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Glutamine synthetase licenses APC/C-mediated mitotic progression to drive cell growth

An Author Correction to this article was published on 30 May 2022

This article has been updated

Abstract

Tumors can reprogram the functions of metabolic enzymes to fuel malignant growth; however, beyond their conventional functions, key metabolic enzymes have not been found to directly govern cell mitosis. Here, we report that glutamine synthetase (GS) promotes cell proliferation by licensing mitotic progression independently of its metabolic function. GS depletion, but not impairment of its enzymatic activity, results in mitotic arrest and multinucleation across multiple lung and liver cancer cell lines, patient-derived organoids and xenografted tumors. Mechanistically, GS directly interacts with the nuclear pore protein NUP88 to prevent its binding to CDC20. Such interaction licenses activation of the CDC20-mediated anaphase-promoting complex or cyclosome to ensure proper metaphase-to-anaphase transition. In addition, GS is overexpressed in human non-small cell lung cancer and its depletion reduces tumor growth in mice and increases the efficacy of microtubule-targeted chemotherapy. Our findings highlight a moonlighting function of GS in governing mitosis and illustrate how an essential metabolic enzyme promotes cell proliferation and tumor development, beyond its main metabolic function.

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Fig. 1: GS depletion but not inhibition impairs cancer cell proliferation and tumor growth.
Fig. 2: GS is required for cancer cell proliferation independently of its catalytic activity.
Fig. 3: The nonmetabolic function of GS governs mitotic progression.
Fig. 4: GS directly interacts with NUP88 independently of its catalytic activity.
Fig. 5: GS licenses APC/C activation by regulating the interaction of NUP88 and CDC20.
Fig. 6: GS depletion sensitizes NSCLC cells to microtubule drugs in vitro.
Fig. 7: GS is overexpressed in NSCLC and regulates sensitivity to microtubule drugs in vivo.

Data availability

All data needed to evaluate the conclusions of this study are included in the Source Data of this paper. Source data are provided with this paper.

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Acknowledgements

We thank Y. Sun (Zhejiang University), L. Jiang (City of Hope) and Z. Li for helpful discussion about the manuscript. We thank S. Feng (Westlake University) and staff at the Mass Spectrometry and Metabolomics Core Facility at Westlake University and Agilent Technologies for help with MS analysis. We thank H. Ji for kindly providing the KP cells. We thank F. Wang for kindly providing the pBOS-H2B-GFP plasmid. We thank Z. Sun (K2 Oncology) for providing important assistance on organoid studies. This study was funded by grants from the Ministry of Science and Technology of the People’s Republic of China (2020YFA0803300, Z.L. and Y.-X.F.), the National Natural Science Foundation of China (31871369 and 32011530396, Y.-X.F.; 82188102, Z.L.), the Zhejiang Provincial Natural Science Foundation of China (LD19H160002, Y.-X.F.), the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (2019R01001, Z.L.), the Czech Science Foundation (18-24473Y, Z.K.) and the EMBO Installation Grant (Z.K.).

Author information

Authors and Affiliations

Authors

Contributions

J.-S.Z. and Y.-X.F. conceived the study and designed the experiments. J.-S.Z. conducted experiments, analyzed the data and wrote the manuscript. S.S., H.-Y.Q., Z.K., R.-Y.M. and Y.-T.S. conducted cell culture studies and biochemical studies. H.-Y.Q., Z.-J.C., L.-X.Y., Y.-M.F. and J.Y. conducted animal studies. S.S. and X.G. conducted MS analysis. T.F. conducted protein structure analysis. J.K., W.L. and D.X. designed and constructed GS domain mapping constructs. X.Y., L.F., Z.S. and T.L. provided clinical samples and conducted patient samples-based experiments. Z.L. analyzed the data and wrote the manuscript. Y.-X.F. conducted animal studies, analyzed the data, wrote the manuscript and oversaw the study.

