title: Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress.
abstract: The interplay between oxidative stress and autophagy is critical for determining the fate of cancer cells exposed to redox-active and cytotoxic chemotherapeutic agents. Mitoquinone (MitoQ), a mitochondrially-targeted redox-active ubiquinone conjugate, selectively kills breast cancer cells over healthy mammary epithelial cells. We reported previously that MitoQ, although a derivative of the antioxidant ubiquinone, can generate excess ROS and trigger the Keap1-Nrf2 antioxidant response in the MDA-MB-231 cell line. Following MitoQ treatment, a greater number of cells underwent autophagy than apoptosis. However, the relationship between MitoQ-induced oxidative stress and autophagy as a primary cellular response was unclear. In this report, we demonstrate that MitoQ induces autophagy related gene 7 (Atg7)-dependent, yet Beclin-1-independent, autophagy marked by an increase in LC3-II. Both the ATG7-deficient human MDA-MB-231 cells and Atg7-knockout mouse embryonic fibroblasts exhibited lower levels of autophagy following MitoQ treatment than their respective wild-type counterparts. Increased apoptosis was confirmed in these autophagy-deficient isogenic cell line pairs, indicating that autophagy was attempted for survival in wild type cell lines. Furthermore, we observed higher levels of ROS in Atg7-deficient cells, as measured by hydroethidine oxidation. In Atg7-deficient cells, redox-sensitive Keap1 degradation was decreased, suggesting autophagy- and Atg7-dependent degradation of Keap1. Conversely, downregulation of Keap1 decreased autophagy levels, increased Nrf2 activation, upregulated cytoprotective antioxidant gene expression, and caused accumulation of p62, suggesting a feedback loop between ROS-regulated Keap1-Nrf2 and Atg7-regulated autophagy. Our data indicate that excessive ROS causes the upregulation of autophagy, and autophagy acts as an antioxidant feedback response triggered by cytotoxic levels of MitoQ.
authors.affiliation: Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration.
authors.forename: Yanira
authors.initials: Y
authors.lastname: Gonzalez
authors.forename: Baikuntha
authors.initials: B
authors.lastname: Aryal
authors.forename: Leena
authors.initials: L
authors.lastname: Chehab
authors.forename: V Ashutosh
authors.initials: VA
authors.lastname: Rao
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071193:Autophagy-Related Protein 7; D000071186:Beclin-1; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000494:pharmacology; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D008565:Membrane Proteins; D051267:NF-E2-Related Factor 2; D009943:Organophosphorus Compounds; D012333:RNA, Messenger; D014451:Ubiquinone; C429014:mitoquinone; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D044764:Ubiquitin-Activating Enzymes