Breast tumor exosomal Jak3BP mediated immunosuppression

描述（由申请方提供）：本提案的目的是确定肿瘤外泌体介导的NK细胞活性抑制的潜在机制。我们的初步数据表明，用TS/A乳腺肿瘤细胞产生的外泌体预处理BALB/c小鼠导致肿瘤生长更快和转移更早。我们已经确定了一种新的乳腺肿瘤外泌体蛋白（Jak 3BP），它能够与NK细胞中的Jak 3结合，导致Jak 3的泛素化。然而，siRNA Jak 3BP敲除结果表明，尽管需要Jak 3BP，但它不足以使Jak 3泛素化。我们现在已经确定了两种与Jak 3BP，Nedd 4和ubiquilin相互作用的外泌体蛋白，它们有可能增强Jak 3的泛素化或保护Jak 3BP免受IL-2刺激的NK细胞的降解。我们的目的是（1）使用siRNA技术来确定在肿瘤细胞中敲除外泌体Jak 3BP是否足以逆转TS/A外泌体介导的NK细胞活化的抑制，或者是否需要其他外泌体蛋白，如Nedd 4和ubiquilin。(2)利用siRNA技术确定与Jak 3BP相互作用的其他外泌体蛋白是否参与NK细胞中Jak 3的泛素化，并确定这些相互作用的分子决定因素。我们将确定Jak 3BP的修饰是否调节其相互作用伴侣的选择，并同时使用生物发光共振能量转移测定技术来鉴定可以阻断Jak 3和Jak 3BP相互作用的肽，从而逆转肿瘤外泌体介导的NK细胞活化抑制。(3)确定哪些肿瘤外泌体蛋白有助于IL-2刺激的NK细胞中Jak 3BP的稳定，并进一步确定这些外泌体蛋白的消除是否导致Jak 3BP的降解，从而导致NK细胞活化抑制的减弱和肿瘤生长的预防。(4)为了确定人类乳腺肿瘤Jak 3BP是否也调节NK细胞Jak 3的泛素化，并确定Jak 3BP是否是 在乳腺导管腺癌患者中过度表达。产生的数据应该阐明肿瘤细胞产生的外泌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.