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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 August 1, 2017; First published July 24, 2017
Citation Information: J Clin Invest. 2017;127(8):3075-3089. https://doi.org/10.1172/JCI89092.
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Categories: Research Article Oncology Stem cells

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

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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 1

NEK2 plays an essential role in the posttranslational regulation of EZH2.

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NEK2 plays an essential role in the posttranslational regulation of EZH2...
(A) OTS167 treatment attenuated tumor growth in a U87 sub-Q xenograft mouse model (n = 6). *P < 0.05, by 1-way ANOVA. (B) Long-term OTS167 treatment induced a resistant population in 83 glioma spheres (n = 6). P < 0.01, by 1-way ANOVA. (C) An in vitro clonogenicity assay using limiting dilution neurosphere formation indicated that the OTS167-resistant population in 83 glioma spheres was more resistant to radiotherapy (IR) (n = 10). **P < 0.01, by ELDA. (D) An in vitro cell viability assay indicated that the OTS167-resistant population in 83 glioma spheres was still sensitive to the EZH2 inhibitor GSK343 (n = 6). P > 0.05, by 1-way ANOVA. (E) qRT-PCR showed that EZH2 expression was dramatically decreased by OTS167 treatment in a U87 tumor from a sub-Q xenograft mouse model (n = 3). ***P < 0.001, by 2-tailed t test. (F) Western blotting showed that EZH2 protein was only modestly affected by OTS167 treatment in a U87 tumor from a sub-Q xenograft mouse model. Replicate samples were run on parallel gels to obtain total EZH2 and phosphorylated EZH2 (p-EZH2) (Ser21) at the same time. (G) Western blotting for a CHX assay of 267 glioma spheres pretreated with OTS167 at the IC50 concentration (10 nM) for 4 weeks. (H) Genome-wide transcriptome microarray analysis (GSE67089) showed that NEK2 was the most upregulated kinase-encoding gene in glioma spheres. (I) NEK2 showed a strong association with EZH2 expression at the protein level in THPA database (EZH2hi samples, n = 69; EZH2lo samples, n = 83). (J) Western blotting indicated that NEK2 expression was increased in a U87 sub-Q xenograft mouse model after OTS167 treatment.
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