Exosomal TRAF2-CSN5 complex mediated inflammation promotes tumor growth

描述（由申请人提供）： 该提案的总体目标是阐明肺肿瘤外泌体促进炎症反应的机制，即巨噬细胞的活化，并促进肺肿瘤的进展和生长。我们的初步数据表明，用TC-1肺肿瘤细胞产生的外泌体或从已用致癌物氨基甲酸乙酯预处理的A/J小鼠的肺组织分离的外泌体预处理A/J小鼠导致肿瘤生长更快和肺癌进展更早。氨基甲酸乙酯处理导致TRAF 2的活化形式募集至外泌体，并且具有活化TRAF 2的外泌体被骨髓来源的巨噬细胞前体摄取，导致它们成熟并随后迁移至肺。激活的TRAF 2向外泌体的募集需要肿瘤细胞中炎症抑制蛋白CYLD的降解;此外，TRAF 2可以介导CYLD的泛素化，从而促进其降解。然而，siRNA TRAF 2敲除的结果表明，TRAF 2是必需的，但不足以泛素化CYLD。我们现在已经确定了两种与TRAF 2相互作用的外泌体蛋白，Itch和CSN 5，它们有可能增强肺肿瘤细胞中CYLD的泛素化。我们最近的数据表明，CYLD并不包装在从肺癌患者的外周血中分离的外来体中，尽管它存在于来自健康志愿者的外来体中，这表明该模型的潜在临床相关性，该模型将致癌物的作用与炎症反应和促进肿瘤发展直接联系起来。我们建议：（1）通过确定TC-1肺肿瘤细胞中TRAF 2的敲除是否足以（i）抑制巨噬细胞分化和（ii）预防肺肿瘤外泌体介导的尿烷诱导的肺癌的增强，确认肺肿瘤外泌体TRAF 2在促进尿烷诱导的肺癌中的作用。(2)确定与CYLD相互作用的其他外泌体蛋白是否参与TC-1肿瘤细胞中CYLD的泛素化，并确定这些蛋白的消除是否稳定外泌体CYLD并抑制外泌体TRAF 2活化，从而抑制巨噬细胞活化并预防肿瘤生长。(3)确定用从肺癌患者分离的肺肿瘤外泌体脉冲的巨噬细胞是否促进NOD-SCID小鼠模型中的肺肿瘤生长。产生的数据应该允许开发一个高度创新的模型的细胞和分子基础外泌体介导的慢性炎症和促进肿瘤生长，并指出新的预防和治疗策略。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to elucidate the mechanisms by which lung tumor exosomes promote an inflammatory response, namely the activation of macrophages, and promote the progression and growth of lung tumors. Our preliminary data indicate that pretreatment of A/J mice with exosomes produced by TC-1 lung tumor cells or exosomes isolated from the lung tissue of A/J mice that have been pretreated with the carcinogen urethane results in more rapid tumor growth and earlier progression of lung carcinomas. Urethane treatment results in the recruitment of the activated form of TRAF2 to the exosomes, and the exosomes with activated TRAF2 are taken up by the bone marrow-derived precursors of macrophages, leading to their maturation and subsequent migration to the lung. The recruitment of the activated TRAF2 to the exosomes requires degradation of the inflammation suppressor protein, CYLD, in the tumor cells; moreover, TRAF2 can mediate ubiquination of CYLD thereby promoting its degradation. The results of siRNA TRAF2 knockout indicated, however, that TRAF2 is required but is not sufficient for ubiquitination of CYLD. We have now identified two exosomal proteins that interact with TRAF2, Itch and CSN5, which have the potential to enhance the ubiquitination of CYLD in lung tumor cells. Our recent data indicating that CYLD is not packed in exosomes isolated from the peripheral blood of lung cancer patients although it is present in exosomes from healthy volunteers indicate the potential clinical relevance of this model that directly links the effects of a carcinogen with an inflammatory response and promotion of tumor development. We propose to: (1) Confirm the role of lung tumor exosomal TRAF2 in the promotion of urethane-induced lung carcinomas by determining whether knockout of TRAF2 in TC-1 lung tumor cells is sufficient for (i) inhibition of macrophage differentiation and (ii) prevention of lung tumor exosome-mediated enhancement of urethane induced lung cancer. (2) Determine if other exosomal proteins that interact with CYLD participate in the ubiquitination of CYLD in TC-1 tumor cells and determine if the elimination of these proteins stabilizes exosomal CYLD and suppresses exosomal TRAF2 activation resulting in the inhibition of macrophage activation and prevention of tumor growth. (3) Determine if macrophages pulsed with lung tumor exosomes isolated from lung cancer patients promote lung tumor growth in a NOD-SCID mouse model. The data generated should permit the development of a highly innovative model of the cellular and molecular basis for exosome-mediated chronic inflammation and promotion of tumor growth and indicate novel preventive and therapeutic strategies.