Exosomal TRAF2-CSN5 complex mediated inflammation promotes tumor growth

描述(由申请人提供)： 这项建议的总体目标是阐明肺肿瘤外切体促进炎症反应的机制，即巨噬细胞的激活，并促进肺肿瘤的进展和生长。我们的初步数据表明，用TC-1肺癌细胞产生的外切体或从A/J小鼠肺组织中分离的外切体经致癌剂乌拉坦预处理后，A/J小鼠的肿瘤生长更快，肺癌进展更早。乌拉坦治疗导致激活形式的TRAF2重新聚集到外切体，具有激活TRAF2的外切体被巨噬细胞的骨髓源前体摄取，导致它们成熟并随后迁移到肺。激活的TRAF2募集到外体需要肿瘤细胞中炎症抑制蛋白CyLD的降解，而且TRAF2可以介导CyLD的泛化，从而促进其降解。然而，siRNA TRAF2基因敲除的结果表明，TRAF2是CyLD泛素化所必需的，但不足以实现泛素化。我们现在已经确定了两种与TRAF2相互作用的外体蛋白，Itch和CSN5，它们有可能增强肺癌细胞中CyLD的泛素化。我们最近的数据表明，尽管从健康志愿者的外切体中存在，但从肺癌患者的外周血中分离出来的外切体中并没有包装CyLD，这表明这一模型具有潜在的临床相关性，该模型直接将致癌物的影响与炎症反应和促进肿瘤发展联系起来。我们建议：(1)通过确定TRAF2在TC-1肺癌细胞中的敲除是否足以(I)抑制巨噬细胞分化和(Ii)防止肺癌外体介导的促进乌拉坦诱导的肺癌，来证实肺癌外体TRAF2在乌拉坦诱导的肺癌中的促进作用。(2)确定其他与CyLD相互作用的外体蛋白是否参与了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了外体CyLD，抑制了外体TRAF2的激活，从而抑制了巨噬细胞的激活，防止了肿瘤的生长。(3)在NOD-SCID小鼠模型中，确定肺癌患者肺肿瘤外切体冲击的巨噬细胞是否促进了肺癌的生长。产生的数据应该允许开发高度创新的细胞和分子基础模型，用于外切体介导的慢性炎症和促进肿瘤生长，并指示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.