Corresponding authors

Correspondence to Jiang-Sha Zhao, Tingbo Liang, Zhimin Lu or Yu-Xiong Feng.

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The authors declare no competing interests.

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Nature Metabolism thanks Sarah-Maria Fendt and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary handling editors: Christoph Schmitt, George Caputa

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Extended data

Extended Data Fig. 1 GS is required for cancer cell proliferation in a glutamine-sufficient condition.

a, Quantification of cell proliferation of H460 cells transduced with shRNAs targeting 35 amino acid metabolic enzymes. The n of shRNAs used for each gene was shown as indicated and in Supplementary Table 1. b, Knockdown (KD) efficacy of shRNAs for the indicated enzymes. (3–5 individual shRNA per gene; shown was the mean of 3 independent biological replicates). c, Expression of GS in the human lung and liver cancer cell lines transduced with control or GS-targeted shRNAs. d, Expression of GS in H460 cells treated with or without MSO in the presence of 2 mM glutamine. e, Expression of mouse GS (mGS) in the mouse lung and liver cancer cell lines transduced with control or mGS-targeted shRNAs. f, Cell proliferation of KP cells transduced with control shRNA or shmGS-1 (left) or treated with solvent control or 1 mM MSO (right) in the presence of 2 mM glutamine. g, Colony formation by the indicated cells in the presence of 2 mM glutamine. h, Cell colonies formed by indicated cells treated with solvent control or 1 mM MSO in the presence of 2 mM glutamine. i, Expression of GS in A549 cells transfected with control siRNA or pooled siGS oligos. j, Cell growth of A549 cells transfected with control siRNA or pooled siGS oligos for 5-day culture with 2 mM glutamine. (n=3 independent biological replicates for f-g, j). k, Expression of GS in two patient-derived organoids transduced with control shRNA or GS-targeted shRNAs. l, Luc2-transduced Huh7 cells were intrahepatically implanted into nude mice. AAV expressing control (shScram) or GS-targeted (shGS) hairpins was delivered by tail vein injection and the expression of GS was measured by western blots. m, Luc2-transduced Huh7 cells were intrahepatically implanted into nude mice, and animals were treated with PBS or MSO (20 mg/kg) daily for two weeks. Tumor growth was monitored by measuring luciferase activity. (n=5 for each group of tumors). Data were presented as mean±s.e.m. Unpaired, two-tailed t-tests were applied for f, g, h, j, and m. *: p<0.05, **: p<0.01.

Source data

Extended Data Fig. 2 GS depletion inhibits tumor growth without rewiring metabolic pathways.

a, Flow cytometry analysis showing apoptosis of H460 cells transduced with control (shScram) or GS-targeting shRNAs (shGS-1 and shGS-2) cultured in glutamine-deprived media. b, Quantification of intracellular glutamine and glutamate in H460 cells transduced with control shRNA or GS-targeted shRNAs in the presence of 2 mM glutamine. (n=3 independent biological replicates). c, A schematic showing the major metabolites involved in the glutamine metabolism network. d, Quantification of the content of GSH and GSSG, components of the TCA cycles, nucleotides and hexosamines in H460 cells transduced with control shRNA or GS-targeted shRNAs in the presence of 2 mM glutamine measured by mass spectrometry analysis. (n=3 independent samples). e, Expression of GS in H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C. f. The catalytic activity of GS in H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C in the presence of 0.5 mM glutamine. g, Relative cell survival of H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C in the presence of 0.5 mM glutamine. h, Colonies formed by A549 (left) and Huh7 (right) cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C in the presence of 2 mM glutamine. (n=3 independent biological replicates for f-h). i, In vivo tumor growth of H460 cells (left) and Huh7 cells (right) transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C by weight. (n=16 for each group of the H460 tumors; for Huh7 tumors, n=8 for the shScram and shGS-1+GS-WT tumors, n=7 for shGS-1 and shGS-1+GS-R324C). Data were presented as mean±s.e.m. Unpaired, two-tailed t-tests were applied for b, d, f, g, h, and i (right panel). Mann-Whitney test was applied for i (left panel). *: p<0.05, **: p<0.01.

