搜

... The item referenced in this repository content can be found by following the link on the descriptive page.  ...

... 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. 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-08-0211 This article cites 53 articles, 12 of which you can access for free at: http://cancerpreventionresearch.aacrjournals.org/content/2/4/385.full#ref-list-1 This article has been cited by 8 HighWire-hosted articles. Access the articles at: http://cancerpreventionresearch.aacrjournals.org/content/2/4/385.full#related-urls Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, use this link http://cancerpreventionresearch.aacrjournals.org/content/2/4/385. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site. Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research.  ...

... REVIEW Cell Research (2012) 22:23-32. 2012 IBCB, SIBS, CAS All rights reserved 1001-0602/12 $ 32.00 www.nature.com/cr Semaphorin signaling in angiogenesis, lymphangiogenesis and cancer Atsuko Sakurai1, Colleen Doci1, J Silvio Gutkind1 1 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Rm. 211, Bethesda, MD 20892, USA Angiogenesis, the formation of new blood vessels from preexisting vasculature, is essential for many physiological processes, and aberrant angiogenesis contributes to some of the most prevalent human diseases, including cancer. Angiogenesis is controlled by delicate balance between pro- and anti-angiogenic signals. While pro-angiogenic signaling has been extensively investigated, how developmentally regulated, naturally occurring anti-angiogenic molecules prevent the excessive growth of vascular and lymphatic vessels is still poorly understood. In this review, we summarize the current knowledge on how semaphorins and their receptors, plexins and neuropilins, control normal and pathological angiogenesis, with an emphasis on semaphorin-regulated anti-angiogenic signaling circuitries in vascular and lymphatic endothelial cells. This emerging body of information may afford the opportunity to develop novel anti-angiogenic therapeutic strategies. Keywords: semaphorin; signaling; angiogenesis; lymphangiogenesis; cancer Cell Research (2012) 22:23-32. doi:10.1038/cr.2011.198; published online 13 December 2011 Introduction Angiogenesis, the formation of new blood vessels from preexisting vasculature, is essential for many physiological processes, such as wound healing, and also plays a critical role in many pathological conditions including diabetic retinopathy, age-related macular degeneration, and tumor growth. Angiogenesis is controlled by delicate balance between pro- and anti-angiogenic signals, thus elucidating the molecular mechanisms underlying normal and aberrant blood vessel growth may provide new therapeutic options for many human diseases [1]. Among the pro-angiogenic molecules, vascular endothelial growth factor (VEGF) has received a considerable amount of attention as a promising target for anti-angiogenic therapy. Anti-angiogenic drugs targeting the VEGF pathway, such as a humanized monoclonal antibody against VEGF, were developed rapidly and used in the clinical settings. However, anti-VEGF treatments often result in only modest improvement in progression-free Correspondence: J Silvio Gutkind Tel: +1-301-496-3695, Fax: +1-301-402-0823 E-mail: sg39v@nih.gov survival, and tumors can eventually acquire resistance to anti-angiogenic therapies. To overcome these problems, a better understanding of the molecular mechanisms responsible for angiogenesis is necessary, which may afford the opportunity to develop new anti-angiogenic therapeutic strategies. The most widely investigated angiogenesis inhibitors are the proteolytic cleavage products of extracellular matrix or serum components, such as endostatin, angiostatin, arresten, and tumstatin (reviewed in [2, 3]). Multiple cytokines can also exert anti-angiogenic properties, including interferons and certain interleukins, which often act indirectly by limiting the expression of pro-angiogenic mediators or inducing anti-angiogenic molecules (reviewed in [2, 3]). In contrast, there are very few known developmentally regulated, naturally occurring anti-angiogenic molecules, which include platelet factor 4 [4], thrombospondin-1 [5], and pigment epithelium-derived factor [6], whose precise mechanism of action is not fully understood. In this regard, emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of the netrin, slit, eph and semaphorin families, also play a critical role in blood vessel guidance during physiological and pathological blood vessel development [7, 8]. In this review, npg npg Semaphorin signaling in angiogenesis 24 we summarize the current knowledge on semaphorin family proteins in angiogenesis, with an emphasis on semaphorin-regulated anti-angiogenic signaling circuitries in endothelial cells. The possible role of semaphorin signaling in supressing lymphangiogenesis will also be discussed. Semaphorins Semaphorins are a family of cell surface and soluble proteins originally identified as axon guidance factors that control the development of central nervous system [9]. All semaphorins are characterized by an aminoterminal 500-amino acid Sema domain that is essential for signaling. Semaphorins are grouped into eight classes based on their structural domains, with classes 3-7 comprising the vertebrate semaphorins (Figure 1). Although initially identified as potent axon chemorepellents, several semaphorins can provide bifunctional guidance cues, functioning as repulsive or attractive molecules depending on the cell types and biological context [10, 11]. The common targets of semaphorins are the actin cytoskeleton and focal adhesions; the latter are dynamic cell-to-extracellular matrix adhesive structures that are assembled upon integrin engagement. Semaphorin signaling affects focal adhesion assembly/disassembly and induces cytoskeletal remodeling, thus consequently af- fecting cell shape, attachment to the extracellular matrix, cell motility, and cell migration [12, 13]. Semaphorin receptors: plexins and neuropilins (Nrps) Semaphorins signal through two major receptor families, plexins and Nrps. In vertebrates, two Nrps (Nrp1 and Nrp2) and nine plexins have been identified [14] (Figure 1). Membrane-bound semaphorins bind directly to plexins, whereas secreted type of semaphorins (class 3 semaphorins; Sema3s) bind to a holoreceptor complex consisting of Nrps as ligand binding and plexins as signal transducing subunit. An exception to this rule is Sema3E, which binds and signals directly through plexin-D1, independently of Nrps [15]. Plexins are single-pass transmembrane receptors and subdivided into four groups, type A, B, C, and D. Similar to semaphorins, plexins have extracellular Sema domains. In addition, plexins have PSI (plexins, Sema, integrins) and IPT (Ig-like, plexins, transcription factors) domains, and share homology in their extracellular segment with the Met family tyrosine kinase receptors [14]. The intracellular domains of plexins have weak sequence similarity to GTPase activating proteins (GAPs) and display GAP activity towards the small GTPase R-Ras [16]. Type A, B, and D plexins require the association of the Figure 1 Human Plexins, Nrps, and Semaphorins: Vertebrates express semaphorin classes 3 through 7, plexins A, B, C, D, and Nrps 1 and 2. Semaphorins and plexins are comprised of a Sema domain and variable repeats of the PSI domain, as indicated. Members of the semaphorin 3 (Sema3, A-G) class are secreted, while the semaphorins 4-7 (Sema4-7) are membrane bound. Plexins contain cytoplasmic tails that contain both GAP and GTPase-binding domains. Additionally, plexins B1, B2, B3, and D1 contain a PDZ binding domain. Nrp1 and Nrp2 are comprised of extracellular complement binding, FV/FVIII, and MAM domains, but have only a short cytoplasmic tail, requiring their association with plexin A1-4 to facilitate signaling. Cell Research | Vol 22 No 1 | January 2012 Atsuko Sakurai et al. npg 25 Rnd family of Rho-related GTPases to function as R-Ras GAP, while plexin-C1 displays GAP activity without Rnd [17]. Plexin-B1 also possesses GAP activity for RRas3/M-Ras [18]. Nrps are transmembrane proteins with a short cytoplasmic domain of about 40 amino acids, and their three C-terminal amino acids (S-E-A) constitute a PDZbinding motif [19]. In addition to Sema3s, Nrps also bind to structurally unrelated molecules, such as VEGF family proteins, and serve as their co-receptors [20, 21]. The extracellular domains of Nrps contain two complementbinding domains (a1/a2), two coagulation factor V/VIII homology domains (b1/b2), and a MAM domain (c). Sema3s principally bind to a1/a2 domains, and VEGFs bind to b1/b2 domains [14]. Genetic studies have shown that Nrp1 is required for vascular morphogenesis. Nrp1knockout mice are embryonically lethal due to vascular remodeling and branching defects [22, 23], whereas Nrp2-knockout mice are viable and their vasculature is grossly normal. However, Nrp2 null mice show absence of or reduced lymphatic vessel sprouting during development [24], suggesting that Nrp2 plays a key role in lymphangiogenesis (discussed below). 37]. The molecular mechanisms underlying the antiangiogenic effects of Sema3A are complex, as its receptor Nrp1 also controls VEGFR2 signaling by binding VEGF165 [38, 39]. Hence, it was initially suggested that Sema3s compete for the binding to Nrp1 with VEGF, thus inhibiting VEGF-induced angiogenesis. However, recent reports have shown that Sema3s and VEGF use different domains on Nrp1 for binding [40]. Consistent with a separate function of Sema3A on Nrp1, Sema3A increases vascular permeability, inhibits endothelial cell proliferation, and induces apoptosis even in the absence of VEGF [34, 41], suggesting Sema3A activates its own signaling routes. Interestingly, Sema3A impairs endothelial cell adhesion and migration by inhibiting integrin function [32]. The molecular mechanisms by which Sema3A regulates integrins are not fully understood. From the studies in neuronal cells, it is likely that activation of plexin-A1 by Sema3A induces the intrinsic R-Ras GAP activity of Plexin-A1, thus resulting in R-Ras inhibition [16, 42]. As R-Ras is known to sustain integrin activation, the inactivation of R-Ras by plexin-A1 may lead to the inactivation of integrins, thereby inhibiting integrinmediated cell adhesion. Anti-angiogenic semaphorins Sema3B Sema3B and Sema3F were identified as tumor suppressors that are deleted or inactivated in lung cancer [43, 44]. Consistently, overexpression of Sema3B suppresses tumorigenesis in adenocarcinoma cell lines [45], and Sema3B also decreases proliferation of lung and breast cancer cell lines [30], suggesting Sema3B exerts direct effects on cancer cells. In addition to the inhibitory effect on cancer cells, Sema3B can repel endothelial cells mainly through Nrp1, and therefore functions as an angiogenesis inhibitor [46]. Interestingly, Sema3B activity is abrogated as a result of proteolytic cleavage by furin-like pro-protein convertases, which is associated with enhanced invasion and proliferation in many cancers [46, 47]. Together, Sema3B may function as a tumor suppressor by working on both tumor cells and endothelial cells. Certainly, more work is necessary to define the precise signaling events that mediate this effect of Sema3B. Sema3s are the only