A mitotic NADPH upsurge promotes chromosome segregation and tumour progression in aneuploid cancer cells.

Redox metabolites have been observed to fluctuate through the cell cycle in cancer cells, but the functional impacts of such metabolic oscillations remain unknown. Here, we uncover a mitosis-specific nicotinamide adenine dinucleotide phosphate (NADPH) upsurge that is essential for tumour progression. Specifically, NADPH is produced by glucose 6-phosphate dehydrogenase (G6PD) upon mitotic entry, which neutralizes elevated reactive oxygen species (ROS) and prevents ROS-mediated inactivation of mitotic kinases and chromosome missegregation. Mitotic activation of G6PD depends on the phosphorylation of its co-chaperone protein BAG3 at threonine 285, which results in dissociation of inhibitory BAG3. Blocking BAG3T285 phosphorylation induces tumour suppression. A mitotic NADPH upsurge is present in aneuploid cancer cells with high levels of ROS, while nearly unobservable in near-diploid cancer cells. High BAG3T285 phosphorylation is associated with worse prognosis in a cohort of patients with microsatellite-stable colorectal cancer. Our study reveals that aneuploid cancer cells with high levels of ROS depend on a G6PD-mediated NADPH upsurge in mitosis to protect them from ROS-induced chromosome missegregation.

Aurora A-mediated pyruvate kinase M2 phosphorylation promotes biosynthesis with glycolytic metabolites and tumor cell cycle progression.

Cancer cells have distinctive demands for intermediates from glucose metabolism for biosynthesis and energy in different cell cycle phases. However, how cell cycle regulators and glycolytic enzymes coordinate to orchestrate the essential metabolic processes are still poorly characterized. Here, we report a novel interaction between the mitotic kinase, Aurora A, and the glycolytic enzyme, pyruvate kinase M2 (PKM2), in the interphase of the cell cycle. We found Aurora A-mediated phosphorylation of PKM2 at threonine 45. This phosphorylation significantly attenuated PKM2 enzymatic activity by reducing its tetramerization and also promoted glycolytic flux and the branching anabolic pathways. Replacing the endogenous PKM2 with a nonphosphorylated PKM2 T45A mutant inhibited glycolysis, glycolytic branching pathways, and tumor growth in both in vitro and in vivo models. Together, our study revealed a new protumor function of Aurora A through modulating a rate-limiting glycolytic enzyme, PKM2, mainly during the S phase of the cell cycle. Our findings also showed that although both Aurora A and Aurora B kinase phosphorylate PKM2 at the same residue, the spatial and temporal regulations of the specific kinase and PKM2 interaction are context dependent, indicating intricate interconnectivity between cell cycle and glycolytic regulators.

Development of a one-plasmid system to replace the endogenous protein with point mutation for post-translational modification studies.

BACKGROUND: Post-translational modification (PTM) is one of the major regulatory mechanism for protein activities. To understand the function of PTMs, mutants that prevent or mimic the modification are frequently utilized. The endogenous proteins are usually depleted while the point mutations are expressed. A common strategy to accomplish these tasks includes two-steps: First, a cell line stably expressing shRNA for protein depletion is generated, then an RNAi-resistance construct is introduced to express mutant. However, these steps are time- and labor-consuming. More importantly, shRNA and mutant protein are frequently expressed in different cells at different time, which significantly disturbs the conclusions. METHODS: To overcome these technical problems, we developed a lentiviral based one-plasmid system that allowed concurrent expression of shRNA and mutant protein. The puromycin-resistant gene was inserted for the selection of stable-expression cells. RESULTS: Using this plasmid, we efficiently replaced the endogenous proteins with comparable levels of exogenous proteins for LDHB and PKM2, two glycolytic enzymes regulated by PTM in cancer cells. The system was also successfully exploited in evaluating the role of phosphorylation of LDHB serine 162 in multiple in vitro and in vivo assays. CONCLUSION: Thus, we have developed an efficient one-plasmid system to replace endogenous protein with point mutations for the functional study of PTM.

Energy restriction causes metaphase delay and chromosome mis-segregation in cancer cells.

ATP metabolism during mitosis needs to be coordinated with numerous energy-demanding activities, especially in cancer cells whose metabolic pathways are reprogramed to sustain rapid proliferation in a nutrient-deficient environment. Although strategies targeting the energy metabolic pathways have shown therapeutic efficacy in preclinical cancer models, how normal cells and cancer cells differentially respond to energy shortage is unclear. In this study, using time-lapse microscopy, we found that cancer cells displayed unique mitotic phenotypes in a dose-dependent manner upon decreasing ATP (i.e. energy) supply. When reduction in ATP concentration was moderate, chromosome movements in mitosis were barely affected, while the metaphase-anaphase transition was significantly prolonged due to reduced tension between the sister-kinetochores, which delayed the satisfaction of the spindle assembly checkpoint. Further reduction in ATP concentration led to a decreased level of Aurora-B at the centromere, resulting in increased chromosome mis-segregation after metaphase delay. In contrast to cancer cells, ATP restriction in non-transformed cells induced cell cycle arrest in interphase, rather than causing mitotic defects. In addition, data mining of cancer patient database showed a correlation between signatures of energy production and chromosomal instability possibly resulted from mitotic defects. Together, these results reveal that energy restriction induces differential responses in normal and cancer cells, with chromosome mis-segregation only observed in cancer cells. This points to targeting energy metabolism as a potentially cancer-selective therapeutic strategy.

Aurora-A mediated phosphorylation of LDHB promotes glycolysis and tumor progression by relieving the substrate-inhibition effect.

Overexpressed Aurora-A kinase promotes tumor growth through various pathways, but whether Aurora-A is also involved in metabolic reprogramming-mediated cancer progression remains unknown. Here, we report that Aurora-A directly interacts with and phosphorylates lactate dehydrogenase B (LDHB), a subunit of the tetrameric enzyme LDH that catalyzes the interconversion between pyruvate and lactate. Aurora-A-mediated phosphorylation of LDHB serine 162 significantly increases its activity in reducing pyruvate to lactate, which efficiently promotes NAD+ regeneration, glycolytic flux, lactate production and bio-synthesis with glycolytic intermediates. Mechanistically, LDHB serine 162 phosphorylation relieves its substrate inhibition effect by pyruvate, resulting in remarkable elevation in the conversions of pyruvate and NADH to lactate and NAD+. Blocking S162 phosphorylation by expression of a LDHB-S162A mutant inhibited glycolysis and tumor growth in cancer cells and xenograft models. This study uncovers a function of Aurora-A in glycolytic modulation and a mechanism through which LDHB directly contributes to the Warburg effect.