Source data

Extended Data Fig. 3 GS depletion causes mitotic arrest.

a, Percentage of cells in G2/M phase of KP cells transduced with control shRNA or mGS-targeted shRNAs in the presence of 2 mM glutamine. (n=3 independent biological replicates). b, Representative images from a time-lapse experiment showing mitotic progression of H460-H2B-GFP cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C in the presence of 2 mM glutamine. The transition from metaphase to anaphase was highlighted in red rectangles. c, Quantification the duration of mitotic progression of H460-H2B-GFP cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C in the presence of 2 mM glutamine. (n=80 individual cells for shScram and shGS-1, n=60 for shGS-1+GS-WT and shGS-1+GS-R324C). d, Quantification of chromosome abnormalities (chromosome lagging, chromosome bridging, chromosome mis-alignment, and multipolar spindle) of H460-H2B-GFP cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C in the presence of 2 mM glutamine. Insert: representative images showing the indicated chromosome abnormalities. (n=3 independent biological replicates). Data were presented as mean±s.e.m. For mitotic progression analysis, data were plotted in the format of a violin plot. Unpaired, two-tailed t-tests were applied for a, c, and d. *: p<0.05, **: p<0.01.

Source data

Extended Data Fig. 4 GS depletion causes polyploidy, but does not induce apoptosis.

a, Flow cytometry analysis of H460, A549, SK-MES-1, and KP cells transduced with control shRNA or GS-targeted (or mGS-targeted) shRNAs using Propidium Iodide (PI) staining. Sub-G0 cells and cells with DNA content >4N were indicated. b, Quantification of apoptotic cells of (a). (n=3 independent biological replicates). c, Fold change of polyploid cells (DNA content > 4n) in KP cells transduced with control shRNA or mGS-targeted shRNAs. (n=3 independent biological replicates). d, Representative Hoechst 33342 staining (left) and bright-field images (right) of H460 (upper) and A549 (lower) cells transduced with shGS-1. Multinucleated cells are highlighted. Scale bar: 30 µm. e, Quantification of multinucleated cells of H460 and SK-MES-1 cells transduced with control shRNA or GS-targeted shRNAs in the presence of 2 mM glutamine. (n=3 independent biological replicates). Data were presented as mean±s.e.m. Unpaired, two-tailed t-tests were applied for b, c, and e. *: p<0.05, **: p<0.01.

Source data

Extended Data Fig. 5 GS directly interacts with NUP88 via a motif of its b-Grasp domain.

a, A subset of the GS-interacting proteins identified by co-IP and LC-MS/MS analyses. b, Co-IP experiments were conducted with the lysate of Huh7 or A549 cells using an anti-GS antibody. c. Co-IP experiments were conducted with the lysate of H460 or A549 cells transduced with control shRNA or shGS-1 using an anti-GS antibody. d, Silver staining for purified FLAG-GS and V5-NUP88. e, In vitro pull-down experiments were conducted with the mixture of purified FLAG-GS and V5-NUP88 protein using an anti-FLAG antibody. NUP98, NUP214 and RAE1 were not detectable in the input (mixture of the indicated purified proteins) or the precipitates from the pull-down experiments. f, Left: Western blots showing the knockdown efficiency of NUP214-targeted siRNA in A549 cells; right: Co-IP experiments were conducted in A549 cells transfected with control or NUP214 siRNA oligos as indicated using an anti-GS antibody. g, Co-IP experiments were conducted with the lysate of H460 cells deprived of glutamine or cultured with 2 mM glutamine. h, Schematic showing the wild-type (WT) and five mutants (D1-D5) of GS. i, Co-IP experiments were conducted with the lysate of H460 cells transduced with the indicated WT or mutant forms of FLAG-tagged GS using an anti-FLAG antibody.

