Breast tumor exosomal Jak3BP mediated immunosuppression

描述(由申请人提供)：本提案的目标是确定肿瘤外切体介导的抑制NK细胞活性的潜在机制。我们的初步数据表明，用TS/A乳腺肿瘤细胞产生的外切体预处理BALB/c小鼠，肿瘤生长更快，转移更早。我们已经鉴定出一种新的乳腺肿瘤外体蛋白(Jak3BP)，它能够与NK细胞中的JAK3结合，导致JAK3的泛素化。然而，siRNA Jak3BP敲除结果表明，虽然Jak3BP是必需的，但它不足以使JAK3泛素化。我们现在已经鉴定了两种与Jak3BP相互作用的外体蛋白，Nedd4和泛素，它们有可能增强JAK3的泛素化或保护Jak3BP在IL-2刺激的NK细胞中不被降解。我们的目标是(1)利用siRNA技术来确定在肿瘤细胞中敲除外体Jak3BP是否足以逆转TS/A外体介导的NK细胞激活抑制，或者是否需要其他外体蛋白，如Nedd4和泛素。(2)利用siRNA技术确定其他与Jak3BP相互作用的外体蛋白是否参与了NK细胞中JAK3的泛素化，并确定了这些相互作用的分子决定因素。我们将确定Jak3BP的修饰是否调节其相互作用伙伴的选择，并同时使用生物发光共振能量转移分析技术来识别能够阻断JAK3和Jak3BP相互作用的多肽，从而逆转肿瘤外体介导的对NK细胞激活的抑制。(3)确定哪些肿瘤外体蛋白参与了IL-2刺激的NK细胞中Jak3BP的稳定，并进一步确定这些外体蛋白的消除是否会导致Jak3BP的降解，从而减弱对NK细胞激活的抑制，从而阻止肿瘤的生长。(4)确定人乳腺肿瘤Jak3BP是否也调节NK细胞JAK3的泛素化，并确定Jak3BP是否 在乳腺导管癌患者中高表达。所产生的数据应该阐明由肿瘤细胞产生的外切体介导的肿瘤免疫抑制的细胞和分子基础，并表明各种治疗策略的可行性。

DESCRIPTION (provided by applicant): The goal of this proposal is to identify the mechanisms underlying tumor exosome-mediated suppression of NK cell activity. Our preliminary data indicate that pretreatment of BALB/c mice with exosomes produced by TS/A breast tumor cells results in more rapid tumor growth and earlier metastasis. We have identified a novel breast tumor exosomal protein (Jak3BP) that is capable of binding to Jak3 in the NK cells, resulting in the ubiquitination of Jak3. siRNA Jak3BP knockout results indicated, however, that although Jak3BP is required, it is not sufficient for ubiquitination of Jak3. We have now identified two exosomal proteins that interact with Jak3BP, Nedd4 and ubiquilin, which have the potential to enhance the ubiquitination of Jak3 or protect Jak3BP against its degradation in IL-2-stimulated NK cells. Our aims are (1) To use siRNA technology to determine if knockout of exosomal Jak3BP in tumor cells is sufficient for reversal of the inhibition of NK-cell activation mediated by TS/A exosomes, or whether other exosomal proteins, such as Nedd4 and ubiquilin, are required. (2) To use siRNA technology to determine if other exosomal proteins that interact with Jak3BP participate in the ubiquitination of Jak3 in NK cells and to identify the molecular determinants of these interaction. We will determine if modification of Jak3BP regulates its choice of interaction partners and, in parallel, use bioluminescence resonance energy transfer assay technology to identify a peptide that can block the interaction of Jak3 and Jak3BP thus reversing the tumor exosome-mediated inhibition of NK-cell activation. (3) To determine which tumor exosomal proteins contribute to the stabilization of Jak3BP in IL-2 stimulated NK cells, and further determine if the elimination of these exosome proteins causes degradation of Jak3BP thus resulting in the attenuation of inhibition of NK cell activation and prevention of tumor growth. (4) To determine if human breast tumor Jak3BP also regulates the ubiquitination of Jak3 of NK cells, and to determine if Jak3BP is over expressed in patients with ductal adenocarcinoma of the breast. The data generated should elucidate the cellular and molecular basis underlying tumor immunosuppression mediated by the exosomes produced by tumor cells and indicate the feasibility of various therapeutic strategies.