Nrf2 (nuclear factor erythroid 2 (NF-E2)-related factor 2) transcription factor is recognized for its pro-survival and cell protective role upon exposure to oxidative, chemical, or metabolic stresses. Nrf2 controls a number of cellular processes such as proliferation, differentiation, apoptosis, autophagy, lipid synthesis, and metabolism and glucose metabolism and is a target of activation in chronic diseases like diabetes, neurodegenerative, and inflammatory diseases. The dark side of Nrf2 is revealed when its regulation is imbalanced (e.g., via oncogene activation or mutations) and under such conditions constitutively active Nrf2 promotes cancerogenesis, metastasis, and radio- and chemoresistance. When there is no stress, Nrf2 is instantly degraded via Keap1-Cullin 3 (Cul3) pathway but despite this, cells exhibit a basal activation of Nrf2 target genes. It is yet not clear how Nrf2 maintains the expression of its targets under homeostatic conditions. Here, we found a stable 105 kDa Nrf2 form that is resistant to Keap1-Cul3-mediated degradation and translocates to the nucleus of lung cancer cells. RNA-Seq analysis indicate that it might originate from the exon 2 or exon 3-truncated transcripts. This stable 105 kDa Nrf2 form might help explain the constitutive activity of Nrf2 under normal cellular conditions.

Department of Mathematical and Statistical Methods Poznań University of Life Sciences 28 Wojska Polskiego St 60 637 Poznań Poland

Department of Medical Biosciences Building 6M Umeå University 901 85 Umeå Sweden

Edinburgh Cancer Research Centre at the Institute of Genetics and Molecular Medicine Edinburgh University Edinburgh EH14 1DJ UK

Inserm UMRS1131 Institut de Génétique Moléculaire Université Paris 7 Hôpital St Louis F 75010 Paris France

International Centre for Cancer Vaccine Science University of Gdansk Kladki 24 80 822 Gdansk Poland

Laboratory of Virus Molecular Biology Intercollegiate Faculty of Biotechnology University of Gdansk Abrahama 58 80 307 Gdansk Poland

RECAMO Masaryk Memorial Cancer Institute Zluty kopec 7 65653 Brno Czech Republic

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Moi P., Chan K., Asunis I., Cao A., Kan Y.W. Isolation of NF-E2-Related Factor 2 (Nrf2), a NF-E2-Like Basic Leucine Zipper Transcriptional Activator that Binds to the Tandem NF-E2/AP1 Repeat of the Β-Globin Locus Control Region. Proc. Natl. Acad. Sci. USA. 1994;91:9926–9930. doi: 10.1073/pnas.91.21.9926. PubMed DOI PMC

Motohashi H., Yamamoto M. Nrf2-Keap1 Defines a Physiologically Important Stress Response Mechanism. Trend. Mol. Med. 2004;10:549–557. doi: 10.1016/j.molmed.2004.09.003. PubMed DOI

Zhang D.D. Mechanistic Studies of the Nrf2-Keap1 Signaling Pathway. Drug. Metab. Rev. 2006;38:769–789. doi: 10.1080/03602530600971974. PubMed DOI

Tonelli C., Chio I.I.C., Tuveson D.A. Transcriptional Regulation by Nrf2. Antioxid. Redox Signal. 2018;29:1727–1745. doi: 10.1089/ars.2017.7342. PubMed DOI PMC

Jaramillo M.C., Zhang D.D. The Emerging Role of the Nrf2-Keap1 Signaling Pathway in Cancer. Genes. Dev. 2013;27:2179–2191. doi: 10.1101/gad.225680.113. PubMed DOI PMC

Kobayashi A., Kang M.-I., Okawa H., Ohtsuji M., Zenke Y., Chiba T., Igarashi K., Yamamoto M. Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase to Regulate Proteasomal Degradation of Nrf2. Mol. Cell. Biol. 2004;24:7130–7139. doi: 10.1128/MCB.24.16.7130-7139.2004. PubMed DOI PMC

