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

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诱导血管生成，即新血管的产生和重塑，以满足快速分裂转化细胞日益增长的代谢需求，因此开发抗血管生成药物是一种很有吸引力的治疗策略。最近的研究发现，在许多不同的原发性和转移性肿瘤中，缺氧诱导因子(HIF)-1的表达增加，这是一种促进血管生成的转录因子，表明其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，我们首次发现Plexin-B1，一种先前被确定为神经元生长锥进展、迁移和神经束束束的调节因子的蛋白质，在内皮细胞中也高度表达，当与其配体Semaphorin 4D （Sema4D）结合时，促进促血管生成反应(3)，而Sema4D在头颈部鳞状细胞癌（HNSCC）和许多其他实体肿瘤中表达，增强其生长和血管性(4)。该应用的广泛和长期目标是阐明Sema4D的调控机制及其对肿瘤诱导血管生成的影响。需要验证的假设是，由于HIF-1介导的途径，Sema4D在缺氧时上调，并且其表达与其他HIF-1调节的基因产物如膜型1-基质金属蛋白酶（MT1-MMP）和血管内皮生长因子（VEGF）一起诱导更血管化，从而更具有侵袭性的肿瘤表型。具体目的是：1)建立Sema4D的转录调控机制及其在缺氧介导的肿瘤发生中的生物学意义。这将通过分析常氧和缺氧细胞中表达降低或组成性活性HIF的Sema4D蛋白和信息水平，对Sema4D基因进行全面的启动子分析，并通过体外和体内血管生成实验和肿瘤异种移植实验来完成；2)确定缺氧介导的MT1-MMP诱导Sema4D诱导血管生成能力的重要性。这将通过分析HIF活性改变的细胞中的MT1-MMP水平和mt1介导的Sema4D加工在肿瘤异种移植模型中的生物学意义来研究。3)在条件敲除小鼠模型中，通过逆转录病毒介导的基因直接转移到肿瘤细胞中，确定VEGF和Sema4D对hnscc诱导的血管生成的贡献。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。

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.