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 3

SCFβ-TrCP E3 ligase controls MFN1 turnover and regulates mitochondrial dynamics.

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SCFβ-TrCP E3 ligase controls MFN1 turnover and regulates mitochondrial d...
(A) 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 with indicated antibodies. α-Tubulin and Tom20 served as markers for cytoplasmic and mitochondrial fractions, respectively. (B and C) HEK293 cells were transfected with indicated plasmids, immunoprecipitated with FLAG-agarose beads, and analyzed by Western blotting to detect exogenous (B) or endogenous (C) β-TrCP1. (D) MDA-MB-231 and SK-BR-3 cells were transfected with si-NC or siRNA targeting β-TrCP1/2 for 48 hours and cells were then analyzed by Western blotting. Asterisks in Western blots indicate nonspecific bands. (E and F) MDA-MB-231 cells were cotransfected with MFN1 and increasing amounts of β-TrCP1 or β-TrCP1ΔF (E), or transfected with increasing amounts of β-TrCP1 or β-TrCP1ΔF alone (F) for 48 hours. Cells were then analyzed by Western blotting. The band density was quantified by ImageJ and normalized to α-tubulin. (G and H) MDA-MB-231 cells were transfected with si-NC or siRNA targeting β-TrCP1/2 for 48 hours, then incubated with CHX for indicated periods of time. (G) Cells were collected and analyzed by Western blotting. (H) MFN1 band density in G was quantified using ImageJ software and normalized to α-tubulin, then normalized to t = 0 time point. (I) HEK293 cells were transfected with indicated plasmids, immunoprecipitated with FLAG-agarose beads and analyzed by Western blotting to detect endogenous β-TrCP1. (J and K) MDA-MB-231 cells were cotransfected with β-TrCP1 and MFN1 or MFN1 S85/85/90A mutant for 48 hours, then incubated with CHX for indicated periods of time. (J) Cells were collected and analyzed by Western blotting. (K) The band density of FLAG-MFN1 in J was quantified using ImageJ software and normalized to α-tubulin, then normalized to t = 0 time point. (L) HEK293 ells were transfected with indicated plasmids and lysed under denaturing conditions, followed by Ni-bead pulldown. Washed beads were boiled and analyzed by Western blotting for MFN1. (M) MDA-MB-231 cells were transfected with either si-NC or siRNA targeting β-TrCP1/2 for 48 hours. Cells were stained with MitoTracker Red, and mitochondrial morphology was photographed by confocal microscopy. The percentage of cells with interconnected filamentous mitochondria was quantified. Results are shown as mean ± SD (n = 3). **P < 0.01 by 1-way ANOVA.