secreted type of semaphorins in vertebrates. Seven Sema3s have been identified (designated by the letters A-G) and multiple Sema3s are reported to control physiological and pathological angiogenesis [25, 26]. Nrps and the type A/D family plexins (plexinA1, -A2, and -A3, and plexin-D1) act as receptors for Sema3s, and each Sema3 family member shows distinct binding preference for Nrps. For example, Sema3A binds to Nrp1 [27, 28], while Sema3F binds exclusively to Nrp2 [29]. Other Sema3s, such as Sema3B, can bind to both Nrps [30, 31]. Each Sema3-Nrp complex associates with specific plexins to mediate downstream signaling. Some Sema3s, such as Sema3A and Sema3F, are expressed in endothelial cells, suggesting an endothelialinitiated autocrine regulation of angiogenesis [32]. Sema3A Sema3A regulates endothelial cell migration and survival in vitro [33, 34], and tumor-induced angiogenesis in vivo [35]. The information emerging from the analysis of Sema3A null mice and mutant mice lacking Sema3A-Nrp1 signaling suggests that Sema3A may not be required for the early stages of developmental angiogenesis, but rather that Sema3A contributes to the reshaping of the vasculature to form a mature vascular network, such as that in the heart and brain [32, 36, www.cell-research.com | Cell Research Sema3E As described above, Sema3E binds directly to and activates plexin-D1 receptor. Plexin-D1 is highly expressed in endothelium during development [48] and also in tumor-associated blood vessels [49]. Interestingly, the expression of plexin-D1 is dynamically regulated and increased in tip cells, which extend numerous filopodia and respond to attractive and repulsive guidance cues at npg Semaphorin signaling in angiogenesis 26 the leading edge of the new branching blood vessels [50]. Sema3E controls vascular patterning during development by inhibiting the expansion of intersomitic vessels into the somites [15, 51], and causes endothelial-tip cell filopodial retraction in growing blood vessels [52]. These data suggest that Sema3E is a potent chemorepellent for plexin-D1-expressing endothelial cells. Indeed, Sema3E displays anti-angiogenic properties in several different in vivo angiogenesis models [52, 53]. Furthermore, Sema3E is highly expressed in metastatic tumors, where a cleaved form of Sema3E may enhance tumor cell motility. Conversely, when wild-type Sema3E is overexpressed in cancer cell lines, Sema3E decreases tumor vessel density and tumor growth [54]. The interplay between cleaved products of Sema3E and potential receptors in cancer cells is an area of active investigation. More progress has been recently made on the study of how Sema3E prevents angiogenesis in endothelial cells, given the obligatory role of plexin-D1 but not of Nrps in this biological response. Sema3E may counteract the pro-angiogenic effects of VEGF by promoting the expression and release of a soluble VEGF receptor, thus inhibiting VEGF function in developing vessels [55]. In a more direct fashion in endothelial cells, upon stimulation with Sema3E, plexin-D1 initiates a two-pronged mechanism involving R-Ras inactivation and Arf6 activation, thereby affecting the activation status of 1 integrin and its intracellular trafficking, respectively [52] (Figure 2). At the molecular level, Sema3E binding to plexin-D1 induces the activation of type I phosphatidylinositol-4-phosphate5-kinase (PIP5K) , which may stimulate the production of phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) locally [56]. PI(4,5)P2 then binds to the PH domain of a guanine nucleotide exchange factor (GEF) for Arf6, guanine nucleotide exchange protein 100, thus increasing its GEF activity and activating Arf6. Sema3E-induced Arf6 activation induces rapid focal adhesion disassembly and endothelial cell collapse, thereby inhibiting angiogenesis [56]. In addition, recent reports revealed that PI(4,5)P2 acts as a second messenger and controls focal adhesion dynamics and actin cytoskeleton [57], both of which are critical for cell migration. In neuronal cells, inhibition of another isoform of PIP5K, PIPKI661, is required for Sema3A-induced axonal repulsion and growth cone collapse [42]. These data suggest that semaphorins may commonly utilize phospholipid-regulated signaling Figure 2 Anti-angiogenic signaling by Sema3E-plexin D1 in endothelial cells. The activation of plexin D1 by Sema3E induces the association of the Ras GAP domain of plexin D1 with R-Ras. This inactivates integrins and enables their subsequent internalization by the plexin D1-mediated activation of Arf6, thus inhibiting endothelial cell adhesion to the extracellular matrix (ECM) by disrupting integrin-mediated adhesive structures, and causing filopodial retraction in endothelial tip cells. The pathway by which Sema3E stimulation of plexin D1 leads to Arf6 activation involves phosphatidylinositol-4-phosphate-5-kinase activation, which generates PI(4,5)P2 locally. PI(4,5)P2 then binds to the PH domain of an Arf6 GEF, guanine nucleotide exchange protein 100, resulting in Arf6 activation. Sema3E-induced Arf6 activation induces rapid focal adhesion (FA) disassembly, integrin internalization, and endothelial cell collapse, thereby inhibiting angiogenesis. Cell Research | Vol 22 No 1 | January 2012 Atsuko Sakurai et al. npg 27 pathways to regulate cell adhesion and migration, by regulating lipid kinases such as PIP5K. Sema3F Sema3F decreases tumor growth in a number of in vivo tumor models, and although Sema3F is capable of interacting with Nrp1, its higher-affinity interaction with Nrp2 appears to be required for its tumor-suppressive activity in many models [58-60]. Sema3F exerts a repulsive effect on breast cancer cells [61] and reduces the growth and metastatic activity of colorectal carcinoma cells by modifying integrin v3 [62], suggesting that Sema3F affects tumor cells directly by controlling cell adhesion and migration. Similar to other Sema3s, the Sema3Fmediated suppression of tumor growth was associated with an anti-angiogenic phenotype, where Sema3F expression significantly decreased the vascularity of tumors [25, 58]. Consistently, Sema3F can induce endothelial cell collapse [63], repel endothelial cells and inhibit their survival [59], and this anti-angiogenic effect is synergistically enhanced by the addition of Sema3A [34]. At the molecular level, it has been shown that Sema3F inhibits multiple signaling pathways in cancer cells [63, 64]; however, whether these pathways are also affected in endothelial cells remains unclear. While it is clear that Sema3F can function as a potent tumor suppressor in vivo, further studies are required to understand the molecular mechanism behind this function and the role that Sema3F expression plays in cancer development and progression. Sema3G Far less is known about the role of Sema3G in cancer and signaling. Sema3G has been identified as a significant prognostic marker in glioma [65]. Sema3G appears to bind to Nrp2, but not Nrp1 [66], and is able to suppress tumor growth and inhibit soft-agar colony formation only in cells expressing high levels of this receptor [25]. However, further work is necessary to determine whether Sema3G has anti-angiogenic effects in vivo and the mechanism by which it achieves its Nrp2-dependent tumor growth-suppressive activity. Pro-angiogenic semaphorins Some plexins are highly expressed in endothelial cells. Among them, plexin-B1 is a receptor for Sema4D, and it has been reported that Sema4D-Plexin-B1 signaling promotes endothelial cell migration and tube formation [67, 68]. Interestingly, Sema4D expression is upregulated in head and neck squamous cell carcinoma as well as in some other solid tumors [69, 70]. Sema4D is processed and released from the membrane by membrane type www.cell-research.com | Cell Research 1-matrix metalloproteinase, which is frequently overexpressed in malignant tumors, and activates plexin-B1 to elicit pro-angiogenic signaling in endothelial cells [71]. Similarly, Sema4D can be released from platelets by the action of metalloprotease ADAM17 (TACE), thereby acting on endothelial cells as well as platelets that also express Sema4D receptors [72]. The pro-angiogenic effect of Sema4D is mediated by the activation of a small GTPase RhoA through Rho-specific GEFs, leukemiaassociated RhoGEF, and PDZ-RhoGEF, which bind to the C-terminal PDZ-binding motif of plexin-B1 [67, 73-76]. Upon ligation of Sema4D, PDZ-RhoGEF and leukemia-associated RhoGEF are recruited to plexin-B1, thus stimulating RhoA and its downstream effector Rho kinase, and Rho kinase activation promotes angiogenic response through a chain of events involving myosin light chain phosphorylation, stress fiber contaction, nonreceptor tyrosine kinase activation, and activation of the Akt and Erk pathways [77] (Figure 3). RhoA/Rho kinase signaling also activates PIP5K and leads to the generation of the lipid second messenger PI(4,5)P2, further supporting the idea that phosphoinositides may represent a common mediator for semaphorin signaling. In the case of Sema4D, PI(4,5)P2 serves as a substrate for PLC and increases intracellular calcium level, which is required for Sema4D-induced tube formation in vitro [78]. In addition to the direct effect of Sema4D on plexin-B1, this semaphorin can also promote endothelial cell migration through the activation of HGF receptor Met [68], while in other cell types, plexin-B1 activates members of the EGF receptor family [79] and inhibits RhoA through p190 RhoGAP [80], resulting in changes in the actin cytoskeleton and cell migration. Lymphangiogenesis and cancer Like angiogenesis, lymphangiogenesis is both a physiological and pathological process required for the formation of new vasculature, and the regulation of this process is mediated by a balance of pro- and anti-lymphangiogenic factors. The lymphatic system is critical for the removal of waste products and transport of cells and proteins, and contributes to the immune response and cancer progression. During embryogenesis, expression of the transcription factor PROX1 leads to the differentiation of venous endothelial cells into lymphatic endothelial cells [81-84]. This differentiation is followed by sprouting and proliferation of the lymphatic vasculature, which is primarily attributed to signaling by VEGF-C through its receptor VEGFR3 [85-87]. VEGF-C binds to receptors VEGFR3 and Nrp2, and loss of these molecules results in a variety of lymphangiogenic defects. Vegfc/ mice npg Semaphorin signaling in angiogenesis 28 Figure 3 Pro-angiogenic signaling by semaphorins in endothelial cells. Membrane-bound Sema4D expressed by cancer cells or by tumor-associated macrophages and platelets is cleaved by matrix metalloproteinases, and soluble Sema4D can then bind and activate plexin-B members expressed on endothelial cells. After stimulation by Sema4D, plexin-B can activate certain receptor tyrosine kinases, such as Met, EGFR, and Her2, in some cellular contexts, hence activating their regulated signaling pathways indirectly. Sema4D binding to plexin-B causes the recruitment and activation of PDZ-RhoGEF and leukemiaassociated RhoGEF through association mediated by their PDZ domains. These GEFs then activate Rho and its downstream effector Rho kinase (ROCK). In turn, ROCK signaling promotes the phosphorylation of myosin light chain. This induces polymerization and contraction of actin/myosin