Glutaminase-1 Mediated Glutaminolysis to Glutathione Synthesis Maintains Redox Homeostasis and Modulates Ferroptosis Sensitivity in Cancer Cells

Abstract

Glutaminase-1 (GLS1) has garnered considerable interest as a metabolic target in cancer due to its heightened involvement and activity. However, the precise fate of glutaminolysis catalysed by GLS1 in cancer cells remains elusive. We found that GLS1 knockout led to significant suppression of cancer cell proliferation, which can be reversed or partially restored by supplementation of glutamate or non-essential amino acids that can be converted into glutamate. The addition of spliceosomal KGA or GAC ameliorates cancer cell growth in vitro and in vivo, providing both simultaneously completely reverse the effect. The primary metabolic fate of glutamate produced through glutaminolysis in cancer cells is mainly used to produce glutathione (GSH) for redox homeostasis, not entering the tricarboxylic acid cycle or synthesising nucleotides. GSH monoethyl ester (GSH-MEE) effectively rescues the inhibition of cancer cell proliferation caused by GLS1 knockout. Deletion of GLS1 results in an elevation of reactive oxygen species (ROS) and malondialdehyde (MDA), a reduction of NADPH/NADP⁺ ratio, and an augmented susceptibility of cells to ferroptosis. Glutathione Peroxidase 4 (GPX4) and GPX1 exhibit complementary roles in redox regulation, with GLS1 knockout promoting GPX4 degradation. Pharmacological inhibition of GLS1 synergises with GPX4 inhibitor to suppress tumour growth. Dual targeting of GPX4 and GPX1 presents a potent anti-cancer strategy. This metabolic mechanism facilitates a deeper comprehension of the abnormal glutamine metabolism in cancer cells, establishing a theoretical basis for the potential clinical utilisation of GLS1 inhibitors and presenting novel perspectives for advancing combinatorial therapeutic approaches.

Keywords

cancer cell / ferroptosis / GLS1 / glutamate / GPX4 / GSH

References

[1] D. Hanahan and R. A. Weinberg, “Hallmarks of Cancer: The Next Generation,” Cell 144 (2011): 646–674.

[2] M. G. Vander Heiden, L. C. Cantley, and C. B. Thompson, “Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation,” Science 324 (2009): 1029–1033.

[3] B. J. Altman, Z. E. Stine, and C. V. Dang, “From Krebs to Clinic: Glutamine Metabolism to Cancer Therapy,” Nature Reviews Cancer 16 (2016): 619–634.

[4] W. H. Yang, Y. Qiu, O. Stamatatos, T. Janowitz, and M. J. Lukey, “Enhancing the Efficacy of Glutamine Metabolism Inhibitors in Cancer Therapy,” Trends in Cancer 7 (2021): 790–804.

[5] J. Zhang, N. N. Pavlova, and C. B. Thompson, “Cancer Cell Metabolism: The Essential Role of the Nonessential Amino Acid, Glutamine,” EMBO Journal 36 (2017): 1302–1315.

[6] Y. Wang, C. Bai, Y. Ruan, et al., “Coordinative Metabolism of Glutamine Carbon and Nitrogen in Proliferating Cancer Cells Under Hypoxia,” Nature Communications 10 (2019): 201.

[7] J. B. Spinelli, H. Yoon, A. E. Ringel, S. Jeanfavre, C. B. Clish, and M. C. Haigis, “Metabolic Recycling of Ammonia via Glutamate Dehydrogenase Supports Breast Cancer Biomass,” Science 358 (2017): 941–946.

[8] M. Liu, Y. Wang, Y. Ruan, et al., “PKM2 Promotes Reductive Glutamine Metabolism,” Cancer Biology & Medicine 15 (2018): 389–399.

[9] C. M. Metallo, P. A. Gameiro, E. L. Bell, et al., “Reductive Glutamine Metabolism by IDH1 Mediates Lipogenesis Under Hypoxia,” Nature 481 (2011): 380–384.

[10] A. R. Mullen, W. W. Wheaton, E. S. Jin, et al., “Reductive Carboxylation Supports Growth in Tumour Cells With Defective Mitochondria,” Nature 481 (2011): 385–388.

[11] A. Méndez-Lucas, W. Lin, P. C. Driscoll, et al., “Identifying Strategies to Target the Metabolic Flexibility of Tumours,” Nature Metabolism 2, no. 4 (2020): 335–350, https://doi.org/10.1038/s42255-020-0195-8.

