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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Alternative splicing; Multiple myeloma; NEK2; Pyruvate kinase.

Fig. 1

NEK2 interacts with hnRNPA1/2 proteins. a Western blots confirm that NEK2 cDNA conjugated with tags HA-3xFLAG are transfected into the myeloma cell line ARP1. b Immunofluorescence analysis of ARP1 cells stained with NEK2 antibody (red), hnRNPA1/2 (green), and DAPI (blue). c HA antibody was used to pull down NEK2, and its interacting proteins were analyzed by Western blotting. The lysates before IP were used as a positive control and IgG pulled down proteins as a negative control

Fig. 2

High NEK2 increases the ratio of PKM2/PKM1. a RNA immunoprecipitation using anti-HA antibody to pull down NEK2 binding RNA in ARP1 NEK2-HA OE cells. Real-time PCR was performed to test the enrichment of intronic sequence flanking exon 9 of PKM pre-mRNA. All values were normalized by genomic GAPDH, and IgG was used as negative control, *p < 0.05. b Western blots were performed to test the levels of NEK2 and PKM2 in NEK2-shRNA ARP1 and OPM2 MM cells. c Real-time PCR analyses of the ratio of PKM2/PKM1 in NEK2 knocked down ARP1 and OPM2 MM cells. Results of real-time PCR were normalized against GAPDH and presented means ± SD of triplicate determinations from an experiment representative of three, *p < 0.05

Fig. 3

NEK2 regulates aerobic glycolysis in myeloma cells. a GEP analysis of NEK2, HK2, ENO1, and LDHA on plasma cells derived from normal healthy donors (n = 22), MGUS patients (n = 44), low-, and high- (n = 305) risk MM patients (n = 46). b Real-time PCR was performed to test the expression of GLUT4, NEK2, HK2, ENO1, and LDHA in NEK2 silenced ARP1 and OPM2 MM cell lines, *p < 0.001. c, d Glucose uptake and lactate production were analyzed in NEK2 knocked down ARP1 MM cells cultured at normoxia (black column) or hypoxia (grey column), *p < 0.05

Fig. 4

NEK2 is a transcriptional target of c-Myc. a Dot-plots present the expression of NEK2, c-Myc, and PKM2 in plasma cells of GEP derived from normal donors (n = 22), MGUS patients (n = 44), low- (n = 305), and high- (n = 46) risk MM patients (p < 0.001). b ChIP PCR detected binding of c-Myc to the promoter of Nek2 in P493-6 cells. IgG antibodies were used as negative control. c Real-time PCR shows the expression of NEK2 in P493-6 cells after silencing c-Myc. d Western blots show protein expression of NEK2, c-Myc, and β-actin in P493-6 cells with knocking down c-Myc. e Kaplan-Meier analyses of event-free survival (top panels) and overall survival (bottom panels) among MM patients with different expression levels of NEK2 and PKM2

Fig. 5

NEK2 mediates c-Myc-regulated aerobic glycolysis. a P493-6 cells with or without NEK2-OE were treated with Dox to inhibit c-Myc expression. Western blots show the protein expression of c-Myc, NEK2, and PKM2. b Real-time PCR shows the relative expression of PKM1 and PKM2 in P493-6 cells with altered expression of c-Myc and NEK2. c Glucose uptake and lactate production were evaluated in P493-6 cells with altered expression of c-Myc and NEK2. d Cell growth was analyzed in P493-6 cells with altered expression of c-Myc and NEK2 by trypan blue staining (*p < 0.05). e Flow cytometry analysis of apoptosis in P393-6 cells with silencing of c-Myc in the presense or absence of NEK2 overexpression using FITC-conjugated annexinV/PI staining. Apoptotic cells were annexinV positive. Representative pictures of FCM were shown with quantification of percentage of cells with apoptosis. Results from 3 independent experiments were shown. f Cell viability was analyzed in P493-6 cells with altered expression of c-Myc and NEK2 using trypan blue staining, *p < 0.05

Fig. 6

Schematic model of NEK2-mediated aerobic glycolysis through splicing of PKM1/2. c-Myc enhances the transcription of NEK2 and hnRNPA1/2, then NEK2 and hnRNPA1/2 complex bind to the intronic sequences flanking exon 9 of PKM pre-mRNA to out splicing exon 10 result in elevated expression of PKM2 and increased aerobic glycolysis