stress fibers. The tension generated by contracting stress fibers promotes the assembly of mature focal adhesion complexes at the sites of cell contact with the extracellular matrix via integrins, thereby activating non-receptor tyrosine kinases such as Pyk2 and Src. Pyk2 activation results in the phosphorylation of phosphatidylinositol 3-kinase and the activation of the Akt and Erk1/2 pathways, leading to increased endothelial cell migration and proliferation, which contribute to promoting an angiogenic response. are able to commit to the lymphatic lineage, but cannot undergo lymphangiogenesis and die in utero [88]. VEGFR3-knockout mice display a more severe phenotype, where they lack lymphatic vasculature entirely and fail to undergo lymphangiogenesis [89]. In contrast, Nrp2 is not required for lymphangiogenesis but appears to be a key regulator of the process. Mice deficient in Nrp2 show a variety of lymphatic defects, including reduction of small lymphatic vessels and capillaries [24]. Further, Nrp2 appears to be selectively expressed in tumor-associated lymphatic vessels, and inhibition of Nrp2 function blocked VEGF-C/VEGFR3-mediated lymphangiogenesis and reduced metastasis in vivo [90, 91]. By exten- sion, this suggests that anti-angiogenic semaphorins like Sema3F can function as anti-lymphangiogenic signaling molecules in vivo through their interaction with Nrp2. Sema3F overexpression has been shown to have a direct chemorepulsive effect on lymphatic endothelial cells [58]. Sema3F appears to compete for Nrp2 binding with VEGF-C and decreases endothelial cell survival and migration [92]. Nevertheless, direct evidence for an antilymphangiogenic role of Sema3F is limited. Indirectly, tumor-induced lymphangiogenesis is correlated with metastasis in a number of cancer models, including colorectal, breast, prostate, head and neck cancer, and melanoma [93-99], and expression of Semaphorin Cell Research | Vol 22 No 1 | January 2012 Atsuko Sakurai et al. npg 29 molecules decreases the incidence of metastasis [58, 62, 100]. Inhibition of tumoral lymphangiogenesis through neutralization of VEGFR3 or knockdown of VEGF-C expression blocks metastasis in vivo [101-103], indicating that this process is essential for metastasis in a variety of cancer models. Interestingly, this inhibition did not affect the normal lymphatic vasculature in adult mice, suggesting that alternative signaling mechanisms are employed after development, and targeting the VEGF-C/VEGFR3 signaling axis in adults may represent a potent means to target the tumor-associated lymphatics [104]. The precise molecular events that signal for lymphangiogenesis are poorly understood. Inhibition of the mTOR pathway decreases lymphangiogenesis and metastasis, and prolongs survival in a head and neck cancer animal model [99]. In support of this, a separate study found that inhibition of phosphatidylinositol 3-kinase/Akt similarly blocked lymphangiogenesis [105]. However, the specific signaling events required to initiate and sustain lymphangiogenesis in the context of tumor progression and metastasis are poorly defined. Given that tumoral lymphangiogenesis is likely a critical event in the progression of metastatic disease, the potential role of semaphorin-mediated regulation in this process warrants further investigation. Concluding remarks Recent studies have revealed the importance of semaphorins, plexins, and Nrps in tumor progression. These molecules directly control the behavior of tumor cells and also affect the other cell types which constitute the tumor microenviroment. In this review, we focused on vascular and lymphatic endothelial cells, both of which contribute to providing a suitable environment for tumor cells to grow and metastasize by inducing angiogenesis and lymphangiogenesis. A large fraction of the current information regarding semaphorin signaling was initially obtained from extensive studies in neuronal and cancer cells. It will be important to analyze whether the pathways deployed by the semaphorin-plexin signaling systems are shared by all cell types, with emphasis on those utilized in vascular and lymphatic endothelial cells to inhibit or promote angiogenesis and lymphangiogenesis. From the studies on Sema3B and Sema3F, loss of semaphorin function may represent an early event in cancer development, thus contributing to the acquisition of the angiogenic phenotype that characterizes most solid tumors. However, a number of questions remain: Which semaphorins are cleaved by furin-like proteases, and how does the proteolytic cleavage of semaphorins affect their function? Can the differential expression of these enzymatic factors account for the discrepancies in signaling www.cell-research.com | Cell Research and biological effects observed among different studies? Does differential expression of semaphorin receptors on tumor and endothelial cells affect their signaling potential? How does expression of competitive factors, such as VEGF, regulate the signaling capabilities of the semaphorins? Further, can their tumor-suppressive and anti-angiogenic roles be strengthened by multi-targeted approaches that take into account both the semaphorins and the regulators of their expression and function? What are the molecular differences in the signaling pathways activated by semaphorin binding, as opposed to the withdrawal of VEGF signaling? Semaphorin expression appears to affect a number of cellular phenotypes, including proliferation, survival, anchorage dependence, angiogenesis, and migration. Which among these represent the critical processes that are central to semaphorin-mediated tumor suppression? Answering these and other questions may be central to understanding the role of semaphorins in angiogenesis and cancer. Acknowledgments This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Dental and Craniofacial Research. References 1 2 3 4 5 6 7 8 9 10 11 Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005; 438:932-936. Folkman J. Angiogenesis: an organizing principle for drug discovery?. Nat Rev Drug Discov 2007; 6:273-286. Nyberg P, Xie L, Kalluri R. Endogenous inhibitors of angiogenesis. Cancer Res 2005; 65:3967-3979. Maione TE, Gray GS, Petro J, et al. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science 1990; 247:77-79. Good DJ, Polverini PJ, Rastinejad F, et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci USA 1990; 87:6624-6628. Dawson DW, Volpert OV, Gillis P, et al. Pigment epitheliumderived factor: a potent inhibitor of angiogenesis. Science 1999; 285:245-248. Carmeliet P, Tessier-Lavigne M. Common mechanisms of nerve and blood vessel wiring. Nature 2005; 436:193-200. Adams RH, Eichmann A. Axon guidance molecules in vascular patterning. Cold Spring Harb Perspect Biol 2010; 2:a001875. Kruger RP, Aurandt J, Guan KL. Semaphorins command cells to move. Nat Rev Mol Cell Biol 2005; 6:789-800. Eichmann A, Makinen T, Alitalo K. Neural guidance molecules regulate vascular remodeling and vessel navigation. Genes Dev 2005; 19:1013-1021. Derijck AA, Van Erp S, Pasterkamp RJ. Semaphorin signaling: molecular switches at the midline. Trends Cell Biol npg Semaphorin signaling in angiogenesis 30 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 2010; 20:568-576. Gelfand MV, Hong S, Gu C. Guidance from above: common cues direct distinct signaling outcomes in vascular and neural patterning. Trends Cell Biol 2009; 19:99-110. Serini G, Napione L, Arese M, Bussolino F. Besides adhesion: new perspectives of integrin functions in angiogenesis. Cardiovasc Res 2008; 78:213-222. Neufeld G, Kessler O. The semaphorins: versatile regulators of tumour progression and tumour angiogenesis. Nat Rev Cancer 2008; 8:632-645. Gu C, Yoshida Y, Livet J, et al. Semaphorin 3E and plexinD1 control vascular pattern independently of neuropilins. Science 2005; 307:265-268. Oinuma I, Ishikawa Y, Katoh H, Negishi M. The Semaphorin 4D receptor plexin-B1 is a GTPase activating protein for RRas. Science 2004; 305:862-865. Uesugi K, Oinuma I, Katoh H, Negishi M. Different requirement for Rnd GTPases of R-Ras GAP activity of plexin-C1 and plexin-D1. J Biol Chem 2009; 284:6743-6751. Saito Y, Oinuma I, Fujimoto S, Negishi M. Plexin-B1 is a GTPase activating protein for M-Ras, remodelling dendrite morphology. EMBO Rep 2009; 10:614-621. Schwarz Q, Ruhrberg C. Neuropilin, you gotta let me know: should I stay or should I go? Cell Adh Migr 2010; 4:61-66. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 1998; 92:735-745. Gluzman-Poltorak Z, Cohen T, Herzog Y, Neufeld G. Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165. J Biol Chem 2000; 275:18040-18045. Kawasaki T, Kitsukawa T, Bekku Y, et al. A requirement for neuropilin-1 in embryonic vessel formation. Development 1999; 126:4895-4902. Mukouyama YS, Gerber HP, Ferrara N, Gu C, Anderson DJ. Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback. Development 2005; 132:941-952. Yuan L, Moyon D, Pardanaud L, et al. Abnormal lymphatic vessel development in neuropilin 2 mutant mice. Development 2002; 129:4797-4806. Kigel B, Varshavsky A, Kessler O, Neufeld G. Successful inhibition of tumor development by specific class-3 semaphorins is associated with expression of appropriate semaphorin receptors by tumor cells. PLoS ONE 2008; 3:e3287. Serini G, Maione F, Bussolino F. Semaphorins and tumor angiogenesis. Angiogenesis 2009 . He Z, Tessier-Lavigne M. Neuropilin is a receptor for the axonal chemorepellent Semaphorin III. Cell 1997; 90:739-751. Kolodkin AL, Levengood DV, Rowe EG, Tai YT, Giger RJ, Ginty DD. Neuropilin is a semaphorin III receptor. Cell 1997; 90:753-762. Chen H, Chedotal A, He Z, Goodman CS, Tessier-Lavigne M. Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III. Neuron 1997; 19:547-559. Castro-Rivera E, Ran S, Thorpe P, Minna JD. Semaphorin 3B (SEMA3B) induces apoptosis in lung and breast cancer, 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 whereas VEGF165 antagonizes this effect. Proc Natl Acad Sci USA 2004; 101:11432-11437. Falk J, Bechara A, Fiore R, et al. Dual functional activity of semaphorin 3B is required for positioning the anterior commissure. Neuron 2005; 48:63-75. Serini G, Valdembri D, Zanivan S, et al. Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function. Nature 2003; 424:391-397. Miao HQ, Soker S, Feiner L, Alonso JL, Raper JA, Klagsbrun M. Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165. J Cell Biol 1999; 146:233-242. Guttmann-Raviv N, Shraga-Heled N, Varshavsky A, Guimaraes-Sternberg C, Kessler O, Neufeld G. Semaphorin3A and semaphorin-3F work together to repel endothelial cells and to inhibit their survival by induction of apoptosis. J Biol Chem 2007; 282:26294-26305. Maione F, Molla F, Meda C, et al. Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models. J Clin Invest 2009; 119:3356-3372. Gu C, Rodriguez ER, Reimert DV, et al. Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development. Dev Cell 2003; 5:45-57. Arese M, Serini G, Bussolino F. Nervous vascular parallels: axon guidance and beyond. Int J Dev Biol 2011; 55:439-445. Soker S, Miao HQ, Nomi M, Takashima S, Klagsbrun M. VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding. J Cell Biochem 2002; 85:357-368. Whitaker GB, Limberg BJ, Rosenbaum JS. Vascular endothelial growth factor receptor-2 and neuropilin-1 form a receptor complex that is responsible for the differential signaling potency of VEGF(165) and VEGF(121). J Biol Chem 2001; 276:25520-25531. Appleton BA, Wu P, Maloney J, et al. Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding. EMBO J 2007; 26:4902-4912. Acevedo LM, Barillas S, Weis SM, Gothert JR, Cheresh DA. Semaphorin 3A suppresses VEGF-mediated angiogenesis yet acts as a vascular permeability factor. Blood 2008; 111:26742680. Toyofuku T, Yoshida J, Sugimoto T, et al. FARP2 triggers signals for Sema3A-mediated axonal repulsion. Nat Neurosci 2005; 8:1712-1719. Sekido Y, Bader S, Latif F, et al. Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns. Proc Natl Acad Sci USA 1996; 93:4120-4125. Tomizawa Y, Sekido Y, Kondo M, et al. Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B. Proc Natl Acad Sci USA 2001; 98:13954-13959. Tse C, Xiang RH, Bracht T, Naylor SL. Human Semaphorin 3B (SEMA3B) located at chromosome 3p21.3 suppresses tumor formation in an adenocarcinoma cell line. Cancer Res 2002; 62:542-546. Varshavsky A, Kessler O, Abramovitch S, et al. SemaphorinCell Research | Vol 22 No 1 | January 2012 Atsuko Sakurai et al. npg 31 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 3B is an angiogenesis inhibitor that is inactivated by furinlike pro-protein convertases. Cancer Res 2008; 68:69226931. Bassi DE, Fu J, Lopez de Cicco R, Klein-Szanto AJ. Proprotein convertases: master switches in the regulation of tumor growth and progression. Mol Carcinog 2005; 44:151-161. van der Zwaag B, Hellemons AJ, Leenders WP, et al. PLEXIN-D1, a novel plexin family member, is expressed in vascular endothelium and the central nervous system during mouse embryogenesis. Dev Dyn 2002; 225:336-343. Roodink I, Raats J, van der Zwaag B, et al. Plexin D1 expression is induced on tumor vasculature and tumor cells: a novel target for diagnosis and therapy?. Cancer Res 2005; 65:83178323. Kim J, Oh WJ, Gaiano N, Yoshida Y, Gu C. Semaphorin 3Eplexin-D1 signaling regulates VEGF function in developmental angiogenesis via a feedback mechanism. Genes Dev 2011; 25:1399-1411. Torres-Vazquez J, Gitler AD, Fraser SD, et al. Semaphorinplexin signaling guides patterning of the developing vasculature. Dev Cell 2004; 7:117-123. Sakurai A, Gavard J, Annas-Linhares Y, et al. Semaphorin 3E initiates antiangiogenic signaling through plexin D1 by regulating Arf6 and R-Ras. Mol Cell Biol 2010; 30:3086-3098. Fukushima Y, Okada M, Kataoka H, et al. Sema3E-plexinD1 signaling selectively suppresses disoriented angiogenesis in ischemic retinopathy in mice. J Clin Invest 2011; 121:19741985. Casazza A, Finisguerra V, Capparuccia L, et al. Sema3Eplexin D1 signaling drives human cancer cell invasiveness and metastatic spreading in mice. J Clin Invest 2010; 120:2684-2698. Zygmunt T, Gay CM, Blondelle J, et al. SemaphorinplexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1. Dev Cell 2011; 21:301-314. Sakurai A, Jian X, Lee CJ, et al. Phosphatidylinositol-4phosphate 5-Kinase and GEP100/Brag2 protein mediate antiangiogenic signaling by semaphorin 3E-plexin-D1 through Arf6 protein. J Biol Chem 2011; 286:34335-34345. van den Bout I, Divecha N. PIP5K-driven PtdIns(4,5)P2 synthesis: regulation and cellular functions. J Cell Sci 2009; 122:3837-3850. Bielenberg DR, Hida Y, Shimizu A, et al. Semaphorin 3F, a chemorepulsant for endothelial cells, induces a poorly vascularized, encapsulated, nonmetastatic tumor phenotype. J Clin Invest 2004; 114:1260-1271. Kessler O, Shraga-Heled N, Lange T, et al. Semaphorin3F is an inhibitor of tumor angiogenesis. Cancer Res 2004; 64:1008-1015. Futamura M, Kamino H, Miyamoto Y, et al. Possible role of semaphorin 3F, a candidate tumor suppressor gene at 3p21.3, in p53-regulated tumor angiogenesis suppression. Cancer Res 2007; 67:1451-1460. Nasarre P, Kusy S, Constantin B, et al. Semaphorin SEMA3F has a repulsing activity on breast cancer cells and inhibits Ecadherin-mediated cell adhesion. Neoplasia 2005; 7:180-189. Wu F, Zhou Q, Yang J, et al. Endogenous axon guiding chemorepulsant semaphorin-3F inhibits the growth and metastasis of colorectal carcinoma. Clin Cancer Res 2011; www.cell-research.com | Cell Research 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 17:2702-2711. Shimizu A, Mammoto A, Italiano JE Jr, et al. ABL2/ARG tyrosine kinase mediates SEMA3F-induced RhoA inactivation and cytoskeleton collapse in human glioma cells. J Biol Chem 2008; 283:27230-27238. Potiron VA, Sharma G, Nasarre P, et al. Semaphorin SEMA3F affects multiple signaling pathways in lung cancer cells. Cancer Res 2007; 67:8708-8715. Karayan-Tapon L, Wager M, Guilhot J, et al. Semaphorin, neuropilin and VEGF expression in glial tumours: SEMA3G, a prognostic marker?. Br J Cancer 2008; 99:1153-1160. Taniguchi M, Masuda T, Fukaya M, et al. Identification and characterization of a novel member of murine semaphorin family. Genes Cells 2005; 10:785-792. Basile JR, Barac A, Zhu T, Guan KL, Gutkind JS. Class IV semaphorins promote angiogenesis by stimulating Rho-initiated pathways through plexin-B. Cancer Res 2004; 64:52125224. Conrotto P, Valdembri D, Corso S, et al. Sema4D induces angiogenesis through Met recruitment by plexin B1. Blood 2005; 105:4321-4329. Basile JR, Castilho RM, Williams VP, Gutkind JS. Semaphorin 4D provides a link between axon guidance processes and tumor-induced angiogenesis. Proc Natl Acad Sci USA 2006; 103:9017-9022. Chng E, Tomita Y, Zhang B, et al. Prognostic significance of CD100 expression in soft tissue sarcoma. Cancer 2007; 110:164-172. Basile JR, Holmbeck K, Bugge TH, Gutkind JS. MT1-MMP controls tumor-induced angiogenesis through the release of semaphorin 4D. J Biol Chem 2007; 282:6899-6905. Zhu L, Bergmeier W, Wu J, et al. Regulated surface expression and shedding support a dual role for semaphorin 4D in platelet responses to vascular injury. Proc Natl Acad Sci USA 2007; 104:1621-1626. Aurandt J, Vikis HG, Gutkind JS, Ahn N, Guan KL. The semaphorin receptor plexin-B1 signals through a direct interaction with the Rho-specific nucleotide exchange factor, LARG. Proc Natl Acad Sci USA 2002; 99:12085-12090. Hirotani M, Ohoka Y, Yamamoto T, et al. Interaction of plexin-B1 with PDZ domain-containing Rho guanine nucleotide exchange factors. Biochem Biophys Res Commun 2002; 297:32-37. Perrot V, Vazquez-Prado J, Gutkind JS. Plexin B regulates Rho through the guanine nucleotide exchange factors leukemia-associated Rho GEF (LARG) and PDZ-RhoGEF. J Biol Chem 2002; 277:43115-43120. Swiercz JM, Kuner R, Behrens J, Offermanns S. PlexinB1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology. Neuron 2002; 35:51-63. Basile JR, Gavard J, Gutkind JS. Plexin-B1 utilizes RhoA and Rho kinase to promote the integrin-dependent activation of Akt and ERK and endothelial cell motility. J Biol Chem 2007; 282:34888-34895. Binmadi NO, Proia P, Zhou H, Yang YH, Basile JR. Rhomediated activation of PI(4)P5K and lipid second messengers is necessary for promotion of angiogenesis by Semaphorin 4D. Angiogenesis 2011; 14:309-319. Swiercz JM, Kuner R, Offermanns S. Plexin-B1/RhoGEF- npg Semaphorin signaling in angiogenesis 32 80 81 82 83 84 85 86 87 88 89 90 91 92 93 mediated RhoA activation involves the receptor tyrosine kinase ErbB-2. J Cell Biol 2004; 165:869-880. Barberis D, Casazza A, Sordella R, et al. p190 Rho-GTPase activating protein associates with plexins and it is required for semaphorin signalling. J Cell Sci 2005; 118:4689-4700. Wigle JT, Oliver G. Prox1 function is required for the development of the murine lymphatic system. Cell 1999; 98:769778. Wigle JT, Harvey N, Detmar M, et al. An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J 2002; 21:1505-1513. Petrova TV, Makinen T, Makela TP, et al. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J 2002; 21:4593-4599. Hong YK, Harvey N, Noh YH, et al. Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate. Dev Dyn 2002; 225:351-357. Karpanen T, Egeblad M, Karkkainen MJ, et al. Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res 2001; 61:17861790. Veikkola T, Jussila L, Makinen T, et al. Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J 2001; 20:12231231. He Y, Kozaki K, Karpanen T, et al. Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling. J Natl Cancer Inst 2002; 94:819-825. Karkkainen MJ, Haiko P, Sainio K, et al. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat Immunol 2004; 5:74-80. Haiko P, Makinen T, Keskitalo S, et al. Deletion of vascular endothelial growth factor C (VEGF-C) and VEGF-D is not equivalent to VEGF receptor 3 deletion in mouse embryos. Mol Cell Biol 2008; 28:4843-4850. Caunt M, Mak J, Liang WC, et al. Blocking neuropilin-2 function inhibits tumor cell metastasis. Cancer Cell 2008; 13:331-342. Xu Y, Yuan L, Mak J, et al. Neuropilin-2 mediates VEGF-Cinduced lymphatic sprouting together with VEGFR3. J Cell Biol 2010; 188:115-130. Favier B, Alam A, Barron P, et al. Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration. Blood 2006; 108:1243-1250. Nathanson SD. Insights into the mechanisms of lymph node metastasis. Cancer 2003; 98:413-423. 94 95 96 97 98 99 100 101 102 103 104 105 Schietroma C, Cianfarani F, Lacal PM, et al. Vascular endothelial growth factor-C expression correlates with lymph node localization of human melanoma metastases. Cancer 2003; 98:789-797. Dadras SS, Lange-Asschenfeldt B, Velasco P, et al. Tumor lymphangiogenesis predicts melanoma metastasis to sentinel lymph nodes. Mod Pathol 2005; 18:1232-1242. Roma AA, Magi-Galluzzi C, Kral MA, Jin TT, Klein EA, Zhou M. Peritumoral lymphatic invasion is associated with regional lymph node metastases in prostate adenocarcinoma. Mod Pathol 2006; 19:392-398. He XW, Yu X, Liu T, Yu SY, Chen DJ. Vector-based RNA interference against vascular endothelial growth factor-C inhibits tumor lymphangiogenesis and growth of colorectal cancer in vivo in mice. Chin Med J 2008; 121:439-444. Das S, Ladell DS, Podgrabinska S, et al. Vascular endothelial growth factor-C induces lymphangitic carcinomatosis, an extremely aggressive form of lung metastases. Cancer Res 2010; 70:1814-1824. Patel V, Marsh CA, Dorsam RT, et al. Decreased lymphangiogenesis and lymph node metastasis by mTOR inhibition in head and neck cancer. Cancer Res 2011; 71:71037112. Casazza A, Fu X, Johansson I, et al. Systemic and targeted delivery of semaphorin 3A inhibits tumor angiogenesis and progression in mouse tumor models. Arterioscler Thromb Vasc Biol 2011; 31:741-749. Roberts N, Kloos B, Cassella M, et al. Inhibition of VEGFR-3 activation with the antagonistic antibody more potently suppresses lymph node and distant metastases than inactivation of VEGFR-2. Cancer Res 2006; 66:2650-2657. Khromova N, Kopnin P, Rybko V, Kopnin BP. Downregulation of VEGF-C expression in lung and colon cancer cells decelerates tumor growth and inhibits metastasis via multiple mechanisms. Oncogene 2011 Aug 1. doi: 10.1038/ onc.2011.330 Kodera Y, Katanasaka Y, Kitamura Y, et al. Sunitinib inhibits lymphatic endothelial cell functions and lymph node metastasis in a breast cancer model through inhibition of vascular endothelial growth factor receptor 3. Breast Cancer Res 2011; 13:R66. Karpanen T, Wirzenius M, Makinen T, et al. Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation. Am J Pathol 2006; 169:708718. Yoo YA, Kang MH, Lee HJ, et al. Sonic hedgehog pathway promotes metastasis via activation of Akt, EMT, and MMP-9 in gastric cancer. Cancer Res 2011; 71:7061-7070. Cell Research | Vol 22 No 1 | January 2012  ...

