Targeting NEK2 attenuates glioblastoma growth and radioresistance by destabilizing histone methyltransferase EZH2
Jia Wang, … , Maode Wang, Ichiro Nakano
Jia Wang, … , Maode Wang, Ichiro Nakano
Published July 24, 2017
Citation Information: J Clin Invest. 2017;127(8):3075-3089. https://doi.org/10.1172/JCI89092.
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Research Article Oncology

Targeting NEK2 attenuates glioblastoma growth and radioresistance by destabilizing histone methyltransferase EZH2

Abstract

Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity–dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.

Authors

Jia Wang, Peng Cheng, Marat S. Pavlyukov, Hai Yu, Zhuo Zhang, Sung-Hak Kim, Mutsuko Minata, Ahmed Mohyeldin, Wanfu Xie, Dongquan Chen, Violaine Goidts, Brendan Frett, Wenhao Hu, Hongyu Li, Yong Jae Shin, Yeri Lee, Do-Hyun Nam, Harley I. Kornblum, Maode Wang, Ichiro Nakano

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Figure 3

NEK2 silencing reduces GSC self-renewal in vitro and tumorigenesis in vivo.

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NEK2 silencing reduces GSC self-renewal in vitro and tumorigenesis in vi...
(A) Western blotting for NEK2 in 528 glioma spheres transduced with shNEK2 no. 1, shNEK2 no. 2, or shNT. (B) In vitro growth assay showed that NEK2 silencing inhibited the cell proliferation of 528 glioma spheres (n = 6). ***P < 0.001, by 1-way ANOVA. (C) An in vitro clonogenicity assay by limiting dilution neurosphere formation indicated that NEK2 silencing decreased the clonogenicity of 528 glioma spheres (n = 10). ***P < 0.001, by ELDA. (D) qRT-PCR for CD133 expression in 528 glioma spheres transduced with shNEK2 no. 1, shNEK2 no. 2, or shNT (n = 3). *P < 0.05 and ***P < 0.001, by 1-way ANOVA followed by Dunnett’s post-hoc test. (E) ICC for NEK2 and GFAP expression in 528 glioma spheres transduced with shNEK2 no. 1, shNEK2 no. 2, or shNT. Scale bars: 5 μm. (F) Representative images of H&E-stained mouse brain sections after the intracranial transplantation of 528 glioma spheres transduced with shNEK2 no. 1, shNEK2 no. 2, or shNT. Scale bars: 1 mm (top) and 100 μm (bottom). (G) Kaplan-Meier analysis of nude mice harboring an intracranial tumor derived from 528 glioma spheres transduced with shNT (n = 6), shNEK2 no. 1 (n = 5), or shNEK2 no. 2 (n = 5). **P = 0.0021, for shNT versus shNEK2 no. 1; **P = 0.0017, for shNT versus shNEK2 no. 2; both by log-rank test.