Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening

• Published in: Oncogene 2015-11, Vol.34 (45), p.5662-5676
• Main Authors: Strohecker, A M, Joshi, S, Possemato, R, Abraham, R T, Sabatini, D M, White, E
• Format: Article
• Language: English
• Subjects: 13/1; 13/89; 14/63; 38/109; 38/35; 38/47; 6-Phosphofructo-2-kinase; 631/67/2327; Adaptor Proteins, Signal Transducing - biosynthesis; Adaptor Proteins, Signal Transducing - genetics; Apoptosis; Autophagy; Autophagy (Cytology); Cancer; Cell Biology; Cell Line, Tumor; Cysteine; Degenerative diseases; Detoxification; Fructose; Gene Deletion; Gene Expression Regulation, Neoplastic; Genetic screening; Genetics; Health aspects; Human Genetics; Humans; Influence; Internal Medicine; Kinases; Male; Medicine; Medicine & Public Health; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Neoplasms - genetics; Neoplasms - metabolism; Neoplasms - pathology; Oncology; original-article; Oxidative Stress; Pentose phosphate pathway; Phagocytosis; Phosphates; Phosphofructokinase-2 - genetics; Phosphofructokinase-2 - metabolism; Prostate cancer; Reactive oxygen species; Reactive Oxygen Species - metabolism; Ribonucleic acid; RNA; RNA, Small Interfering; Sequestosome-1 Protein; Substrates; Up-Regulation
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/onc.2015.23

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.