Jiang T., Chen N., Zhao F., Wang X.J., Kong B., Zheng W., Zhang D.D. High Levels of Nrf2 Determine Chemoresistance in Type II Endometrial Cancer. Cancer Res. 2010;70:5486–5496. doi: 10.1158/0008-5472.CAN-10-0713. PubMed DOI PMC

Mitsuishi Y., Taguchi K., Kawatani Y., Shibata T., Nukiwa T., Aburatani H., Yamamoto M., Motohashi H. Nrf2 Redirects Glucose and Glutamine into Anabolic Pathways in Metabolic Reprogramming. Cancer Cell. 2012;22:66–79. doi: 10.1016/j.ccr.2012.05.016. PubMed DOI

Mitsuishi Y., Motohashi H., Yamamoto M. The Keap1–Nrf2 system in Cancers: Stress Response and Anabolic Metabolism. Front. Oncol. 2012;2:1–13. doi: 10.3389/fonc.2012.00200. PubMed DOI PMC

Lau A., Tian W., Whitman S.A., Zhang D.D. The Predicted Molecular Weight of Nrf2: It Is what it Is Not. Antioxid. Redox Signal. 2013;18:91–93. doi: 10.1089/ars.2012.4754. PubMed DOI PMC

Kemmerer Z.A., Ader N.R., Mulroy S.S., Eggler A.L. Comparison of Human Nrf2 Antibodies: A Tale of Two Proteins. Toxicol. Lett. 2015;238:83–89. doi: 10.1016/j.toxlet.2015.07.004. PubMed DOI PMC

Pi J., Bai Y., Reece J.M., Williams J., Liu D., Freeman M.L., Fahl W.E., Shugar D., Liu J., Qu W., et al. Molecular Mechanism of Human Nrf2 Activation and Degradation: Role of Sequential Phosphorylation by Protein Kinase CK2. Free Radic. Biol. Med. 2007;42:1797–1806. doi: 10.1016/j.freeradbiomed.2007.03.001. PubMed DOI PMC

Huang H.C., Nguyen T., Pickett C.B. Regulation of the Antioxidant Response Element by Protein Kinase C-Mediated Phosphorylation of NF-E2-Related Factor 2. Proc. Natl. Acad. Sci. USA. 2000;97:12475–12480. doi: 10.1073/pnas.220418997. PubMed DOI PMC

Apopa P.L., 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;22:63–76. doi: 10.1002/jbt.20212. PubMed DOI

Goldstein L.D., Lee J., Gnad F., Klijn C., Schaub A., Reeder J., Daemen A., Bakalarski C.E., Holcomb T., Shames D.S., et al. Recurrent Loss of NFE2L2 Exon 2 Is a Mechanism for Nrf2 Pathway Activation in Human Cancers. Cell Rep. 2016;16:2605–2617. doi: 10.1016/j.celrep.2016.08.010. PubMed DOI

Bolger A.M., Lohse M., Usadel B. Trimmomatic: A Flexible Trimmer for Illumina Sequence Data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Law W.D., Warren R.L., McCallion A.S. Establishment of an eHAP1 Human Haploid Cell Line Hybrid Reference Genome Assembled from Short and Long Reads. Genomics. 2020;112:2379–2384. doi: 10.1016/j.ygeno.2020.01.009. PubMed DOI PMC

Kim D., Paggi J.M., Park C., Bennett C., Salzberg S.L. Graph-Based Genome Alignment and Genotyping with HISAT2 and HISAT-Genotype. Nat. Biotechnol. 2019;37:907–915. doi: 10.1038/s41587-019-0201-4. PubMed DOI PMC

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. The Sequence Alignment/Map Format and SAMtools. Bioinformatics. 2009;25:2078–2079. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

European Environment Agency R Core Team. [(accessed on 4 May 2021)];2020 Available online: www.eea.europa.eu/data-and-maps/indicators/oxygen-consuming-substances-in-rivers/r-development-core-team-2006.

