NEK2 Promotes Aerobic Glycolysis in Multiple Myeloma Through Regulating Splicing of Pyruvate Kinase

Zhimin Gu,Jiliang Xia,Fenghuang Zhan,Guido Tricot,Ivana Frech,Hongwei Xu

doi:10.1186/s13045-017-0392-4

Zhimin Gu, Jiliang Xia + Show 4 more

Open Access

https://doi.org/10.1186/s13045-017-0392-4

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Abstract

BackgroundAerobic 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.MethodsTandem 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.ResultsNEK2 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.ConclusionsOur 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.

Highlights

Aerobic glycolysis, a hallmark of cancer, is characterized by increased metabolism of glucose and production of lactate in normaxia
NEK2 and its binding proteins were pulled down using sequential HA and Flag antibodies immunoprecipitation, and proteins bound to NEK2 were identified by mass spectrometry
NEK2, hnRNPA1, and hnRNPA2 were detected in HA immunoprecipitated proteins but not in IgG control. These results indicate that NEK2 binds to hnRNPA1 and hnRNPA2 proteins

Summary

Introduction

A hallmark of cancer, is characterized by increased metabolism of glucose and production of lactate in normaxia. In the 1920s, Dr Otto Heinrich Warburg observed that cancer cells uptake more glucose compared with normal tissues and metabolize glucose via glycolysis, a low efficient pathway for generating ATP, rather than mitochondrial oxidative phosphorylation, regardless of oxygen availability [1,2,3]. This process is known as “Warburg effect” or aerobic glycolysis. PKM1 is constitutively activated and expressed in terminally differentiated tissues to promote oxidative phosphorylation, whereas PKM2 is highly expressed in embryonic and cancer cells, which is an allosteric isoform and exhibits a dimer with low affinity for PEP. The splicing factors of heterogeneous nuclear ribonucleoproteins (hnRNP) A1/2 and polypyrimidine-tract binding (PTB) protein, which mediate c-Myc enhanced PKM2/PKM1, drive alternate splicing of PKM pre-mRNA by selectively inclusion of exon 10 and the exclusion of exon 9 [37,38,39]

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