O-GlcNAcylation promotes malignancy and cisplatin resistance of lung cancer by stabilising NRF2

Abstract

Background: The transcription factor NRF2 plays a significant role in regulating genes that protect cells from oxidative damage. O-GlcNAc modification, a type of posttranslational modification, is crucial for cellular response to stress. Although the involvement of both NRF2 and O-GlcNAc in maintaining cellular redox balance and promoting cancer malignancy has been demonstrated, the potential mechanisms remain elusive.

Methods: The immunoblotting, luciferase reporter, ROS assay, co-immunoprecipitation, and immunofluorescence was used to detect the effects of global cellular O-GlcNAcylation on NRF2. Mass spectrometry was utilised to map the O-GlcNAcylation sites on NRF2, which was validated by site-specific mutagenesis and O-GlcNAc enzymatic labelling. Human lung cancer samples were employed to verify the association between O-GlcNAc and NRF2. Subsequently, the impact of NRF2 O-GlcNAcylation in lung cancer malignancy and cisplatin resistance were evaluated in vitro and in vivo.

Results: NRF2 is O-GlcNAcylated at Ser103 residue, which hinders its binding to KEAP1 and thus enhances its stability, nuclear localisation, and transcription activity. Oxidative stress and cisplatin can elevate the phosphorylation of OGT at Thr444 through the activation of AMPK kinase, leading to enhanced binding of OGT to NRF2 and subsequent elevation of NRF2 O-GlcNAcylation. Both in cellular and xenograft mouse models, O-GlcNAcylation of NRF2 at Ser103 promotes the malignancy of lung cancer. In human lung cancer tissue samples, there was a significant increase in global O-GlcNAcylation, and elevated levels of NRF2 and its O-GlcNAcylation compared to paired adjacent normal tissues. Chemotherapy promotes NRF2 O-GlcNAcylation, which in turn decreases cellular ROS levels and drives lung cancer cell survival.

Conclusion: Our findings indicate that OGT O-GlcNAcylates NRF2 at Ser103, and this modification plays a role in cellular antioxidant, lung cancer malignancy, and cisplatin resistance.

Keywords

drug resistance / lung cancer / NRF2 / O-GlcNAc / ROS

References

[1] Qin S, Jiang J, Lu Y, et al. Emerging role of tumor cell plasticity in modifying therapeutic response. Signal Transduct Target Ther. 2020; 5(1): 228.

[2] Prasad S, Gupta SC, Tyagi AK. Reactive oxygen species (ROS) and cancer: role of antioxidative nutraceuticals. Cancer Lett. 2017; 387: 95-105.

[3] Reczek CR, Chandel NS. ROS promotes cancer cell survival through calcium signaling. Cancer Cell. 2018; 33(6): 949-951.

[4] Cheung EC, Vousden KH. The role of ROS in tumour development and progression. Nat Rev Cancer. 2022; 22(5): 280-297.

[5] Yamamoto M, Kensler TW, Motohashi H. The KEAP1-NRF2 system: a thiol-based sensor-effector apparatus for maintaining redox homeostasis. Physiol Rev. 2018; 98(3): 1169-1203.

[6] Zhang Q, Wang L, Wang S, et al. Signaling pathways and targeted therapy for myocardial infarction. Signal Transduct Target Ther. 2022; 7(1): 78.

[7] Panieri E, Santoro MM. ROS homeostasis and metabolism: a dangerous liason in cancer cells. Cell Death Dis. 2016; 7(6): e2253.

[8] Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal. 2012; 24(5): 981-990.

[9] Moloney JN, Cotter TG. ROS signalling in the biology of cancer. Semin Cell Dev Biol. 2018; 80: 50-64.

[10] Hayes JD, Dinkova-Kostova AT, Tew KD. Oxidative stress in cancer. Cancer Cell. 2020; 38(2): 167-197.

[11] Dinkova-Kostova AT, Abramov AY. The emerging role of Nrf2 in mitochondrial function. Free Radic Biol Med. 2015; 88(Pt B): 179-188.

[12] Vomund S, Schafer A, Parnham MJ, Brune B, von Knethen A. Nrf2, the master regulator of anti-oxidative responses. Int J Mol Sci. 2017; 18(12): 2772.

[13] Torrente L, DeNicola GM. Targeting NRF2 and its downstream processes: opportunities and challenges. Annu Rev Pharmacol Toxicol. 2022; 62(1): 279-300.

[14] Tebay LE, Robertson H, Durant ST, et al. Mechanisms of activation of the transcription factor Nrf2 by redox stressors, nutrient cues, and energy status and the pathways through which it attenuates degenerative disease. Free Radic Biol Med. 2015; 88(Pt B): 108-146.

