Gu Zhimin, Xia Jiliang, Xu Hongwei, Frech Ivana, Tricot Guido, Zhan Fenghuang
Department of Medicine, Division of Hematology, Oncology and Blood and Marrow Transplantation and Holden Comprehensive Cancer Center, University of Iowa, 585 Newton Rd, 52242, Iowa City, IA, USA.
Institute of Cancer Research, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.
J Hematol Oncol. 2017 Jan 13;10(1):17. doi: 10.1186/s13045-017-0392-4.
Aerobic glycolysis, a hallmark of cancer, is characterized by increased metabolism of glucose and production of lactate in normaxia. Recently, pyruvate kinase M2 (PKM2) has been identified as a key player for regulating aerobic glycolysis and promoting tumor cell proliferation and survival.
Tandem affinity purification followed up by mass spectrometry (TAP-MS) and co-immunoprecipitation (Co-IP) were used to study the interaction between NIMA (never in mitosis gene A)-related kinase 2 (NEK2) and heterogeneous nuclear ribonucleoproteins (hnRNP) A1/2. RNA immunoprecipitation (RIP) was performed to identify NEK2 binding to PKM pre-mRNA sequence. Chromatin-immunoprecipitation (ChIP)-PCR was performed to analyze a transcriptional regulation of NEK2 by c-Myc. Western blot and real-time PCR were executed to analyze the regulation of PKM2 by NEK2.
NEK2 regulates the alternative splicing of PKM immature RNA in multiple myeloma cells by interacting with hnRNPA1/2. RIP shows that NEK2 binds to the intronic sequence flanking exon 9 of PKM pre-mRNA. Knockdown of NEK2 decreases the ratio of PKM2/PKM1 and also other aerobic glycolysis genes including GLUT4, HK2, ENO1, LDHA, and MCT4. Myeloma patients with high expression of NEK2 and PKM2 have lower event-free survival and overall survival. Our data indicate that NEK2 is transcriptionally regulated by c-Myc in myeloma cells. Ectopic expression of NEK2 partially rescues growth inhibition and cell death induced by silenced c-Myc.
Our studies demonstrate that NEK2 promotes aerobic glycolysis through regulating splicing of PKM and increasing the PKM2/PKM1 ratio in myeloma cells which contributes to its oncogenic activity.
有氧糖酵解是癌症的一个标志，其特征是在正常氧条件下葡萄糖代谢增加和乳酸生成增多。最近，丙酮酸激酶M2（PKM2）已被确定为调节有氧糖酵解以及促进肿瘤细胞增殖和存活的关键因子。
采用串联亲和纯化后进行质谱分析（TAP-MS）和免疫共沉淀（Co-IP）来研究NIMA（有丝分裂中从不出现基因A）相关激酶2（NEK2）与不均一核核糖核蛋白（hnRNP）A1/2之间的相互作用。进行RNA免疫沉淀（RIP）以鉴定NEK2与PKM前体mRNA序列的结合。进行染色质免疫沉淀（ChIP）-PCR以分析c-Myc对NEK2的转录调控。进行蛋白质免疫印迹和实时PCR以分析NEK2对PKM2的调控。
NEK2通过与hnRNPA1/2相互作用来调节多发性骨髓瘤细胞中PKM未成熟RNA的可变剪接。RIP显示NEK2与PKM前体mRNA外显子9侧翼的内含子序列结合。敲低NEK2会降低PKM2/PKM1的比例以及其他有氧糖酵解相关基因（包括GLUT4、HK2、ENO1、LDHA和MCT4）的比例。NEK2和PKM2高表达的骨髓瘤患者无事件生存期和总生存期较低。我们的数据表明，在骨髓瘤细胞中，NEK2受c-Myc转录调控。NEK2的异位表达部分挽救了由c-Myc沉默诱导的生长抑制和细胞死亡。
我们的研究表明，NEK2通过调节PKM的剪接并增加骨髓瘤细胞中PKM2/PKM1的比例来促进有氧糖酵解，这有助于其致癌活性。
