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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. Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer 1953;6:9638. 5. Lippman SM, Hong WK. Second malignant tumors in head and neck squamous cell carcinoma: the overshadowing threat for patients with earlystage disease. Int J Radiat Oncol Biol Phys 1989; 17:6914. 6. Kelloff GJ, Lippman SM, Dannenberg AJ, et al. Progress in chemoprevention drug development: the promise of molecular biomarkers for prevention of intraepithelial neoplasia and cancera plan to move forward. Clin Cancer Res 2006;12:366197. 7. Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005;438:9326. 8. Lingen MW, DiPietro LA, Solt DB, Bouck NP, Polverini PJ. The angiogenic switch in hamster buccal pouch keratinocytes is dependent on TGF-1 and is unaffected by ras activation. Carcinogenesis 1997;18:32938. 9. Carlile J, Harada K, Baillie R, et al. Vascular endothelial growth factor (VEGF) expression in oral tissues: possible relevance to angiogenesis, tumour progression and field cancerisation. J Oral Pathol Med 2001;30:44957. 10. Pazouki S, Chisholm DM, Adi MM, et al. The association between tumour progression and vascularity in the oral mucosa. J Pathol 1997;183:3943. 11. Jin Y, Tipoe GL, White FH, Yang L. A quantitative investigation of immunocytochemically stained blood vessels in normal, benign, premalignant and malignant human oral cheek epithelium. Virchows Arch 1995;427:14551. 12. Steidler NE, Reade PC. Experimental induction of oral squamous cell carcinomas in mice with 4-nitroquinolone-1-oxide. Oral Surg Oral Med Oral Pathol 1984;57:52431. 13. Hawkins BL, Heniford BW, Ackermann DM, Leonberger M, Martinez SA, Hendler FJ. 4NQO carcinogenesis: a mouse model of oral cavity squamous cell carcinoma. Head Neck 1994;16:42432. 14. von Pressentin MM, Kosinska W, Guttenplan JB. Mutagenesis induced by oral carcinogens in lacZ mouse (MutaMouse) tongue and other oral tissues. Carcinogenesis 1999;20:216770. 15. Thomas GR, Chen Z, Oechsli MN, Hendler FJ, Van Waes C. Decreased expression of CD80 is a marker for increased tumorigenicity in a new murine model of oral squamous-cell carcinoma. Int J Cancer 1999;82:37784. 16. Kim TW, Chen Q, Shen X, et al. Oral mucosal carcinogenesis in SENCAR mice. Anticancer Res 2002;22:273340. 17. Tang XH, Knudsen B, Bemis D, Tickoo S, Gudas LJ. Oral cavity and esophageal carcinogenesis modeled in carcinogen-treated mice. Clin Cancer Res 2004;10:30113. 18. Miyamoto S, Yasui Y, Kim M, et al. A novel rasH2 mouse carcinogenesis model that is highly susceptible to 4-NQO-induced tongue and esophageal carcinogenesis is useful for preclinical chemoprevention studies. Carcinogenesis 2008;29:41826. 19. Brudno M, Poliakov A, Salamov A, et al. Automated whole-genome multiple alignment of rat, mouse, and human. Genome Res 2004;14:68592. www.aacrjournals.org 20. Hancock JM. A bigger mouse? The rat genome unveiled. Bioessays 2004;26:103942. 21. Twigger SN, Shimoyama M, Bromberg S, Kwitek AE, Jacob HJ. The Rat Genome Database, update 2007easing the path from disease to data and back again. Nucleic Acids Res 2007;35:D65862. 22. O'Brien SJ, Menotti-Raymond M, Murphy WJ, et al. The promise of comparative genomics in mammals. Science 1999;286:45862, 47981. 23. Haviv F, Bradley MF, Kalvin DM, et al. Thrombospondin-1 mimetic peptide inhibitors of angiogenesis and tumor growth: design, synthesis, and optimization of pharmacokinetics and biological activities. J Med Chem 2005;48:283846. 24. Dawson DW, Volpert OV, Pearce SF, et al. Three distinct d-amino acid substitutions confer potent antiangiogenic activity on an inactive peptide derived from a thrombospondin-1 type 1 repeat. Mol Pharmacol 1999;55:3328. 25. Anderson JC, Grammer JR, Wang W, et al. ABT510, a modified type 1 repeat peptide of thrombospondin, inhibits malignant glioma growth in vivo by inhibiting angiogenesis. Cancer Biol Ther 2007;6: 45462. 26. Yang Q, Tian Y, Liu S, et al. Thrombospondin-1 peptide ABT-510 combined with valproic acid is an effective antiangiogenesis strategy in neuroblastoma. Cancer Res 2007;67:171624. 27. Rusk A, McKeegan E, Haviv F, Majest S, Henkin J, Khanna C. Preclinical evaluation of antiangiogenic thrombospondin-1 peptide mimetics, ABT526 and ABT-510, in companion dogs with naturally occurring cancers. Clin Cancer Res 2006;12: 744455. 28. Yap R, Veliceasa D, Emmenegger U, et al. Metronomic low-dose chemotherapy boosts CD95-dependent antiangiogenic effect of the thrombospondin peptide ABT-510: a complementation antiangiogenic strategy. Clin Cancer Res 2005;11:667885. 29. Reiher FK, Volpert OV, Jimenez B, et al. Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics. Int J Cancer 2002; 98:6829. 30. Abbey LM, Kaugars GE, Gunsolley JC, et al. Intraexaminer and interexaminer reliability in the diagnosis of oral epithelial dysplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;80:18891. 31. Warnakulasuriya S, Reibel J, Bouquot J, Dabelsteen E. Oral epithelial dysplasia classification systems: predictive value, utility, weaknesses and scope for improvement. J Oral Pathol Med 2008; 37:12733. 32. Hasina R, Whipple ME, Martin LE, Kuo WP, Ohno-Machado L, Lingen MW. Angiogenic heterogeneity in head and neck squamous cell carcinoma: biological and therapeutic implications. Lab Invest 2008;88:34253. 33. Hasina R, Pontier AL, Fekete MJ, et al. NOL7 is a nucleolar candidate tumor suppressor gene in cervical cancer that modulates the angiogenic phenotype. Oncogene 2006;25:58898. 34. Cuzick J. A Wilcoxon-type test for trend. Stat Med 1985;4:8790. 35. Lingen MW. Angiogenesis in the development of head and neck cancer and its inhibition by chemopreventive agents. Crit Rev Oral Biol Med 1999;10: 15364. 36. Heniford BW, Shum-Siu A, Leonberger M, Hendler FJ. Variation in cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. Nucleic Acids Res 1993;21:315966. 393 37. Takeuchi S, Nakanishi H, Yoshida K, et al. Isolation of differentiated squamous and undifferentiated spindle carcinoma cell lines with differing metastatic potential from a 4-nitroquinoline N-oxide-induced tongue carcinoma in a F344 rat. Jpn J Cancer Res 2000;91:121121. 38. Yuan B, Heniford BW, Ackermann DM, Hawkins BL, Hendler FJ. Harvey ras (H-ras) point mutations are induced by 4-nitroquinoline-1-oxide in murine oral squamous epithelia, while squamous cell carcinomas and loss of heterozygosity occur without additional exposure. Cancer Res 1994;54:53107. 39. Chang SE, Bhatia P, Johnson NW, et al. Ras mutations in United Kingdom examples of oral malignancies are infrequent. Int J Cancer 1991; 48:40912. 40. Homberger F. Chemical carcinogenesis in Syrian hamsters. Prog Exp Tumor Res 1972;16:15275. 41. Eveson JW, MacDonald DG. Quantitative histological changes during early experimental carcinogenesis in the hamster cheek pouch. Br J Dermatol 1978;98:63944. 42. Vered M, Yarom N, Dayan D. 4NQO oral carcinogenesis: animal models, molecular markers and future expectations. Oral Oncol 2005;41:3379. 43. Kanojia D, Vaidya MM. 4-Nitroquinoline-1-oxide induced experimental oral carcinogenesis. Oral Oncol 2006;42:65567. 44. Folkman J, Hanahan D. Princess Takamatsu Symposium. 1991, p. 33947. 45. Volpert OV, Dameron KM, Bouck N. Sequential development of an angiogenic phenotype by human fibroblasts progressing to tumorigenicity. Oncogene 1997;14:1495502. 46. Liss C, Fekete MJ, Hasina R, Lam CD, Lingen MW. Paracrine angiogenic loop between headand-neck squamous-cell carcinomas and macrophages. Int J Cancer 2001;93:7815. 47. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005;438:96774. 48. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2002;2:72739. 49. Albini A, Noonan DM, Ferrari N. Molecular pathways for cancer angioprevention. Clin Cancer Res 2007;13:43205. 50. Sharma RA, Harris AL, Dalgleish AG, Steward WP, O'Byrne KJ. Angiogenesis as a biomarker and target in cancer chemoprevention. Lancet Oncol 2001;2:72632. 51. Ebbinghaus S, Hussain M, Tannir N, et al. Phase 2 study of ABT-510 in patients with previously untreated advanced renal cell carcinoma. Clin Cancer Res 2007;13:668995. 52. Markovic SN, Suman VJ, Rao RA, et al. A phase II study of ABT-510 (thrombospondin-1 analog) for the treatment of metastatic melanoma. Am J Clin Oncol 2007;30:3039. 53. Gietema JA, Hoekstra R, de Vos FY, et al. A phase I study assessing the safety and pharmacokinetics of the thrombospondin-1-mimetic angiogenesis inhibitor ABT-510 with gemcitabine and cisplatin in patients with solid tumors. Ann Oncol 2006;17:13207. 54. Hoekstra R, de Vos FY, Eskens FA, et al. Phase I study of the thrombospondin-1-mimetic angiogenesis inhibitor ABT-510 with 5-fluorouracil and leucovorin: a safe combination. Eur J Cancer 2006; 42:46772. 55. Hoekstra R, de Vos FY, Eskens FA, et al. Phase I safety, pharmacokinetic, and pharmacodynamic study of the thrombospondin-1-mimetic angiogenesis inhibitor ABT-510 in patients with advanced cancer. J Clin Oncol 2005;23:518897. 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 ABT-510 Is an Effective Chemopreventive Agent in the Mouse 4-Nitroquinoline 1-Oxide Model of Oral Carcinogenesis Rifat Hasina, Leslie E. Martin, Kristen Kasza, et al. Cancer Prev Res 2009;2:385-393. Published OnlineFirst March 31, 2009. 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... Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 RESEARCH ARTICLE Open Access Identification and functional analysis of NOL7 nuclear and nucleolar localization signals Guolin Zhou, Colleen L Doi, Mark W Lingen* Abstract Background: NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated. Results: An in vitro import assay demonstrated that NOL7 requires cytosolic machinery for active nuclear transport. Using sequence homology and prediction algorithms, four putative nuclear localization signals (NLSs) were identified. NOL7 deletion constructs and cytoplasmic pyruvate kinase (PK) fusion proteins confirmed the functionality of three of these NLSs. Site-directed mutagenesis of PK fusions and full-length NOL7 defined the minimal functional regions within each NLS. Further characterization revealed that NLS2 and NLS3 were critical for both the rate and efficiency of nuclear targeting. In addition, four basic clusters within NLS2 and NLS3 were independently capable of nucleolar targeting. The nucleolar occupancy of NOL7 revealed a complex balance of rapid nucleoplasmic shuttling but low nucleolar mobility, suggesting NOL7 may play functional roles in both compartments. In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7. Conclusions: These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus. Further, this work characterizes the relative contribution of each sequence to NOL7 nuclear and nucleolar dynamics, the subnuclear constituents that participate in this targeting, and suggests a functional role for NOL7 in both compartments. Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus. Background NOL7 is a predicted 29 kDa, 257 amino acid protein with no significant homologies to other characterized proteins that localizes to the nucleus and nucleoli of cells. NOL7 localizes to 6p23, a region with frequent loss of heterozygosity (LOH) in a number of cancers, including hormone-refractory breast carcinoma, leukemia, lymphoma, osteosarcoma, retinoblastoma, nasopharyngeal carcinoma and cervical cancer (CC) [1-19]. Using CC as a model for investigation, where LOH of * Correspondence: Mark.Lingen@uchospitals.edu Contributed equally Departments of Pathology, Medicine and Radiation and Cellular Oncology, The University of Chicago, Chicago, IL, USA 6p23 is the most common allelic loss in this neoplasm [20-25], we demonstrated that reintroduction of NOL7 suppresses in vivo tumor growth by 95% [26]. This suppression is due in part to the induction of an anti-angiogenic phenotype via decreased expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and increased expression of the inhibitor of angiogenesis Thrombospondin-1 (TSP-1). One of the important features that differentiate eukaryotic from prokaryotic cells is the presence of intracellularly distinct compartments and organelles such as the nucleus, nucleolus and mitochondria. The rapid exchange of proteins between the cytoplasm and the nucleus is a vital process in eukaryotic cells, and this 2010 Zhou et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 occurs through the nuclear pore complex (NPC), a large macromolecular structure embedded in the double membrane of the nuclear envelope [27-29]. Small molecules such as ions and some small proteins can move from the cytoplasm to the nucleus by passive diffusion through the NPC. However, proteins larger than ~20 kDa typically cross the NPC in a carrier-mediated fashion [30]. This active nuclear transport of proteins is mediated by specific amino acids sequences, which are referred to as nuclear localization signals (NLS) and nuclear export signals (NESs). The classical NLSs contain a cluster of basic amino acids, while classical NESs contain stretches of hydrophobic, leucine-rich residues. The best-described nuclear import pathway is driven by the so-called classical NLS (cNLS). This signal is typically lysines (K) or arginines (R), that are organized as a single-stretch monopartite NLS: (K/R) 4-6 , or as a bipartite NLS in which there are two small clusters typically separated by ten to twelve amino acids (K/R)2X10-12(KR)3. The SV40 large-T antigen (PKKKRKV) and nucleoplasmin (KRPAATKKAGQAKKKK) cNLS are the prototypical mono- and bipartite cNLS [31,32]. Recently, a tripartite NLS, consisting of three clusters of basic residues separated by two spacer peptides, has also been described [33-38]. Finally, a fourth type of NLS contains two to four dispersed basic residues contiguous to hydrophobic amino acids [39,40]. The active transport of proteins between the cytoplasm and nucleus is facilitated by the karyopherin/importin family of carrier proteins. During classical nuclear import, NLSs are typically recognized in the cytoplasm by a heterodimeric complex consisting of importin a and b with the a-subunit providing the NLS binding site. The NLS protein-receptor complex docks to the nuclear pore complex via importin b and is subsequently translocated through the pore by an energydependent mechanism [41]. Once the import complex reaches the nucleus, it is dissociated by RanGTP. Binding of RanGTP to importin b can cause a conformational change, resulting in the release of the importin a/cargo complex [42]. Recent modeling studies have shown that some cargo proteins can also bind directly to b -karyopherins [43]. While active transport mechanisms are required for nuclear localization, targeting to the nucleolus has been shown to depend on interactions with nucleolar constituents. Nucleolar localization sequences (NoLSs) have been shown to represent binding domains with resident nucleolar proteins, rRNA, and other nucleolar components and function more as retention rather than targeting signals [44-50]. The affinity and stability of these interactions among nucleolar proteins has been shown to have functional consequences that are reflected in the dynamics of their nucleolar localization. Many ribosomal proteins show higher immobility and slower recovery Page 2 of 20 compared to processing and transcriptional factors, and these differences have been attributed to the stability and duration of their nucleolar functions [51,52]. Mutations, truncations, and changes in posttranslational modifications that have functional consequences have also been shown to affect the immobility and recovery of classic nucleolar proteins such as NPM [53]. Together, these observations suggest the nucleolar localization is a factor of the affinity, stability, and abundance of nucleolar interactions and that the dynamics of nucleolar occupancy are a reflection of potential functions within the nucleus and nucleolus. The purpose of this work was to determine the mechanism by which NOL7 was transported into the nucleus, identify the minimal functional sequences required for NOL7 nuclear translocation, and the relative influence each of these sequences may have on the rate and efficiency of localization. We further wished to define the elements responsible for the nucleolar localization of NOL7 and characterize the dynamics of this targeting. Together, the aim of this paper was to define the mechanism responsible for localization of NOL7 within the cell and the functional consequences of the different components that comprise that mechanism. Results NOL7 is imported into the nucleus via an energydependent, nucleoporin-mediated mechanism NOL7 is predicted to have a molecular weight of approximately 29 kDa. Some proteins of this size have been reported to enter the nucleus through passive diffusion, while others require active NLS-mediated transport. To distinguish between an active transport and a passive diffusion mechanism of nuclear localization for NOL7, we subjected GFP-tagged NOL7 to an in vitro transport assay using permeabilized HeLa cells [54]. In this assay, the cytoplasmic membranes are permeabilized with digitonin, which depletes the cells of their soluble endogenous cytosolic factors while leaving the nuclear membrane intact. In this fashion, the nuclear import of GFP-tagged proteins can be studied under various conditions. In these experiments, digitonin-permeabilized HeLa cells were incubated with GFP-tagged NOL7 under various conditions to assay the mechanism of its localization. NOL7-GFP alone was not sufficient for nuclear import (Figure 1, first panel). Addition of cytosolic extract or ATP alone was also insufficient for transport (Figure 1, second and third panels), suggesting that NOL7 localization is energy and complex dependent. At 4C or in the presence of heat-inactivated cytosol, NOL7-GFP was restricted to the cytoplasm (Figure 1, fourth and fifth panels), demonstrating that NOL7 requires cytosolic proteins and is localized via an active transport mechanism. At physiologic temperature, Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 3 of 20 Figure 1 NOL7 requires cytosolic factors for efficient nuclear localization. HeLa cells permeabilized with digitonin were incubated at 4C or 37C as indicated with (+) or without (-) full length NOL7 expressing a C-terminal GFP tag, cytosol, heat-inactivated cytosol, ATP, or WGA. Localization of NOL7 was confirmed by visualization of GFP. combination of cytosol and ATP was sufficient for NOL7 nuclear localization (Figure 1, sixth panel). However, this was blocked by pretreatment with wheat germ agglutinin (WGA) (Figure 1, seventh panel). Together, these controls demonstrate that NOL7 is actively targeted to the nucleus and nucleolus via an energy-dependent, nucleoporin-mediated mechanism. Protein prediction programs identify distinct biochemical domains and putative NLSs in NOL7 Database analysis of the full length sequence of human NOL7, utilizing the protein domain prediction programs PSORT [55], TargetP [56], SAPS [57] and NetNES [58], identified five distinct biochemical domains and four putative NLSs within NOL7 (Figure 2). SAPS analysis revealed the existence of four highly basic regions and one acidic domain which correlates with its high pI of 9.7. Stretches of highly basic residues have been shown to participate in nucleic acid binding, nuclear transport, and may contribute to the tumor suppressive function and localization of NOL7 (Figure 2A). While NOL7 lacks homology to other known proteins and domains, the significant sequence conservation among its orthologs suggests a consistent evolutionary role. In particular, four long stretches of basic amino acids are particularly conserved throughout evolution (Figure 2B). Not coincidently, the prediction programs also identified potential NLSs within these basic biochemical domains. However, NOL7 was not predicted to contain any NESs. Putative Sequence (PS) 1, amino acids 1-10 appeared to be a monopartite NLS, while PS2, amino acids 88-112 was predicted to be an example of the recently described tripartite NLS. PS3, amino acids 144-162 appeared to be bipartite NLS and PS4, amino acids 242257, was predicted to be a bipartite NLS (Figure 2B). Analysis of potential sequence conservation of each of the four putative NOL7 NLSs among its orthologs was performed using BLAST and aligned by the ClustalW method (Figure 2B). Significant evolutionary conservation between mammalian species Homo sapiens, Pan troglodytes, Macaca mulatta, Bos taurus, Canis familiaris, Rattus norvegicus, and Mus musculus was observed not only for the NLS regions for the full-length protein as well. Less striking but still significant homology was also seen in Gallus gallus, Tetraodon nigroviridis, Danio rerio, and Saccharomyces cerevisiae. While some stretches of the full-length protein showed typical divergence of the sequence, the basic stretches of residues that comprised the putative NLSs were remarkably conserved. In particular, PS1, which is composed of three basic residues contiguous to hydrophobic amino acids, showed highly significant homology between all of the NOL7 orthologs. This suggests that PS1 may target NOL7 to the nucleus despite its dispersed amino acid sequence. Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 4 of 20 Figure 2 NOL7 is composed of distinct biochemical domains and multiple putative NLSs that show evolutionary conservation. (A) Multiple sequential analysis programs confirmed the existence of four basic (blue) and one acidic (green) region in the full-length sequence of NOL7. Putative NLSs identified in sequence analysis programs are shown in red. (B) Sequence conservation between human NOL7 and its putative orthologs was analyzed for each of the putative NLSs and the alignment is shown. Black shaded boxes indicate identical amino acid conservation, while grey boxes signify similar amino acids to Homo sapiens. Numbers correspond to residues within the RefSeq sequences listed in the Materials and Methods 2.7. Interestingly, no homology could be detected between NOL7 and any other proteins or domains. Further, despite the significant conservation between human NOL7 and its putative orthologs, no functional role can be extrapolated for NOL7. With the exception of S. cerevisiae ortholog Bud21, no functional data exists for any of these proteins. In the case of the putative yeast ortholog, initial studies suggest that the U3 snoRNA function of Bud21 is not conserved, as NOL7 is incapable of interacting with major Bud21 cofactors that regulate its activity (unpublished data). Further, no similarity could be detected between NOL7 and other characterized Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 proteins of any species, either in the context of the fulllength NOL7 or for shorter stretches of the protein. Taken together, this data suggests that NOL7 is a critical protein in higher eukaryotes that may function in a specialized manner. Furthermore, the correlation between the regions of strongest sequence conservation and predicted NLSs suggests the localization and function of NOL7 may be linked. NOL7 contains three functional NLSs that translocate cytoplasmic PK into the nucleus In order to determine the functionality of the four putative NLSs, we first generated a series of N- and C- terminal deletion mutants of NOL7 with an HA tag that were transiently expressed in HeLa and 293T cells (Figure 3A). The subcellular localization of the truncations was visualized by immunofluorescence with DAPI costaining of the nucleus. While the majority of the deletion constructs retained nuclear localization, a deletion construct lacking all four PSs [1-10, 88-257] was cytoplasmic (Figure 3B). Further, PS1 and PS2 were shown to function individually as NLSs, as constructs missing PS2, PS3, and PS4 [88-257] or PS1, PS3, and PS4 [1-10, 113-257] were nuclear localized. While these results demonstrated that PS1 and PS2 were sufficient to target NOL7 to the nucleus, we sought to further clarify the role of PS3 and PS4. Therefore, five additional truncations were cloned together with inactivating mutations in the region of PS1 [PS1 (-)]. PS4 was found to be sufficient for nuclear localization of NOL7 [PS1(-),88-216 and PS1(-),88-241] but a construct containing only an intact PS3 [PS1(-),88112,217-257] remained in the cytoplasm, suggesting that the putative sequence of PS3 is not a functional NLS. To specifically determine if the three candidate NLSs were sufficient to target proteins for nuclear import and further confirm that PS3 was not a functional NLS, the localization of a series of fusion constructs containing each putative NLS sequence and the cytoplasmic protein PK was evaluated. PS1, 2, 3 and 4 were cloned in frame with the C- terminus of the PK bearing an N-terminal myc tag and transiently transfected into HeLa