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... Bone loss after acute sex-hormone removal via gonadectomy prior to skeletal maturity most striking in male but not female animals 1,2 1 1 1,3 Nick Momeni , Alyson Essex , Padmini Deosthale , Lilian Plotkin of Anatomy, Cell Biology, & Physiology, Indiana University School of Medicine, Indianapolis, IN 46202; 2 Marian University College of Osteopathic Medicine, Indianapolis, IN 46222, 3Roudebush Veterans Administration Medical Center, Indianapolis, IN 46202. BV/TV 0.10 35 30 25 20 # 15 * 10 0 5 * 0.02 0.00 0.25 Sham OVX Sham OVX 2 wk post-op 4 wk post-op Female Sham ORX Sham ORX 2 wk post-op 4 wk post-op Male Figure 1 Bone Volume/ Tissue Volume, Trabecular Thickness, Trabecular Number, and Volumetric Tissue Mineral Density (v-TMD) from male and female sham and operated animals 2 and 4 weeks post gonadectomy. N = 4-5/group. Volumetric -Tissue Mineral Density (v-TMD) 4 0.20 # * 2 Sham ORX 2 wk post-op Sham ORX 4 wk post-op Male * * * 1 0.15 0.10 # * * Sham OVX Sham OVX 2 wk post-op 4 wk post-op Female 0.00 Sham OVX Sham OVX 2 wk post-op 4 wk post-op Female Sham ORX Sham ORX 2 wk post-op 4 wk post-op Male Figure 2 4 weeks post-gonadectomy, males lose cortical bone volume, but both males and females lose TMD TMD 1.2 * 1.0 * Sham OVX Sham OVX 2 wk post-op 4 wk post-op Female Male 0.4 7 0.2 6 Endocortical BS Male Female 3 INDIANA UNIVERSITY SCHOOL OF MEDICINE Future studies will be needed to assess the cellular mechanisms responsible for this sex-dependent bone volume and mineral density loss with acute sex hormone removal. 1) Mohamad, Nur Vaizura, et al. A Concise Review of Testosterone and Bone Health. Clinical Interventions in Aging, Volume 11, 2016, pp. 13171324., https://doi.org/10.2147/cia.s115472. 1 0 Sex hormones, particularly androgens in males, are crucial for proper development of the skeletal system. Testosterone seems to play a role in maintaining cancellous bone in males. The large decreases in bone volume and tissue mineral density suggest without proper exposure to sex hormone levels, bone achieves a lower peak bone mineral density. References 4 2 Figure 2 Tissue Mineral Density (TMD), Cortical Area, and Endocortical Bone Surface from male and female sham and operated animals 2 and 4 weeks post gonadectomy. N = 5/group. * = p <0.05 via Two-way ANOVA vs. Sham , #= p <0.05 via Two-Way ANOVA. Conclusions * 5 Sham OVX Sham OVX 2 wk post-op 4 wk post-op Male mice demonstrated a larger decline in both cancellous and cortical bone after removal of sex hormones. * Sham ORX Sham ORX 2 wk post-op 4 wk post-op 0.6 Sham ORX Sham ORX 2 wk post-op 4 wk post-op The acute removal of sex hormones prior to skeletal maturity negatively impacts the tissue mineral density of bone in both male and female mice. Cortical Area 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.8 0.0 * = p <0.05 via Two-way ANOVA vs. Sham , #= p <0.05 via Two-Way ANOVA. 0.05 mm2 3 Summary Cancellous bone is more sensitive than cortical bone to the decline in circulating sex hormones. 0.04 Trabecular Number Materials & Methods Images of the femurs were obtained using a SkyScan 1176 micoCT. The microCT images of the individual femurs were then reconstructed using Nrecon and DataViewer software. To analyze the cortical and trabecular bone separately, region of interests (ROIs) were drawn around the trabecular and cortical areas of each femur. Analysis programs were then executed to report the data of the cortical and trabecular bone volume. Tissue mineral density (TMD) was normalized to a phantom using houndsafield units. * Sham OVX Sham OVX 2 wk post-op 4 wk post-op Female Sham ORX Sham ORX 2 wk post-op 4 wk post-op Male g/cm3 Females undergo a rapid depletion of their estrogen levels once they enter menopause. It is understood that this decline in estrogen levels leads to increased activity of osteoclasts, more bone resorption, and ultimately a higher likelihood of developing osteoporosis. Males do not undergo a similar rapid loss of testosterone. This difference in depletion of sex hormones explains why women age 50 and older have a