Glioblastoma Cells Elongation Factor-2 Kinase Regulates Autophagy in Human

Elongation factor-2 kinase (eEF-2 kinase), also known as Ca/ calmodulin–dependent kinase III, regulates protein synthesis by controlling the rate of peptide chain elongation. The activity of eEF-2 kinase is increased in glioblastoma and other malignancies, yet its role in neoplasia is uncertain. Recent evidence suggests that autophagy plays an important role in oncogenesis and that this can be regulated by mammalian target of rapamycin (mTOR). Because eEF-2 kinase lies downstream of mTOR, we studied the role of eEF-2 kinase in autophagy using human glioblastoma cell lines. Knockdown of eEF-2 kinase by RNA interference inhibited autophagy in glioblastoma cell lines, as measured by light chain 3 (LC3)-II formation, acidic vesicular organelle staining, and electron microscopy. In contrast, overexpression of eEF-2 kinase increased autophagy. Furthermore, inhibition of autophagy markedly decreased the viability of glioblastoma cells grown under conditions of nutrient depletion. Nutrient deprivation increased eEF-2 kinase activity and decreased the activity of S6 kinase, suggesting an involvement of mTOR pathway in the eEF-2 kinase regulation of autophagy. These results suggest that eEF-2 kinase plays a regulatory role in the autophagic process in tumor cells; and eEF-2 kinase is a downstream member of the mTOR signaling; eEF-2 kinase may promote cancer cell survival under conditions of nutrient deprivation through regulating autophagy. Therefore, eEF-2 kinase may be a part of a survival mechanism in glioblastoma and targeting this kinase may represent a novel approach to cancer treatment. (Cancer Res 2006; 66(6): 3015-23) Introduction Elongation factor-2 kinase (eEF-2 kinase), also known as Ca/ calmodulin–dependent kinase III, was first identified by Nairn et al. (1) and shown to regulate many cellular processes through its essential role in protein translation (2). eEF-2 kinase controls the movement of the elongating peptide chain by specifically phosphorylating eEF-2 at Thr56, which decreases the affinity of the elongation factor and cognate peptide for the ribosome (3, 4). eEF-2 kinase was identified in glioblastoma cell lines as an increased Ca/calmodulin–dependent enzyme activity (5, 6) and was later shown to be up-regulated in many human cancers (7, 8). Inhibition of eEF-2 kinase by selective (9) and nonselective (10) inhibitors produced cancer cell death in vitro and in vivo . Yet the precise role of this kinase in malignancy is not fully understood. The activity of eEF-2 kinase is regulated at several levels. These include activation by Ca/calmodulin (2, 4, 5) and by mitogenic growth factors (11, 12). The kinase is inactivated by phosphorylation of Ser366 by S6 kinase and by autophosphorylation (13–15). S6 kinase is part of the signaling pathway by which the mammalian target of rapamycin (mTOR) senses nutrient deprivation and regulates autophagy (16). Work by Browne et al. (17) showed that eEF2 kinase could also be directly phosphorylated by insulindependent mTOR activity at Ser78, a site adjacent to the calmodulin binding domain. Thus, eEF-2 kinase seems to be regulated by mTOR, providing links to several cellular functions including cell transformation and autophagic degradation. Autophagy is a highly conserved cellular process for large-scale degradation of proteins and organelles (18). It is activated in yeast in response to nutrient deprivation and is believed to protect cell viability by recycling digested proteins for emergency energy utilization (19). In mammals, autophagy may be essential for surviving under conditions of transient nutrient deprivation (20, 21). Furthermore, withdrawal of growth factors from hematopoietic cells induces autophagy, and this seems to protect cell viability (22). In contrast, under circumstances of severe nutrient depletion, this pathway can result in autophagocytic cell death. Recent studies point to the importance of autophagy in cancer. For example, genetic manipulation of this pathway can increase the appearance of cancer in mice (23); autophagy is regulated by the AKT/phosphatidylinositol 3-kinase and mTOR pathways (24); and treatment with certain cytotoxic drugs (25) or withdrawal of obligate growth factors may induce autophagic cell death (26). Several lines of evidence suggest that eEF-2 kinase may be involved in autophagy. First, eEF-2 kinase regulates protein synthesis, a major consumer of cellular energy. Second, eEF-2 kinase lies downstream of mTOR, a known negative regulator of autophagy. Third, growth factor deprivation can markedly reduce the activity of eEF2 kinase. Yet, a role for eEF-2 kinase in autophagy has not been identified. Therefore, in the current study, we investigated the effects of eEF-2 kinase on autophagy in human glioblastoma cell lines. We found that this inhibitor of protein translation can induce autophagy and protect cancer cell viability under conditions of nutrient depletion. Materials and Methods Reagents and antibodies. pSUPER.retro.neo+green fluorescent protein (GFP) vector was purchased from OligoEngine, Inc. (Seattle, WA). Restriction enzymes, T4 DNA ligase, Oligofectamine, Lipofectamine 2000, and G418 were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA). Acridine orange, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and anti-a-actin and anti-h-tubulin antibodies were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-eEF-2 kinase, anti-phospho-eEF-2 kinase, anti-eEF-2, and anti-phospho-eEF-2 antibodies Requests for reprints: Jin-Ming Yang and William N. Hait, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08901. Phone: 732-2358075; Fax: 732-235-8094; E-mail: [email protected] and [email protected]. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-1554 www.aacrjournals.org 3015 Cancer Res 2006; 66: (6). March 15, 2006 Research Article American Association for Cancer Research Copyright © 2006 on February 21, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-05-1554