... Oncogene (2013) 32, 43774386 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE The novel tumor suppressor NOL7 post-transcriptionally regulates thrombospondin-1 expression CL Doci, G Zhou and MW Lingen Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis whose expression suppresses tumor growth in vivo. Like many angiogenesis-related genes, TSP-1 expression is tightly controlled by various mechanisms, but there is little data regarding the contribution of post-transcriptional processing to this regulation. NOL7 is a novel tumor suppressor that induces an antiangiogenic phenotype and suppresses tumor growth, in part through upregulation of TSP-1. Here we demonstrate that NOL7 is an mRNA-binding protein that must localize to the nucleoplasm to exert its antiangiogenic and tumor suppressive effects. There, it associates with the RNA-processing machinery and specically interacts with TSP-1 mRNA through its 30 UTR. Reintroduction of NOL7 into SiHa cells increases luciferase expression through interaction with the TSP-1 30 UTR at both the mRNA and protein levels. NOL7 also increases endogenous TSP-1 mRNA half-life. Further, NOL7 post-transcriptional stabilization is observed in a subset of angiogenesis-related mRNAs, suggesting that the stabilization of TSP-1 may be part of a larger novel mechanism. These data demonstrate that NOL7 signicantly alters TSP-1 expression and may be a master regulator that coordinates the posttranscriptional expression of key signaling factors critical for the regulation of the angiogenic phenotype. Oncogene (2013) 32, 43774386; doi:10.1038/onc.2012.464; published online 22 October 2012 Keywords: NOL7; TSP-1; angiogenesis; post-transcriptional INTRODUCTION Angiogenesis is one of the hallmarks of cancer and is required for tumors to grow beyond a diffusion gradient.1,2 It is regulated by a balance of pro-and antiangiogenic factors, and, in many cancers, the differential regulation of these factors is a precursor to the angiogenic switch and subsequent malignant transformation.35 Current therapies aimed at disrupting this process have been hindered by off-target effects owing to the diversity of angiogenic molecules and their downstream signaling pathways.69 This diversity is mediated in part by the post-transcriptional processing of many of these angiogenesis-related transcripts, where alternative splicing, polyadenylation, stability, and translational control contributes to their expression level and functionality. In some cases, as in the alternative splicing of vascular endothelial growth factor (VEGF) and its receptor, VEGF receptor 1, these processes can change the function of the molecule itself.1013 In other cases, these processes can inuence the bioavailability through modulation of secretory domains or mRNA stability.1418 The post-transcriptional regulation of mRNA stability is particularly critical for the rapid cellular response to internal and external stimuli and is therefore a crucial mechanism in the control of growth factors, cytokines, and angiogenic molecules.19 While advances in our understanding of the post-transcriptional regulation of proangiogenic factors have emerged, considerably less is known about the post-transcriptional processing of antiangiogenic molecules. The rst endogenous antiangiogenic molecule reported was thrombospondin-1 (TSP-1).20,21 TSP-1 suppresses angiogenesis by inhibiting cell migration, inducing apoptosis, and modulating the signaling of growth factors.2224 TSP-1 expression is downregulated in a number of cancers, and reintroduction of TSP-1 has been shown to signicantly suppress tumor growth.2528 Owing to its central role in physiological and pathological angiogenesis, expression of TSP-1 mRNA is tightly controlled, with no known alternative splicing or polyadenylation isoforms. TSP-1 mRNA is post-transcriptionally regulated by TGF-b stimulation, hypoxia, or heat shock, but the underlying mechanism is poorly understood.2934 Post-transcriptional regulation is achieved in part through interaction with RNA-binding proteins (RBPs) such as HuR. HuR regulates a number of angiogenesis-related transcripts, including VEGF, COX-2, and HIF-1a3538 and recently has been shown to bind to the 30 UTR of TSP-1.39 While many of the well-characterized mRNA-stabilizing RBPs such as HuR function in the cytoplasm, the existence of signicant parallel mechanisms for nuclear RNA turnover argues for similar stabilizing roles in this compartment. Many of the decay pathways active in the nucleus are tied to processing events such that degradation of aberrant transcripts and further processing of mRNAs occur co-transcriptionally and are tied to the RNA polymerase II machinery in ribonucleoprotein (RNP) complexes.4043 These RNPs, including nuclear-processing factors such as XRN2, EXOSC10, TRAMP, and others are critical for appropriate expression tied to transcriptional termination, 30 end processing, and mRNA quality control.4455 Within the nucleus, RBPs have a central role in coordinating mRNA quality control through alternative decay, stabilization, and export.5659 NOL7 is a novel tumor suppressor that induces an antiangiogenic phenotype through differential regulation of VEGF and TSP-1.60 However, the mechanism underlying this function is unknown. In this study, we demonstrate that NOL7 must reside in the nucleoplasm to upregulate TSP-1 and induce an antiangiogenic and tumor suppressive phenotype. There, it functions as an RBP that associates with the RNA-processing machinery. NOL7 interacts with polyadenylated transcripts, specically with TSP-1 mRNA through its 30 UTR. Reintroduction of NOL7 increases Departments of Pathology, Medicine and Radiation, and Cellular Oncology, University of Chicago, Chicago, IL, USA. Correspondence: Dr MW Lingen, Department of Pathology, Medicine and Radiation, and Cellular Oncology, University of Chicago, 5841S. Maryland Avenue MC6101, Chicago, IL 60637, USA. E-mail: mark.lingen@uchospitals.edu Received 18 April 2012; revised 22 August 2012; accepted 24 August 2012; published online 22 October 2012 NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4378 reporter gene expression through this interaction at both the mRNA and protein levels. This is also observed in endogenous TSP-1, where reintroduction of NOL7 signicantly increases the TSP-1 transcript half-life. NOL7 post-transcriptional stabilization is limited to a distinct subset of mRNAs through a novel mechanism. Together, these data demonstrate that NOL7 is a critical regulator of TSP-1 expression and may participate in the coordination of cellular signaling pathways through post-transcriptional mRNA regulation to control the angiogenic process. RESULTS NOL7 must reside in the nucleoplasm to induce an antiangiogenic phenotype and suppress tumor growth NOL7 is a highly basic protein that suppresses tumor growth and induces an antiangiogenic phenotype in part through upregulation of TSP-1 expression.60 NOL7 is localized exclusively to the nucleus and nucleolus of cells and completely absent from the cytoplasm (Supplementary Figure S1), but it is not known if NOL7 is sequestered or functional within these compartments. To determine the compartment in which NOL7 must reside for its function, we made a series of mutational constructs that targeted NOL7 to the nucleolus (NOL7 wild-type), nucleoplasm (N23(  )) or cytoplasm (N123(  )) (Zhou et al 61; Figure 1a). In SiHa cells, which essentially lack endogenous NOL7,60,62 reintroduction of wild-type nucleolar NOL7 was able to signicantly upregulate endogenous TSP-1 expression compared with GFP controls, while cytoplasmic NOL7 had no effect on TSP-1 levels (Figure 1b). However, nucleoplasmic NOL7 was still able to upregulate endogenous TSP-1 to the same levels as wild-type NOL7 (Figure 1b). In the same fashion, there was no difference in endothelial cell migration between cells treated with conditioned media from GFP control or cytoplasmic NOL7 (Figure 1c). However, conditioned media from wild-type NOL7 and nucleoplasmic NOL7 were able to suppress Figure 1. NOL7 must reside in the nucleus to modulate TSP-1 expression, inhibit endothelial cell migration and suppress tumor growth. (a) Cells were transfected with GFP-tagged wild-type NOL7 or mutants that target NOL7 to the nucleoplasm (N23(  ), clones 1 and 2) or the cytoplasm (N123(  ), clones 1 and 2). Cell nuclei were counterstained with DAPI and imaged by fluorescence microscopy. (b) Conditioned media from transfected cells was analyzed by enzyme-linked immunosorbent assay and concentration calculated from a standard curve. Data are represented as means.e.m. Significance was calculated using Students t-test from three independent experiments. *Po0.005; **Po0.002; ***Po0.001; n.s., not significant. (c) Serum-free conditioned media from parental SiHa, GFP and NOL7 wt- and mutant-transfected clones were functionally tested for their ability to stimulate endothelial cell migration. Data are represented as means.e.m. Significance was calculated using Students t-test from four independent experiments. *Po1  10  5; **Po1  10  8; n.s., not significant. (d) SiHa parental, GFP and NOL7 wtand mutant-transfected cells were subcutaneously injected into nude mice and monitored over a period of 30 (n 6 animals per group). Data are represented as means.e.m. Significance was calculated using two-way analysis of variance. *Po0.001; **Po0.0001; n.s., not significant. (e) Tumor angiogenesis was assessed by CD31 staining, followed by microvessel density quantification. Data are represented as means.e.m. Significance was calculated using Students t-test (n 6 animals per group). *Po2  10  6; ** Po1  10  10; n.s., not significant. Oncogene (2013) 4377 4386 & 2013 Macmillan Publishers Limited NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4379 Figure 2. NOL7 interacts with a large RNP complex in an RNA-dependent manner. NOL7-transfected lysate was separated on a 1030% gradient. Individual fractions were collected and plotted on the basis of their absorbance at 260 nm. Individual fractions were also subjected to western blotting for NOL7. The co-migration of NOL7 with a large RNP complex is marked with a box. (a) Total lysate was evaluated for association of NOL7 into RNP complexes. (b) Lysate was either mock-treated or digested with RNase A before separation. endothelial cell migration to the same degree (Figure 1c). This correlated with a signicant suppression in tumor growth, where both nucleoplasmic and nucleolar NOL7 demonstrated 90% less tumor volume than GFP control or cytoplasmic NOL7 (Figure 1d). Tumor angiogenesis, as assessed by CD31 microvessel density, revealed that wild-type and nucleoplasmic NOL7 suppressed tumor angiogenesis, whereas GFP control and the cytoplasmic NOL7 mutant displayed robust angiogenesis in these tumors (Figure 1e). Taken together, this suggests that NOL7 must reside in the nucleoplasm to induce an antiangiogenic phenotype and suppress tumor growth. NOL7 interacts with a large ribonucleoprotein complex in an RNAdependent manner To determine the potential mechanism by which NOL7 suppresses tumor growth and induces an antiangiogenic phenotype in the nucleoplasm, we hypothesized that NOL7 may be part of a nucleic-acid-interacting complex. To test this, we performed sucrose gradient ultracentrifugation of NOL7-expressing lysate (Figure 2a). NOL7 co-migrated with a large, B70S complex (Figure 2a, box). To determine if the association of NOL7 was dependent upon RNA, lysate was either mock-treated or treated with RNase, and separated as before (Figure 2b). Treatment with RNase caused NOL7 to revert completely to the soluble fraction & 2013 Macmillan Publishers Limited (Figure 2b, arrow). This demonstrates that NOL7 interacts with a large RNP complex in an RNA-dependent manner. NOL7 interacts with mRNA-processing factors To identify the RNP complex and protein cofactors of NOL7, mass spectroscopic (MS) analysis was performed on comigrating NOL7 immunoprecipitates (Figure 3a). This sample was highly enriched for RNA-processing factors, particularly those involved in mRNA maturation. To conrm the putative cofactors identied by mass spectroscopy, coimmunoprecipitation of NOL7 or controls was performed and analyzed by western blot against the endogenous MS-identied proteins. To determine if these putative cofactors interact in an RNA-dependent or -independent manner, lysates were mock-treated or digested with RNase A before immunoprecipitation (IP). SR proteins SF2-ASF and SRp40, RNAprocessing factor NCL, and the 30 -processing and decay factors XRN2, CNOT3, and CPSF2 were found to specically associate with NOL7 in either the presence of absence of RNA (Figure 3b). In addition, the multifunctional RBPs