cells (Figure 4A). The subcellular localization of the chimeric proteins was visualized using an a-myc monoclonal antibody and Cy3-conjugated secondary, with DAPI costaining for visualization of the nucleus. Both wild-type Myc-tagged PK protein and the PS3-PK fusion were seen exclusively in the cytoplasm (Figure 4B). In contrast, PK-PS1, PKPS2, and PK-PS4 localized predominately to the nucleus (Figure 4B). Taken together, these results from the truncation and PS-PK fusion experiments confirm that PS3 is not a functional NLS while PS1, PS2, and PS4 are functional nuclear localization signals. Furthermore, the data Page 5 of 20 demonstrate that each NLS is capable of translocating a cytoplasmic protein into the nucleus independently. From this point forward, we will therefore refer to PS1, PS2, and PS4 as NLS1, NLS2, and NLS3, respectively. Identification of the amino acids required for functionality of each NLS The deletion and PK fusion constructs demonstrated the regions within NOL7 that are individually capable of driving nuclear localization, but do not define the specifc sequence elements that comprise the individual NLSs. To specifically define the minimal amino acids required for functionality of each NLS, site-directed mutagenesis was performed to convert the basic amino acids of interest (arginine and lysine) to the nonpolar, electrically neutral amino acid alanine in each of the NLSs. Within each sequence, three individual basic amino acids (NLS1) or clusters of basic amino acids (NLS2 and NLS3) were identified and mutated individually to determine their relative contribution to the functionality of the NLS. Each intact NLS and the mutant NLSs were cloned in-frame with PK bearing an N-terminal myc tag (Figure 5A). As before, fusion with all three wild type NLSs resulted in nuclear localization of PK (Figure 5B-D). Mutation of any one of the three basic residues of NLS1 abolished nuclear localization, suggesting that each of these amino acids is critical for functionality of NLS1 (Figure 5B). For NLS2, loss of the second or third cluster of basic residues resulted in cytoplasmic localization, while loss of the first basic cluster had no effect on nuclear localization of PK (Figure 5C). These mutations suggest that the minimal region required for nuclear localization directed by NLS2 resides within residues 95-112. Consistent with our predictions, both basic clusters of NLS3 were required for nuclear localization (Figure 5D), suggesting that NLS1 is a monopartite sequence while both NLS2 and NLS3 are bipartite sequences. Taken together, these data confirm the functionality of the NLSs and define the specific amino acids present in each of the individual NLS that are required for the nuclear import of PK. While experiments using individual NLS fused to PK are useful, there are several limitations to these types of studies. For example, Burgess et al [59] demonstrated that EBNA3B has three functional NLSs when investigated in truncation experiments but only two were found to be functional in the context of the full-length protein. To determine the contributions of each NLS within full-length GFP-tagged NOL7, the arginine and lysine residues in each NLS were mutated to alanine (Figure 6A). The subcellular localization of the constructs was visualized using by GFP fluorescence, with DAPI costaining of the nucleus. Mutation of all three Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 6 of 20 Figure 3 NOL7 contains three separate NLSs that are necessary for nuclear localization. (A) Schematic representation of deletion constructs of NOL7 used to determine which regions of NOL7 are required for nuclear localization. Results as demonstrated in (B) are summarized in the column to the right, where No designates nucleolar localization, Np designates nucleoplasmic localization, and C designates cytoplasmic localization. (B) Localization of the constructs was confirmed in HeLa cells by immunofluorescence using an a-HA primary and FITC-coupled secondary antibody. NLSs resulted in cytoplasmic localization of NOL7 but retention of only one NLS was sufficient for nuclear localization (Figure 6B). Taken together, these results demonstrate that NOL7 has three functional NLS that can independently cause translocation of full length NOL7. Each NLS contributes differentially to the rate and efficiency of NOL7 nuclear import While each NLS was shown to be independently capable of directing nuclear transport of NOL7, it was unclear what the relative contribution of each signal to this function might be. To address this question, we Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 A Page 7 of 20 PK-WT Myc PS1 Myc PK PS2 Myc PK PS3 Myc PK PS3 C PS4 Myc PK PS4 Np B PK-WT PK PS1 C PS1 PS2 PS2 PS3 Np Np PS4 DAPI a-Myc Merge Figure 4 NOL7 contains three NLSs that are sufficient for nuclear localization. (A) Schematic representing the three different NLSs cloned in-frame with the cytoplasmic protein PK bearing a c-myc tag. Results demonstrated in (B) are summarized in the column to the right, where Np designates nucleoplasmic localization and C designates cytoplasmic localization. (B) Localization of the constructs in Hela cells was confirmed by immunofluorescence using an a-myc primary and Cy-3 conjugated secondary antibody. Costaining of the nucleus with DAPI is shown in blue. determined the rate and efficiency of nuclear import for each NLS construct. While fluorescence recovery after photobleaching (FRAP) has been employed previously for measuring the rate of import, this method is limited to small bleaching areas and measures a combination of active nuclear import and nucleoplasmic diffusion, the magnitude of which can vary greatly between proteins [60-62]. In most cases, the nuclear diffusion can be considered equivalent among different constructs of the same protein. However, subnuclear targeting of proteins within the nucleus affects their nuclear diffusion and can no longer be discounted in the calculation of rate by FRAP. As mutant constructs of NOL7 are differentially localized to the nucleoplasm and nucleolus, a different approach needed to be applied to investigate the role of different NLSs in the rate of nuclear import that would not be influenced by mobility within the nuclear compartment. Therefore, two complementary methods were adapted to measure both rate and efficiency of NOL7 nuclear import, based on quantitative immunofluorescence methods previously established in the literature [63-67]. In both cases, HA-tagged NOL7 constructs were transfected into HeLa cells and imaged by immunofluorescence against the HA tag. Using ImageJ software, the fluorescence intensity was measured and reported as a ratio of nuclear to total fluorescence. While previous reports typically utilize the nuclear to cytoplasmic ratio, we normalized to total cell fluorescence to accommodate differences in subnuclear localization and expression level between the different mutant constructs. For the efficiency experiments, data was collected twenty hours after transfection, when import had reached steady-state equilibrium (Figure 7A). It was found that WT NOL7 was most efficiently localized to the nucleus, and the strictly nucleoplasmic mutant N23(-), was least efficiently targeted. The single mutants demonstrated nearly 10% more efficient nuclear targeting than the double mutants, with a p-value of 2.1310-7. The most dramatic loss in targeting efficiency was observed upon the Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 8 of 20 Figure 5 Basic residues within each of the NLSs are required for nuclear localization of PK. (A) Schematic representing the three different NLSs bearing neutralizing mutations in the basic residues were cloned in-frame with the cytoplasmic protein PK bearing a c-Myc tag. Results demonstrated in (B-D) are summarized in the column to the right, where Np designates nucleoplasmic localization and C designates cytoplasmic localization. (BD) Subcellular localization was determined by immunofluorescence in HeLa cells using an a-myc primary and either a HRP or Cy-3 conjugated secondary antibody. Costaining of the nucleus with DAPI is shown in blue. (B) Mutations in either the first (NLS1-sub1), second (NLS1-sub2), or third (NLS1-sub3) basic cluster of NLS1 were mutated in alanine and visualized for nuclear localization. (C) Expression and localization of NLS2 mutants lacking either the first (NLS2-sub1), second (NLS2-sub2), or third (NLS2-sub3) basic cluster. (D) Expression and localization of the constructs bearing mutations in the first (NLS3-sub1) or second (NLS3-sub2) basic cluster of NLS3. combined loss of NLS2 and NLS3, with over a 15% decrease in efficiency for N23(-) compared to WT NOL7 and over 10% decrease compared to all other mutants. This decrease was highly significant, with a p-value of 9.4510 -9 compared to WT NOL7 and 2.6810 -5 and 1.2410-4 compared to the other double mutants N12(-) and N13(-), respectively. Together, these observations suggest that NLS2 and NLS3 are the major sequences involved in the efficient targeting of NOL7 to the nucleus. To determine the rate of nuclear import, a similar approach was used, this time measuring the relative nuclear fluorescence intensity over a four-hour time course. To ensure that this rate represented strictly nuclear import, the increase in fluorescence intensity was measured prior to the establishment of steady-state NOL7 levels. To determine the time frame for measurement, we transfected cells with GFP-tagged wild type NOL7 and measured the time posttransfection when fluorescent signal can first be detected until it accumulates to a steady-state level. It was determined that NOL7 protein is detectable 5 hours posttransfection, and its accumulation reaches steady-state levels approximately 10 hours after transfection (Additional file 1: Supplementary Movie 1). Therefore, the rate was calculated as the change in fluorescence intensity at 5, 6, 7, and 8 hours posttransfection (Figure 7B). Loss of even one NLS had a significant effect on the change in rate, regardless of identity. The single NLS mutants had a significantly higher rate of import than the double mutants (4.94 0.34 versus 3.05 0.40, p = 6.1910-8). The most dramatic decrease was observed with the N23(-) mutant, which was imported at a rate nearly 80% less than WT NOL7, with a p-value of 6.1310-17. This suggests that each NLS plays a unique Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 9 of 20 Figure 6 Basic residues within each of the NLSs are required for nuclear localization of full-length NOL7. (A) Schematic representing the different mutant constructs used to evaluate nuclear localization in the context of the full length protein. Results demonstrated in (B) are summarized in the column on the right, where No designates nucleolar localization, Np designates nucleoplasmic localization, and C designates cytoplasmic localization. (B) Localization of the GFP-tagged constructs in HeLa cells was confirmed by fluorescent microscopy and costaining of the nucleus with DAPI is shown in blue. role in the targeting of NOL7. Together with efficiency, this suggests that NLS2 and NLS3 in combination are critical for efficient and rapid targeting of NOL7 to the nucleus. NLS2 and NLS3 of NOL7 comprise domains that are required for nucleolar localization It has been previously demonstrated that NOL7 localizes to the nucleolus via colocalization with the nucleolar protein NPM [26]. In the analysis of the NLSs, it was noted that loss of NLS2 and NLS3 together abolished the nucleolar but not nuclear localization of NOL7 (Figure 6B). Interestingly, NLS23(-) also significantly decreased both the rate and efficiency of NOL7 nuclear transport (Figure 7A and 7B). To identify the possible NoLS(s) within these signals, systematic mutation of the basic residues within NLS2 or NLS3 was undertaken (Figure 8A). Restoration of any basic cluster in NLS2 or in NLS3 was sufficient to restore nucleolar localization, suggesting that these regions are capable of individually functioning as NoLSs (Figure 8B). Thus, NOL7 contains at least four separate NoLSs within its nuclear targeting sequences that are individually capable of directing nucleolar localization. NOL7 demonstrates rapid recovery but low mobility within the nucleolus Protein occupancy and complex assembly in subnuclear bodies has been shown to relate to function for a majority