[15] Loboda A, Damulewicz M, Pyza E, Jozkowicz A, Dulak J. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cell Mol Life Sci. 2016; 73(17): 3221-3247.

[16] Tonelli C, Chio IIC, Tuveson DA. Transcriptional regulation by Nrf2. Antioxid Redox Signal. 2018; 29(17): 1727-1745.

[17] Pillai R, Hayashi M, Zavitsanou A-M, Papagiannakopoulos T. NRF2: KEAPing tumors protected. Cancer Discov. 2022; 12(3): 625-643.

[18] Itoh K, Wakabayashi N, Katoh Y, et al. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 1999; 13(1): 76-86.

[19] Wang J, Lu Q, Cai J, et al. Nestin regulates cellular redox homeostasis in lung cancer through the Keap1-Nrf2 feedback loop. Nat Commun. 2019; 10(1): 5043.

[20] Dinkova-Kostova AT, Holtzclaw WD, Cole RN, et al. Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci USA. 2002; 99(18): 11908-11913.

[21] Itoh K, Wakabayashi N, Katoh Y, Ishii T, O’Connor T, Yamamoto M. Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles. Genes Cells. 2003; 8(4): 379-391.

[22] Lau A, Wang XJ, Zhao F, et al. A noncanonical mechanism of Nrf2 activation by autophagy deficiency: direct interaction between Keap1 and p62. Mol Cell Biol. 2010; 30(13): 3275-3285.

[23] Komatsu M, Kurokawa H, Waguri S, et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nat Cell Biol. 2010; 12(3): 213-223.

[24] Jain AK, Jaiswal AK. GSK-3beta acts upstream of Fyn kinase in regulation of nuclear export and degradation of NF-E2 related factor 2. J Biol Chem. 2007; 282(22): 16502-16510.

[25] Apopa PL, He X, Ma Q. Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR-32 neuroblastoma cells. J Biochem Mol Toxicol. 2008; 22(1): 63-76.

[26] Niture SK, Jain AK, Jaiswal AK. Antioxidant-induced modification of INrf2 cysteine 151 and PKC-delta-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance. J Cell Sci. 2009; 122(Pt 24): 4452-4464.

[27] Sun Z, Chin YE, Zhang DD. Acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 during the antioxidant response. Mol Cell Biol. 2009; 29(10): 2658-2672.

[28] Kawai Y, Garduno L, Theodore M, Yang J, Arinze IJ. Acetylation-deacetylation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) regulates its transcriptional activity and nucleocytoplasmic localization. J Biol Chem. 2011; 286(9): 7629-7640.

[29] Liu X, Li H, Liu L, et al. Methylation of arginine by PRMT1 regulates Nrf2 transcriptional activity during the antioxidative response. Biochim Biophys Acta. 2016; 1863(8): 2093-2103.

[30] Torres CR, Hart GW. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J Biol Chem. 1984; 259(5): 3308-3317.

[31] Gao Y, Wells L, Comer FI, Parker GJ, Hart GW. Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain. J Biol Chem. 2001; 276(13): 9838-9845.

[32] Haltiwanger RS, Holt GD, Hart GW. Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphospho-N-acetylglucosamine:peptide beta-N-acetylglucosaminyltransferase. J Biol Chem. 1990; 265(5): 2563-2568.

[33] Yang X, Qian K. Protein O-GlcNAcylation: emerging mechanisms and functions. Nat Rev Mol Cell Biol. 2017; 18(7): 452-465.

[34] Ma J, Hou C, Wu C. Demystifying the O-GlcNAc code: a systems view. Chem Rev. 2022; 122(20): 15822-15864.

[35] Song H, Ma J, Bian Z, et al. Global profiling of O-GlcNAcylated and/or phosphorylated proteins in hepatoblastoma. Signal Transduct Target Ther. 2019; 4: 40.

[36] Chatham JC, Zhang J, Wende AR. Role of O-linked N-acetylglucosamine protein modification in cellular (patho)physiology. Physiol Rev. 2020; 101(2): 427-493.

[37] Bolanle IO, Riches-Suman K, Williamson R, Palmer TM. Emerging roles of protein O-GlcNAcylation in cardiovascular diseases: insights and novel therapeutic targets. Pharmacol Res. 2021; 165: 105467.

[38] Ma J, Banerjee P, Whelan SA, et al. Comparative proteomics reveals dysregulated mitochondrial O-GlcNAcylation in diabetic hearts. J Proteome Res. 2016; 15(7): 2254-2264.

[39] Lee BE, Kim HY, Kim HJ, et al. O-GlcNAcylation regulates dopamine neuron function, survival and degeneration in Parkinson disease. Brain. 2020; 143(12): 3699-3716.