Source data

Extended Data Fig. 6 GS directly interacts with NUP88 through a charge-charge interaction.

a, Surface electrostatic potential of the crystal structure of GS (PDB code: 2QC8) showing the two charged patches for potential protein-protein interaction. The surface electrostatic potential was calculated in PyMOL. b, A schematic showing the design of GS-del6 mutant in which the first six amino acids of the K103-R119 motif are deleted. c, Co-IP experiments were conducted with the lysate of 293T cells transfected with V5-tagged NUP88 and FLAG-tagged GS-WT or GS-del6 using anti-FLAG antibody. d, Left: In vitro pull-down experiments were conducted with the mixture of purified V5-NUP88 and FLAG-GS-WT or FLAG-GS-m4 protein using anti-FLAG or anti-V5 antibodies; right: Western blot showing V5-NUP88, FLAG-GS-WT and FLAG-GS-m4 protein purified from 293T cells transfected with V5-NUP88 and FLAG-GS expressing constructs. e, Silver staining for purified FLAG-GS-m4 proteins. f, Western blot showing the expression of GS in H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-m4. g, Quantification of the duration of mitotic progression of H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-m4 in the presence of 2 mM glutamine. (n=60 individual cells for each group of cells). Data from mitotic analysis were plotted in the format of a violin plot. Unpaired, two-tailed t-tests were applied for g. *: p<0.05, **: p<0.01.

Source data

Extended Data Fig. 7 GS depletion does not affect the interaction of NUP88-RAE1-NUP98, but augments the interaction of NUP88 and CDC20.

a, Co-IP experiments conducted with the lysate of H460 cells as indicated using anti-RAE1, anti-NUP98, and anti-NUP88 antibodies. b, Expression of PLK1 in H460 cells transduced with control shRNA, shGS-1, shGS-1 combined with overexpression of wild-type GS (GS-WT), or shGS-1 combined with overexpression of GS-R324C. c, H460 cells transduced with control shRNA or GS-targeted shRNA were arrested at mitotic prometaphase (PrM), metaphase (Met), or anaphase (Ana) by indicated treatment. Cell lysate was collected for Co-IP experiments using an anti-NUP88 antibody. d, Content of the purified proteins used in Fig. 5b. e, Co-IP experiments conducted with the lysate of H460 cells of b using an anti-NUP88 antibody. Cells were treated with nocodazole for 18 hours before M-phase cells harvested for co-IP experiments. f, Upper: expression of GS in H460 cells transduced with control shRNA, shGS-1 in the presence of 0.5 mM or 2 mM glutamine. Lower: H460 cells transduced with control shRNA or GS-targeted shRNA were cultured with 0.5 mM or 2 mM glutamine for 8 hours then treated with nocodazole for 18 hours. Floating cells were collected and treated with MG132 for another hour. Cell lysate was collected for co-IP experiments using an anti-CDC27 antibody. g, In vitro pull-down experiments conducted with the mixture of the purified Myc-CDC20, HA-CDC27 and different dosages of V5-NUP88 protein using an anti-HA antibody. h, Co-IP experiments conducted with the lysate of H460 cells cultured in 0.5 mM or 2 mM glutamine using an anti-NUP88 antibody. i. Percentage of G2/M cells of H460 cells transduced with control shRNA or GS-targeted shRNA after released from nocodazole treatment as indicated time points. (n=3 independent biological replicates). j, Expression of CDC20 in H460 cells transduced with control shRNA, control shRNA combined with overexpression of CDC20, shGS-1, and shGS-1 combined with overexpression of wild-type CDC20. k, The fold change of polyploid cells in H460 cells of j. (n=3 independent biological replicates). Data were presented as mean±s.e.m. Unpaired, two-tailed t-tests were applied for i and k. *: p<0.05, **: p<0.01.

