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 2

Mitochondrial dynamics regulators mediate MLN4924-induced mitochondrial fusion.

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Mitochondrial dynamics regulators mediate MLN4924-induced mitochondrial ...
(A and B) MDA-MB-231 cells were treated with various concentrations of MLN4924 for 24 or 48 hours (A), or treated with 300 nM MLN4924 for indicated time periods (B) and analyzed by Western blotting using the indicated antibodies. Asterisks in all Western blots indicate nonspecific bands. (C) MDA-MB-231 cells were treated with 300 nM MLN4924 for 24 hours and mitochondria were isolated. Cytoplasmic (CY) and mitochondrial (MT) fractions were analyzed by Western blotting. α-Tubulin and Tom20 served as markers for cytoplasmic and mitochondrial fractions, respectively. SE, short exposure; LE, long exposure. (D) DRP1 expression levels in cytosol and mitochondria in C were normalized to α-tubulin and Tom20, respectively. Results are shown as mean ± SD (n = 3). (E) Phosphorylated DRP1S616 levels in C were normalized to total DRP1 levels in indicated cellular fractions (mean ± SD, n = 3). (F and G) MDA-MB-231 cells were transfected with indicated siRNAs against MFN1 or MFN2. Forty-eight hours after transfection, cells were harvested and analyzed by Western blotting. (H and I) MDA-MB-231 cells were transfected with indicated siRNAs (H) or plasmids expressing HA-vector (I) for 24 hours, and then treated with 300 nM MLN4924 for another 24 hours. Cells were then stained with MitoTracker Red, and mitochondrial morphology was photographed by confocal microscopy. The interconnected filamentous mitochondria are quantified (mean ± SD, n = 3). (J) MDA-MB-231 cells were transfected with HA-vector or HA-DRP1 for 24 hours, followed by treatment with various concentrations of MLN4924 for another 24 hours before being analyzed by Western blotting. (K) MDA-MB-231 cells were transfected with indicated siRNAs against MFN1 or MFN2 and plasmids expressing HA-vector for 24 hours, treated with 100 or 300 nM MLN4924, followed by FACS analysis after 24 hours. Cells at the G2/M phase were plotted (mean ± SD, n = 3). (L) MDA-MB-231 cells were transfected with si-NC, si-MFN1-2, or si-MFN2-2 for 24 hours, then treated with 100 or 300 nM MLN4924, followed by trypan blue exclusion assay for cell viability after 48 hours (mean ± SD, n = 3). (M) MDA-MB-231 cells were transfected with HA-vector and HA-DRP1 for 24 hours, then treated with 100 or 300 nM MLN4924, followed by trypan blue exclusion assay for cell viability after 48 hours (mean ± SD, n = 3). *P < 0.05, **P < 0.01 by 1-way ANOVA.