of proteins [68-70]. Therefore, the nucleolar occupancy of NOL7 was evaluated by FRAP. The occupancy was described by the recovery half life (t1/2) and mobile fraction (Mf) of GFP-fusion constructs. In order to define an upper and lower limit for nucleolar protein mobility, NOL7 was evaluated with the controls NCL, a freely diffusing nuclear/nucleolar shuttle with functions in both the Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 10 of 20 Figure 7 Each NLS contributes differently to the rate and efficiency of NOL7 nuclear localization. The steady-state efficiency and rate of import for NLS mutants was evaluated to determine their relative contribution to the subcellular localization of NOL7 in HeLa cells. (A) Twenty hours after transfection, mutants were imaged by immunofluorescence against the HA tags and costained with DAPI and WGA to delineate the nucleus and cytoplasm. Using ImageJ, the nuclear-to-total cell fluorescence ratio was calculated for twenty cells per construct. Error bars represent standard error. (B) Cells were transfected with the different NOL7 NLS constructs and imaged at 5, 6, 7, and 8 hours post-transfection. The nuclear accumulation was measured by a-HA immunofluorescence and the rates were calculated as the change in nuclear signal over time. Bars represent the average rate for ten cells and error bars are representative of the standard error. nucleus and nucleolus, and RPS5, a low-mobility resident nucleolar protein (Figure 9A). These proteins represent typical controls within the literature and allow for comparison to other dynamic studies [71]. The t1/2 of NOL7 was found to be most similar to a shuttling protein such as NCL, suggesting that NOL7 can freely exchange with the nucleoplasm (Figure 9B). Conversely, the immobile fraction (Mf) of NOL7 was found to be most similar to an immobile, complexed nucleolar protein like RPS5 (Figure 9B). This is consistent with previous reports describing the nucleolar occupation of a number of nucleolar proteins, including NPM, NCL, and RPS5 [71,72]. This suggests that a large pool of nucleolar NOL7 is functionally occupied in a nucleolar complex, while the free protein is able to shuttle rapidly between subnuclear compartments. Compared to literature reports, this data indicates that NOL7 is most similar to proteins with multiple nuclear and nucleolar roles like NPM, which is both a nucleolar shuttle and associates in functional nucleolar complexes, than either NCL or RPS5. Further, these data suggest that NOL7 shuttles between the nucleolus and nucleoplasm and may play a functional role in both compartments. NOL7 localization is dynamically regulated by changes in RNA composition The shuttling of NOL7 between the nucleus and nucleolus suggested that specific interactions within these compartments may regulate the nucleolar occupancy of Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 11 of 20 Figure 8 Basic residues within NLS2 and NLS3 are required for nucleolar localization of NOL7. Basic residues within each of the NLSs are required for nucleolar localization of full length NOL7. A) Schematic representation of the different mutant constructs used to evaluate nucleolar localization. Results demonstrated in (B) are summarized in the column on the right, where No designates nucleolar localization and Np designates nucleoplasmic localization. B) Localization of the constructs in HeLa cells was confirmed by GFP visualization. Costaining of the nucleus with DAPI is shown in blue. NOL7. Due to the highly basic nature of the protein, it was hypothesized that subnuclear localization of NOL7 may be due to interactions with nucleic acids. To investigate, various cell treatments were employed to change the abundance of different nucleic acid species (Figure 10). Cells overexpressing GFP-tagged NOL7 were treated with RNase, DNase, actinomycin D (ActD), or a-amanitin and visualized by fluorescence microscopy for changes in subcellular localization. RNase treatment resulted in nucleolar loss and nucleoplasmic accumulation of NOL7, while cells treated with DNase did not show any significant change. Culture of mammalian cells in low doses of ActD selectively inhibit rRNA synthesis while having no effect on tRNA, 5S rRNA, nuclear RNA and mRNA synthesis [73,74]. Similarly, treatment with low doses of a-amanitin inhibits RNAPII and subsequent mRNA synthesis without affecting the abundances of other RNA species. Loss of these specific RNA species has been shown to selectively deplete their RNA-binding protein counterparts from different cellular compartments, enabling visualization of binding activities that may participate in protein localization [75-77]. Upon treatment with ActD, NOL7 was found to translocate to the nucleoplasm. Upon treatment with Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 12 of 20 Figure 9 FRAP analysis of NOL7 nucleolar occupancy demonstrates rapid recovery but low mobility within the nucleolus. (A) The fluorescence recovery within the nucleolus was measured over time for HeLa cells transfected with GFP-tagged NOL7, the high mobility shuttle NCL, and the low mobility resident protein RPS5. Measurements represent thirteen different cells per protein. The curves were fit to the line curve F(t) = F(1-et). (B) The nucleolar occupancy was plotted as a function of recovery versus mobility. The mobile fraction was used as a measurement of free versus complexed protein within the nucleolus and calculated from the regression values in (A) using the formula Mf = (F-F0)/(Fi-F0). The half-time to maximal recovery was calculated using the formula t1/2 = ln(0.5)/ and used as a measurement of shuttling between the nucleolus and nucleoplasm. All error bars represent the standard error of the measurement. a-amanitin, no change in the nucleolar localization of NOL7 was observed. However, the nucleoplasmic localization of NOL7 previously observed was absent. This data suggests that targeting of NOL7 to both the nucleus and nucleolus results in multiple RNA-dependent interactions. Discussion Active nuclear transport involves complex interactions between the transport machinery and protein cargo, mediated in part through NLSs. Typically composed of discrete patterns of basic residues, these sequences are recognized by the transport machinery and can vary in their affinity, rate, and efficiency of localization, which in turn can influence the function and biologic relevance of the cargo protein in different physiologic settings. Here, we have shown that NOL7 is targeted to the nucleus via an energy- and nucleoporin-dependent mechanism. This transport is mediated by three evolutionarily conserved but distinct NLSs. In addition, each NLS was found to be independently capable of directing the nuclear localization of the cytoplasmic protein PK or full length NOL7. Each NLS individually and additively contributed to the rate and efficiency of NOL7 nuclear targeting, suggesting that each of the NLSs has differential effects in driving the localization kinetics, likely reflecting differences in the regulation of import. Taken together, these data indicate that NOL7 localization is tightly regulated and may contribute to functions in various cellular compartments. Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 13 of 20 Figure 10 NOL7 subnuclear localization is dynamically regulated by changes in RNA composition. 293T cells were stably transfected with NOL7-GFP and treated with RNase A (100 g/ml, 2 hours), DNase I (100 g/ml, 2 hours), actinomycin D (0.05 g/ml, 4 hours), or a-amanitin (50 g/ml, 4 hours) to specifically deplete individual nucleic acid species. Treatment with DNase (total DNA), RNase (total RNA), ActD (rRNA), or aamanitin (mRNA) was performed and localization of NOL7 was confirmed by fluorescent microscopy of the GFP tag. The transport of proteins and RNAs into the nucleus occurs through the NPC and is an important step in regulating the subcellular location of a number of different proteins, including transcription factors, signalling proteins, and various enzymes. Although alternative mechanisms exist, the classic nuclear import pathway appears to be the predominate method of transport into the nucleus. A recent survey of Saccharomyces cerevisiae screened over 5800 genomic sequences and found that 45% contained classic NLSs and nearly 60% of nuclear proteins contained monopartite or bipartite sequences [78]. This is likely true across species, as a number of studies have found that the nuclear transport machinery for essential proteins is highly conserved between Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 animals, yeast and plants [30-32]. This observation is certainly true for the three NLSs present in NOL7, where sequence alignment of the three NLSs demonstrated significant evolutionary conservation and aided in the identification of putative targeting sequences. It further suggested that targeting may play a significant role in the regulation and function of NOL7, as these sequences were highly conserved across species but demonstrated little similarity to other proteins or domains. NOL7s three functional NLSs are located in the N terminus, middle, and C terminus of the protein. While a single functional NLS is sufficient for most proteins, the presence of multiple functional NLSs is seen frequently among proteins whose function is critically determined by its localization. Proteins such as p53 [79], E2F1 [80], c-Abl [81], p14 ARF , HPV E6 [82], BRCA2 [83], most ribosomal proteins including RPS7 [84,85], b-myb [86], ATF2/c-jun heterodimer [87], PAK-1 [88] and others have been demonstrated to contain more than one NLS. Interestingly, many of these proteins are also implicated in cancer, and aberrant or mislocalized protein plays a significant role in the development and progression of the disease. As such, nuclear localization, and the rate and efficiency at which it occurs, has been shown to have many downstream functional consequences for proteins [89-92]. Terry, et al, have proposed a hierarchical regulation to classical nuclear transport via NLSs, with multiple mechanisms acting at the level of the cargo, receptors, and NPC [93]. The existence of multiple NLSs within a single protein may therefore provide a mechanism to exploit these different targeting controls for proteins whose nuclear localization is critical for function [93]. The first level of regulation involves the NPC, and the permeability, stability, and expression of the proteins that comprise this complex can affect the efficiency and targeting of cargo. The existence of multiple NLSs within NOL7 may therefore be used to achieve nuclear localization despite cellular conditions where NPC is less accessible. The next level of regulation involves the transport receptors. Here, differing accessibility, affinity, competition, and expression of the importins in various cell types and under different cellular conditions can affect transport [40,94-99]. In this case, the existence of multiple NLSs can increase likelihood of transporter interaction regardless of environment, coordinate for better efficiency and rate of localization, or outcompete other NLS-bearing proteins for these receptors. Indeed, combined loss of NLS2 and NLS3 significantly impact both the rate and efficiency of NOL7 localization, and the presence of more than one NLS results in a statistically significant increase in NOL7 nuclear accumulation (Figure 7). Finally, at the Page 14 of 20 level of the cargo, modifications and interactions of the cargo protein itself regulates its own localization. Inter- and intramolecular interactions can provide or preclude access to NLSs, and modifications within NLSs can also affect transport, either inhibiting or promoting import to the nucleus [79,100]. The differential rate and efficiency of localization observed among NOL7 mutants, particularly in the N23(-) mutant, suggests each NLS may participate in different levels of this regulation. In addition, many NLSs have also been shown to harbor subnuclear targeting sequences such as NoLSs. NoLSs typically represent interaction motifs between nucleolar constituents, making nucleolar localization a dynamic, multidirectional process compared to nuclear targeting [44-50,101,102]. Our results have shown that NLS2 and NLS3 include four NoLSs. These sequences are composed of basic clusters and each is capable of individually driving