NEK2上调Beclin-1诱导自噬参与多发性骨髓瘤细胞耐药的分子机制探讨

The increase of autophagy in multiple myeloma(MM) cells is one of the novel mechanisms of drug resistance in MM. Our previous studies demonstrated NEK2 mediates drug resistance through upregulation of multidrug resistance proteins(MDR) by activation of AKT/NF-κB and Wnt-β-catenin signaling pathways in MM cells. In addition, NEK2 can upregulate the expression of Beclin-1 and induce autophagy in MM cells. However, the underlying mechanisms of autophagy induced by NEK2 are not well understood. Moreover, It's unclear whether autophagy is involved in NEK2 mediated drug resistance in MM. In this study, we will firstly elucidate whether NEK2 upregulates Beclin-1 through activation of NF-κB thereby promoting the formation of Beclin-1-Vps34 complex and induces autophagy in MM cells. Then, we will analyze the clinical correlation between NEK2, autophagy and drug resistance in new diagnosed, relapsed and sequential MM patient specimens. Finally, we will explore the relationship between NEK2 induced autophagy and drug resistance in MM and to investigate whether the combination of Bortezomib and autophagy inhibitor can overcomes NEK2 mediated drug resistance by in vitro and in vivo studies. This study will reveal the molecular mechanisms of drug resistance in MM cells mediated by NEK2 induced autophagy and provide novel insights into overcoming NEK2 mediated drug resistance in MM.

多发性骨髓瘤（Multiple Myeloma,MM）细胞内自噬增加是MM耐药新机制之一。我们前期研究发现NEK2一方面激活AKT/NF-κB和Wnt-β-catenin信号通路上调多药耐药蛋白促进MM细胞耐药，另一方面上调Beclin-1诱导MM细胞自噬，但NEK2诱导MM细胞自噬的分子机制不清楚，以及NEK2是否通过诱导自噬介导MM耐药不明确。本课题拟首先研究NEK2是否通过激活NF-κB上调Beclin-1从而促进Beclin-1-Vps34复合物形成并最终诱导MM细胞自噬；然后收集初诊、复发及序贯MM临床样本，分析NEK2、自噬与耐药的临床相关性；最后通过体内外实验研究NEK2诱导自噬与MM耐药的相关性，探讨Bortezomib联合自噬抑制剂是否能克服NEK2介导的耐药。本项目的开展将揭示NEK2诱导自噬参与MM细胞耐药的分子机制，为克服NEK2介导的MM耐药提供新思路。

耐药是导致多发性骨髓瘤（Multiple myeloma, MM）患者复发的主要原因之一，因此探讨MM细胞的耐药机制具有重要意义。NEK2基因与MM患者耐药复发密切相关，高表达NEK2的MM患者总生存率显著低于低表达患者，提示NEK2可能是克服MM患者耐药复发的理想靶点。本项目在前期研究基础上进一步探讨了NEK2通过增强自噬促进MM细胞硼替佐米（Bortezomib, BTZ）耐药的分子机制。研究结果表明NEK2能够增强MM细胞自噬水平，且自噬抑制剂氯喹显著增加NEK2过表达MM细胞对BTZ的敏感性。Western blot和qRT-PCR结果表明NEK2能够在蛋白水平影响自噬启动基因Beclin1，而不影响其RNA水平。CO-IP实验结果表明NEK2、Beclin1和去泛素化酶USP7三者在MM细胞存在相互作用。进一步的机制研究证实NEK2通过USP7介导Beclin1去泛素化，进而稳定和上调Beclin1蛋白。敲低Beclin1显著降低NEK2过表达MM细胞的自噬水平，因此NEK2通过上调Beclin1表达增强MM细胞自噬水平。此外，体内外实验结果证实敲低Beclin1显著降低NEK2过表达MM细胞对BTZ的耐药性。本项目研究进一步丰富了NEK2促进MM细胞耐药的分子机制，为治疗NEK2高表达的复发难治MM患者提供了新方案。