[40] Baldini SF, Lefebvre T. O-GlcNAcylation and the metabolic shift in high-proliferating cells: all the evidence suggests that sugars dictate the flux of lipid biogenesis in tumor processes. Front Oncol. 2016; 6: 6.

[41] Ferrer CM, Sodi VL, Reginato MJ. O-glcnacylation in cancer biology: linking metabolism and signaling. J Mol Biol. 2016; 428(16): 3282-3294.

[42] Ferrer CM, Reginato MJ. Sweet connections: o-GlcNAcylation links cancer cell metabolism and survival. Mol Cell Oncol. 2015; 2(1): e961809.

[43] Shi Q, Shen Q, Liu Y, et al. Increased glucose metabolism in TAMs fuels O-GlcNAcylation of lysosomal Cathepsin B to promote cancer metastasis and chemoresistance. Cancer Cell. 2022; 40(10): 1207-1222. e10.

[44] Verhaak RG, Hoadley KA, Purdom E, et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010; 17(1): 98-110.

[45] Mi W, Gu Y, Han C, et al. O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy. Biochim Biophys Acta. 2011; 1812(4): 514-519.

[46] He X, Li Y, Chen Q, et al. O-GlcNAcylation and stablization of SIRT7 promote pancreatic cancer progression by blocking the SIRT7-REGγ interaction. Cell Death Differ. 2022; 29(10): 1970-1981.

[47] Zachara NE, O’Donnell N, Cheung WD, Mercer JJ, Marth JD, Hart GW. Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress: a survival response of mammalian cells. J Biol Chem. 2004; 279(29): 30133-30142.

[48] Chen P-H, Chi J-TA, Boyce M. Functional crosstalk among oxidative stress and O-GlcNAc signaling pathways. Glycobiology. 2018; 28(8): 556-564.

[49] Bullen JW, Balsbaugh JL, Chanda D, et al. Cross-talk between two essential nutrient-sensitive enzymes: o-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK). J Biol Chem. 2014; 289(15): 10592-10606.

[50] Kao SH, Wang WL, Chen CY, et al. Analysis of protein stability by the cycloheximide chase assay. Bio Protoc. 2015; 5(1): e1374.

[51] Ding N, Ping L, Shi Y, et al. Thiamet-G-mediated inhibition of O-GlcNAcase sensitizes human leukemia cells to microtubule-stabilizing agent paclitaxel. Biochem Biophys Res Commun. 2014; 453(3): 392-397.

[52] Ortiz-Meoz RF, Jiang J, Lazarus MB, et al. A small molecule that inhibits OGT activity in cells. ACS Chem Biol. 2015; 10(6): 1392-1397.

[53] Maier T, Guell M, Serrano L. Correlation of mRNA and protein in complex biological samples. FEBS Lett. 2009; 583(24): 3966-3973.

[54] Deng L, Meng T, Chen L, Wei W, Wang P. The role of ubiquitination in tumorigenesis and targeted drug discovery. Signal Transduct Target Ther. 2020; 5(1): 11.

[55] Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci USA. 2004; 101(36): 13132-13137.

[56] Ramakrishnan B, Qasba PK. Structure-based design of beta 1, 4-galactosyltransferase I (beta 4Gal-T1) with equally efficient N-acetylgalactosaminyltransferase activity: point mutation broadens beta 4Gal-T1 donor specificity. J Biol Chem. 2002; 277(23): 20833-20839.

[57] Gao H, Shi M, Wang R, et al. A widely compatible expression system for the production of highly O-GlcNAcylated recombinant protein in Escherichia coli. Glycobiology. 2018; 28(12): 949-957.

[58] Lee DY, Song MY, Kim EH. Role of oxidative stress and Nrf2/KEAP1 signaling in colorectal cancer: mechanisms and therapeutic perspectives with phytochemicals. Antioxidants (Basel). 2021; 10(5): 743.

[59] Song G, Wang J, Liu J, Ruan Y. Dimethyl fumarate ameliorates erectile dysfunction in bilateral cavernous nerve injury rats by inhibiting oxidative stress and NLRP3 inflammasome-mediated pyroptosis of nerve via activation of Nrf2/HO-1 signaling pathway. Redox Biol. 2023; 68: 102938.

[60] Mu K, Kitts DD. Intestinal polyphenol antioxidant activity involves redox signaling mechanisms facilitated by aquaporin activity. Redox Biology. 2023; 68: 102948.

[61] Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017; 45(W1): W98-W102.

[62] Tomczak K, Czerwińska P, Wiznerowicz M. The Cancer Genome Atlas (TCGA): an immeasurable source of knowledge. Contemporary Oncology/Współczesna Onkologia. 2015; 19(1A): 68-77.

[63] Schumacker PT. Reactive oxygen species in cancer cells: live by the sword, die by the sword. Cancer Cell. 2006; 10(3): 175-176.