nucleolar localization of NOL7. Whether these regions represent unique binding domains or are functionally redundant to ensure efficient interaction with nucleolar cofactors is unknown at this time. Investigation of the nucleolar occupancy of proteins under various cellular conditions has demonstrated that the kinetics are often highly similar for functionally related proteins [75]. In particular, FRAP analysis of the recovery and mobility of proteins within this compartment has been shown to reflect their functional roles. The nucleolar mobility is typically viewed as a reflection of the stability of the interactions and size of the interaction complex within that compartment, while the recovery reflects the shuttling characteristics of a protein between the nucleoplasm and nucleolus. While many ribosomal proteins are highly immobile within the nucleolus, proteins such as NCL, UBF, and NPM have higher mobility and rapid recovery, due to their multiple functional roles in the nucleus and nucleolus [60,62,71,72]. FRAP analysis of the nucleolar occupancy of NOL7 demonstrates that a large fraction of nucleolar NOL7 is involved in a relatively stable complex, as evidenced by its small Mf. Interestingly, free NOL7 protein rapidly shuttles between compartments. These dynamics, with low Mf and high t1/2, have been demonstrated in the literature to be unique to proteins that functionally interact with ribonucleoproteins (RNPs) in both the nucleus and nucleolus such as NPM [60-62,68-72]. Together, this suggests that NOL7 may interact in RNP complexes in both compartments. Further support for the potential nuclear and nucleolar interactions of NOL7 can be observed by the changes in localization for NOL7 upon specific depletion of nucleic acid species. The pattern of NOL7 expression is significantly altered by loss of RNA but not loss of DNA, suggesting that NOL7 is an RNA-associated protein, either Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 directly or through RNP complexes. Further, changes in rRNA and mRNA abundance affected the abundance of NOL7 in the nucleolus and nucleoplasm, respectively, suggesting that NOL7 may be participating in distinct functional complexes within each compartment. Whether this is a direct effect of rRNA and mRNA interaction, or an indirect consequence of changes in the transcriptome of the cell remains to be investigated. However, together these observations indicate that the RNA abundance within the cell can influence the localization of NOL7 protein, and the dynamics of this localization is similar to the kinetics of proteins that play functional roles in nuclear and nucleolar RNP complexes. While it is unknown what, if any, function NOL7 may have in either compartment, it suggests that its localization is actively regulated and this differential targeting may influence its role in cancer development and progression. Localization and function within multiple cellular compartments has previously been observed for many proteins. In addition, regulation of protein function through localization mechanisms is known to be employed in multiple cancer signaling pathways, including the Wnt, TGFb, and Hh pathways. Oncogenes and tumor suppressors such as Rb, c-Myc, p53, VHL, and p14Arf have multiple, different functions depending on their localization or sequestration [103-117]. Our evidence suggests that like many of these oncogenes and tumor suppressors, NOL7 may have be regulated through its subcellular localization, and its targeting may be critically linked to its tumor suppressive activity. Conclusions In summary, we have found that NOL7 requires cytosolic proteins for active transport into the nucleus, consistent with a classical import mechanism. We have identified three functional NLSs within NOL7, each of which is independently capable of directing the nuclear localization of the cytoplasmic protein PK or full length NOL7 and contribute to different degrees to the rate and efficiency of NOL7 nuclear import. Further, these sequences harbor at least four NoLSs that are independently capable of mediating nucleolar localization. The nucleolar occupancy of NOL7 is balanced by its rapid recovery and low mobility, similar to other proteins that play multiple functional roles in both the nucleus and nucleolus. Further, the nucleolar localization of NOL7 is dependent upon the presence of rRNA, while the nucleoplasmic localization of NOL7 is mediated by the abundance of mRNA. This work provides the basis for further investigation into the levels, activity, and mechanism of regulation for NOL7 and elucidation of its role in tumor growth suppression. Page 15 of 20 Methods Deletion Mutant Constructs Constructs were cloned as described. Template and primer sequences are listed in Additional file 2: Supplementary Table 1. NOL7 Deletion Constructs Each deletion construct was cloned by PCR and inserted into the pcDNA3.1/Hygro(+) vector (Invitrogen, Carlsbad CA). For the mutants, residues were mutated using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla CA). NLS-PK Fusion Proteins Myc-tagged chicken muscle PK expression DNA was obtained from Gideon Dreyfuss (University of Pennsylvania, Howard Hughes Medical Institute) [118]. It was cloned in frame with individual NLSs to create fusion constructs. GFP-fusion Constructs Fusion constructs were generated using the GFP Fusion TOPO TA Cloning kit (Invitrogen, Carlsbad CA). Briefly, the full-length NOL7 DNA fragments were TOPO cloned into the plasmid vector pcDNA3.1/NTGFP-TOPO, and the cloning reaction was transformed into chemically competent cells provided in the kit. The plasmids were purified with Qiagen Plasmid Mini kit (Qiagen, Valencia CA), and sequenced for verification of insert orientation. Mutation of individual residues within the NLSs were constructed by using Quikchange XL site-directed mutagenesis kit (Stratagene, La Jolla CA). NOL7-GFP Purification Construct For nuclear import assays, NOL7-GFP was cloned with tandem C-terminal GFP-V5-His6 tags using the Gatweway cloning system from Invitrogen (Carlsbad, CA). All TOPO and LR cloning reactions were performed as described by the manufacturer. First, wild-type NOL7 was PCR amplified and TOPO cloned into pENTR-SDD-TOPO. The pENTR-NOL7 construct was transferred to the pcDNA-DEST47 vector, resulting in a C-terminal GFP tag. The NOL7-GFP fusion was PCR amplified and TOPO cloned into the pENTR-SD-D-TOPO vector and this time transferred to the pcDNA-DEST40 vector, thereby expressing NOL7 in frame with a tandem Cterminal GFP-V5-His6 tag. Tissue Culture HeLa cells were grown in minimum essential medium supplemented with 10% fetal bovine serum (FBS), 100 g/ml penicillin and streptomycin. 293T cells were grown in DMEM supplemented with 10% FBS, 100 g/ ml penicillin and streptomycin. Transfections for HeLa, and 293T cells were done using Lipofectin following the manufacturers directions (Invitrogen, Carlsbad CA) in 75 mm2 dishes when the cells were approximately 80% Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 confluent. Five hours after addition of the DNA precipitate, cells were washed and refed with minimum essential medium or Dubeccos Modified Essential media plus 10% FBS. For stable cell lines, cells were selected in 400 g/ml G418 (Invitrogen, Carlsbad CA) for three weeks. For transient expression experiments, cell extracts were prepared 20-36 hours after transfection. Immunofluorescence For immunofluorescence staining, cells were plated on 4-well chamber slides and were transfected using Lipofectin for HeLa and 293T cells according to the manufacturers instructions. Expression of all constructs was validated by western blot. Cells were fixed and stained as previously described [119] thirty-six hours post-transfection unless otherwise stated. Immunostaining was performed using the following primary antibodies: Rabbit a-HA (Invitrogen, Carlsbad CA), 1:4000; Mouse a-cMyc (Ab-1) (Calbiochem, Gibbstown NJ), 1:500. Secondary antibodies were fluorescein isothiocyanate (FITC) AffiniPure F(ab) 2 Fragment Goat Anti-Rabbit IgG (H +L) (Jackson ImmunoResearch Labs, West Grove PA), 1:500; Cy3 AffiniPure F(ab)2 Fragment Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, West Grove PA), 1:500. Cells were mounted in DAPI-containing media (Vector Labs, Burlingame CA) according to the manufacturers instructions. WGA staining (Invitrogen, Carlsbad CA) was performed at a concentration of 5.0 g/ml according to the manufacturers instructions. Protein Purification 293T cells were transfected with the NOL7-GFP purification construct and positive clones were selected and maintained as described. For purification, approximately 1 10 8 cells were collected by trypsinization and washed twice with ice cold PBS. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X100; Roche Complete EDTA-free protease inhibitor tablet). Cell pellets were sonicated 6 30 s at 30% power. Lysates were then cleared by centrifugation and the supernatant was collected and filtered through a 0.45 m filter. Size-exclusion chromatography was performed using a 0.7 cm 50 cm Econo-column (BioRad, Hercules CA) that was packed with 5-100 kDA polyacrylamide beads (Bio-gel P-100, 45-90 M, BioRad, Hercules CA) according to manufacturers instructions. Fractions of approximately 300 l were collected and tested for the presence of NOL7 by SDS-PAGE followed by silver staining and western blot using mouse a-V5 monoclonal antibody (Invitrogen, Carlsbad CA). Positive fractions were further purified by affinity chromatography against the His6 tag of NOL7 using the ProPur IMAC Kit (Nunc, Rochester NY) under native Page 16 of 20 conditions with 30 mM imidazole washes. The column was washed five times and five elution fractions were collected. NOL7-containing fractions were verified by silver stain and western blot against the V5 tag of NOL7. Positive fractions were concentrated and dialyzed against transport buffer (20 mM Hepes-KOH, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 1 mM EGTA, Roche complete mini protease inhibitor) for use in the import assay. Preparation of Cytosol Fractions Exponentially growing cultures of HeLa cells were collected by low speed centrifugation and washed twice with cold PBS, pH 7.4, by resuspension and centrifugation. The cells were then washed with 10 mM Hepes, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT and pelleted. The cell pellet was gently resuspended in 1.5 volumes of lysis buffer (5 mM Hepes, pH 7.3, 10 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, 20 M cytochalasin B, 1 mM PMSF, and 1 g/ml each aprotinin, leupeptin, and pepstatin) and swelled for 10 min on ice. The cells were lysed with a homogenizer. The resulting homogenates were centrifuged at 1,500g for 15 min to remove nuclei and cell debris. The supernatants were then sequentially centrifuged at 15,000g for 20 min and 100,000g for 30 min. The final supernatants were dialyzed against transport buffer (20 mM HEPES, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT, and 1 g/ml each aprotinin, leupeptin, and pepstatin) and frozen in aliquots in liquid nitrogen before storage at -80C. Cell Permeabilization and In Vitro Transport Assay Import assays was performed essentially as previously described [54]. Cells plated on 4-well chamber slides were rinsed in cold transport buffer (20 mM Hepes, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM DTT, 1.0 mM EGTA, and 1 g/ml each aprotinin, leupeptin, and pepstain). Wells were immersed in ice cold transport buffer containing 40 g/ml digitonin (Calbiochem, Gibbstown NJ). The cells were allowed to permeabilize for 5 min, after which the digitonin-containing buffer was removed and replaced with cold transport buffer. For each assay, 150 l of transport buffer was supplemented with 10 g/ml NOL7-GFP and incubated for 30 minutes at either 37C or 4C. Where indicated, assays were supplemented with 1 mM ATP and 15 mg/ml cytosol. For heat-inactivated cytosol, extracts were boiled at 95C for 5 min, chilled, and then added to the import assay. For WGA treatment, cells were pre-incubated with 50 ug/ml WGA for 15 minutes at 20C, washed, and then the import assay was performed as described above. After the 30 min incubation, Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 all slides were washed, fixed with 4.0% paraformaldehyde and analyzed directly by fluorescence microscopy. Protein analysis, Domain prediction, and