SIGNALING MECHANISMS IN REGULATION OF TUMOR ANGIOGENESIS

描述(申请人提供)：癌症是目前美国最常见的死亡原因之一。一种主要的治疗方案旨在减缓癌症的进展(例如：在结肠癌中使用抗血管内皮生长因子)。因此，必须重新努力寻找相关的分子途径，以利用这些途径作为治疗的靶点。许多类型的癌症的进展与几种促血管生成因子(HIF-1a、血管内皮生长因子、碱性成纤维细胞生长因子、PDGF和基质金属蛋白酶等)的上调有关。促进肿瘤血管生成。这些血管生成因子激活不同的信号通路，调节细胞的增殖、迁移和凋亡。我的实验室的长期目标是了解来自细胞外基质(通常存在于血液中的IV型胶原NC1域)的血管抑制物的复杂信号机制，并确定它们在抗血管生成信号中的作用。最近，我的实验室确定A1(IV)NC1介导多种信号机制来调节肿瘤血管生成。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被了解。目前的拨款建议是专门针对阐明A1(IV)NC1抑制促血管生成分子表达的机制，以及它在抑制肿瘤生长中的作用。在这项研究中，我们的目的是验证我们的假设：A1(IV)NC1通过A1？1整合素促进内皮细胞凋亡，并通过整合素非依赖的途径抑制基质金属蛋白酶-2的激活。这些活性对A1(IV)NC1的抗血管生成和抗肿瘤活性至关重要。为此，我们建立了A1(IV)NC1转基因小鼠模型和腺病毒，克隆并在杆状病毒表达系统中表达了A1(IV)NC1，并将使用该蛋白进行特定目的的研究。(1)研究A1(IV)NC1抑制血管生成因子表达的信号转导机制，包括可能的生化途径。(2)研究A1(IV)NC1/MMP2之间的复杂相互作用，确定参与这种相互作用和功能的互补结合部位。(3)研究A1(IV)NC1对A1整合素缺失和A1(IV)NC1转基因小鼠肿瘤生长的抑制作用。上述特定目的将使用细胞培养、A1整合素缺失、A1(IV)NC1转基因小鼠和携带A1(IV)NC1域表达框的腺病毒来实现。我们将使用免疫印迹、免疫共沉淀、免疫组织化学、Matrigel Plug和肿瘤研究来分析这些模型。这项拟议工作的成功完成将提供有价值的见解，可能有助于开发新的抗血管生成肿瘤疗法。 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，必须重新努力寻找无毒的内源性循环抗癌分子作为治疗靶点。我的实验室已经克隆并表达了一个这样的循环分子，目前正在细胞培养系统和有肿瘤的活小鼠身上进行测试。从这些拟议的研究中开发的数据具有治疗实体肿瘤生长(癌症)的潜在应用，在实体肿瘤生长中，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.