Identification of 6-Phosphofructo-2-Kinase/Fructose-2, 6-Bisphosphatase As a Novel Autophagy Regulator by High Content Shrna Screening
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Oncogene (2015) 34, 5662–5676 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE Identification of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase as a novel autophagy regulator by high content shRNA screening AM Strohecker1,2,3,4,10, S Joshi1,2,10, R Possemato5,6,7,8, RT Abraham9, DM Sabatini5,6,7 and E White1,2 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. Oncogene (2015) 34, 5662–5676; doi:10.1038/onc.2015.23; published online 16 March 2015 INTRODUCTION to target a metabolic vulnerability in aggressive cancers. Autophagy maintains protein and organelle homeostasis and Although targeted small molecule modulators of autophagy are enables survival in the absence of exogenous nutrients. Although in development, the optimal targets remain unknown. active in most tissues in low levels, autophagy is markedly induced Studies assessing the functional consequences of pharma- by stress and starvation where it eliminates damaged proteins and cologic inhibition of autophagy in multiple cancers using the organelles, and recycles their components into metabolic and lysosomotropic agents and chloroquine (CQ) and hydroxychlo- biosynthetic pathways. Autophagy is critical to survive starvation roquine (HCQ) are underway. These inhibitors block autophagy at and to prevent the toxic buildup of damaged or superfluous the terminal step of cargo degradation by interfering with intracellular components.1 lysosome function. CQ and HCQ suppress tumor growth in mouse Autophagy-mediated preservation of mitochondrial metabolism models of cancer and in human cancer cell line xenografts, and is exploited by tumor cells and is associated with promotion of have shown efficacy in patient-derived xenograft models alone – mammary, lung and pancreatic cancers, suggesting that and in combination with cancer therapeutics.9,24 32 These results autophagy-inhibiting strategies may have therapeutic efficacy in have prompted clinical trials with HCQ to block autophagy these cancer subtypes.2–12 In other settings, autophagy may and enhance the activity of cancer therapy in refractory suppress tumorigenesis by mitigating chronic tissue damage,13–17 malignancies.33,34 Although the early findings of these trials are – leading to the general consensus that the role of autophagy in promising35 40 there is an unmet need for more specific tool cancer is context dependent.12 Defective autophagy has been compounds to probe actionable targets in the autophagy linked to neurodegenerative disorders such as Alzheimer’s and pathway. The central problem lies in the fact that HCQ Parkinson’s diseases, as well as liver disease, all characterized by nonspecifically interferes with lysosome function and likely has the accumulation of toxic protein aggregates.18,19 Autophagy off-target effects.41 The current challenge is to identify the genes prevents muscle wasting, and impaired autophagy has been and regulatory points in the autophagy pathway that merit – implicated in various models of muscular dystrophy.20 23 Taken anticancer drug development. together, this suggests that autophagy-modulating therapies To identify new autophagy regulators, we designed and will be of great clinical interest: autophagy stimulators for executed a novel image-based high-throughput short hairpin neurodegenerative and degenerative musculo-skeletal disorders RNA (shRNA) screen based on functional elimination of and perhaps also cancer prevention; and autophagy inhibitors the autophagy cargo receptor and substrate p62/SQSTM1. 1Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA; 2Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA; 3Departments of Molecular Virology, Immunology, and Medical Genetics, and Surgery, The Ohio State University, Columbus, OH, USA; 4The Comprehensive Cancer Center and Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, OH, USA; 5Whitehead Institute for Biomedical Research, Cambridge, MA, USA; 6Broad Institute of Harvard and MIT, Cambridge, MA, USA; 7Department of Biology, MIT, Cambridge, MA, USA; 8Department of Pathology, NYU, New York, NY, USA and 9Pfizer, Oncology Research Unit, San Diego, CA, USA. Correspondence: Dr E White, Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA. E-mail: [email protected] or [email protected] 10These authors contributed equally to this work. Received 23 October 2014; revised 20 January 2015; accepted 20 January 2015; published online 16 March 2015 PFKFB4 is a novel autophagy regulator AM Strohecker et al 5663 p62 localizes to the site of autophagosome formation where it homozygous deletion of Atg5 exhibit a greater impairment recognizes ubiquitinated cargo via its ubiquitin-binding domain in p62 elimination than cells with allelic loss of Becn1).15,48 and delivers it to the autophagosome via its LC3 interaction region Thus, monitoring the levels of p62 in cells following stress and – domain.42 44 Importantly, p62 is an autophagy substrate that is recovery can be used as a surrogate of autophagy functionality. degraded along with its cargo; thus, monitoring p62 levels can be We designed our autophagy screen differently from those used as a surrogate of the functional state of autophagy in a given conducted previously.56–62 Our assay used murine immortalized cell. Deletion of the essential autophagy genes Atg5 or Atg7 in epithelial cells possessing an allelic loss of the essential autophagy mice leads to profound p62 accumulation, often in large gene, Becn1, chosen over 293T or HeLa cells that are common aggregates.12 Screening shRNA libraries for genes that alter the choices for cell-based lentiviral screens, as the tumorigenic ability of cells to eliminate aggregated p62 following metabolic potential of the mouse epithelial cells is known and high-priority stress and recovery is a novel approach to identify autophagy candidate genes can be rapidly validated in syngeneic mouse regulators upstream of the terminal substrate degradation step in models. Moreover, the Becn1 heterozygous cells display a partial the pathway. Although our screen was designed to identify defect in autophagy that amplifies the p62 signal, which enables autophagy regulators, it is likely our candidate gene lists also the identification of both positive and negative regulators and contain p62 modulators, which are of interest independent of maximizes the discovery potential of the screen. their regulation of autophagy given the importance of p62 in Our assay stimulus, metabolic stress (1% oxygen, and glucose cancer. deprivation) (Figure 1a), is highly physiologically relevant; p62 is a multi-functional protein that has key roles in nuclear autophagy localizes to hypoxic regions of tumors thus identifying factor-kB signaling (via TRAF6), bone remodeling (mutations in genes capable of regulating autophagy under these conditions p62 are associated with Paget’s disease) and cancer where its would be advantageous for cancer therapy.13 Importantly, our – − − overexpression has been linked to tumorigenesis.15,45 49 p62 / screen provides a functional readout of autophagy by measuring mice are obesity prone and develop diabetes with age.50 More clearance of fluorescently tagged autophagy substrate EGFP-p62 recently, p62 has been linked to amino-acid sensing via TORC1, containing aggregates during recovery, differentiating it from LC3- controlling the localization and activity of the kinase via its based screens that are limited to the detection of autophagosome interaction with TRAF6.51,52 Thus, p62 is emerging as a nutrient formation at the initial step of autophagy activation. Thus, our sensor regulating redox balance and mammalian target of assay enables the identification of autophagy modulators at both rapamycin (mTOR) activation, as well as autophagy. the beginning and throughout the late stages of the pathway in a We used the ability of cells to eliminate p62 following ischemic single screen in addition to identifying modulators of p62 stress as a readout of the functional autophagy status of the cell. homeostasis. We identified 186 positive and 67 negative regulators of p62 We first evaluated the consequences of lentiviral infection and accumulation from function-based libraries encompassing the selection with puromycin on the elimination of p62 from protein kinome, components