sequence alignment Protein analysis of NOL7 was carried out using a variety of prediction programs on the following accession sequences: Homo sapiens, NP_057251.2; Pan troglodytes, XP_518245.2; Macaca mulatta, XP_001092572.1; Bos taurus, NP_001029556.1; Canis familiaris, XP_535892.2; Rattus norvegicus, XP_573999.2; Mus musculus, NP_076043.2; Gallus gallus, XP_418926.1; Tetraodon nigroviridis, CAF97792.1; Danio rerio, XP_687281.1; Saccharomyces cerevisiae, NP_014721.1. Alignment of sequences was doing using MegAlign software under the ClustalW parameters. Page 17 of 20 Transport Rate Experiments HeLa cells were fixed and stained with rabbit a-HA primary (Invitrogen, Carlsbad CA, 1:4000 dilution) and FITC AffiniPure F(ab)2 Fragment Goat Anti-Rabbit IgG (H+L) secondary (Jackson ImmunoResearch Labs, West Grove PA, 1:500 dilution) as described 5, 6, 7, and 8 hours after transfection. Immunofluorescent images were captured by using Zeiss Axiovert 200 M microscope system. Image analysis was performed using Image J to quantify the nuclear staining intensity per unit of area. Ten high power fields were selected for analysis of each construct. The rate of import was calculated as the slope of the fluorescent intensity versus time and the statistical significance of this data was evaluated using Students t-test. Drug treatment and fluorescence microscopy FRAP Photobleaching, Imaging, and Quantitation Approximately forty-eight hours after transfection, HeLa cells were maintained in MEM supplemented with 30 mM Hepes, pH 7.1, to stabilize the pH of the medium during imaging. FRAP was performed on a DM4000 microscope (Leica Microsystems, Wetzlar Germany) equipped with a MicroPoint Laser System (Photonic Instruments, St. Charles, IL), a Roper Coolsnap HQ camera (Princeton Instruments, Trenton NJ), and a Leica 63X HCX PL APO L U-V-I aqueous immersion objective (Molecular Devices, Sunnyvale CA). Fluorescence intensity was measured using Metamorph imaging software (Universal Imaging Corp, West Chester PA). The average intensities of the areas of interest, including before, immediately after, and a series of time points after bleaching, were measured under the same condition for each data set. Data was analyzed using SigmaPlot software and fit to the curve F(t) = F(1-et). From the regression values, the half-maximal recovery [t1/2 = ln (0.5)/] and mobile fraction [M f = (F -F 0 )/(F i -F 0 )] were calculated for each replicate and statistical significance was determined using Students t-test. Transport Efficiency Experiments HeLa cells were fixed and stained with rabbit a-HA primary (Invitrogen, Carlsbad CA, 1:4000 dilution) and FITC AffiniPure F(ab)2 Fragment Goat Anti-Rabbit IgG (H+L) secondary (Jackson ImmunoResearch Labs, West Grove PA, 1:500 dilution) as described twenty hours after transfection. Immunofluorescent images were captured by using Zeiss Axiovert 200 M microscope system. Image analysis was performed using Image J to quantify per unit area staining intensity in the total cell and nucleus. Twenty high power fields were selected for analysis of each stain. The efficiency was calculated as the ratio of nuclear to total intensity and Statistics were evaluated using Students t-test. HeLa cells were stably transfected with wild-type NOL7GFP and plated on 2-well chamber slides. When cells were approximately 70% confluent, media was replaced with serum-free DMEM containing 0.05 g/ml actinomycin D (Sigma-Aldrich, St. Louis MO) or 50 g/ml aamanitin (Sigma-Aldrich, St. Louis MO) and incubated for 4 hours at 37C in 5% CO 2. Cells were then fixed with 4% paraformaldehyde, washed with PBS, and mounted with DAPI-containing media (Vector Labs, Burlingame CA). For nuclease treatment, cells were first washed with PBS and permeabilized with ice-cold methanol for 10 min and then incubated with 100 g/ ml RNase A (Sigma-Aldrich, St. Louis MO) or 100 g/ ml DNase I (Sigma-Aldrich, St. Louis MO) at 37C for 2 hours. Cells were then fixed and mounted in the same manner. All cells were imaged on a Zeiss Axioplan microscope. Additional material Additional file 1: Supplementary Movie 1 - Detection of NOL7-GFP Posttransfection. HeLa cells were transfected with WT NOL7 in frame with a GFP fusion tag and imaged every fifteen minutes from the time fluorescent signal can first be detected, approximately five hours after transfection, until it reaches steady-state intensity, approximately nine hours after transfection. Time zero corresponds to five hours posttransfection. Additional file 2: Supplementary Table 1 - Primers used to clone the constructs used in this study. Each construct is listed, along with the forward and reverse PCR primers and template for cloning PCR reaction able legend text Abbreviations NLS(s): nuclear localization signal(s); CC: cervical cancer; HA: hemagglutinin; PK: pyruvate kinase; CHX: cycloheximide; VEGF: vascular endothelial growth factor; TSP-1: thrombospondin-1; NPM: nucleophosmin; NCL: nucleolin; RPS5: ribosomal protein 5; ribonucleoprotein: RNP; NPC: nuclear pore comples; NES: nuclear export signal; cNLS: classical nuclear localization signal; NoLS: nucleolar localization signal; WGA: wheat germ agglutinin; FBS: fetal bovine Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 serum; FITC: fluorescein isothiocyanate; FRAP: (fluorescence recovery after photobleaching). Acknowledgements We would like to thank Dr. Jerrold Turner for his assistance with the FRAP experiments. We would also like to acknowledge Dr. Vytas Bindokas for his advice regarding the nuclear transport rate and efficiency experiments. This work was supported in part by Illinois Department of Public Health Penny Severns Cancer Research Fund and the National Institutes for Health grant DE00470 (MWL). Authors contributions GZ performed plasmid construction, localization immunofluorescence, import assays, transport rate and efficiency experiments, and FRAP. CLD performed plasmid construction, sequence analysis, protein purification, drug treatment fluorescent microscopy, statistical analysis, and participated in the design of the study and manuscript preparation. MWL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Received: 22 April 2010 Accepted: 27 September 2010 Published: 27 September 2010 References 1. Lung ML, Choi CV, Kong H, Yuen PW, Kwong D, Sham J, Wei WI: Microsatellite allelotyping of chinese nasopharyngeal carcinomas. Anticancer Res 2001, 21(4B):3081-3084. 2. Liao SK, Perng YP, Shen YC, Chung PJ, Chang YS, Wang CH: Chromosomal abnormalities of a new nasopharyngeal carcinoma cell line (NPC-BM1) derived from a bone marrow metastatic lesion. Cancer Genet Cytogenet 1998, 103(1):52-58. 3. Mutirangura A, Tanunyutthawongese C, Pornthanakasem W, Kerekhanjanarong V, Sriuranpong V, Yenrudi S, Supiyaphun P, Voravud N: Genomic alterations in nasopharyngeal carcinoma: loss of heterozygosity and Epstein-Barr virus infection. Br J Cancer 1997, 76(6):770-776. 4. Lim G, Karaskova J, Vukovic B, Bayani J, Beheshti B, Bernardini M, Squire JA, Zielenska M: Combined spectral karyotyping, multicolor banding, and microarray comparative genomic hybridization analysis provides a detailed characterization of complex structural chromosomal rearrangements associated with gene amplification in the osteosarcoma cell line MG-63. Cancer Genet Cytogenet 2004, 153(2):158-164. 5. Takeshita A, Naito K, Shinjo K, Sahara N, Matsui H, Ohnishi K, Beppu H, Ohtsubo K, Horii T, Maekawa M, et al: Deletion 6p23 and add(11)(p15) leading to NUP98 translocation in a case of therapy-related atypical chronic myelocytic leukemia transforming to acute myelocytic leukemia. Cancer Genet Cytogenet 2004, 152(1):56-60. 6. Amare Kadam PS, Ghule P, Jose J, Bamne M, Kurkure P, Banavali S, Sarin R, Advani S: Constitutional genomic instability, chromosome aberrations in tumor cells and retinoblastoma. Cancer Genet Cytogenet 2004, 150(1):33-43. 7. Fan YS, Rizkalla K: Comprehensive cytogenetic analysis including multicolor spectral karyotyping and interphase fluorescence in situ hybridization in lymphoma diagnosis. a summary of 154 cases. Cancer Genet Cytogenet 2003, 143(1):73-79. 8. Batanian JR, Cavalli LR, Aldosari NM, Ma E, Sotelo-Avila C, Ramos MB, Rone JD, Thorpe CM, Haddad BR: Evaluation of paediatric osteosarcomas by classic cytogenetic and CGH analyses. Mol Pathol 2002, 55(6):389-393. 9. Starostik P, Patzner J, Greiner A, Schwarz S, Kalla J, Ott G, MullerHermelink HK: Gastric marginal zone B-cell lymphomas of MALT type develop along 2 distinct pathogenetic pathways. Blood 2002, 99(1):3-9. 10. Giagounidis AA, Hildebrandt B, Heinsch M, Germing U, Aivado M, Aul C: Acute basophilic leukemia. Eur J Haematol 2001, 67(2):72-76. 11. Achuthan R, Bell SM, Roberts P, Leek JP, Horgan K, Markham AF, MacLennan KA, Speirs V: Genetic events during the transformation of a tamoxifen-sensitive human breast cancer cell line into a drug-resistant clone. Cancer Genet Cytogenet 2001, 130(2):166-172. 12. Shao JY, Wang HY, Huang XM, Feng QS, Huang P, Feng BJ, Huang LX, Yu XJ, Li JT, Hu LF, et al: Genome-wide allelotype analysis of sporadic primary nasopharyngeal carcinoma from southern China. Int J Oncol 2000, 17(6):1267-1275. Page 18 of 20 13. Chen Z, Issa B, Brothman LJ, Hendricksen M, Button D, Brothman AR: Nonrandom rearrangements of 6p in malignant hematological disorders. Cancer Genet Cytogenet 2000, 121(1):22-25. 14. Nakase K, Wakita Y, Minamikawa K, Yamaguchi T, Shiku H: Acute promyelocytic leukemia with del(6)(p23). Leuk Res 2000, 24(1):79-81. 15. Nagai H, Kinoshita T, Suzuki H, Hatano S, Murate T, Saito H: Identification and mapping of novel tumor suppressor loci on 6p in diffuse large Bcell non-Hodgkins lymphoma. Genes Chromosomes Cancer 1999, 25(3):277-283. 16. Nemani M, Bellanne-Chantelot C, Cohen D, Cann HM: Detection of triplet repeat sequences in yeast artificial chromosomes using oligonucleotide probes: application to the SCA1 region in 6p23. Cytogenet Cell Genet 1996, 72(1):5-8. 17. Jadayel D, Calabrese G, Min T, van Rhee F, Swansbury GJ, Dyer MJ, Maitland J, Palka G, Catovsky D: Molecular cytogenetics of chronic myeloid leukemia with atypical t(6;9) (p23;q34) translocation. Leukemia 1995, 9(6):981-987. 18. Hoyle CF, Sherrington P, Hayhoe FG: Translocation (3;6)(q21;p21) in acute myeloid leukemia with abnormal thrombopoiesis and basophilia. Cancer Genet Cytogenet 1988, 30(2):261-267. 19. Fleischman EW, Prigogina EL, Iljinskaja GW, Konstantinova LN, Puchkova GP, Volkova MA, Frenkel MA, Balakirev SA: Chromosomal rearrangements with a common breakpoint at 6p23 in five cases of myeloid leukemia. Hum Genet 1983, 64(3):254-256. 20. Huettner PC, Gerhard DS, Li L, Gersell DJ, Dunnigan K, Kamarasova T, Rader JS: Loss of heterozygosity in clinical stage IB cervical carcinoma: relationship with clinical and histopathologic features. Hum Pathol 1998, 29(4):364-370. 21. Kersemaekers AM, Kenter GG, Hermans J, Fleuren GJ, van de Vijver MJ: Allelic loss and prognosis in carcinoma of the uterine cervix. Int J Cancer 1998, 79(4):411-417. 22. Mitra AB, Murty VV, Li RG, Pratap M, Luthra UK, Chaganti RS: Allelotype analysis of cervical carcinoma. Cancer Res 1994, 54(16):4481-4487. 23. Mullokandov MR, Kholodilov NG, Atkin NB, Burk RD, Johnson AB, Klinger HP: Genomic alterations in cervical carcinoma: losses of chromosome heterozygosity and human papilloma virus tumor status. Cancer Res 1996, 56(1):197-205. 24. Rader JS, Gerhard DS, OSullivan MJ, Li Y, Li L, Liapis H, Huettner PC: Cervical intraepithelial neoplasia III shows frequent allelic loss in 3p and 6p. Genes Chromosomes Cancer 1998, 22(1):57-65. 25. Rader JS, Li Y, Huettner PC, Xu Z, Gerhard DS: Cervical cancer suppressor gene is within 1 cM on 6p23. Genes Chromosomes Cancer 2000, 27(4):373-379. 26. Hasina R, Pontier AL, Fekete MJ, Martin LE, Qi XM, Brigaudeau C, Pramanik R, Cline EI, Coignet LJ, Lingen MW: NOL7 is a nucleolar candidate tumor suppressor gene in cervical cancer that modulates the angiogenic phenotype. Oncogene 2006, 25(4):588-598. 27. Fried H, Kutay U: Nucleocytoplasmic transport: taking an inventory. Cell Mol Life Sci 2003, 60(8):1659-1688. 28. Mosammaparast N, Pemberton LF: Karyopherins: from nuclear-transport mediators to nuclear-function regulators. Trends Cell Biol 2004, 14(10):547-556. 29. Weis K: Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 2003, 112(4):441-451. 30. Breeuwer M, Goldfarb DS: Facilitated nuclear transport of histone H1 and other small nucleophilic proteins. Cell 1990, 60(6):999-1008. 