title: Alkylating Agent-Induced NRF2 Blocks Endoplasmic Reticulum Stress-Mediated Apoptosis via Control of Glutathione Pools and Protein Thiol Homeostasis.
abstract: Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000-14. ©2016 AACR.
authors.affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas.|Departamento de Farmacologia, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.|Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Alfeu
authors.initials: A
authors.lastname: Zanotto-Filho
authors.affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas.|Children's Medical Research Institute, Westmead, New South Wales, Australia.
authors.forename: V Pragathi
authors.initials: VP
authors.lastname: Masamsetti
authors.affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas.
authors.forename: Eva
authors.initials: E
authors.lastname: Loranc
authors.affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas.|Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas.
authors.forename: Sonal S
authors.initials: SS
authors.lastname: Tonapi
authors.affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas.|Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas.
authors.forename: Aparna
authors.initials: A
authors.lastname: Gorthi
authors.affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas.
authors.forename: Xavier
authors.initials: X
authors.lastname: Bernard
authors.affiliation: Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Rosângela Mayer
authors.initials: RM
authors.lastname: Gonçalves
authors.affiliation: Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: José C F
authors.initials: JC
authors.lastname: Moreira
authors.affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas.|Department of Epidemiology and Biostatistics, University of Texas Health Science Center at San Antonio, San Antonio, Texas.
authors.forename: Yidong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, Texas. bishopa@uthscsa.edu.|Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas.
authors.forename: Alexander J R
authors.initials: AJ
authors.lastname: Bishop
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D016000:Cluster Analysis; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects* / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism*; D006706:Homeostasis / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000401:mortality; D011379:Prognosis; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D019892:eIF-2 Kinase / Q000378:metabolism
chemical_list: D018906:Antineoplastic Agents, Alkylating; D051267:NF-E2-Related Factor 2; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D019892:eIF-2 Kinase; D005978:Glutathione