SIGNALING MECHANISMS IN REGULATION OF TUMOR ANGIOGENESIS

描述（由申请人提供）：癌症目前是美国最常见的死亡原因之一。一种主要的治疗选择旨在减缓癌症的进展（例如：在结肠癌中使用抗VEGF）。因此，必须重新努力，以确定相关的分子通路，这可能是开发作为治疗靶点。许多类型的癌症的进展与几种促血管生成因子（HIF-1 α、VEGF、bFGF、PDGF和MMP's等）的上调有关。其促进肿瘤血管生成。这些血管生成因子激活调节细胞增殖、迁移和凋亡的不同信号通路。我实验室的长期目标是了解细胞外基质（IV型胶原NC 1结构域，通常存在于血液中）的血管生成抑制剂的复杂信号传导机制，并确定它们在抗血管生成信号传导中的作用。最近，我的实验室确定，a1（IV）NC 1介导多种信号传导机制，以调节肿瘤血管生成。然而，它对肿瘤血管生成和肿瘤生长的复杂影响还远未完全了解.本授权提案特别针对阐明α 1（IV）NC 1抑制促血管生成分子表达的机制及其在抑制肿瘤生长中的作用。在这项研究中，我们的目的是测试我们的假设：“a1（IV）NC 1促进内皮细胞凋亡通过a1 <$1整合素和抑制MMP-2激活通过整合素的独立途径。这些活性对于α 1（IV）NC 1-的抗血管生成和抗肿瘤生成活性至关重要。为此，我们已经产生了一个a1（IV）NC 1转基因小鼠模型和腺病毒，克隆和表达a1（IV）NC 1的杆状病毒表达系统，并将使用这种蛋白质的研究概述的具体目标。(1)研究a1（IV）NC 1抑制血管生成因子表达的血管抑制信号机制，包括可能的生化途径的研究。(2)研究a1（IV）NC 1/MMP-2之间的复杂相互作用，并确定参与这种相互作用和功能的互补结合位点。(3)研究α 1（IV）NC 1对α 1-整合素缺失和α 1（IV）NC 1转基因小鼠肿瘤生长的抑制作用。上述具体目的将使用细胞培养物、α 1整联蛋白缺失、α 1（IV）NC 1转基因小鼠和携带α 1（IV）NC 1结构域表达盒的腺病毒来实现。我们将使用免疫印迹、免疫共沉淀、免疫组织化学、基质胶栓塞和肿瘤研究来分析这些模型。成功完成拟议的工作将提供有价值的见解，可能促进新的抗血管生成肿瘤疗法的发展。 公共卫生相关性：癌症目前是美国最常见的死亡原因之一，目前的治疗方案仅旨在减缓癌症的进展。因此，必须重新努力，以确定无毒的内源性循环抗癌分子，可以利用作为治疗目标。我的实验室已经克隆并表达了一种这样的循环分子，目前正在细胞培养系统和患有肿瘤的活小鼠中进行测试。从这些拟议的研究中开发的数据具有治疗实体瘤生长（癌症）的潜在应用，其中血管生成有助于疾病阶段。

DESCRIPTION (provided by applicant): Cancer is currently one of the most prevalent causes of death in the U.S.A. A major therapeutic option aims to slow the progression of cancer (example: use of anti-VEGF in colon cancer). Therefore, a renewed effort must be made to identify relevant molecular pathways, which could be exploited as therapeutic targets. Progression of many types of cancers is associated with up-regulation of several proangiogenic factors (HIF- 1a, VEGF, bFGF, PDGF, and MMP's etc.) which promotes tumor angiogenesis. These angiogenic factors activate different signaling pathways that regulate cell proliferation, migration and apoptosis. The long-term goals of my laboratory aims to understand the complex signaling mechanisms of angioinhibitors from extracellular matrix (type IV collagen NC1 domains, which are normally present in the blood), and to determine their role in antiangiogenic signaling. Recently my laboratory determined that a1(IV)NC1 mediates multiple signaling mechanisms to regulate tumor angiogenesis. However, its complex influences on tumor- angiogenesis and tumor growth are far from being fully understood. The present grant proposal is specifically directed at elucidating the mechanism whereby a1(IV)NC1 inhibits expression of the pro-angiogenic molecules, and its role in inhibition of tumor growth. In this study we aim to test our hypothesis: "a1(IV)NC1 promotes endothelial cell apoptosis through a1¿1 integrin and inhibits MMP-2 activation through an integrin independent pathway. These activities are critical for the antiangiogenic and antitumorogenic activity of a1(IV)NC1". Towards this end, we have generated an a1(IV)NC1 transgenic mouse model and adenoviruses, cloned and expressed a1(IV)NC1 in the baculovirus expression system and will use this protein for studies outlined in the specific aims. (1) Investigate the angioinhibitory signaling mechanisms by which a1(IV)NC1 inhibits the expression of angiogenic factors, including investigation of possible biochemical pathways. (2) Investigate the complex interactions between a1(IV)NC1/MMP-2 and identify the complementary binding sites involved in this interaction and function. (3) To investigate inhibition of tumor growth by a1(IV)NC1 in a1-integrin null and a1(IV)NC1 transgenic mice. The above Specific aims will be accomplished using cell cultures, a1 integrin null, a1(IV)NC1 transgenic mice and adenoviruses harboring a1(IV)NC1 domain expression cassettes. We will analyze these models using immunoblotting, co-immunoprecipitation, immunohistochemistry, Matrigel plug, and tumor studies. Successful completion of the proposed work will provide valuable insights that may facilitate the development of new antiangiogenic tumor therapies. PUBLIC HEALTH RELEVANCE: Cancer is currently one of the most prevalent causes of death in the USA, and current therapeutic options aim only to slow the progression of cancer. Therefore, a renewed effort must be made to identify nontoxic endogenous circulating anticancer molecules which could be exploited as therapeutic targets. My laboratory has cloned and expressed one such circulating molecule, and is presently testing it in a cell culture system and in live mice having tumors. The data being developed from these proposed studies have potential applications to cure solid tumor growth (cancers) in which angiogenesis contributes to the disease phase.