[12] D. Tang, X. Chen, R. Kang, and G. Kroemer, “Ferroptosis: Molecular Mechanisms and Health Implications,” Cell Research 31 (2021): 107–125.

[13] G. Lei, L. Zhuang, and B. Gan, “Targeting Ferroptosis as a Vulnerability in Cancer,” Nature Reviews Cancer 22, no. 7 (2022): 381–396, https://doi.org/10.1038/s41568-022-00459-0.

[14] D. Liang, A. M. Minikes, and X. Jiang, “Ferroptosis at the Intersection of Lipid Metabolism and Cellular Signaling,” Molecular Cell 82 (2022): 2215–2227.

[15] H. Zhang, F. Yang, Y. Xiao, Y. Jiang, and Z. Shao, “Conceptualizing the Complexity of Ferroptosis to Treat Triple-Negative Breast Cancer: Theory-To-Practice,” Cancer Biology & Medicine 20 (2023): 98–103.

[16] X. Jiang, B. R. Stockwell, and M. Conrad, “Ferroptosis: Mechanisms, Biology and Role in Disease,” Nature Reviews. Molecular Cell Biology 22 (2021): 266–282.

[17] J. D. Hayes, A. T. Dinkova-Kostova, and K. D. Tew, “Oxidative Stress in Cancer,” Cancer Cell 38, no. 2 (2020): 167–197, https://doi.org/10.1016/j.ccell.2020.06.001.

[18] Y. Xu, Y. Li, J. Li, and W. Chen, “Ethyl Carbamate Triggers Ferroptosis in Liver Through Inhibiting GSH Synthesis and Suppressing Nrf2 Activation,” Redox Biology 53 (2022): 102349.

[19] P. Koppula, L. Zhuang, and B. Gan, “Cystine Transporter SLC7A11/xCT in Cancer: Ferroptosis, Nutrient Dependency, and Cancer Therapy,” Protein & Cell 12 (2021): 599–620.

[20] X. Zhang, K. Yu, L. Ma, et al., “Endogenous glutamate determines ferroptosis sensitivity via ADCY10-dependent YAP suppression in lung adenocarcinoma,” Theranostics 11 (2021): 5650–5674.

[21] N. E. Sanjana, O. Shalem, and F. Zhang, “Improved Vectors and Genome-Wide Libraries for CRISPR Screening,” Nature Methods 11 (2014): 783–784.

[22] J. Y. Zhang, B. Zhou, R. Y. Sun, et al., “The Metabolite α-KG Induces GSDMC-Dependent Pyroptosis Through Death Receptor 6-Activated Caspase-8,” Cell Research 31 (2021): 980–997.

[23] A. M. Hosios, V. C. Hecht, L. V. Danai, et al., “Amino Acids Rather Than Glucose Account for the Majority of Cell Mass in Proliferating Mammalian Cells,” Developmental Cell 36 (2016): 540–549.

[24] J. B. Wang, J. W. Erickson, R. Fuji, et al., “Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation,” Cancer Cell 18 (2010): 207–219.

[25] J. Bi, T. A. Ichu, C. Zanca, et al., “Oncogene Amplification in Growth Factor Signaling Pathways Renders Cancers Dependent on Membrane Lipid Remodeling,” Cell Metabolism 30 (2019): 525–538.e528.

[26] M. Bhardwaj, J. J. Lee, A. M. Versace, et al., “Lysosomal Lipid Peroxidation Regulates Tumor Immunity,” Journal of Clinical Investigation 133 (2023): e164596.

[27] T. Nakamura, C. Hipp, A. Santos Dias Mourão, et al., “Phase Separation of FSP1 Promotes Ferroptosis,” Nature 619, no. 7969 (2023): 371–377, https://doi.org/10.1038/s41586-023-06255-6.

[28] H.-Q. Ju, J.-F. Lin, T. Tian, D. Xie, and R.-H. Xu, “NADPH Homeostasis in Cancer: Functions, Mechanisms and Therapeutic Imp Lications,” Signal Transduction and Targeted Therapy 5 (2020): 231.

[29] P. Koppula, L. Zhuang, and B. Gan, “Cystine Transporter SLC7A11/xCT in Cancer: Ferroptosis, Nutrient Depen Dency, and Cancer Therapy,” Protein & Cell 12, no. 8 (2021): 599–620, https://doi.org/10.1007/s13238-020-00789-5.