31. Wagner P, Hall MN: Nuclear protein transport is functionally conserved between yeast and higher eukaryotes. FEBS Lett 1993, 321(2-3):261-266. 32. Dingwall C, Laskey RA: Nuclear targeting sequencesa consensus? Trends Biochem Sci 1991, 16(12):478-481. 33. Savory JG, Hsu B, Laquian IR, Giffin W, Reich T, Hache RJ, Lefebvre YA: Discrimination between NL1- and NL2-mediated nuclear localization of the glucocorticoid receptor. Mol Cell Biol 1999, 19(2):1025-1037. 34. Turlure F, Maertens G, Rahman S, Cherepanov P, Engelman A: A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin in vivo. Nucleic Acids Res 2006, 34(5):1653-1675. 35. Wang AH, Yang XJ: Histone deacetylase 4 possesses intrinsic nuclear import and export signals. Mol Cell Biol 2001, 21(17):5992-6005. Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 36. Pokorska A, Drevet C, Scazzocchio C: The analysis of the transcriptional activator PrnA reveals a tripartite nuclear localisation sequence. J Mol Biol 2000, 298(4):585-596. 37. Picard D, Kumar V, Chambon P, Yamamoto KR: Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell Regul 1990, 1(3):291-299. 38. El Alami M, Feller A, Pierard A, Dubois E: Characterisation of a tripartite nuclear localisation sequence in the regulatory protein Lys14 of Saccharomyces cerevisiae. Curr Genet 2000, 38(2):78-86. 39. Luo M, Pang CW, Gerken AE, Brock TG: Multiple nuclear localization sequences allow modulation of 5-lipoxygenase nuclear import. Traffic 2004, 5(11):847-854. 40. Hall MN, Hereford L, Herskowitz I: Targeting of E. coli beta-galactosidase to the nucleus in yeast. Cell 1984, 36(4):1057-1065. 41. Nigg EA: Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 1997, 386(6627):779-787. 42. Lee SJ, Matsuura Y, Liu SM, Stewart M: Structural basis for nuclear import complex dissociation by RanGTP. Nature 2005, 435(7042):693-696. 43. Riddick G, Macara IG: A systems analysis of importin-{alpha}-{beta} mediated nuclear protein import. J Cell Biol 2005, 168(7):1027-1038. 44. DAgostino DM, Ciminale V, Pavlakis GN, Chieco-Bianchi L: Intracellular trafficking of the human immunodeficiency virus type 1 Rev protein: involvement of continued rRNA synthesis in nuclear retention. AIDS Res Hum Retroviruses 1995, 11(9):1063-1071. 45. Fujiwara T, Suzuki S, Kanno M, Sugiyama H, Takahashi H, Tanaka J: Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25. Exp Cell Res 2006, 312(10):1703-1712. 46. Heine MA, Rankin ML, DiMario PJ: The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization. Mol Biol Cell 1993, 4(11):1189-1204. 47. Horke S, Reumann K, Schweizer M, Will H, Heise T: Nuclear trafficking of La protein depends on a newly identified nucleolar localization signal and the ability to bind RNA. J Biol Chem 2004, 279(25):26563-26570. 48. Michael WM, Dreyfuss G: Distinct domains in ribosomal protein L5 mediate 5 S rRNA binding and nucleolar localization. J Biol Chem 1996, 271(19):11571-11574. 49. Maeda Y, Hisatake K, Kondo T, Hanada K, Song CZ, Nishimura T, Muramatsu M: Mouse rRNA gene transcription factor mUBF requires both HMG-box1 and an acidic tail for nucleolar accumulation: molecular analysis of the nucleolar targeting mechanism. Embo J 1992, 11(10):3695-3704. 50. Spanopoulou E, Cortes P, Shih C, Huang CM, Silver DP, Svec P, Baltimore D: Localization, interaction, and RNA binding properties of the V(D)J recombination-activating proteins RAG1 and RAG2. Immunity 1995, 3(6):715-726. 51. Misteli T: Going in GTP cycles in the nucleolus. J Cell Biol 2005, 168(2):177-178. 52. Hernandez-Verdun D: Nucleolus: from structure to dynamics. Histochem Cell Biol 2006, 125(1-2):127-137. 53. Negi SS, Olson MO: Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23. J Cell Sci 2006, 119(Pt 17):3676-3685. 54. Adam SA, Marr RS, Gerace L: Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol 1990, 111(3):807-816. 55. Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, Nakai K: WoLF PSORT: protein localization predictor. Nucleic Acids Res 2007, , 35 Web Server: W585-587. 56. Emanuelsson O, Nielsen H, Brunak S, von Heijne G: Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 2000, 300(4):1005-1016. 57. Brendel V, Bucher P, Nourbakhsh IR, Blaisdell BE, Karlin S: Methods and algorithms for statistical analysis of protein sequences. Proc Natl Acad Sci USA 1992, 89(6):2002-2006. 58. la Cour T, Kiemer L, Molgaard A, Gupta R, Skriver K, Brunak S: Analysis and prediction of leucine-rich nuclear export signals. Protein Eng Des Sel 2004, 17(6):527-536. 59. Burgess A, Buck M, Krauer K, Sculley T: Nuclear localization of the EpsteinBarr virus EBNA3B protein. J Gen Virol 2006, 87(Pt 4):789-793. Page 19 of 20 60. Dundr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum LI, Phair RD, Misteli T: A kinetic framework for a mammalian RNA polymerase in vivo. Science 2002, 298(5598):1623-1626. 61. Phair RD, Misteli T: Kinetic modelling approaches to in vivo imaging. Nat Rev Mol Cell Biol 2001, 2(12):898-907. 62. Phair RD, Misteli T: High mobility of proteins in the mammalian cell nucleus. Nature 2000, 404(6778):604-609. 63. Noursadeghi M, Tsang J, Haustein T, Miller RF, Chain BM, Katz DR: Quantitative imaging assay for NF-kappaB nuclear translocation in primary human macrophages. J Immunol Methods 2008, 329(1-2):194-200. 64. Seibel NM, Eljouni J, Nalaskowski MM, Hampe W: Nuclear localization of enhanced green fluorescent protein homomultimers. Anal Biochem 2007, 368(1):95-99. 65. Timney BL, Tetenbaum-Novatt J, Agate DS, Williams R, Zhang W, Chait BT, Rout MP: Simple kinetic relationships and nonspecific competition govern nuclear import rates in vivo. J Cell Biol 2006, 175(4):579-593. 66. Efthymiadis A, Shao H, Hubner S, Jans DA: Kinetic characterization of the human retinoblastoma protein bipartite nuclear localization sequence (NLS) in vivo and in vitro. A comparison with the SV40 large T-antigen NLS. J Biol Chem 1997, 272(35):22134-22139. 67. Hu W, Jans DA: Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import. Differential role of NTF2. J Biol Chem 1999, 274(22):15820-15827. 68. Hendzel MJ, Kruhlak MJ, MacLean NA, Boisvert F, Lever MA, BazettJones DP: Compartmentalization of regulatory proteins in the cell nucleus. J Steroid Biochem Mol Biol 2001, 76(1-5):9-21. 69. Houtsmuller AB, Vermeulen W: Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching. Histochem Cell Biol 2001, 115(1):13-21. 70. Lewis JD, Tollervey D: Like attracts like: getting RNA processing together in the nucleus. Science 2000, 288(5470):1385-1389. 71. Chen D, Huang S: Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells. J Cell Biol 2001, 153(1):169-176. 72. Dundr M, Misteli T, Olson MO: The dynamics of postmitotic reassembly of the nucleolus. J Cell Biol 2000, 150(3):433-446. 73. Perry RP, Kelley DE: Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species. J Cell Physiol 1970, 76(2):127-139. 74. Hill RN, McConkey EH: Coordination of ribosomal RNA synthesis in vertebrate cells. J Cell Physiol 1972, 79(1):15-26. 75. Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, Mann M: Nucleolar proteome dynamics. Nature 2005, 433(7021):77-83. 76. Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, Mann M, Lamond AI: Directed proteomic analysis of the human nucleolus. Curr Biol 2002, 12(1):1-11. 77. Scherl A, Coute Y, Deon C, Calle A, Kindbeiter K, Sanchez JC, Greco A, Hochstrasser D, Diaz JJ: Functional proteomic analysis of human nucleolus. Mol Biol Cell 2002, 13(11):4100-4109. 78. Lange A, Mills RE, Lange CJ, Stewart M, Devine SE, Corbett AH: Classical nuclear localization signals: definition, function, and interaction with importin alpha. J Biol Chem 2007, 282(8):5101-5105. 79. Liang SH, Clarke MF: Regulation of p53 localization. Eur J Biochem 2001, 268(10):2779-2783. 80. Ivanova IA, Vespa A, Dagnino L: A novel mechanism of E2F1 regulation via nucleocytoplasmic shuttling: determinants of nuclear import and export. Cell Cycle 2007, 6(17):2186-2195. 81. Wen ST, Jackson PK, Van Etten RA: The cytostatic function of c-Abl is controlled by multiple nuclear localization signals and requires the p53 and Rb tumor suppressor gene products. Embo J 1996, 15(7):1583-1595. 82. Tao M, Kruhlak M, Xia S, Androphy E, Zheng ZM: Signals that dictate nuclear localization of human papillomavirus type 16 oncoprotein E6 in living cells. J Virol 2003, 77(24):13232-13247. 83. Yano K, Morotomi K, Saito H, Kato M, Matsuo F, Miki Y: Nuclear localization signals of the BRCA2 protein. Biochem Biophys Res Commun 2000, 270(1):171-175. 84. Wool IG, Chan YL, Gluck A: Structure and evolution of mammalian ribosomal proteins. Biochem Cell Biol 1995, 73(11-12):933-947. 85. Ko JR, Wu JY, Kirby R, Li IF, Lin A: Mapping the essential structures of human ribosomal protein L7 for nuclear entry, ribosome assembly and function. FEBS Lett 2006, 580(16):3804-3810. Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 86. Takemoto Y, Tashiro S, Handa H, Ishii S: Multiple nuclear localization signals of the B-myb gene product. FEBS Lett 1994, 350(1):55-60. 87. Liu H, Deng X, Shyu YJ, Li JJ, Taparowsky EJ, Hu CD: Mutual regulation of c-Jun and ATF2 by transcriptional activation and subcellular localization. Embo J 2006, 25(5):1058-1069. 88. Singh RR, Song C, Yang Z, Kumar R: Nuclear localization and chromatin targets of p21-activated kinase 1. J Biol Chem 2005, 280(18):18130-18137. 89. OKeefe K, Li H, Zhang Y: Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination. Mol Cell Biol 2003, 23(18):6396-6405. 90. Wang SC, Hung MC: Cytoplasmic/nuclear shuttling and tumor progression. Ann N Y Acad Sci 2005, 1059:11-15. 91. Meyer T, Vinkemeier U: Nucleocytoplasmic shuttling of STAT transcription factors. Eur J Biochem 2004, 271(23-24):4606-4612. 92. Fabbro M, Henderson BR: Regulation of tumor suppressors by nuclearcytoplasmic shuttling. Exp Cell Res 2003, 282(2):59-69. 93. Terry LJ, Shows EB, Wente SR: Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science 2007, 318(5855):1412-1416. 94. Weis K: Importins and exportins: how to get in and out of the nucleus. Trends Biochem Sci 1998, 23(5):185-189. 95. Nadler SG, Tritschler D, Haffar OK, Blake J, Bruce AG, Cleaveland JS: Differential expression and sequence-specific interaction of karyopherin alpha with nuclear localization sequences. J Biol Chem 1997, 272(7):4310-4315. 96. Hahn MA, Marsh DJ: Identification of a functional bipartite nuclear localization signal in the tumor suppressor parafibromin. Oncogene 2005, 24(41):6241-6248. 97. Liu MT, Hsu TY, Chen JY, Yang CS: Epstein-Barr virus DNase contains two nuclear localization signals, which are different in sensitivity to the hydrophobic regions. Virology 1998, 247(1):62-73. 98. Chan CK, Hubner S, Hu W, Jans DA: Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery. Gene Ther 1998, 5(9):1204-1212. 99. Kaffman A, Rank NM, OShea EK: Phosphorylation regulates association of the transcription factor Pho4 with its import receptor Pse1/Kap121. Genes Dev 1998, 12(17):2673-2683. 100. OBrate A, Giannakakou P: The importance of p53 location: nuclear or cytoplasmic zip code? Drug Resist Updat 2003, 6(6):313-322. 101. LaCasse EC, Lefebvre YA: Nuclear localization signals overlap DNA- or RNA-binding domains in nucleic acid-binding proteins. Nucleic Acids Res 1995, 23(10):1647-1656. 102. Mears WE, Lam V, Rice SA: Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27. J Virol 1995, 69(2):935-947. 103. Angus SP, Solomon DA, Kuschel L, Hennigan RF, Knudsen ES: Retinoblastoma tumor suppressor: analyses of dynamic behavior in living cells reveal multiple modes of regulation. Mol Cell Biol 2003, 23(22):8172-8188. 104. Jiao W, Lin HM, Datta J, Braunschweig T, Chung JY, Hewitt SM, Rane SG: Aberrant nucleocytoplasmic localization of the retinoblastoma tumor suppressor protein in human cancer correlates with moderate/poor tumor differentiation. Oncogene 2008, 27(22):3156-3164. 105. den Besten W, Kuo ML, Williams RT, Sherr CJ: Myeloid leukemia-associated nucleophosmin mutants perturb p53-dependent and independent activities of the Arf tumor suppressor protein. Cell Cycle 2005, 4(11):1593-1598. 106. Maggi LB Jr, Weber JD: Nucleolar adaptation in human cancer. Cancer Invest 2005, 23(7):599-608. 107. Zimber A, Nguyen QD, Gespach C: Nuclear bodies and compartments: functional roles and cellular signalling in health and disease. Cell Signal 2004, 16(10):1085-1104. 