Source data

Extended Data Fig. 8 GS depletion sensitizes cancer cells to microtubule drugs in vitro.

a, Percentage of apoptotic cells (sub-G0 phase) of H460 cells treated with nocodazole (Noc) for 2 days. (n=3 independent biological replicates). b, Quantification of cell death in parental H460 cells or H460 cells transduced with control shRNA or GS-targeted shRNAs treated with solvent control, vincristine (20 nM), or nocodazole (50 ng/ml) as indicated for three days. (n=3 independent biological replicates). c, Dose-response curves showing the response of SK-MES-1 and A549 cells transduced with control shRNA or shGS-1 treated with various concentrations of vincristine, paclitaxel, or nocodazole for three days. (n=3 independent biological replicates). d, Dose-response curves showing the response of H460 and A549 cells transduced with control shRNA or shGS-1 treated with various concentrations of doxorubicin for three days. (n=3 independent biological replicates). e, Dose-response curves showing the response of SK-MES-1 and A549 cells treated with various concentrations of vincristine or nocodazole in the presence or absence of 1 mM MSO for three days. (n=3 independent biological replicates). f, Western blots showing the knockdown efficacy of GS in two primary lung cancer cells. g, Dose-response curves showing the response of two freshly isolated primary lung cancer cells transduced with control shRNA or shGS-1 treated with various concentrations of vincristine or nocodazole for three days. (n=2 independent biological replicates). Data were presented as mean±s.e.m. Unpaired, two-tailed t-tests were applied for a and b. Two-way ANOVA was applied for c, d, e, and g. *: p<0.05, **: p<0.01.

Source data

Extended Data Fig. 9 GS is overexpressed in human NSCLC and contributes to resistance to microtubule drugs in vivo.

a, Quantification of the body weight of the H460 tumor-bearing nude mice in both solvent control and paclitaxel treatment cohorts. (n=10 for each group of the H460-innoculated mice). b, Quantification of weight of tumors dissected from nude mice injected with the indicated cells and treated with solvent control (PBS) or paclitaxel (5 mg/kg) every other day in vivo for 16 days. (n=10 for each group of the H460-innoculated mice). c, Representative images of tumors of b. d, Quantification of GS expression in a tissue array containing 16 normal lung tissues and 86 lung cancer samples (right). Data were presented as mean±s.e.m. Unpaired, two-tailed t-test was applied for b. χ2 test was applied for d. *: p<0.05, **: p<0.01.

Source data

Supplementary information

Supplementary Information

Supplementary Fig. 1

Reporting Summary

Supplementary Table 1

Supplementary Table 1. Target sequence of the hairpins for the 35 essential amino acid metabolism enzymes. Supplementary Table 2. Peptides identified from the co-IP LC–MS experiment. Supplementary Table 3. Sequence of the siRNA oligonucleotides targeting GS and NUP214. Supplementary Table 4. Patient metadata for NSCLC samples

Supplementary Video 1

Mitotic progression of H460-shScram cells.

Supplementary Video 2

Mitotic progression of H460-shGS-1 cells.

Supplementary Video 3

Mitotic progression of H460-shGS + WT GS cells.

Supplementary Video 4

Mitotic progression of H460-shGS + GS-R324C cells.

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Source Data Extended Data Fig. 7

Uncropped western blots.

Source Data Extended Data Fig. 7

Statistical Source Data.

Source Data Extended Data Fig. 8

Uncropped western blots.

Source Data Extended Data Fig. 8

Statistical Source Data.

Source Data Extended Data Fig. 9

Statistical Source Data.

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Zhao, JS., Shi, S., Qu, HY. et al. Glutamine synthetase licenses APC/C-mediated mitotic progression to drive cell growth. Nat Metab 4, 239–253 (2022). https://doi.org/10.1038/s42255-021-00524-2

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