Curcumin reverses tumor exosomes-mediated inhibition of myeloid differentiation

描述(申请人提供)：自然产生的多酚，姜黄素，已被证明在各种肿瘤模型中是一种有效的化疗药物；然而，它对肿瘤相关免疫抑制的影响还没有得到很大程度的研究。未成熟髓系细胞的积聚是癌症的一个标志，我们的数据表明，肿瘤产生的外切体可以抑制未成熟髓系细胞(IMCs)的分化。我们现在发现，姜黄素治疗可以抑制肿瘤外泌体介导的对小鼠IMCs分化的抑制，防止它们在脾内聚集，并延缓肿瘤的生长。用蛋白质组学方法分析肿瘤外切体的组成，发现CSN5是一个候选的调节分子，CSN5的siRNA敲除导致了肿瘤外切体介导的脾内IMCs诱导的减弱，提示外切体CSN5在肿瘤外切体介导的免疫抑制中起关键作用。我们现在发现，姜黄素处理会破坏外体CSN5的稳定性。当与磷酸化的STAM1结合时，外体CSN5是稳定的。姜黄素处理导致STAM1去磷酸化，允许E3连接酶BARD1/BRCA1重新聚集到CSN5复合体，导致CSN5降解。我们的体外激酶分析结果表明，姜黄素介导的STAM1与CSN5复合体的解离很可能是由于抑制了CSN5相关的激酶活性。这些研究的临床相关性得到了我们最近的数据的支持，这些数据表明CSN5包装在从乳腺癌患者的外周血中分离出来的外体中，而不是健康受试者中。我们建议：(1)利用siRNA技术来确定肿瘤外体CSN5是否是姜黄素预防乳腺癌外切体介导的免疫抑制的主要靶点；(2)确定CSN5相关的激酶活性是否可以阻止TS/A肿瘤细胞中BARD1/BRCA1 E3复合体降解CSN5；以及(3)确定姜黄素处理人肿瘤细胞是否导致CSN5的降解和阻止CD33(相当于小鼠GDI Lb)GRL细胞的分化。我们将进一步确定携带CSN5的外切体是否在乳腺癌患者的外周血中循环，以及外切体的过度生产是否与乳腺癌患者中iMCs的积累有关。相关性：产生的数据应该确定姜黄素作用的新机制，并确定其在防止CSN5/肿瘤外切体介导的阻止髓系细胞分化的分子靶点。

DESCRIPTION (provided by applicant): The naturally occurring polyphenol, curcumin, has been shown to be a potent chemotherapeutic agent in a variety of tumor models; however, its effects on tumor-associated immunosuppression have not been investigated to any great extent. The accumulation of immature myeloid cells is a hallmark of cancer and our data indicate that exosomes produced by tumors can suppress the differentiation of immature myeloid cells (iMCs). We have now found that curcumin treatment inhibits the tumor exosome-mediated suppression of differentiation of IMCs in mice, preventing their accumulation in the spleen and delaying tumor growth. Analysis of the composition of the tumor exosomes using a proteomics approach identified CSN5 as a candidate regulatory molecule and siRNA knockdown of CSN5 resulted in attenuation of the tumor exosome- mediated induction of IMCs in the spleen, suggesting that exosomal CSN5 plays a key role in tumor exosome-mediated immune suppression. We have now found that curcumin treatment destabilizes exosomal CSN5. The exosomal CSN5 is stable when associated with phosphorylated STAM1. Curcumin treatment leads to the dephosphorylation of STAM1 permitting the recruitment of an E3 ligase BARD1/BRCA1 to the CSN5 complex, resulting in CSN5 degradation. Our in vitro kinase assay results indicate that the curcumin- mediated disassociation of STAM1 from CSN5 complex is most likely due to the inhibition of CSN5- associated kinase activity. The clinical relevance of these studies is supported by our recent data that indicate that CSN5 is packed in exosomes isolated from the peripheral blood of breast cancer patients but not healthy subjects. We propose: (1) To use siRNA technology to determine if tumor exosomal CSN5 is a primary target of curcumin in its prevention of breast tumor exosome-mediated immunosuppression; (2) To determine whether CSN5-associated kinase activity prevents BARD1/BRCA1 E3 complex degradation of CSN5 in TS/A tumor cells; and (3) To determine whether curcumin treatment of human tumor cells results in degradation of CSN5 and prevents blocking of differentiation of CD33 (equivalent to mouse GDI lb+) Grl+ cells. We will further determine if exosomes packed with CSN5 circulate in the peripheral blood of patients with breast cancer and if overproduction of exosomes is correlated with the accumulation of iMCs in patients with breast cancer. RELEVANCE: The data generated should identify a novel mechanism for the actions of curcumin and identify its molecular targets in preventing CSN5/tumor exosome-mediated blockade of myeloid cell differentiation.