108. Olson MO, Hingorani K, Szebeni A: Conventional and nonconventional roles of the nucleolus. Int Rev Cytol 2002, 219:199-266. 109. Olson MO, Dundr M: The moving parts of the nucleolus. Histochem Cell Biol 2005, 123(3):203-216. 110. Elam C, Hesson L, Vos MD, Eckfeld K, Ellis CA, Bell A, Krex D, Birrer MJ, Latif F, Clark GJ: RRP22 is a farnesylated, nucleolar, Ras-related protein with tumor suppressor potential. Cancer Res 2005, 65(8):3117-3125. Page 20 of 20 111. Simmons HM, Ruis BL, Kapoor M, Hudacek AW, Conklin KF: Identification of NOM1, a nucleolar, eIF4A binding protein encoded within the chromosome 7q36 breakpoint region targeted in cases of pediatric acute myeloid leukemia. Gene 2005, 347(1):137-145. 112. Arabi A, Wu S, Ridderstrale K, Bierhoff H, Shiue C, Fatyol K, Fahlen S, Hydbring P, Soderberg O, Grummt I, et al: c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription. Nat Cell Biol 2005, 7(3):303-310. 113. Sanders JA, Gruppuso PA: Nucleolar localization of hepatic c-Myc: a potential mechanism for c-Myc regulation. Biochim Biophys Acta 2005, 1743(1-2):141-150. 114. Hannan KM, Kennedy BK, Cavanaugh AH, Hannan RD, HirschlerLaszkiewicz I, Jefferson LS, Rothblum LI: RNA polymerase I transcription in confluent cells: Rb downregulates rDNA transcription during confluenceinduced cell cycle arrest. Oncogene 2000, 19(31):3487-3497. 115. Rizos H, McKenzie HA, Ayub AL, Woodruff S, Becker TM, Scurr LL, Stahl J, Kefford RF: Physical and functional interaction of the p14ARF tumor suppressor with ribosomes. J Biol Chem 2006, 281(49):38080-38088. 116. Gjerset RA, Bandyopadhyay K: Regulation of p14ARF through subnuclear compartmentalization. Cell Cycle 2006, 5(7):686-690. 117. Mekhail K, Gunaratnam L, Bonicalzi ME, Lee S: HIF activation by pHdependent nucleolar sequestration of VHL. Nat Cell Biol 2004, 6(7):642-647. 118. Siomi H, Dreyfuss G: A nuclear localization domain in the hnRNP A1 protein. J Cell Biol 1995, 129(3):551-560. 119. Rubbi CP, Milner J: Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses. Embo J 2003, 22(22):6068-6077. doi:10.1186/1471-2121-11-74 Cite this article as: Zhou et al.: Identification and functional analysis of NOL7 nuclear and nucleolar localization signals. BMC Cell Biology 2010 11:74. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color figure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit  ...

... 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. Int J Radiat Oncol Biol Phys 1989;17:6914. 5. Rennemo E, Zatterstrom U, Boysen M. Impact of second primary tumors on survival in head and neck cancer: an analysis of 2,063 cases. Laryngoscope 2008;118:13506. 6. Kelloff GJ, Lippman SM, Dannenberg AJ, et al. Progress in chemoprevention drug development: the promise of molecular biomarkers for prevention of intraepithelial neoplasia and cancera plan to move forward. Clin Cancer Res 2006;12:366197. 7. Wrangle JM, Khuri FR. Chemoprevention of squamous cell carcinoma of the head and neck. Curr Opin Oncol 2007;19:1807. 8. Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005;438:9326. 9. Saba NF, Shin DM, Khuri FR. Targeting angiogenesis in head and neck cancer. Curr Cancer Drug Targets 2007;7:6439. 10. Lingen MW, DiPietro LA, Solt DB, Bouck NP, Polverini PJ. The angiogenic switch in hamster buccal pouch keratinocytes is dependent on TGF-1 and is unaffected by ras activation. Carcinogenesis 1997;18:32938. 11. Carlile J, Harada K, Baillie R, et al. Vascular endothelial growth factor (VEGF) expression in oral tissues: possible relevance to angiogenesis, tumour progression and field cancerisation. J Oral Pathol Med 2001;30:44957. 12. Pazouki S, Chisholm DM, Adi MM, et al. The association between tumour progression and vascularity in the oral mucosa. J Pathol 1997;183:3943. www.aacrjournals.org 13. Jin Y, Tipoe GL, White FH, Yang L. A quantitative investigation of immunocytochemically stained blood vessels in normal, benign, premalignant and malignant human oral cheek epithelium. Virchows Arch 1995;427:14551. 14. Ciardiello F, Tortora G. Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. Eur J Cancer 2003;39:134854. 15. Goldman CK, Kim J, Wong WL, King V, Brock T, Gillespie GY. Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology. Mol Biol Cell 1993;4:12133. 16. Gille J, Swerlick RA, Caughman SW. Transforming growth factor-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA binding and transactivation. EMBO J 1997;16:7509. 17. Hennequin LF, Stokes ES, Thomas AP, et al. Novel 4-anilinoquinazolines with C-7 basic side chains: design and structure activity relationship of a series of potent, orally active, VEGF receptor tyrosine kinase inhibitors. J Med Chem 2002;45:130012. 18. Wedge SR, Ogilvie DJ, Dukes M, et al. ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration. Cancer Res 2002;62:464555. 19. Ciardiello F, Bianco R, Caputo R, et al. Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy. Clin Cancer Res 2004;10:78493. 20. Ciardiello F, Caputo R, Damiano V, et al. Antitumor effects of ZD6474, a small molecule vascular endothelial growth factor receptor tyrosine kinase inhibitor, with additional activity against epidermal growth factor receptor tyrosine kinase. Clin Cancer Res 2003;9:154656. 21. Beaudry P, Nilsson M, Rioth M, et al. Potent antitumor effects of ZD6474 on neuroblastoma via dual targeting of tumor cells and tumor endothelium. Mol Cancer Ther 2008;7:41824. Cancer Prev Res; 3(11) November 2010 Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. 1501 Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Zhou et al. 22. Bianco R, Rosa R, Damiano V, et al. Vascular endothelial growth factor receptor-1 contributes to resistance to anti-epidermal growth factor receptor drugs in human cancer cells. Clin Cancer Res 2008; 14:506980. 23. Conrad C, Ischenko I, Kohl G, et al. Antiangiogenic and antitumor activity of a novel vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor ZD6474 in a metastatic human pancreatic tumor model. Anticancer Drugs 2007;18:56979. 24. Gustafson DL, Frederick B, Merz AL, Raben D. Dose scheduling of the dual VEGFR and EGFR tyrosine kinase inhibitor vandetanib (ZD6474, Zactima) in combination with radiotherapy in EGFRpositive and EGFR-null human head and neck tumor xenografts. Cancer Chemother Pharmacol 2008;61:17988. 25. Natale RB. Dual targeting of the vascular endothelial growth factor receptor and epidermal growth factor receptor pathways with vandetinib (ZD6474) in patients with advanced or metastatic non-small cell lung cancer. J Thorac Oncol 2008;3:S12830. 26. Naumov GN, Nilsson MB, Cascone T, et al. Combined vascular endothelial growth factor receptor and epidermal growth factor receptor (EGFR) blockade inhibits tumor growth in xenograft models of EGFR inhibitor resistance. Clin Cancer Res 2009;15:348494. 27. Rich JN, Sathornsumetee S, Keir ST, et al. ZD6474, a novel tyrosine kinase inhibitor of vascular endothelial growth factor receptor and epidermal growth factor receptor, inhibits tumor growth of multiple nervous system tumors. Clin Cancer Res 2005;11:814557. 28. Shibuya K, Komaki R, Shintani T, et al. Targeted therapy against VEGFR and EGFR with ZD6474 enhances the therapeutic efficacy of irradiation in an orthotopic model of human non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 2007;69:153443. 29. Troiani T, Serkova NJ, Gustafson DL, et al. Investigation of two dosing schedules of vandetanib (ZD6474), an inhibitor of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling, in combination with irinotecan in a human colon cancer xenograft model. Clin Cancer Res 2007;13:64508. 30. Xiao X, Wu J, Zhu X, et al. Induction of cell cycle arrest and apoptosis in human nasopharyngeal carcinoma cells by ZD6474, an inhibitor of VEGFR tyrosine kinase with additional activity against EGFR tyrosine kinase. Int J Cancer 2007;121:2095104. 31. Morabito A, Piccirillo MC, Falasconi F, et al. Vandetanib (ZD6474), a dual inhibitor of vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) tyrosine kinases: current status and future directions. Oncologist 2009;14:37890. 32. Herbst RS, Sun Y, Eberhardt WE, et al. Vandetanib plus docetaxel versus docetaxel as second-line treatment for patients with advanced non-small-cell lung cancer (ZODIAC): a double-blind, randomised, phase 3 trial. Lancet Oncol 2010;11:61926. 33. Robinson BG, Paz-Ares L, Krebs A, Vasselli J, Haddad R. Vandetanib (100 mg) in patients with locally advanced or metastatic hereditary medullary thyroid cancer. J Clin Endocrinol Metab 2010;95: 266471. 34. Wells SA, Jr., Gosnell JE, Gagel RF, et al. Vandetanib for the treatment of patients with locally advanced or metastatic hereditary medullary thyroid cancer. J Clin Oncol 2010;28:76772. 35. Hasina R, Martin LE, Kasza K, Jones CL, Jalil A, Lingen MW. ABT-510 is an effective chemopreventive agent in the mouse 4nitroquinoline 1-oxide model of oral carcinogenesis. Cancer Prev Res (Phila Pa) 2009;2:38593. 36. Abbey LM, Kaugars GE, Gunsolley JC, et al. Intraexaminer and interexaminer reliability in the diagnosis of oral epithelial dysplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;80:18891. 37. Warnakulasuriya S, Reibel J, Bouquot J, Dabelsteen E. Oral epithelial 1502 Cancer Prev Res; 3(11) November 2010 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. dysplasia classification systems: predictive value, utility, weaknesses and scope for improvement. J Oral Pathol Med 2008;37:12733. Dougherty U, Sehdev A, Cerda S, et al. Epidermal growth factor receptor controls flat dysplastic aberrant crypt foci development and colon cancer progression in the rat azoxymethane model. Clin Cancer Res 2008;14:225362. Wedam SB, Low JA, Yang SX, et al. Antiangiogenic and antitumor effects of bevacizumab in patients with inflammatory and locally advanced breast cancer. J Clin Oncol 2006;24:76977. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009;139:87190. Thomson S, Buck E, Petti F, et al. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res 2005;65:945562. Frederick BA, Helfrich BA, Coldren CD, et al. Epithelial to mesenchymal transition predicts gefitinib resistance in cell lines of head and neck squamous cell carcinoma and non-small cell lung carcinoma. Mol Cancer Ther 2007;6:168391. Fuchs BC, Fujii T, Dorfman JD, et al. Epithelial-to-mesenchymal transition and integrin-linked kinase mediate sensitivity to epidermal growth factor receptor inhibition in human hepatoma cells. Cancer Res 2008;68:23919. Iwatsuki M, Mimori K, Yokobori T, et al. Epithelial-mesenchymal transition in cancer development and its clinical significance. Cancer Sci 2010;101:2939. Moreno-Bueno G, Peinado H, Molina P, et al. The morphological and molecular features of the epithelial-to-mesenchymal transition. Nat Protoc 2009;4:1591613. Alferez D, Wilkinson RW, Watkins J, et al. Dual inhibition of VEGFR and EGFR signaling reduces the incidence and size of intestinal adenomas in Apc(Min/+) mice. Mol Cancer Ther 2008;7:5908. Ko J, Ross J, Awad H, Hurwitz H, Klitzman B. The effects of ZD6474, an inhibitor of VEGF signaling, on cutaneous wound healing in mice. J Surg Res 2005;129:2519. Day GL, Blot WJ. Second primary tumors in patients with oral cancer. Cancer 1992;70:149. Slaughter DP, Southwick HW, Smejkal W. Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer 1953;6:9638. Bergers G, Hanahan D. Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 2008;8:592603. Paez-Ribes M, Allen E, Hudock J, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 2009;15:22031. Chen LF, Cohen EE, Grandis JR. New strategies in head and neck cancer: understanding resistance to epidermal growth factor receptor inhibitors. Clin Cancer Res 16:248995. Kweon MH, Adhami VM, Lee JS, Mukhtar H. Constitutive overexpression of Nrf2-dependent heme oxygenase-1 in A549 cells contributes to resistance to apoptosis induced by epigallocatechin 3-gallate. J Biol Chem 2006;281:3376172. Yan M, Myung SJ, Fink SP, et al. 15-Hydroxyprostaglandin dehydrogenase inactivation as a mechanism of resistance to celecoxib chemoprevention of colon tumors. Proc Natl Acad Sci U S A 2009;106: 940913. Freemantle SJ, Spinella MJ, Dmitrovsky E. Retinoids in cancer therapy and chemoprevention: promise meets resistance. Oncogene 2003;22:730515. Lee MJ, Kim YS, Kummar S, Giaccone G, Trepel JB. Histone deacetylase inhibitors in cancer therapy. Curr Opin Oncol 2008;20:63949. 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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