The Role of Semaphorin 4D and Plexin-B1 in Tumor-Induced Angiogenesis.

描述(申请人提供)：口腔癌在发达国家的男性中很常见，也是最难治疗的肿瘤之一。所有实体瘤的生长和转移都需要血管生成的诱导、新血管的生成和重塑，以满足快速分裂转化细胞日益增长的代谢需求，从而使抗血管生成药物的开发成为一种吸引人的治疗策略。最近的研究发现，缺氧诱导因子(HIF)-1，一种促进血管生成的转录因子，在许多不同的原发和转移肿瘤中表达增加，这表明它的激活在人类癌症中很常见(1)。我们现在知道，参与传递轴突引导信号的蛋白质也可以在肿瘤诱导的血管生成中发挥作用。例如，我们第一个展示了Plexin-B1，一种先前被认为是神经元生长锥体推进和迁移以及神经束束束形成的调节蛋白，在内皮细胞中也高表达，并在与其配体Semaphorin 4D(Sema4D)(3)结合时促进促血管生成反应，Sema4D是一种在头颈部鳞状细胞癌(HNSCC)和许多其他实体肿瘤中表达的蛋白质，可促进其生长和血管生成(4)。这一应用的广泛、长期的目标是阐明Sema4D的调节机制以及对肿瘤诱导的血管生成的影响。需要检验的假设是，由于HIF-1介导的途径，Sema4D在缺氧条件下上调，其表达与其他HIF-1调节的基因产物，如膜型1-基质金属蛋白酶(MT1-MMPs)和血管内皮生长因子(VEGF)共同作用，诱导更多的血管，从而更具侵袭性的肿瘤表型。其具体目的是：1)建立Sema4D转录调控机制及其在低氧介导的肿瘤发生中的生物学意义。这将通过分析正常和低氧细胞中表达减少或固有活性HIF的Sema4D蛋白和消息水平，彻底分析Sema4D基因的启动子，以及通过体外和体内血管生成试验和肿瘤异种移植实验来实现；2)确定低氧介导的MT1-MMP诱导对Sema4D诱导血管生成能力的重要性。这将通过分析HIF活性改变的细胞中MT1-MMP水平和MT1介导的Sema4D在肿瘤异种移植模型中的生物学意义来研究；以及3)在条件基因敲除小鼠模型中，通过逆转录病毒介导的基因直接进入肿瘤细胞，确定VEGF和Sema4D在HNSCC诱导的血管生成中的作用。我相信，这些对Sema4D/Plexin-B1介导的血管生成的研究将支持一种新的肿瘤诱导血管生成模型，并为癌症治疗提供可能的新靶点。 与公共卫生相关：从本质上讲，肿瘤细胞在遗传上是不稳定的，这一特征导致它们能够不适当地增殖，避免自然防御，并对化疗产生抗药性。开发能够阻断肿瘤血液供应的抗血管生成药物已成为绕过这一问题的有趣方法。如果成功，我们提出的项目可能揭示肿瘤诱导血管生成的重要机制，从而为治疗侵袭性肿瘤的抗血管生成疗法的开发提供新的靶点。

DESCRIPTION (provided by applicant): Oral cancer is common among men in the developed world and among the most difficult neoplasms to treat. The growth and metastasis of all solid tumors requires induction of angiogenesis, the creation and remodeling of new blood vessels, to meet the increasing metabolic demands of rapidly dividing transformed cells, thus making the development of anti-angiogenic agents an appealing treatment strategy. Recent studies have identified increased expression of hypoxia- inducible factor (HIF)-1, a transcription factor that promotes angiogenesis, in many different primary and metastatic tumors, suggesting that its activation is common in human cancer (1). We now know that proteins involved in transmitting axonal guidance cues can also play a role in tumor-induced angiogenesis. For example, we were the first to show that Plexin-B1, a protein previously identified as a regulator of neuron growth cone progression and migration and nerve bundle fasciculation, is also highly expressed in endothelial cells and promotes a pro-angiogenic response when bound by its ligand Semaphorin 4D (Sema4D) (3), a protein expressed in head and neck squamous cell carcinomas (HNSCC) and many other solid tumors that enhances their growth and vascularity (4). The broad, long-term objectives of this application are to elucidate the mechanisms of regulation of Sema4D and the implications for tumor- induced angiogenesis. The hypothesis to be tested is that Sema4D is upregulated in hypoxia due to HIF-1- mediated pathways, and that its expression acts with other HIF-1-regulated gene products such as membrane type 1-matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF) to induce a more vascular and consequently a more aggressive tumor phenotype. The specific aims are: 1) to establish the mechanism of transcriptional regulation of Sema4D and its biological relevance in hypoxia- mediated tumorigenesis. This will be accomplished by analyzing Sema4D protein and message levels in normoxic and hypoxic cells expressing reduced or constitutively active HIF, thorough promoter analysis of the Sema4D gene and through in vitro and in vivo angiogenesis assays and tumor xenograft experiments; 2) to determine the importance of hypoxia-mediated induction of MT1-MMP on the ability of Sema4D to induce angiogenesis. This will be studied through analysis of MT1-MMP levels in cells with altered HIF activity and the biological significance of MT1-mediated processing of Sema4D in a tumor xenograft model, and; 3) to determine VEGF and Sema4D contributions to HNSCC-induced angiogenesis through retroviral-mediated gene transfer directly into tumor cells in a conditional knockout mouse model. I believe that these investigations into Sema4D/Plexin-B1-mediated angiogenesis will support a newly emerging model of tumor-induced angiogenesis and present possible new targets for cancer therapy. PUBLIC HEALTH RELEVANCE: By their very nature, tumor cells are genetically unstable, a trait that contributes to their ability to proliferate inappropriately, avoid natural defenses and acquire resistance to chemotherapy. The development of anti-angiogenic agents capable of blocking a tumor's blood supply has become an intriguing way to circumvent this problem. If successful, our proposed project may uncover an important mechanism for tumor-induced angiogenesis, thereby presenting new targets for the development of anti- angiogenic therapies for the management of aggressive tumors.