Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but, rather, functions as a global protein synthesis inhibitor. The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but, rather, functions as a global protein synthesis inhibitor. The KEAP1 1The abbreviations used are:KEAP1kelch-like ECH-associated protein 1DCMdichloromethaneNMRnuclear magnetic resonanceRTroom temperatureACNacetonitrileIDinner diameterSPSsynchronous precursor selectionGAPDHglyceraldehyde-3-phosphate dehydrogenaseVSVGvescicular stomatitis virus glycoproteinCMVcytomegalovirusRABL5Rab-like protein 5HPCL1Hippocalcin-like protein 1PYRG2pyrimidine reductaseG2AREanti-oxidant response elementCETSAcellular thermal shift assayNADPHnicotinamide adenine dinucleotide phosphateNSCLCnon-small cell lung cancerTMTtandem mass tagsUTRuntranslated region./Nrf2 pathway is one of the most important cellular mechanisms to react and respond to oxidative stresses. Under normal physiological conditions, Nrf2 protein is maintained at low levels due to constitutive ubiquitination and degradation. This is achieved through recruitment to the Cul3 ubiquitin ligase complex by the substrate adaptor protein KEAP1. KEAP1 exists as a dimer, which binds a single Nrf2 molecule through two peptide motifs at its N terminus, a high affinity GluThrGlyGLU motif and a lower affinity AspLeuGly motif. This positions Nrf2 for effective ubiquitin transfer. Polyubiquitinated Nrf2 is subsequently degraded by the proteasome. In response to oxidative stress or electrophiles, key cysteine residues in KEAP1 become oxidized, thereby impairing its ability to recruit and ubiquitinate Nrf2 (1.Rachakonda G. Xiong Y. Sekhar K.R. Stamer S.L. Liebler D.C. Freeman M.L. Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3.Chem. Res. Toxicol. 2008; 21: 705-710Crossref PubMed Scopus (160) Google Scholar). This results in Nrf2 stabilization, allowing it to translocate to and accumulate in the nucleus (reviewed in (2.Tong K.I. Kobayashi A. Katsuoka F. Yamamoto M. Two-site substrate recognition model for the Keap1-Nrf2 system: A hinge and latch mechanism.Biol. Chem. 2006; 387: 1311-1320Crossref PubMed Scopus (387) Google Scholar)). Nrf2 binds specific DNA sequences in complex with the small Maf proteins, which were originally identified as viral oncogenes containing leucine zipper motifs (3.Nishizawa M. Kataoka K. Goto N. Fujiwara K.T. Kawai S. v-maf, a viral oncogene that encodes a “leucine zipper” motif.Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 7711-7715Crossref PubMed Scopus (220) Google Scholar). Hundreds of target genes have been identified using ChIP-seq and RNAseq experiments (4.Malhotra D. Portales-Casamar E. Singh A. Srivastava S. Arenillas D. Happel C. Shyr C. Wakabayashi N. Kensler T.W. Wasserman W.W. Biswal S. Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis.Nucleic Acids Res. 2010; 38: 5718-5734Crossref PubMed Scopus (569) Google Scholar, 5.Chorley B.N. Campbell M.R. Wang X. Karaca M. Sambandan D. Bangura F. Xue P. Pi J. Kleeberger S.R. Bell D.A. Identification of novel NRF2-regulated genes by ChIP-Seq: Influence on retinoid X receptor alpha.Nucleic Acids Res. 2012; 40: 7416-7429Crossref PubMed Scopus (393) Google Scholar, 6.Hirotsu Y. Katsuoka F. Funayama R. Nagashima T. Nishida Y. Nakayama K. Engel J.D. Yamamoto M. Nrf2-MafG heterodimers contribute globally to antioxidant and metabolic networks.Nucleic Acids Res. 2012; 40: 10228-10239Crossref PubMed Scopus (268) Google Scholar), highlighting the importance of Nrf2 in reducing levels of oxidative stress by increasing glutathione and NADPH synthesis, and up-regulating drug efflux pumps. Therefore, this pathway senses and responds to oxidative stress to maintain cellular redox homeostasis. kelch-like ECH-associated protein 1 dichloromethane nuclear magnetic resonance room temperature acetonitrile inner diameter synchronous precursor selection glyceraldehyde-3-phosphate dehydrogenase vescicular stomatitis virus glycoprotein cytomegalovirus Rab-like protein 5 Hippocalcin-like protein 1 pyrimidine reductaseG2 anti-oxidant response element cellular thermal shift assay nicotinamide adenine dinucleotide phosphate non-small cell lung cancer tandem mass tags untranslated region. When dysregulated, this pathway also contributes to many human pathologies including cardiovascular and neurodegenerative diseases, inflammation and cancer. Large-scale genome sequencing efforts have identified mutations in both KEAP1 and Nrf2 in several human tumor indications, but especially in non-small cell lung cancers (NSCLC). Mutations in KEAP1 are distributed across the gene and occur in ∼20 and ∼12% of the adenomatous tumors and squamous NSCLC, respectively (7.Cancer Genome Atlas Research, N. Comprehensive molecular profiling of lung adenocarcinoma.Nature. 2014; 511: 543-550Crossref PubMed Scopus (3557) Google Scholar). In addition to KEAP1, ∼15% of squamous NSCLC cases present with mutations in Nrf2 (8.Cancer Genome Atlas Research, N. Comprehensive genomic characterization of squamous cell lung cancers.Nature. 2012; 489: 519-525Crossref PubMed Scopus (2925) Google Scholar), supporting the hypothesis that Nrf2 degradation is frequently dysregulated in this indication (9.Hast B.E. Cloer E.W. Goldfarb D. Li H. Siesser P.F. Yan F. Walter V. Zheng N. Hayes D.N. Major M.B. Cancer-derived mutations in KEAP1 impair NRF2 degradation but not ubiquitination.Cancer Res. 2014; 74: 808-817Crossref PubMed Scopus (92) Google Scholar). Nrf2 mutations cluster in two hotspot regions, ∼10 amino acids each, near the DLG and ETGE KEAP1 interacting regions, resulting in constitutive Nrf2 activation. A reasonable expectation is that these tumors become addicted to the high levels of Nrf2 activity, and some data have been presented that supports this hypothesis (10.Singh A. Boldin-Adamsky S. Thimmulappa R.K. Rath S.K. Ashush H. Coulter J. Blackford A. Goodman S.N. Bunz F. Watson W.H. Gabrielson E. Feinstein E. Biswal S. RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases of Res. 2008; PubMed Scopus Google Scholar). Identification of a inhibitor of this pathway be of for which to the of brusatol as Nrf2 inhibitor. Brusatol is a from a of natural product Recent have that it can decrease the levels of Nrf2 and to D. T. T. A. Brusatol the of by the Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, A. K. M. 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PubMed Scopus Google have been to inhibit Nrf2 activity in and the expression of Nrf2 target Brusatol as a Nrf2 as its at Nrf2 protein levels at its in both and mutant and its of cancer to in and in D. T. T. A. Brusatol the of by the Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, A. K. M. Brusatol a and of Nrf2 and to for of 2014; PubMed Scopus Google Scholar, S. Wang S. A. E. by Res. 2014; 74: PubMed Scopus Google Scholar). brusatol was to inhibit protein synthesis in M. of protein synthesis by PubMed Scopus Google Scholar), the used were high the that mechanisms were at the lower used to inhibit Nrf2 protein A. K. M. 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