HuR, NPM, and EXOSC10 associated with NOL7 only in the presence of RNA. These data demonstrate that NOL7 interacts with mRNA-processing complexes and specically associates with proteins involved in mRNA maturation. Oncogene (2013) 4377 4386 NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4380 Figure 3. NOL7 interacts with 30 end-processing proteins. (a) Lysate from SiHa cells stably expressing GFP-V5 or NOL7-V5 was separated by gradient ultracentrifugation and the large 70S fractions were pooled, immunoprecipitated and separated by SDSpolyacrylamide gel electrophoresis and stained with Coomassie before analysis by mass spectroscopy. Data was curated from the mass spectroscopy results to identify putative functional cofactors of NOL7. (b) GFP-V5 or NOL7-V5 lysate was mock-treated (  ) or digested with RNase ( ). Lysates were immunoprecipitated using a-V5-conjugated beads and coimmunoprecipitating proteins were analyzed by western blot. As a control for RNase digestion, RNA was extracted from the lysates after treatment, reverse transcribed and RTPCR against the 18S rRNA was performed. NOL7 interacts specically with mRNA The protein cofactors, identied suggested that NOL7 may be involved in mRNA processing. To determine if NOL7 interacts with mRNA, total lysate overexpressing GFP control or NOL7 was incubated with oligo(dT) beads to precipitate polyadenylated transcripts and coprecipitated proteins were visualized by western blotting. NOL7, but not GFP, interacted with the polyA transcripts, demonstrating that this was not an artifact of overexpression (Figure 4, lanes 3 and 4). This was not due to nonspecic binding to the beads, as treatment of the lysate with RNase before incubation completely abolished NOL7 binding (Figure 4, lane 5). Finally, to demonstrate that this interaction was mRNA-specic and not a random, charge-based interaction, the competition of the bound proteins with either polyA or polyC at ve or ten times the input RNA was performed. NOL7 binding was lost in the polyA treatments (Figure 4, lanes 6 and 8) but unaffected by polyC competition (Figure 4, lanes 7 and 9), indicating that NOL7 interacts specically with mRNA. NOL7 interacts with TSP-1 mRNA through its 30 UTR NOL7 induces an antiangiogenic phenotype in the nucleus through upregulation of TSP-1 (Figure 1). To determine if NOL7 interacts with TSP-1 mRNA, total lysate overexpressing GFP, NOL7, or the TSP-1 mRNA-binding protein HuR was immunoprecipitated and the associated mRNAs were analyzed by northern blotting (Figure 5a). TSP-1 mRNA was signicantly associated with both NOL7 and HuR, and absent from GFP (Figure 5a). This demonstrates that NOL7 is capable of specically interacting with TSP-1 mRNA. The association of NOL7 with polyadenylated transcripts and 30 -processing factors suggested that NOL7 might interact with TSP-1 through its 30 UTR. To determine if NOL7 might bind in this region, the 30 UTR of TSP-1 or R01, a random, nongenic sequence of the same length were in vitro transcribed and Oncogene (2013) 4377 4386 biotinylated. Total lysate from cells overexpressing GFP, NOL7, or HuR were incubated with increasing amounts of biotinylated transcript, precipitated using streptavidin beads, and analyzed by western blotting (Figure 5b). GFP did not bind to either transcript at any of the concentrations assayed. Both NOL7 and HuR bound to TSP-1 30 UTR in a dose-dependent manner, but was not detected in the negative-control lanes (Figure 5b). Together, this demonstrates that NOL7 interacts specically with TSP-1 mRNA through its 30 UTR. The 30 UTR of TSP-1 is sufcient for NOL7-mediated posttranscriptional upregulation Interaction of NOL7 with the 30 UTR of TSP-1 suggested it may have a role in its post-transcriptional regulation. To determine if this interaction can affect downstream expression levels, luciferase reporters were cloned in-frame with the 30 UTR of TSP-1, positivecontrol SV40 late polyA signal (EMP), or a negative-control nongenic (R01) sequence. SiHa cells express extremely low levels of endogenous NOL7, such that reintroduction in stable cell lines restored NOL7 to near-endogenous levels observed in 293T cells, and expression of exogenous NOL7 or HuR did not affect endogenous levels of either gene (Supplementary Figure S2). Luciferase levels for clones bearing the TSP-1 30 UTR were signicantly increased in NOL7- and HuR-expressing cells (Figure 6). No difference was observed between GFP, NOL7, or HuR in EMP or R01 constructs, indicating that the increase in luciferase was due specically to regulation through the TSP-1 30 UTR. To determine if this upregulation was due to an increase in mRNA levels or through an increase in translational rate or protein stability, luciferase levels were analyzed by real-time PCR. The upregulation of luciferase was observed at the mRNA as well as protein levels, suggesting that the NOL7 regulates expression at the level of mRNA abundance. & 2013 Macmillan Publishers Limited NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4381 Figure 4. NOL7 specifically interacts with mRNA. HEK293T cells were transfected with GFP control or NOL7. Proteins associated with poly(A) mRNA were pulled down using oligo(dT) cellulose. Input lanes represent 10% of total input. As controls, lysate was digested with RNase A before incubation or bound proteins were competed using five or ten times input RNA of polyA or polyC. Coprecipitation of GFP and NOL7 was evaluated by western blot. Data are represented as means.e.m. Significance was calculated using Students t-test relative to NOL7 input (a, b) and relative to the NOL7-bound (c, d) fractions. aPo0.002; bPo4  10  4; cPo0.001; d Po0.0001. Statistical significance between groups was also calculated (bars). *Po0.001, **Po1  10  4. Data are represented as means.e.m. from three independent assays. NOL7 post-transcriptionally regulates TSP-1 expression To determine if NOL7 could post-transcriptionally upregulate endogenous TSP-1, two clones each from SiHa cells stably expressing GFP, NOL7 or HuR were treated with a-amanitin to block RNA Pol II-mediated mRNA transcription. TSP-1 expression levels were measured by real-time PCR for increasing durations of a-amanitin treatment. In cells expressing GFP, TSP-1 expression fell to almost half its original level within 1 hour. However, cells expressing NOL7 or HuR decreased only slightly throughout the timecourse (Figure 7a). Reintroduction of NOL7 doubled the halflife of endogenous TSP-1 mRNA (Figure 7b), suggesting that NOL7 regulates TSP-1 expression by enhancing the post-transcriptional stability of its mRNA. NOL7 may stabilize a specic subset of angiogenesis-related mRNAs To determine if NOL7 was capable of post-transcriptionally stabilizing other transcripts in addition to TSP-1, we assessed steady-state and post-transcriptional mRNA levels in a panel of angiogenesis-related mRNAs. SiHa cells stably expressing GFP or NOL7 were left untreated or transcriptionally inhibited with a-amanitin for 4 hours, when the maximal effect of NOL7 posttranscriptional stabilization was observed for TSP-1 (Figure 7). Using the TaqMan 384-well Human Angiogenesis Array, we found approximately one-third of the mRNAs analyzed were differentially expressed at a statistically signicant level between the untreated samples, suggesting that NOL7 can modulate the steady-state expression of angiogenesis-related genes at the mRNA level (Figure 8a). Within these genes, a specic subset of mRNAs was post-transcriptionally stabilized upon reintroduction of NOL7 (Figure 8b). Further, over half of the post-transcriptionally stabilized genes were also upregulated and functionally associated with antiangiogenic and antitumorigenic cell phenotypes & 2013 Macmillan Publishers Limited Figure 5. NOL7 coimmunoprecipitates TSP-1 mRNA. (a) Lysate from cells expressing GFP, NOL7 or HuR were immunoprecipitated with a-V5 beads and RNA was extracted. RNA was then subjected to northern blotting using a probe against the 30 UTR of TSP-1. Western blotting to confirm equivalent protein IP was performed. Densitometry scanning was performed on northern bands and normalized to protein IP lanes. Data are represented as means.e.m. from three independent experiments. *Po0.01. (b) The 30 UTR of TSP-1 and a negative-control sequence R01 were in vitro transcribed and biotinylated. Increasing amounts of transcript were incubated with lysate expressing GFP, NOL7 or HuR. Transcripts were precipitated using streptavidin beads and coprecipitating proteins were analyzed by western blot. (Figure 8c). Together, this indicates that NOL7 is capable of modulating the expression of a specic subset of mRNAs posttranscriptionally, while others may be differentially regulated as a consequence of NOL7 modulation of upstream targets in linked signaling pathways. Finally, it indicates that for some genes, such as TSP-1, this post-transcriptional regulation may signicantly alter the available mRNA pool and inuence downstream function and phenotype. DISCUSSION NOL7 is a novel tumor suppressor that signicantly suppresses in vivo tumor growth and induces an antiangiogenic phenotype in part through upregulation of TSP-1. In this work, we characterize NOL7 as a novel RBP that increases TSP-1 expression through post-transcriptional mRNA stabilization. NOL7 must reside in the nucleoplasm to exert its anticancer phenotype. Within the nucleus, NOL7 is component of a large, RNA-dependent RNP comprised of mRNA-processing factors. NOL7 specically interacted with a number of these putative cofactors as well as polyadenylated transcripts. NOL7 was shown to interact specically with the TSP-1 transcript through binding to its 30 UTR. Reporter constructs bearing the TSP-1 30 UTR were signicantly upregulated at both the mRNA and protein level, and endogenous TSP-1 mRNA was stabilized in cells re-expressing NOL7. Finally, this post-transcriptional regulation was demonstrated to be specic to a subset of angiogenesis-related mRNAs. Taken together, this demonstrates that NOL7 is a novel RBP that post-transcriptionally upregulates TSP-1 through an increase in mRNA stability. Further, this suggests that NOL7 may regulate the antiangiogenic phenotype and suppress tumor growth through post-transcriptional modulation of gene expression. Interaction of NOL7 with components of the RNA-processing machinery in a large RNP suggests that NOL7 may contribute to Oncogene (2013) 4377 4386 NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4382 Figure 6. The 30 UTR of TSP-1 is sufficient for NOL7-mediated post-transcriptional upregulation. Two clones each from SiHa cells stably co-expressing GFP, NOL7 or HuR and luciferase-EMP, -R01 or TSP-1 were assayed for luciferase expression at the mRNA (dark-gray bar) or protein (light-gray bar). To control for vector expression artifacts, values were normalized to luciferase-EMP and reported as a percentage of GFP expression for each set of constructs. Data are represented as means.e.m. from four independent experiments. *Po3  10  5; **Po9  10  5; ***Po2  10  7. Figure 7. NOL7 post-transcriptionally stabilizes TSP-1 mRNA. SiHa cells stably expressing GFP, NOL7 or HuR were treated for 0, 2, 4 or 6 with 5 mg/ml a-amanitin. Endogenous TSP-1 levels were assayed by real-time PCR and calculated via DDCt method. Half-life calculations were calculated from the nonlinear regression of the exponential decay curve N0 N(t)e  lt, where the TSP-1 mRNA half-life t1/2  ln(2)/l. Data are represented as means.e.m. from four independent experiments. *Po0.03; **Po0.001; ***Po0.0001. the co-transcriptional processing of mRNA at multiple levels. These data characterize the role of NOL7 during the 30 end processing and maturation of TSP-1 mRNA, but do not exclude the possibility that NOL7 may contribute to other aspects of mRNA metabolism. It has been demonstrated that the efcacy of downstream processing steps can form feedbacks that inuence upstream transcriptional initiation and elongation.63,64 In addition, some nuclear mRNA complexes are active both in late-maturation and early-initiation steps of processing. One such complex, the CCR4-Not complex, has roles in mRNA transcription and degradation and its activity can inuence cellular signaling pathways.40,41 NOL7 was recently identied in a proteomic study of the CCR4-Not complex as a factor that interacts specically with multiple core subunits of the complex,65 validated in this work by evidence that NOL7 specially interacts with the subunit CNOT3 (Figure 3). Similarly, NOL7 also interacts specically with the 50 -30 endonuclease complex and nuclear exosome via its binding with XRN2 and EXOSC10. While it is unclear if NOL7 functions as a part of these complexes, it does suggest that NOL7 could be