Mechanism of angiogenin-induced angiogenesis

描述（由申请人提供）：血管生成素（ANG）是一种14 kDa的血管生成核糖核酸酶，在多种人类癌症中上调。最近在肌萎缩侧索硬化症（ALS）患者中发现了ANG编码区的功能缺失突变。在上一个资助期进行的研究已经证明ANG在核糖体RNA（rRNA）合成中的重要作用，这对许多基本生物过程（包括细胞生长，增殖和存活）至关重要。我们已经建立了条件性Ang1基因敲除小鼠，并表明Ang1基因敲除小鼠血管生成反应降低，对应激诱导的细胞凋亡的易感性增加。从机制上讲，我们已经证明ANG的生物活性依赖于其亚细胞目的地。在生长条件下，ANG易位到细胞核，在那里它刺激核糖体RNA（rRNA）转录和加工，从而促进细胞生长和增殖。在应激下，ANG被转移到应激颗粒，在那里它增强了tiRNA（tRNA衍生的、应激诱导的小RNA）的产生，该tiRNA重新编程蛋白质翻译，从而促进细胞存活。我们还鉴定了丛蛋白B2作为一种功能性ANG受体，其对于介导ANG的生物活性是必要且充分的。我们已经表明，ANG和神经丛蛋白B2的另一种配体脑信号蛋白4C（Sema4C）在非重叠区域结合并触发不同的信号。该提案的目的是了解ANG在刺激血管生成和癌症进展中的作用机制。这一目标将通过实现以下四个具体目标来实现。(1)评估抗ANG受体（丛蛋白B2）抗体的治疗活性。我们将在异种移植和转基因动物模型中研究抗丛蛋白B2单克隆抗体对血管生成和肿瘤进展的影响。(2)表征ANG和Sema4C在血管生成和肿瘤进展中的差异效应。我们将评估敲低丛蛋白B2和Sema4C对Akt诱导的前列腺上皮内（PIN）的影响，并将结果与ANG敲低的结果进行比较。(3)确定ANG在应激调节蛋白翻译和细胞存活中的作用。我们将确定的信号，直接易位的ANG进入应激颗粒和进入核。我们还将描述ANG介导的tiRNA在重编程蛋白翻译和促进细胞存活中的作用。(4)表征Ang1敲除和ANG过表达在血管生成和肿瘤生长中的作用。我们将研究ANG在癌症对环境和遗传因素造成的致癌损伤的易感性中的作用。我们还将研究ANG的自分泌与旁分泌作用及其各自对肿瘤血管生成和癌细胞增殖的贡献。我们预期这些实验的结果将阐明ANG如何与其细胞表面受体相互作用，信号转导途径如何转导，以及ANG如何促进细胞存活和增殖。这些结果将为全面了解ANG在血管生成和癌症进展中的功能和机制提供帮助。

DESCRIPTION (provided by applicant): Angiogenin (ANG) is a 14 kDa angiogenic ribonuclease that is upregulated in a variety of human cancers. Loss-of-function mutations in the coding region of ANG have recently been found in patients with amyotrophic lateral sclerosis (ALS). Studies carried out in the previous funding period have demonstrated an important role of ANG in ribosomal RNA (rRNA) synthesis, which is essential for many fundamental biological processes including cell growth, proliferation, and survival. We have created conditional Ang1 knockout mice and have shown that Ang1 null mice have reduced angiogenic response and increased susceptibility to stress-induced apoptosis. Mechanistically, we have demonstrated that the biological activity of ANG relies on its subcellular destination. Under growth condition, ANG is translocated to the nucleus where it stimulates ribosomal RNA (rRNA) transcription and processing thereby promoting cell growth and proliferation. Under stress, ANG is translocated to the stress granules where it enhances the production of tiRNA (tRNA-derived, stress-induced small RNA) that reprogram protein translation thereby promoting cell survival. We have also identified PlexinB2 as a functional ANG receptor that is both necessary and sufficient for mediating biological activity of ANG. We have shown that ANG and Semaphorin 4C (Sema4C), the other ligand of PlexinB2, bind at non-overlapping regions and trigger different signals. The objective of this proposal is to understand the mechanism of action of ANG in stimulating angiogenesis and cancer progression. This objective will be achieved by addressing the following four specific aims. (1) Assess the therapeutic activity of anti-ANG receptor (PlexinB2) antibody. We will examine the effect of anti-PlexinB2 monoclonal antibodies on angiogenesis and tumor progression in both xenograft and transgenic animal models. (2) Characterize the differential effect of ANG and Sema4C in angiogenesis and tumor progression. We will assess the effect of knockdown PlexinB2 and Sema4C on Akt- induced prostate intraepithelial (PIN) and compare the results with that from ANG knockdown. (3) Determine the role of ANG in stress-regulated protein translation and cell survival. We will identify the signals that direct translocation of ANG into stress granules and into nucleus. We will also characterize the role of ANG-mediated tiRNA in reprogramming protein translation and promoting cell survival. (4) Characterize the effect of Ang1 knockout and ANG overexpression in angiogenesis and tumor growth. We will examine the function of ANG in cancer susceptibility to oncogenic insults inflicted by environmental and genetic factors. We will also study autocrine vs. paracrine action of ANG and their respective contributions toward tumor angiogenesis and cancer cell proliferation. We anticipate that the results of these experiments will elucidate how ANG interacts with its cell surface receptor, how the signaling pathway is transduced, and how ANG promotes cell survival and proliferation. These results will provide a comprehensive understanding of the function and mechanism of ANG in angiogenesis and in cancer progression.