Pertea M., Kim D., Pertea G.M., Leek J.T., Salzberg S.L. Transcript-Level Expression Analysis of RNA-Seq Experiments with HISAT, StringTie and Ballgown. Nat. Protoc. 2016;11:1650–1667. doi: 10.1038/nprot.2016.095. PubMed DOI PMC

Thorvaldsdóttir H., Robinson J.T., Mesirov J.P. Integrative Genomics Viewer (IGV): High-Performance Genomics Data Visualization and Exploration. Brief. Bioinform. 2013;14:178–192. doi: 10.1093/bib/bbs017. PubMed DOI PMC

Singh A., Misra V., Thimmulappa R.K., Lee H., Ames S., Hoque M.O., Herman J.G., Baylin S.B., Sidransky D., Gabrielson E., et al. Dysfunctional KEAP1-NRF2 Interaction in Non-Small-Cell Lung Cancer. PLoS Med. 2006;3:e420. doi: 10.1371/journal.pmed.0030420. PubMed DOI PMC

Peng K.J., Wang J.H., Su W.T., Wang X.C., Yang F.T., Nie W.H. Characterization of Two Human Lung Adenocarcinoma Cell Lines by Reciprocal Chromosome Painting. Dongwuxue Yanjiu. 2010;31:113–121. doi: 10.3724/SP.J.1141.2010.02113. PubMed DOI

Bialk P., Wang Y., Banas K., Kmiec E.B. Functional Gene Knockout of NRF2 Increases Chemosensitivity of Human Lung Cancer A549 Cells In Vitro and in a Xenograft Mouse Model. Mol. Ther. Oncolytics. 2018;11:75–89. doi: 10.1016/j.omto.2018.10.002. PubMed DOI PMC

Homma S., Ishii Y., Morishima Y., Yamadori T., Matsuno Y., Haraguchi N., Kikuchi N., Satoh H., Sakamoto T., Hizawa N., et al. Nrf2 Enhances Cell Proliferation and Resistance to Anticancer Drugs in Human Lung Cancer. Clin. Cancer Res. 2009;15:3423–3432. doi: 10.1158/1078-0432.CCR-08-2822. PubMed DOI

Stewart D., Killeen E., Naquin R., Alam S., Alam J. Degradation of Transcription Factor Nrf2 via the Ubiquitin-Proteasome Pathway and Stabilization by Cadmium. J. Biol. Chem. 2003;278:2396–2402. doi: 10.1074/jbc.M209195200. PubMed DOI

McMahon M., Itoh K., Yamamoto M., Hayes J.D. Keap1-Dependent Proteasomal Degradation of Transcription Factor Nrf2 Contributes to the Negative Regulation of Antioxidant Response Element-Driven Gene Expression. J. Biol. Chem. 2003;278:21592–21600. doi: 10.1074/jbc.M300931200. PubMed DOI

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;8:379–391. doi: 10.1046/j.1365-2443.2003.00640.x. PubMed DOI

Enchev R.I., Schulman B.A., Peter M. Protein Neddylation: Beyond Cullin-RING Ligases. Nat. Rev. Mol. Cell Biol. 2015;16:30–44. doi: 10.1038/nrm3919. PubMed DOI PMC

Lan H., Tang Z., Jin H., Sun Y. Neddylation Inhibitor MLN4924 Suppresses Growth and Migration of Human Gastric Cancer Cells. Sci. Rep. 2016;6 doi: 10.1038/srep24218. PubMed DOI PMC

Tong S., Si Y., Yu H., Zhang L., Xie P., Jiang W. MLN4924 (Pevonedistat), a Protein Neddylation Inhibitor, Suppresses Proliferation and Migration of Human Clear Cell Renal Cell Carcinoma. Sci. Rep. 2017;7 doi: 10.1038/s41598-017-06098-y. PubMed DOI PMC

