Inhibiting neddylation modification alters mitochondrial morphology and reprograms energy metabolism in cancer cells
Qiyin Zhou, Hua Li, Yuanyuan Li, Mingjia Tan, Shaohua Fan, Cong Cao, Feilong Meng, Ling Zhu, Lili Zhao, Min-Xin Guan, Hongchuan Jin, Yi Sun
Qiyin Zhou, Hua Li, Yuanyuan Li, Mingjia Tan, Shaohua Fan, Cong Cao, Feilong Meng, Ling Zhu, Lili Zhao, Min-Xin Guan, Hongchuan Jin, Yi Sun
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Research Article Cell biology Metabolism

Inhibiting neddylation modification alters mitochondrial morphology and reprograms energy metabolism in cancer cells

Abstract

Abnormal activation of neddylation modification and dysregulated energy metabolism are frequently seen in many types of cancer cells. Whether and how neddylation modification affects cellular metabolism remains largely unknown. Here, we showed that MLN4924, a small-molecule inhibitor of neddylation modification, induces mitochondrial fission-to-fusion conversion in breast cancer cells via inhibiting ubiquitylation and degradation of fusion-promoting protein mitofusin 1 (MFN1) by SCFβ-TrCP E3 ligase and blocking the mitochondrial translocation of fusion-inhibiting protein DRP1. Importantly, MLN4924-induced mitochondrial fusion is independent of cell cycle progression, but confers cellular survival. Mass-spectrometry-based metabolic profiling and mitochondrial functional assays reveal that MLN4924 inhibits the TCA cycle but promotes mitochondrial OXPHOS. MLN4924 also increases glycolysis by activating PKM2 via promoting its tetramerization. Biologically, MLN4924 coupled with the OXPHOS inhibitor metformin, or the glycolysis inhibitor shikonin, significantly inhibits cancer cell growth both in vitro and in vivo. Together, our study links neddylation modification and energy metabolism, and provides sound strategies for effective combined cancer therapies.

Authors

Qiyin Zhou, Hua Li, Yuanyuan Li, Mingjia Tan, Shaohua Fan, Cong Cao, Feilong Meng, Ling Zhu, Lili Zhao, Min-Xin Guan, Hongchuan Jin, Yi Sun

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Figure 6

MLN4924 promotes glycolysis.

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MLN4924 promotes glycolysis. (A and B) MDA-MB-231 cells were treated wit...
(A and B) MDA-MB-231 cells were treated with indicated concentrations of MLN4924 for 24 and 48 hours, and then subjected to analysis for ECAR, which was recorded simultaneously while obtaining OCR data in Figure 5, A and B. (C–F) MDA-MB-231 cells were treated with indicated concentrations of MLN4924 for 24 and 48 hours, and then the culture media were collected to analyze glucose consumption (C), pyruvate (D), and lactate (E) production, whereas cells were collected to measure pyruvate kinase activity (F) (mean ± SD, n = 3). (G) MDA-MB-231 cells were transfected with scrambled control siRNA (si-NC), or 2 independent siRNAs targeting PKM2 (si-PKM2-1, si-PKM2-2). Forty-eight hours after transfection, cells were harvested for Western blotting. (H) MDA-MB-231 cells were transfected with indicated siRNAs for 24 hours, then treated with various concentrations of MLN4924 for another 24 hours, and then collected to measure pyruvate kinase activity (mean ± SD, n = 3). (I) MDA-MB-231 cells were treated with indicated concentrations of MLN4924 for 24 hours, crosslinked using glutaraldehyde, and analyzed by Western blotting. GA, glutaraldehyde. (J) Purification of PKM2. Bacterially expressed PKM2 was induced by IPTG, followed by sonication, Ni-column purification and elution. An aliquot of each fraction was subjected to PAGE, followed by Coomassie blue staining. M, molecular marker; NI, noninduced cells; I, induced cells; CL, cleared cell lysate; PF, precipitate fraction; FT, flow through; W, wash fraction; E, elution. (K) Pyruvate kinase activity of purified human PKM2 (from elution fractions 1 and 2) in the presence of increasing concentrations of MLN4924. (L) MDA-MB-231 cells were transfected with either si-NC or si-MFN2-2 for 24 hours, and then treated with various concentrations of MLN4924 for 72 hours and cell viability was detected by trypan blue exclusion assay (mean ± SD, n = 3). (M) MDA-MB-231 cells were transfected with either si-NC or si-MFN2-2, and then treated with DMSO control or 5 nM MLN4924 for 10 days. Colonies were stained and counted. Data shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01 by 1-way ANOVA.