsmall cell lung cancer, although the mechanisms for EGFR resistance in the context of OSCC seem to be different and remain unclear (52). Conversely, there are limited data concerning the potential mechanisms of acquired resistance in response to long-term chemoprevention therapy using targeted agents (5355). In the 4-NQO model of OSCC, 12% of the mice chronically treated with ZD6474 developed a form of acquired resistance to the drug. This resistance correlated with a loss of E-cadherin and a gain in vimentin protein expression, suggesting that these tumors began to express an EMT phenotype (44). Conversely, none of the control group OSCC expressed EMT markers. This correlation between resistance to ZD6474 and the expression of an EMT phenotype suggests a novel mechanism of acquired resistance in the chemopreventive 1500 Cancer Prev Res; 3(11) November 2010 setting. The expression of EMT transitions is well appreciated in embryology and various types of pathophysiology (40). Recently, there has been an increased interest in the role of EMT in areas of cancer progression as well as resistance to chemotherapy and radiation therapy (40, 44). At this time, we do not know if there is a causal link between the expression of EMT markers and resistance to ZD6474. However, the fact that EMT markers were not expressed in control OSCC provides compelling preliminary evidence worthy of further investigation. In addition, we do not know the timing of the gain of expression of the EMT phenotype. As designed, this prevention study harvested all tissues after 24 weeks of ZD6474 therapy. Therefore, to investigate the dynamics of EMT marker expression, one could sacrifice subsets of mice at specified intervals after the initiation of ZD6474 treatment to determine the incidence and timing of EMT marker expression at the stages of hyperkeratosis, dysplasia, and OSCC. It is also important to determine if one or both receptor pathways are mediating the expression of the EMT phenotype. Resistance to EGFR inhibitors erlotinib, gefitinib and cetuximab has been reported to induce an EMT transition (4143). Similarly, the induction of hypoxia has also been shown to induce EMT (40). Therefore, it is possible that ZD6474 may drive the expression of EMT via both pathways. In addition, the downstream mechanisms of the expression of the EMT phenotype are unknown. The transcription Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 ZD6474 Chemoprevention of Oral Cancer factors Twist, Snail, and Slug are major mediators of EMT and have been shown to repress E-cadherin expression (40). Further investigation into the altered expression of these and other EMT-related regulatory factors may aid in our understanding of how the expression of an EMT phenotype occurs in the setting of chemoprevention. In addition, the biological and clinical implications of EMT expression in ZD6474-resistant tumors requires further investigation, as the expression of the EMT phenotype can lead to resistance to multiple drugs and potentially lead to the progression of tumors (30). However, the potential for altered clinical behavior following a tyrosine kinase inhibitorbased chemopreventive treatment is not limited to this class of drugs, as resistance towards other types of chemopreventive agents has also been described (3941). Finally, if the pattern of EMT development can be modeled, one could envision using EMT markers as diagnostic beacons to herald the expression of acquired resistance. Such beacons may be useful as they could be used as indicators for when it would be most efficacious to switch to an alternative chemopreventive agent. For example, the expression of EMT markers might dictate a switch to a histone deacetylase inhibitor, as these have been shown to reverse the EMT phenotype (56). By doing so, one might hypothesize that the histone deacetylase could thereby restore sensitivity to ZD6474s and prolong its chemopreventive activity. We believe that the mouse 4-NQO model is an excellent model system to pursue each of these important preclinical questions. In conclusion, our data provides novel evidence that ZD6474, a combined inhibitor of the EGFR and VEGFR-2 pathways, holds promise as a chemopreventive agent for OSCC. They further suggest that the development of acquired resistance to ZD6474 may be mediated by the expression of an EMT phenotype. Finally, the data suggests that the 4-NQO model of OSCC is a useful preclinical platform to investigate the mechanisms of acquired resistance in the chemopreventive setting. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Grant Support In part by the NIH (DE012322). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received 06/16/2010; revised 08/12/2010; accepted 09/02/2010; published OnlineFirst 10/26/2010. References 1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:7196. 2. In: Ries LAG, Melbert D, Krapcho M, et al, editors. SEER cancer statistics review, 19752005. Bethesda, MD: National Cancer Institute; 2008. 3. Murphy GP LW, Lenhhardt RE. American cancer society textbook of clinical oncology. 2nd ed. Atlanta: American Cancer Society; 1995. 4. Lippman SM, Hong WK. Second malignant tumors in head and neck squamous cell carcinoma: the overshadowing threat for patients with early-stage disease. 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Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Dual Inhibition of Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor is an Effective Chemopreventive Strategy in the Mouse 4-NQO Model of Oral Carcinogenesis Guolin Zhou, Rifat Hasina, Kristen Wroblewski, et al. Cancer Prev Res 2010;3:1493-1502. Published OnlineFirst October 26, 2010. Updated version Cited articles Citing articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: doi:10.1158/1940-6207.CAPR-10-0135 This article cites 53 articles, 22 of which you can access for free at: http://cancerpreventionresearch.aacrjournals.org/content/3/11/1493.full#ref-list-1 This article has been cited by 4 HighWire-hosted articles. 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