翻译起始因子EIF4G1在多发性内分泌腺瘤病2型中的作用及机制研究

Multiple endocrine neoplasia type 2 (MEN2) is one of the most important hereditary endocrine diseases. RET proto-oncogene mutations play a critical role in MEN2 tumoriogenesis. However, the frequency of non-RET genetic events, their impacts and molecular mechanisms remain to be discovered. We performed next-generation-sequencing of the exome in paired MEN2 tumors, namely medullary thyroid carcinoma (MTC), pheochromocytoma and parathyroid. We found recurrent alterations (SNV and CNV) of EIF4G1, eukaryotic translation initiation factor 4 gamma, in multiple MEN2 tumors. Over 60% of MEN2 tumors showed positive EIF4G1 immunostaining. Furthermore, we confirmed the oncogenic roles of mutant EIF4G1 in MEN2 tumor cells (TT, MZ-CRC1 and PC12 cell lines) by showing its upregulation of cell proliferation as well as phosphorylation of RET, MAPK and AKT. Based upon these findings, the current study is designed to investigate the roles of EIF4G1 in MEN2 tumorigenesis clinically, in vitro and in vivo. We also aim to establish the possible correlation between MEN2 and mTOR, apply high-resolution transcriptome-scale ribosome profiling to reveal the translation target of EIF4G1 in MEN2 tumors. Finally, our study will reveal the type and frequency of its genetic variations in MEN2 and control population, as well as the correlation of these variations with MEN2 phenotype. Our study will provide important information of how eukaryotic translation initiation factor impact MEN2 tumorigenesis, eventually leading to the discovery of novel therapy.

多发性内分泌腺瘤病2型(MEN2)是最为重要的遗传性内分泌疾病之一，已知的病因是RET基因突变，但对其多器官肿瘤本身的调控却了解甚少，且表型的异质性提示MEN2中存在RET外的重要调控机制。我们前期用二代测序技术对MEN2的配对肿瘤进行了外显子组测序，发现翻译起始因子EIF4G1在MEN2多个肿瘤中不同方式的变异；并证实EIF4G1促进肿瘤细胞增殖和RET磷酸化，激活MAPK及AKT通路。本研究拟在此基础上，在临床、细胞及动物三个层面系统分析EIF4G1对MEN2相关肿瘤发生发展的影响；进一步建立MEN2与mTOR可能的联系，并运用高通量测序分析mRNA翻译谱寻找MEN2中直接受EIF4G1调控翻译的下游分子或途径；最后分析EIF4G1变异的遗传特征及临床相关性。最终阐明EIF4G1介导的真核翻译过程对MEN2的影响与分子机制，揭示除RET之外的分子事件，为MEN2的治疗提供新的可能途径。

多发性内分泌腺瘤病（MEN）特征为在同一个体发生涉及两个及两个以上内分泌腺的肿瘤，分为MEN1和MEN2。本研究旨在建立中国人群MEN2队列及突变谱，探讨EIF4G1在MEN2肿瘤中的生物学效应，对MEN2肿瘤发生和发展过程的影响。主要成果如下：1）该课题建立了MEN生物样本库，目前入库标本（液氮标本/石蜡标本/外周血 DNA/血清）超过300例，从而为进一步进行MEN1和MEN2相关的转化医学研究提供了可靠的信息和材料。2）MEN2系RET基因胚系突变所致，中国人群RET突变近80.0%位于634位点。突变位点的ATA危险等级与髓样癌疾病进展具有相关性。危险等级越高，发病年龄越早（MTC中位诊断年龄HST vs H vs MOD: 23.0， 32.0，39.0岁），疾病进展越快；危险等级越高，治疗反应越差，生化治愈率越低（HST vs H vs MOD: 0, 66.7%, 37.1%）。3）对MEN2 的相关肿瘤组分进行外显子组测序结果发现存在 13个重复的基因改变，包括EIF4G1。对EIF4G1进行体外功能研究发现，EIF4G1 (p. E1147V)突变可使细胞内RET(905)、AKT（Ser473）以及ERK（Thy202/Tyr204）磷酸化水平上调；而干扰其内源性表达后，RET/MAPK磷酸化程度降低，同时细胞增殖受抑制。提示 EIF4G1可能通过调控 RET 激酶活性参与 MEN2 肿瘤的形成过程。4）对MEN1的疾病特征及发病机制进行初步探究，详细描绘了MEN1的不典型肿瘤组分。其中胸腺类癌作为MEN1的不典型肿瘤组分，发病率低而致死率高，为 MEN1 患者死亡的重要原因。诊断年龄较大(HR = 1.4, 95% CI = 1.0-1.8, P =0 .03)、肿瘤最大径越大(HR = 1.5, 95% CI = 1.0-2.3, P =0 .04)或合并肿瘤转移(HR = 1.6, 95% CI = 1.0-2.5, P =0 .04)为预后不良的高危因素，需积极处理。肾上腺皮质癌可能是MEN1的罕见肿瘤组分，可出现家族聚集现象，肿瘤极具侵袭性、进展迅速、预后差。本研究是对MEN2的基因型与临床特征、相关肿瘤发病机制的探讨，同时对MEN1的不典型肿瘤成分进行了初步探究，为临床诊治及后续机制的深入探讨提供了线索。