higher lifetime risk of fracture than men age 50 and older (1). However, it is common for males to lose testosterone levels as they age, and this loss of testosterone impacts bone mineral density. A previous study found that older men with higher testosterone levels better maintained their bone mineral density and had lower fracture risks (2). Other studies have postulated that testosterone stimulates osteoblasts to produce trabecular bone and aids osteocytes in preventing trabecular bone loss (3). While this provides insight into how chronic loss of testosterone impacts bone health, this study was aimed at discovering how acute removal of sex hormones impacted male and female mice. * * 5 0.06 * mm % # 0.08 0 Background Trabecular Thickness mm Menopause, an age-related loss of sex hormone production in women, is one of the most common causes of osteoporosis. Previous work has established that this loss of sex hormones, in particular estrogen, causes dramatic loss of bone volume and strength. Similarly, removal of sex steroids results in acute loss of bone mass in adult animals. Mouse models of sex steroid-deficiency include surgery removing the sex organs (orchidectomy, ORX, for males; ovariectomy, OVX, for females) are commonly used to understand the role of sex steroids in bone, but are typically preformed at animal maturity (16 weeks of age) and are analyzed six weeks post-operation. This study aimed to determine whether acute removal of the male or female sex hormones prior to maturity would impact the cortical and trabecular bone volume. Gonadectomy or sham operations were performed on mice at 11 weeks of age, and femurs were then harvested either 2 weeks (13 weeks of age) or 4 weeks postsurgery (15 weeks of age). Analysis of the cortical and cancellous bone volume of the femur were assessed by microCT. In cancellous bone, male animals two and four weeks ORX demonstrated decreases in the following parameters compared to sham operated, agematched controls (2 week; 4 week): bone volume (BV/TV, -70.9%; -86.6%), tissue mineral density (V-TMD, -8.69%; -17.9%), trabecular thickness (TbTh, -31.9%; -27.8%), and trabecular number (TbN, -57.5%; -81.4%). In cancellous bone, female animals two and four weeks OVX demonstrated decreases in the following parameters compared to sham operated, agematched controls: BV/TV (-61.2%; -41.0%), V-TMD (-30.7%; -15.6%), and TbN (-64.0%; 42.4%). In cortical bone, male animals four weeks ORX demonstrated decreases in the following parameters compared to sham operated, age-matched controls: cortical area (13.8%), Endocortical bone surface (-7.10%), and TMD (-10.2%). In cortical bone, female animals four weeks OVX demonstrated decreases in only TMD (-7.50%) compared to sham operated, age-matched controls. In summary, the acute removal of sex hormones has a larger impact on cancellous bone in both males and females, with male animals showing increasing bone loss as time progressed. Further studies are needed to understand the underlying mechanisms behind the progressive bone loss seen in males after sex hormone depletion. Figure 1 Trabecular bone loss in both male and female animals after both 2 and 4 weeks post gonadectomy, but only males have decreased volumetric tissue mineral density g/mm3 Abstract 1/mm 1 Department 2) Chin, Kok-Yong, and Soelaiman Ima-Nirwana. Sex Steroids and Bone Health Status in Men. International Journal of Endocrinology, vol. 2012, 2012, pp. 17., https://doi.org/10.1155/2012/208719. Sham ORX Sham ORX 2 wk post-op 4 wk post-op Male Sham OVX OVX Sham 2 wk post-op 4 wk post-op Female 3) Golds, Gary, et al. Male Hypogonadism and Osteoporosis: The Effects, Clinical Consequences, and Treatment of Testosterone Deficiency in Bone Health. International Journal of Endocrinology, vol. 2017, 2017, pp. 115., https://doi.org/10.1155/2017/4602129. DEPARTMENT OF ANATOMY, CELL BIOLOGY, & PHYSIOLOGY  ...

... 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. 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... 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? 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