playing a role as a master regulator of gene expression through regulation of mRNA maturation and degradation, and subsequent control of critical signaling molecules on multiple levels. This may be particularly signicant in its regulation of the angiogenic phenotype, as feedback mechanisms and integrated signaling can have a major role in driving the angiogenic switch. Oncogene (2013) 4377 4386 Angiogenesis is critical in cancer development and represents a promising target for therapy. However, the diversity and redundancy of many angiogenic molecules, coupled with the complexity of angiogenic signaling, have hindered progress in the eld. Particularly, the ability of post-transcriptional regulation to rapidly and signicantly alter the signaling capability of many of these factors, and in some cases change the functionality of these molecules entirely has been overlooked.12,6670 Proangiogenic molecules such as broblast growth factor 2 (FGF-2), VEGF and COX-2 all undergo alternative post-transcriptional processing that results in modulation of their half-life and downstream functionality.17,18,7175 Importantly, some major angiogenic signaling factors such as TGF-b have been found to modulate gene expression through a dual regulation of transcriptional activity and mRNA turnover.68 Coupled with constitutive low-level expression of many of these mRNAs, rapid changes in mRNA stability can signicantly alter the steadystate pool of a given mRNA in response to stimulus19 and represent a key target for modulating the angiogenic phenotype in cancer. While post-transcriptional regulation of proangiogenic factors has been described in the literature, evidence regarding alternative processing and stability and angiogenic inhibitors is lacking. Despite decades of research focused on TSP-1, few reports address the issue of its post-transcriptional regulation. The evidence available suggests that post-transcriptional regulation is tied to conditions that promote or suppress tumorigenesis in vivo, such as & 2013 Macmillan Publishers Limited NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4383 Figure 8. NOL7 specifically regulates distinct subset of angiogenic transcripts. Total complementary DNA from GFP- or NOL7-expressing cells untreated or transcriptionally inhibited with a-amanitin were analyzed by real-time PCR on TaqMan angiogeniesis array cards. Data are represented as means.e.m. from three independent experiments. (a) Steady-state expression (untreated NOL7 normalized to untreated GFP). Dashed line indicates the GFP control normalization. aPo0.05; bPo0.01; cPo0.001; dPo0.0001. (b) Post-transcriptional expression (a-amanitin-treated GFP/NOL7 normalized to untreated controls). *Po0.05, **Po0.01. (c) Genes significantly upregulated (yellow), unchanged (white), or downregulated (blue) are represented schematically based on their functional annotations, ligand-receptor interactions, and signaling. Those genes that were post-transcriptionally stabilized by NOL7 are outlined in red. hypoxia or oncogene expression. For example, TSP-1 mRNA is destabilized by overexpression of the oncogene Myc or hypoxic conditions, while its half-life is increased by heat shock or TGF-b stimulation.2934 Interestingly, TGF-b is activated by TSP-1, indicating that the post-transcriptional stabilization may be part of a feedback loop to suppress tumor growth. To the best of our knowledge, no studies have investigated any tumor-associated somatic mutations within the TSP-1 30 UTR. The contribution of 30 UTR cis-elements to the overall regulation of TSP-1 expression and the trans-factors that bind to them, such as AUF1 and HuR, are also not well characterized. Here, we demonstrate that NOL7 is a novel TSP-1 30 UTR-interacting protein that stabilizes the TSP-1 transcript. NOL7 may represent a novel type of RBP in cancer, because of its unique domain structure and role as a tumor suppressor in vivo. & 2013 Macmillan Publishers Limited NOL7 lacks any sequence similarity to known proteins or domains, suggesting NOL7 may employ a novel method of interaction with its targets. While the data here are insufcient to identify a NOL7interacting cis-element, it does rule out classic binding elements such as AREs, as VEGF, FGF-2, and interleukin 8 are not posttranscriptionally regulated by NOL7. Nonetheless, these data suggest that NOL7 binds and regulates a specic subset of mRNAs, and for some of these transcripts, that stabilization may signicantly alter the steady-state expression level. In addition, some genes were differentially regulated only at the steady-state, suggesting that for some genes the effect of NOL7 expression may be propagated through signaling pathways or be secondary effects of NOL7s function on upstream targets. Assessment of mRNA levels over a transcriptional inhibition timecourse will be necessary to conrm the modulation of mRNA half-life and Oncogene (2013) 4377 4386 NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4384 expansion of these results to include more genes and pathways is necessary. For example, a major angiogenic transcription factor, HIF-1a, was not proled on this array, and could contribute signicantly to the differential expression of NOL7 targets, including downstream genes affected only at the steady state. The differentially regulated genes further suggest that NOL7 may be having a role in mediating focal adhesion, remodeling of the extracellular matrix and epithelial to mesenchymal (EMT) transition through its post-transcriptional mechanisms, pathways that are critical for tumor growth suppression, metastasis and antiangiogenic therapy. Acquisition of an EMT phenotype is associated with acquired resistance to angiogenic therapies, metastasis, and has recently been tied to global enrichment of ARE-containing mRNAs.7678 Therefore, the role of NOL7 in posttranscriptional regulation of these phenotypes may be critical in avoiding acquired resistance and increasing the efcacy of current angiogenic therapies. In conclusion, NOL7 is a novel tumor suppressor that must reside in the nucleoplasm to suppress in vivo tumor growth. Reintroduction of NOL7 induces an antiangiogenic phenotype drive in part by the post-transcriptional stabilization of TSP-1. This is achieved through specic interaction with the TSP-1 30 UTR, which is sufcient to post-transcriptionally upregulate gene expression at the mRNA and protein levels. Finally, this posttranscriptional regulation was demonstrated to be specic to a small subset of mRNAs. Taken together, this demonstrates that NOL7 is a novel RBP that post-transcriptionally upregulates TSP-1 through an increase in mRNA stability. Further characterization of the mechanism underlying this function and the phenotypic consequences will illustrate the potential role of NOL7 as a master regulator of the angiogenic phenotype through post-transcriptional modulation of gene expression. 2% sucrose, protease inhibitors). RNA digestion was performed by incubation with 500 mg/ml RNase A and 50 mg/ml EDTA at 37 1C for 1 h. All gradients were separated at 27 500 rpm at 4 1C on Beckman LM-80 Centrifuge (Beckman-Coulter, Inc., Brea, CA, USA) for 8 h. Equivalent fractions were collected manually measured for absorbance at 260 nm. Immunoprecipitation and pulldowns Cells stably expressing GFP-V5 or NOL7-V5 were resuspended in sucrose lysis buffer and lysed by freezethaw. RNase digestion was performed, as described above. For IP, 250 mg total protein was mock-treated or RNase digested and incubated with 25 ml a-V5 agarose beads (SigmaAldrich, St Louis, MO, USA). Puried complementary DNA was reverse transcribed using the Superscript III one-step RT-PCR kit and amplied with 18S specic primers (Invitrogen, Carlsbad, CA, USA). For oligo(dT) pulldowns, 500 A260 units were bound to 10 mg oligo(dT) cellulose beads (Ambion) and incubated with buffer alone or buffer containing 2500 or 5000 A260 units of polyadenylic or polycytidylic acid (SigmaAldrich). For 30 UTR pulldowns, the 30 UTR of TSP-1 or R01 was in vitro transcribed using the MEGAscript kit from Ambion (Ambion/Applied Biosystems, Austin, TX, USA). RNA was incubated with 100 mg lysate and bound to streptavidin Dynabeads (Invitrogen). In all cases, beads were washed thoroughly and bound proteins were eluted by boiling in SDS sample buffer. Results were quantied using Bio-Rad QuantityOne software (Bio-Rad) and normalized to input. Statistical signicance was determined from three independent assays using Students t-test. 30 UTR luciferase assays Two clones each of SiHa cells stably co-expressing GFP, NOL7, or HuR and EMP, R01, or TSP-1 30 UTR luciferase reporter constructs were measured using the Steady-Glo luciferase assay system (Promega, Madison, WI, USA), according to manufacturers instructions. Values for GFP, NOL7 and HuR clones were averaged, normalized to luciferase control, and reported as a percentage of GFP. Luciferase mRNA levels were measured by quantitative PCR. Statistical signicance was calculated using Students t-test from four independent assays against log values to control for normalization bias. MATERIALS AND METHODS Further details and additional methods can be found in the Supplementary section. Cell culture and uorescence microscopy HEK293T and SiHa cells were obtained from the ATCC (Manassas, VA, USA) and cultured, as previously described.60,61 Fluorescence microcopy was performed as described.61 Quantitative real-time PCR RNA levels were measured by real-time quantitative PCR using the Ag-Path One Step RTPCR kit (Ambion/Applied Biosystems). For each, 30 ng total RNA was amplied on the CFX-1000 (Bio-Rad) and detected using TaqMan probes against target transcripts (Applied Biosystems). Relative expression levels were calculated using the DDCt method relative to 18S. Statistical differences were calculated as indicated. In vivo tumor studies, ELISA and migration assay Conditioned media from the SiHa parental, GFP control, NOL7 wild-type or NOL7 mutant cells were assayed for TSP-1 and VEGF by enzyme-linked immunosorbent assay (ELISA), as described by the manufacturer (R&D Systems, Minneapolis, MN, USA). The migration assay was performed, as previously described.79 For tumor studies, 107 cells in PBS were injected subcutaneously into 68-week-old female nu/nu mice (Charles River Laboratories, Wilmington, MA, USA) (n 6 per group). Tumor growth was monitored with a caliper. Statistics were calculated using two-way analysis of variance. Microvessel density was calculated from CD31 staining, as previously described.60 Measurement of post-transcriptional mRNA abundance Two clones each of SiHa cells stably co-expressing GFP, NOL7 or HuR were plated in six-well plates, washed with PBS and transcriptionally inhibited in complete media containing 5 mg/ml a-amanitin for 0, 2, 4 or 6 hours. After treatment, RNA was collected from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Post-transcriptional TSP-1 abundance was measured by real-time quantitative PCR. TSP-1 mRNA levels were normalized to time zero and plotted as a function of a-amanitin treatment duration such that N0 N(t)e  lt. Half-life was calculated as t1/2  ln(2)/l. Statistical signicance was calculated using two-way analysis of variance and Students t-test from four independent assays. Northern and western blotting Northern blotting was performed using the NorthernMax kit (Ambion, Austin, TX, USA). The TSP-1-specic RNA probe was in vitro transcribed and labeled with [a-32P]-UTP (PerkinElmer, Waltham, MA, USA) using the MAXIscript kit, according to the manufacturers instructions (Ambion). For westerns, proteins were separated on SDSpolyacrylamide gel electrophoresis gels, transferred to ImmunoBlot PVDF membrane (Bio-Rad, Hercules CA, USA), blocked in 5% milk-TBST and probed overnight. Secondary was probed at 10 ng/ml for 1 h at room temperature. Blots were visualized using Pierce SuperSignal West Dura substrate. Sucrose gradient ultracentrifugation Lysate was separated on 1030% continuous gradients prepared manually in sucrose gradient buffer (50 mM TrisHCl, 80 mM KCl, 5 mM Mg(C2H3O2)2, Oncogene (2013) 4377 4386 TaqMan Array SiHa cells stably expressing GFP or NOL7 were untreated or transcriptionally inhibited with 5 mg/ml a-amanitin for 4 h. After treatment, RNA was collected from cells using the RNeasy Mini Kit (Qiagen) and reverse transcribed with Superscript VILO (Invitrogen). 