[64] Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinog. 2006; 5: 14.

[65] Zhao J. Cancer stem cells and chemoresistance: the smartest survives the raid. Pharmacol Ther. 2016; 160: 145-158.

[66] Panieri E, Buha A, Telkoparan-Akillilar P, et al. Potential applications of NRF2 modulators in cancer therapy. Antioxidants (Basel). 2020; 9(3): 193.

[67] Wang Z, Liu P, Chen Q, et al. Targeting AMPK signaling pathway to overcome drug resistance for cancer therapy. Curr Drug Targets. 2016; 17(8): 853-864.

[68] Chong LW, Chou RH, Liao CC, et al. Saturated fatty acid induces cancer stem cell-like properties in human hepatoma cells. Cell Mol Biol (Noisy-le-grand). 2015; 61(6): 85-91.

[69] Wu T, Wang MC, Jing L, et al. Autophagy facilitates lung adenocarcinoma resistance to cisplatin treatment by activation of AMPK/mTOR signaling pathway. Drug Des Devel Ther. 2015; 9: 6421-6431.

[70] Chen PH, Smith TJ, Wu J, et al. Glycosylation of KEAP1 links nutrient sensing to redox stress signaling. EMBO J. 2017; 36(15): 2233-2250.

[71] Thompson JW, Griffin ME, Hsieh-Wilson LC. Methods for the detection, study, and dynamic profiling of O-GlcNAc glycosylation. Methods Enzymol. 2018; 598: 101-135.

[72] Zhang F, Su K, Yang X, Bowe DB, Paterson AJ, Kudlow JE. O-GlcNAc modification is an endogenous inhibitor of the proteasome. Cell. 2003; 115(6): 715-725.

[73] Zeidan Q, Hart GW. The intersections between O-GlcNAcylation and phosphorylation: implications for multiple signaling pathways. J Cell Sci. 2010; 123(Pt 1): 13-22.

[74] Robledinos-Antón N, Fernández-Ginés R, Manda G, Cuadrado A. Activators and inhibitors of NRF2: a review of their potential for clinical development. Oxid Med Cell Long. 2019; 2019: 1-20.

[75] Dinkova-Kostova AT, Copple IM. Advances and challenges in therapeutic targeting of NRF2. Trends Pharmacol Sci. 2023; 44(3): 137-149.

[76] Meng F, Liang Z, Zhao K, Luo C. Drug design targeting active posttranslational modification protein isoforms. Med Res Rev. 2021; 41(3): 1701-1750.

[77] Lee JB, Pyo K-H, Kim HR. Role and function of O-GlcNAcylation in cancer. Cancers. 2021; 13(21): 5365.

[78] Xia M, Wang S, Qi Y, et al. Inhibition of O-GlcNAc transferase sensitizes prostate cancer cells to docetaxel. Front Oncol. 2022; 12: 993243.

[79] Chang Y-H, Weng C-L, Lin K-I. O-GlcNAcylation and its role in the immune system. J Biomed Sci. 2020; 27(1): 57.

[80] Ouyang M, Yu C, Deng X, Zhang Y, Zhang X, Duan F. O-GlcNAcylation and its role in cancer-associated inflammation. Front Immunol. 2022; 13: 861559.

[81] A Heieis G, Everts B. O-GlcNAcylation at the center of antitumor immunity. Curr Opin Biotechnol. 2023; 84: 103009.

[82] Alteen MG, Tan HY, Vocadlo DJ. Monitoring and modulating O-GlcNAcylation: assays and inhibitors of O-GlcNAc processing enzymes. Curr Opin Struct Biol. 2021; 68: 157-165.

[83] Zhang N, Jiang H, Zhang K, et al. OGT as potential novel target: structure, function and inhibitors. Chem Biol Interact. 2022; 357: 109886.

[84] Borodkin VS, Rafie K, Selvan N, et al. O-GlcNAcase fragment discovery with fluorescence polarimetry. ACS Chem Biol. 2018; 13(5): 1353-1360.

[85] Zhuang S, Liu Z, Wu J, et al. Can O-GIcNAc transferase (OGT) complex be used as a target for the treatment of hematological malignancies? Pharmaceuticals. 2024; 17(6): 664.

[86] Hou C, Wu C, Zhu W, Pei H, Ma J. Systematic pan-cancer analysis reveals OGT and OGA as potential biomarkers for tumor microenvironment and therapeutic responses. Genes Dis. 2024; 11(4): 101089.

[87] He X-F, Hu X, Wen G-J, Wang Z, Lin W-JO. GlcNAcylation in cancer development and immunotherapy. Cancer Lett. 2023; 566: 216258.

RIGHTS & PERMISSIONS

2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.