[30] J. Zheng and M. Conrad, “The Metabolic Underpinnings of Ferroptosis,” Cell Metabolism 32, no. 6 (2020): 920–937, https://doi.org/10.1016/j.cmet.2020.10.011.

[31] X. Chen, R. Kang, G. Kroemer, and D. Tang, “Broadening Horizons: The Role of Ferroptosis in Cancer,” Nature Reviews. Clinical Oncology 18 (2021): 280–296.

[32] H. Lee, F. Zandkarimi, Y. Zhang, et al., “Energy-Stress-Mediated AMPK Activation Inhibits Ferroptosis,” Nature Cell Biology 22 (2020): 225–234.

[33] C. Yang, Y. Zhao, L. Wang, et al., “De Novo Pyrimidine Biosynthetic Complexes Support Cancer Cell Proliferation and Ferroptosis Defence,” Nature Cell Biology 25 (2023): 836–847.

[34] L. Qian, F. Zhang, M. Yin, and Q. Lei, “Cancer Metabolism and Dietary Interventions,” Cancer Biology & Medicine 19 (2021): 163–174.

[35] H. Saeed, B. J. Leibowitz, L. Zhang, and J. Yu, “Targeting Myc-Driven Stress Addiction in Colorectal Cancer,” Drug Resistance Updates 69 (2023): 100963, https://doi.org/10.1016/j.drup.2023.100963.

[36] G. Bergers and S.-M. Fendt, “The Metabolism of Cancer Cells During Metastasis,” Nature Reviews Cancer 21, no. 3 (2021): 162–180, https://doi.org/10.1038/s41568-020-00320-2.

[37] U. E. Martinez-Outschoorn, M. Peiris-Pagés, R. G. Pestell, F. Sotgia, and M. P. Lisanti, “Cancer Metabolism: A Therapeutic Perspective,” Nature Reviews Clinical Oncology 14 (2017): 11–31.

[38] G. Wang, J. J. Wang, X. N. Xu, F. Shi, and X. L. Fu, “Targeting Cellular Energy Metabolism- Mediated Ferroptosis by Small Molecule Compounds for Colorectal Cancer Therapy,” Journal of Drug Targeting 30 (2022): 819–832.

[39] P. K. Robinson, “Enzymes: Principles and Biotechnological Applications,” Essays in Biochemistry 59 (2015): 1–41.

[40] K. M. Lemberg, S. S. Gori, T. Tsukamoto, R. Rais, and B. S. Slusher, “Clinical Development of Metabolic Inhibitors for Oncology,” Journal of Clinical Investigation 132 (2022): e148550.

[41] A. Daemen, B. Liu, K. Song, et al., “Pan-Cancer Metabolic Signature Predicts co-Dependency on Glutaminase and De Novo Glutathione Synthesis Linked to a High-Mesenchymal Cell State,” Cell Metabolism 28 (2018): 383–399.e389.

[42] S. Ahola and T. Langer, “Ferroptosis in Mitochondrial Cardiomyopathy,” Trends in Cell Biology 34, no. 2 (2024): 150–160, https://doi.org/10.1016/j.tcb.2023.06.002.

[43] N. Kang, S. Son, S. Min, et al., “Stimuli-Responsive Ferroptosis for Cancer Therapy,” Chemical Society Reviews 52, no. 12 (2023): 3955–3972, https://doi.org/10.1039/d3cs00001j.

[44] D. Liang, Y. Feng, F. Zandkarimi, et al., “Ferroptosis Surveillance Independent of GPX4 and Differentially Regulated by Sex Hormones,” Cell 186 (2023): 2748–2764.e2722.

[45] G. R. Steinberg and D. G. Hardie, “New Insights Into Activation and Function of the AMPK,” Nature Reviews Molecular Cell Biology 24, no. 4 (2023): 255–272, https://doi.org/10.1038/s41580-022-00547-x.

[46] M. Li, Y. Wang, X. Wei, et al., “AMPK Targets PDZD8 to Trigger Carbon Source Shift From Glucose to Glutamine,” Cell Research 34 (2024): 683–706.

[47] U. Karunakaran, S. Elumalai, J. S. Moon, and K. C. Won, “Pioglitazone-Induced AMPK-Glutaminase-1 Prevents High Glucose-Induced Pancreatic β-Cell Dysfunction by Glutathione Antioxidant System,” Redox Biology 45 (2021): 102029.

RIGHTS & PERMISSIONS

2025 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.