Huang H.C., Nguyen T., Pickett C.B. Phosphorylation of Nrf2 at Ser-40 by Protein Kinase C Regulates Antioxidant Response Element-Mediated Transcription. J. Biol. Chem. 2002;277:42769–42774. doi: 10.1074/jbc.M206911200. PubMed DOI

Nguyen T., Sherratt P.J., Huang H.C., Yang C.S., Pickett C.B. Increased Protein Stability as a Mechanism that Enhances Nrf2-Mediated Transcriptional Activation of the Antioxidant Response Element: Degradation of Nrf2 by the 26 S Proteasome. J. Biol. Chem. 2003;278:4536–4541. doi: 10.1074/jbc.M207293200. PubMed DOI

Cullinan S.B., Zhang D., Hannink M., Arvisais E., Kaufman R.J., Diehl J.A. Nrf2 Is a Direct PERK Substrate and Effector of PERK-Dependent Cell Survival. Mol. Cell. Biol. 2003;23:7198–7209. doi: 10.1128/MCB.23.20.7198-7209.2003. PubMed DOI PMC

Sun Z., Huang Z., Zhang D.D. Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response. PLoS ONE. 2009;4:e6588. doi: 10.1371/journal.pone.0006588. PubMed DOI PMC

Joo M.S., Kim W.D., Lee K.Y., Kim J.H., Koo J.H., Kim S.G. AMPK Facilitates Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550. Mol. Cell. Biol. 2016;36:1931–1942. doi: 10.1128/MCB.00118-16. PubMed DOI PMC

Zhang D.D., Hannink M. Distinct Cysteine Residues in Keap1 Are Required for Keap1-Dependent Ubiquitination of Nrf2 and for Stabilization of Nrf2 by Chemopreventive Agents and Oxidative Stress. Mol. Cell. Biol. 2003;23:8137–8151. doi: 10.1128/MCB.23.22.8137-8151.2003. PubMed DOI PMC

Furukawa M., Xiong Y. BTB Protein Keap1 Targets Antioxidant Transcription Factor Nrf2 for Ubiquitination by the Cullin 3-Roc1 Ligase. Mol. Cell. Biol. 2005;25:162–171. doi: 10.1128/MCB.25.1.162-171.2005. PubMed DOI PMC

Wakabayashi N., Itoh K., Wakabayashi J., Motohashi H., Noda S., Takahashi S., Imakado S., Kotsuji T., Otsuka F., Roop D.R., et al. Keap1-Null Mutation Leads to Postnatal Lethality Due to Constitutive Nrf2 Activation. Nat. Genet. 2003;35:238–245. doi: 10.1038/ng1248. PubMed DOI

Bryan H.K., Olayanju A., Goldring C.E., Park B.K. The Nrf2 Cell Defence Pathway: Keap1-Dependent and -Independent Mechanisms of Regulation. Biochem. Pharmacol. 2013;85:705–717. doi: 10.1016/j.bcp.2012.11.016. PubMed DOI

Nguyen T., Sherratt P.J., Nioi P., Yang C.S., Pickett C.B. Nrf2 Controls Constitutive and Inducible Expression of ARE-Driven Genes Through a Dynamic Pathway Involving Nucleocytoplasmic Shuttling by Keap1. J. Biol. Chem. 2005;280:32485–32492. doi: 10.1074/jbc.M503074200. PubMed DOI

Sun Z., Zhang S., Chan J.Y., Zhang D.D. Keap1 Controls Postinduction Repression of the Nrf2-Mediated Antioxidant Response by Escorting Nuclear Export of Nrf2. Mol. Cell. Biol. 2007;27:6334–6349. doi: 10.1128/MCB.00630-07. PubMed DOI PMC

Watai Y., Kobayashi A., Nagase H., Mizukami M., Mcevoy J., Singer J.D., Itoh K., Yamamoto M. Subcellular Localization and Cytoplasmic Complex Status of Endogenous Keap1. Genes Cells. 2007;12:1163–1178. doi: 10.1111/j.1365-2443.2007.01118.x. PubMed DOI