YOD1促进非小细胞肺癌干细胞自我更新的靶蛋白鉴定及分子机制

Lung cancer stem cells (LCSCs) are responsible for non small cell lung cancer (NSCLC) initiation, metastasis, relapse and drug-resistance. Self-renewal, the key 'stemness' property unique to LCSCs, is the breakthrough of LCSCs-targeted therapy. By using primary NSCLC stem cells-derived retroviral library, we found a potential target of self-renewal of NSCLC stem cells - deubiquitinase YOD1. Though deubiquitinases play an important role in the initiation and development of cancers, studies on the role of YOD1 in cancers have not been reported. We previously described that YOD1 was aberrantly expressed in NSCLC stem cells and promoted the self-renewal of NSCLC stem cells, but its subtrate and molecular mechanism are unclear. In this project we will 1) identify the substrate for YOD1 promoting NSCLC stem cells self-renewal using tandem affinity purification coupled with mass-spectrometry (TAP/MS) analysis, yeast two-hybrid (Y2H) screening and protein-protein interaction platform, 2) employ in vitro assay of sphere initiation and in vivo assay of tumor formation to assess the role of YOD1 and its substrate in the self-renewal of NSCLC stem cells, 3) utilize high-thoughput microarray to analyze the signal network of YOD1 and its substrate and use western blotting to detect the effect of YOD1 on canonical stem cell regulatory factors and signal pathways, and 4) collect NSCLC samples to establish the relationship among YOD1, its substrate and prognosis. Our study will provide a novel therapeutic target for NSCLC stem cells.

肺癌干细胞是非小细胞肺癌发生、转移、复发与耐药主要原因之一。自我更新是肺癌干细胞最关键"干性"特征，也是靶向清除肺癌干细胞突破口。我们采用原代肺癌干细胞逆转录病毒文库筛选到肺癌干细胞自我更新潜在靶点-去泛素化酶YOD1。已知去泛素化酶家族在肿瘤发生发展中起重要作用，但YOD1在肿瘤中尚无文献报道。我们在前期工作中发现YOD1在非小细胞肺癌干细胞中异常高表达并促进肺癌干细胞自我更新，但其靶蛋白和分子机制尚不明确。本研究拟在前期基础上，通过亲和串联质谱和酵母双杂交等技术和蛋白-蛋白作用平台鉴定YOD1促进非小细胞肺癌干细胞自我更新的靶蛋白；采用体外克隆形成和体内致瘤等方法明确YOD1及其靶蛋白促进肺癌干细胞自我更新；运用高通量芯片分析其调控信号通路并用蛋白印迹等检测其对经典干细胞调节因子和信号通路影响；收集肺癌标本研究其与患者预后相关性。本研究将为非小细胞肺癌干细胞定向清除提供新的治疗靶点。

YOD1是属于卵巢肿瘤（ovarian tumor，OTU）家族的去泛素化酶之一，其在肺癌的发生发展机制当中的作用尚无相关报道。项目申请人的前期研究工作表明，YOD1在肺癌干细胞当中表达增高，提示YOD1可能影响肺癌干细胞的自我更新，但涉及的靶分子及信号通路并不明确。本项目在前期研究的基础上，通过RT-PCR及蛋白免疫印迹观察到YOD1在肺癌细胞系和肿瘤标本当中低表达，使用甲基转移酶抑制剂抑制甲基化能逆转YOD1的低表达；免疫荧光定位结果YOD1可能通过自我去泛素化来维持自身稳定；通过亲和串联质谱以及免疫共沉淀筛选和验证得到ZENF512B以及VCP为与YOD1存在相互作用的蛋白分子；肺癌细胞系中过表达YOD1不但能下调TGF-β等肿瘤生长信号通路，更加能够协同VCP抑制剂来抑制肿瘤干性因子OCT-4和Nanog；动物致瘤实验提示YOD1的过表达能够抑制肺癌细胞的致瘤能力，同时预后分析也提示高表达YOD1表达的患者预后更好。本项目首次探究了YOD1在肺癌发生发展当中的作用，资助发表论文5篇，其中SCI收录4篇，在投文章1篇，共培养了1名博士研究生，2名硕士研究生。