100 ng complementary DNA from treated and untreated GFP and NOL7 expressing cells was combined with TaqMan Gene Expression Master Mix, loaded onto the Human Angiogenesis Array, and analyzed on the 7900HT Fast real-time PCR system (Applied Biosystems). For steady state, expression was calculated between untreated samples relative to GFP. For post-transcriptional expression, mRNA levels were normalized to untreated samples and compared directly. Statistical signicance was calculated using Students t-test from an average of three independent assays. & 2013 Macmillan Publishers Limited NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4385 CONFLICT OF INTEREST The authors declare no conict of interest. ACKNOWLEDGEMENTS We wish to thank Drs Ira Wool and Yuen-Ling Chan for their assistance in sucrose gradient ultracentrifugation. This work was supported in part by Illinois Department of Public Health Penny Severns Cancer Research Fund (MWL). REFERENCES 1 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 5770. 2 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell (Research Support, NIH, Extramural Review) 2011; 144: 646674. 3 Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407: 249257. 4 Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol 2002; 29(6 Suppl 16): 1518. 5 Carmeliet P. Angiogenesis in health and disease. Nat Med 2003; 9: 653660. 6 Bergers G, Hanahan D. Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 2008; 8: 592603. 7 Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005; 438: 967974. 8 Heath VL, Bicknell R. Anticancer strategies involving the vasculature. Nat Rev Clin Oncol 2009; 6: 395404. 9 Quesada AR, Medina MA, Alba E. Playing only one instrument may be not enough: limitations and future of the antiangiogenic treatment of cancer. Bioessays 2007; 29: 11591168. 10 Nowak DG, Woolard J, Amin EM, Konopatskaya O, Saleem MA, Churchill AJ et al. Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors. J Cell Sci 2008; 121(Pt 20): 34873495. 11 Rennel E, Waine E, Guan H, Schuler Y, Leenders W, Woolard J et al. The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice. Br J Cancer 2008; 98: 12501257. 12 He Y, Smith SK, Day KA, Clark DE, Licence DR, Charnock-Jones DS. Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT-1) pre-mRNA is important for the regulation of VEGF activity. Mol Endocrinol 1999; 13: 537545. 13 Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci 2001; 114(Pt 5): 853865. 14 Harper SJ, Bates DO. VEGF-A splicing: the key to anti-angiogenic therapeutics? Nat Rev Cancer 2008; 8: 880887. 15 Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997; 18: 425. 16 Yamazaki Y, Morita T. Molecular and functional diversity of vascular endothelial growth factors. Mol Divers 2006; 10: 515527. 17 Hall-Pogar T, Zhang H, Tian B, Lutz CS. Alternative polyadenylation of cyclooxygenase-2. Nucleic Acids Res 2005; 33: 25652579. 18 Lukiw WJ, Bazan NG. Cyclooxygenase 2 RNA message abundance, stability, and hypervariability in sporadic Alzheimer neocortex. J Neurosci Res 1997; 50: 937945. 19 Hargrove JL, Schmidt FH. The role of mRNA and protein stability in gene expression. FASEB J 1989; 3: 23602370. 20 Rastinejad F, Polverini PJ, Bouck NP. Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 1989; 56: 345355. 21 Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci USA 1990; 87: 66246628. 22 Volpert OV. Modulation of endothelial cell survival by an inhibitor of angiogenesis thrombospondin-1: a dynamic balance. Cancer Metastasis Rev 2000; 19: 8792. 23 Ren B, Yee KO, Lawler J, Khosravi-Far R. Regulation of tumor angiogenesis by thrombospondin-1. Biochim Biophys Acta 2006; 1765: 178188. 24 Lawler J, Detmar M. Tumor progression: the effects of thrombospondin-1 and -2. Int J Biochem Cell Biol 2004; 36: 10381045. 25 Volpert OV, Stellmach V, Bouck N. The modulation of thrombospondin and other naturally occurring inhibitors of angiogenesis during tumor progression. Breast Cancer Res Treat 1995; 36: 119126. 26 Bleuel K, Popp S, Fusenig NE, Stanbridge EJ, Boukamp P. Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc Natl Acad Sci USA 1999; 96: 20652070. 27 Streit M, Velasco P, Brown LF, Skobe M, Richard L, Riccardi L et al. Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas. Am J Pathol 1999; 155: 441452. & 2013 Macmillan Publishers Limited 28 Weinstat-Saslow DL, Zabrenetzky VS, VanHoutte K, Frazier WA, Roberts DD, Steeg PS. Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Cancer Res 1994; 54: 65046511. 29 Kang JH, Kim SA, Hong KJ. Induction of TSP1 gene expression by heat shock is mediated via an increase in mRNA stability. FEBS Lett 2006; 580: 510516. 30 Okamoto M, Ono M, Uchiumi T, Ueno H, Kohno K, Sugimachi K et al. Up-regulation of thrombospondin-1 gene by epidermal growth factor and transforming growth factor beta in human cancer cells--transcriptional activation and messenger RNA stabilization. Biochim Biophys Acta 2002; 1574: 2434. 31 Phelan MW, Forman LW, Perrine SP, Faller DV. Hypoxia increases thrombospondin-1 transcript and protein in cultured endothelial cells. J Lab Clin Med 1998; 132: 519529. 32 Janz A, Sevignani C, Kenyon K, Ngo CV, Thomas-Tikhonenko A. Activation of the myc oncoprotein leads to increased turnover of thrombospondin-1 mRNA. Nucleic Acids Res 2000; 28: 22682275. 33 Laderoute KR, Alarcon RM, Brody MD, Calaoagan JM, Chen EY, Knapp AM et al. Opposing effects of hypoxia on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic inducer vascular endothelial growth factor. Clin Cancer Res 2000; 6: 29412950. 34 Penttinen RP, Kobayashi S, Bornstein P. Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability. Proc Natl Acad Sci USA 1988; 85: 11051108. 35 Levy NS, Chung S, Furneaux H, Levy AP. Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J Biol Chem 1998; 273: 64176423. 36 Sengupta S, Jang BC, Wu MT, Paik JH, Furneaux H, Hla T. The RNA-binding protein HuR regulates the expression of cyclooxygenase-2. J Biol Chem 2003; 278: 2522725233. 37 Shein LG, Zou AP, Spaulding SW. Androgens regulate the binding of endogenous HuR to the AU-rich 3UTRs of HIF-1alpha and EGF mRNA. Biochem Biophys Res Commun 2004; 322: 644651. 38 Dixon DA, Tolley ND, King PH, Nabors LB, McIntyre TM, Zimmerman GA et al. Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells. J Clin Invest 2001; 108: 16571665. 39 Mazan-Mamczarz K, Hagner PR, Corl S, Srikantan S, Wood WH, Becker KG et al. Post-transcriptional gene regulation by HuR promotes a more tumorigenic phenotype. Oncogene 2008; 27: 61516163. 40 Collart MA, Timmers HT. The eukaryotic Ccr4-not complex: a regulatory platform integrating mRNA metabolism with cellular signaling pathways? Prog Nucleic Acid Res Mol Biol 2004; 77: 289322. 41 Denis CL, Chen J. The CCR4-NOT complex plays diverse roles in mRNA metabolism. Prog Nucleic Acid Res Mol Biol 2003; 73: 221250. 42 Azzouz N, Panasenko OO, Colau G, Collart MA. The CCR4-NOT complex physically and functionally interacts with TRAMP and the nuclear exosome. PLoS ONE 2009; 4: e6760. 43 Garneau NL, Wilusz J, Wilusz CJ. The highways and byways of mRNA decay. Nat Rev Mol Cell Biol 2007; 8: 113126. 44 West S, Gromak N, Proudfoot NJ. Human 5 --4 3 exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. Nature 2004; 432: 522525. 45 Teixeira A, Tahiri-Alaoui A, West S, Thomas B, Ramadass A, Martianov I et al. Autocatalytic RNA cleavage in the human beta-globin pre-mRNA promotes transcription termination. Nature 2004; 432: 526530. 46 Kim M, Krogan NJ, Vasiljeva L, Rando OJ, Nedea E, Greenblatt JF et al. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 2004; 432: 517522. 47 Kawauchi J, Mischo H, Braglia P, Rondon A, Proudfoot NJ. Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination. Genes Dev 2008; 22: 10821092. 48 El Hage A, Koper M, Kufel J, Tollervey D. Efcient termination of transcription by RNA polymerase I requires the 5 exonuclease Rat1 in yeast. Genes Dev 2008; 22: 10691081. 49 Richard P, Manley JL. Transcription termination by nuclear RNA polymerases. Genes Dev 2009; 23: 12471269. 50 Hilleren P, McCarthy T, Rosbash M, Parker R, Jensen TH. Quality control of mRNA 3-end processing is linked to the nuclear exosome. Nature 2001; 413: 538542. 51 de Almeida SF, Garcia-Sacristan A, Custodio N, Carmo-Fonseca M. A link between nuclear RNA surveillance, the human exosome and RNA polymerase II transcriptional termination. Nucleic Acids Res 2010; 38: 80158026. 52 Anderson JT, Wang X. Nuclear RNA surveillance: no sign of substrates tailing off. Crit Rev Biochem Mol Biol 2009; 44: 1624. 53 Glaunsinger BA, Lee YJ. How tails dene the ending: divergent roles for polyadenylation in RNA stability and gene expression. RNA BIOL 2010; 7: 1317. Oncogene (2013) 4377 4386 NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4386 54 Mukherjee D, Gao M, OConnor JP, Raijmakers R, Pruijn G, Lutz CS et al. The mammalian exosome mediates the efcient degradation of mRNAs that contain AU-rich elements. EMBO J 2002; 21: 165174. 55 Houseley J, LaCava J, Tollervey D. RNA quality control by the exosome. Nat Rev Mol Cell Biol 2006; 7: 529539. 56 Moore MJ. Nuclear RNA turnover. Cell 2002; 108: 431434. 57 Vasudevan S, Peltz SW. Nuclear mRNA surveillance. Curr Opin Cell Biol 2003; 15: 332337. 58 Brennan CM, Gallouzi IE, Steitz JA. Protein ligands to HuR modulate its interaction with target mRNAs in vivo. J Cell Biol 2000; 151: 114. 59 Gallouzi IE, Brennan CM, Stenberg MG, Swanson MS, Eversole A, Maizels N et al. HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc Natl Acad Sci USA 2000; 97: 30733078. 60 Hasina R, Pontier AL, Fekete MJ, Martin LE, Qi XM, Brigaudeau C et al. NOL7 is a nucleolar candidate tumor suppressor gene in cervical cancer that modulates the angiogenic phenotype. Oncogene 2006; 25: 588598. 61 Zhou G, Doci CL, Lingen MW. Identication and functional analysis of NOL7 nuclear and nucleolar localization signals. BMC Cell Biol (Research Support, NIH, Extramural Research Support, Non-US Govt) 2010; 11: 74. 62 Doci CL, Mankame TP, Langerman A, Ostler KR, Kanteti R, Best T et al. Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer. Int J Gynecol Pathol (Research Support, NIH, Extramural Research Support, Non-US Govt) 2012; 31: 1524. 63 Neugebauer KM. On the importance of being co-transcriptional. J Cell Sci 2002; 115(Pt 20): 38653871. 64 Zorio DA, Bentley DL. The link between mRNA processing and transcription: communication works both ways. Exp Cell Res 2004; 296: 9197. 65 Lau NC, Kolkman A, van Schaik FM, Mulder KW, Pijnappel WW, Heck AJ et al. Human Ccr4-Not complexes contain variable deadenylase subunits. Biochem J 2009; 422: 443453. 66 Ladomery MR, Harper SJ, Bates DO. Alternative splicing in angiogenesis: the vascular endothelial growth factor paradigm. Cancer Lett 2007; 249: 133142. 67 Qiu Y, Hoareau-Aveilla C, Oltean S, Harper SJ, Bates DO. The anti-angiogenic isoforms of VEGF in health and disease. Biochem Soc Trans 2009; 37(Pt 6): 12071213. 68 Dibrov A, Kashour T, Amara FM. The role of transforming growth factor beta signaling in messenger RNA stability. Growth Factors 2006; 24: 111. 69 Hashimoto-Uoshima M, Yan YZ, Schneider G, Aukhil I. The alternatively spliced domains EIIIB and EIIIA of human bronectin affect cell adhesion and spreading. J Cell Sci 1997; 110: Pt 18 22712280. 70 Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C et al. Alternative isoform regulation in human tissue transcriptomes. Nature 2008; 456: 470476. 71 Touriol C, Roussigne M, Gensac MC, Prats H, Prats AC. Alternative translation initiation of human broblast growth factor 2 mRNA controlled by its 3-untranslated region involves a Poly(A) switch and a translational enhancer. J Biol Chem 2000; 275: 1936119367. 72 Claffey KP, Shih SC, Mullen A, Dziennis S, Cusick JL, Abrams KR et al. Identication of a human VPF/VEGF 3 untranslated region mediating hypoxia-induced mRNA stability. Mol Biol Cell 1998; 9: 469481. 73 Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996; 271: 27462753. 74 Levy AP, Levy NS, Wegner S, Goldberg MA. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem 1995; 270: 1333313340. 75 Levy NS, Goldberg MA, Levy AP. Sequencing of the human vascular endothelial growth factor (VEGF) 3 untranslated region (UTR): conservation of ve hypoxiainducible RNA-protein binding sites. Biochim Biophys Acta 1997; 1352: 167173. 76 Kanies CL, Smith JJ, Kis C, Schmidt C, Levy S, Khabar KS et al. Oncogenic Ras and transforming growth factor-beta synergistically regulate AU-rich element-containing mRNAs during epithelial to mesenchymal transition. Mol Cancer Res 2008; 6: 11241136. 77 Chung JH, Rho JK, Xu X, Lee JS, Yoon HI, Lee CT et al. Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFRTKIs. Lung Cancer 2011; 73: 176182. 78 Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009; 139: 871890. 79 Lingen MW. Endothelial cell migration assay. A quantitative assay for prediction of in vivo biology. Methods Mol Med (Research Support, US Govt, PHS) 2003; 78: 337347. Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc) Oncogene (2013) 4377 4386 & 2013 Macmillan Publishers Limited  ...