ORIGINAL ARTICLE The E6AP E3 ubiquitin ligase regulates the cellular response to oxidative stress K Wolyniec 1,2 , Y Levav-Cohen 3 , Y-H Jiang 4 , S Haupt 1,2 and Y Haupt 1,2,5,6 The E6AP E3 ubiquitin ligase has been linked to the regulation of cell growth and to the cellular stress response. However, the specific stress conditions that are controlled by E6AP have not been defined. An important stress condition that controls cell growth is oxidative stress, where the levels of intracellular reactive oxygen species (ROS) regulate the appropriate cellular response. Here, we describe a novel role for E6AP in the control of oxidative stress response. Cells lacking E6AP expression have reduced capacity to accumulate ROS, and oxidative DNA damage, in response to 20% cell culture oxygen levels, treatment with hydrogen peroxide and expression of oncogenic RAS. This effect of E6AP is associated with the regulation of the anti-oxidant enzyme, Prx1, a previously identified target of E6AP, and can be corrected by downregulation of Prx1 or by reconstitution of E6AP expression. Consequently, cells with compromised E6AP have impaired senescent and apoptotic response to sub-lethal and lethal doses of oxidative stress, respectively. In a xenograft model, downregulation of E6AP renders transplanted tumours refractory to growth-suppressive effects of hydrogen peroxide. Our results provide the first demonstration that E6AP is an important regulator of ROS-mediated cellular senescence and cell death. Oncogene (2013) 32, 3510–3519; doi:10.1038/onc.2012.365; published online 17 September 2012 Keywords: reactive oxygen species; cellular senescence; anti-oxidants; oxidative stress; cell death; peroxiredoxin 1 INTRODUCTION Aerobic metabolism is intimately linked to the generation of reactive oxygen species (ROS), which are important regulators of key physiological processes, such as the control of cell growth. Therefore, organisms have evolved a tight regulatory mechanism that controls ROS levels. These include multiple anti-oxidant enzymes, such as catalase, superoxide dysmutase and peroxir- edoxins, 1,2 which maintain redox homoeostasis in the cell. 3 Deregulation of these anti-oxidant enzymes alters ROS levels. Excessive generation of ROS triggers oxidative stress, associated with oxidative DNA damage and consequently the induction of cell cycle arrest, cellular senescence or cell death, depending on the severity of the cellular damage and type of cell. 4 For example, the ablation of peroxiredoxin 1 (Prx1) in mouse embryo fibroblasts results in elevated ROS levels, oxidative DNA damage and proliferative arrest; 5,6 whereas, overexpression of Prx1 confers resistance to oxidative stress. 7 Cellular senescence and cell death have vital roles in tumour suppression by irreversibly arresting or by eradicating damaged cells with cancerous potential, respectively. 8,9 Intracellular ROS levels increase in response to a variety of intrinsic and extrinsic factors. 1,10 A common source of oxidative stress is a rise in ambient oxygen concentrations. Primary cells, such as murine embryonic fibroblasts (MEFs) exposed to standard culturing conditions (20% oxygen), accumulate oxidative DNA damage and consequently undergo premature senescence. 10–12 Similarly, direct exposure of MEFs to sub-lethal doses of hydrogen peroxide (H 2 O 2 ) results in the induction of cellular senescence. 13–16 Interestingly, the senescent response can be prevented by culturing the cells under physiological oxygen concentrations (3%), or by supplementing the medium with a hydrogen peroxide- scavenging agents, such as N-acetylcysteine. 10–12 An important trigger of ROS levels are activated oncogenes, such as H-Ras V12 , AML1-ETO or oncogenic EGFR orthologue, which require ROS to trigger cellular senescence. 17–20 Therefore, ROS emerges as a major player in the induction of cellular senescence, thereby contributing to the physiological barrier that suppresses tumourigenesis. Despite the significant body of experimental evidence for the role of ROS in cellular senescence and cell death, the underlying mechanism that regulates ROS levels in this context is largely unknown. E6AP is an E3 ubiquitin ligase being the prototype of the HECT sub-type of E3 ligases. 21 E6AP was originally identified as the cellular protein that is recruited by the human papilloma virus to promote the degradation of p53. 22–24 E6AP is encoded by the Ube3A locus, which is mutated in a neuro-developmental disorder called Angelman Syndrome (AS). 25 Recent studies have linked E6AP to the regulation of the cell cycle and the cellular response to stress. We have recently reported that E6AP is required for cellular senescence in MEFs, 26 whereas Nasu et al. 27 have identified the anti-oxidant enzyme, Prx1, as a novel E6AP target. In the current study, we identified a novel role for E6AP in the regulation of the cellular response to oxidative stress. We found that cells deficient for E6AP are defected in their ability to accumulate ROS levels, and consequently to respond to oxidative stress-induced senescence and cell death. This impairment is 1 Tumour Suppression Laboratory, The Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; 2 Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Victoria, Australia; 3 The Hebrew University Hadassah Medical School, Jerusalem, Israel; 4 Division of Medical Genetics, Department of Pediatrics and Neurobiology, Duke University School of Medicine, Durham, NC, USA; 5 Department of Pathology, University of Melbourne, Melbourne, Victoria, Australia and 6 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. Correspondence: Professor Y Haupt, Tumour Suppression Laboratory, The Peter MacCallum Cancer Centre, St Andrew’s Place, East Melbourne, Melbourne, Victoria 3002, Australia. E-mail: ygal.haupt@petermac.org Received 14 February 2012; revised 20 June 2012; accepted 4 July 2012; published online 17 September 2012 Oncogene (2013) 32, 3510–3519 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc