title: Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening.
abstract: Deregulation of autophagy has been linked to multiple degenerative diseases and cancer, thus the identification of novel autophagy regulators for potential therapeutic intervention is important. To meet this need, we developed a high content image-based short hairpin RNA screen monitoring levels of the autophagy substrate p62/SQSTM1. We identified 186 genes whose loss caused p62 accumulation indicative of autophagy blockade, and 67 genes whose loss enhanced p62 elimination indicative of autophagy stimulation. One putative autophagy stimulator, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), drives flux through pentose phosphate pathway. Knockdown of PFKFB4 in prostate cancer cells increased p62 and reactive oxygen species (ROS), but surprisingly increased autophagic flux. Addition of the ROS scavenger N-acetyl cysteine prevented p62 accumulation in PFKFB4-depleted cells, suggesting that the upregulation of p62 and autophagy was a response to oxidative stress caused by PFKFB4 elimination. Thus, PFKFB4 suppresses oxidative stress and p62 accumulation, without which autophagy is stimulated likely as a ROS detoxification response.
authors.affiliation: Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA.|Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA.|Departments of Molecular Virology, Immunology, and Medical Genetics, and Surgery, The Ohio State University, Columbus, OH, USA.|The Comprehensive Cancer Center and Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, OH, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Strohecker
authors.affiliation: Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA.|Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA.
authors.forename: S
authors.initials: S
authors.lastname: Joshi
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA, USA.|Broad Institute of Harvard and MIT, Cambridge, MA, USA.|Department of Biology, MIT, Cambridge, MA, USA.|Department of Pathology, NYU, New York, NY, USA.
authors.forename: R
authors.initials: R
authors.lastname: Possemato
authors.affiliation: Pfizer, Oncology Research Unit, San Diego, CA, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Abraham
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA, USA.|Broad Institute of Harvard and MIT, Cambridge, MA, USA.|Department of Biology, MIT, Cambridge, MA, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Sabatini
authors.affiliation: Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA.|Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA.
authors.forename: E
authors.initials: E
authors.lastname: White
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis / Q000235:genetics; D001343:Autophagy; D045744:Cell Line, Tumor; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D025481:Phosphofructokinase-2 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D015854:Up-Regulation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D009363:Neoplasm Proteins; C506672:PFKFB4 protein, human; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D025481:Phosphofructokinase-2