Autophagy is an evolutionally conserved process employed by cells to degrade proteins and organelles in response to metabolic stress. Cells can recycle amino acids, fatty acids and nucleotides for macromolecular biosynthesis and ATP generation, and by sequestering damaged organelles can prevent the release or accumulation of toxic substances. Autophagy is commonly observed in cancer cells of various origins in response to nutrient deprivation. We described elongation factor-2 kinase (eEF-2 kinase; a.k.a calmodulin-dependent protein kinase III) as a structurally and functionally unique enzyme (Ryazanov et al, PNAS, 1997) that is activated by starvation and phosphorylates and inactivates eEF-2, thereby terminating peptide chain elongation. Since protein synthesis is a major energy-consuming process, decreasing protein elongation by activating eEF-2 kinase could be an energy-saving survival strategy. Our laboratory reported that the activity of eEF-2 kinase was increased in many malignant cell lines and certain cancer tissues (Bagaglio et al., Cancer Res., 1993), and that knockdown of eEF-2 kinase blocked autophagy and accelerated cell death in human glioblastoma cells (Wu et al, Cancer Res, 2006). We now test the hypothesis that eEF-2 kinase plays a critical role in the ability of cancer cells to survive oxygen and nutrient deprivation. MCF-7 human breast cancer cells were transfected with a GFP-tagged LC3 expression vector to track the formation of autophagosomes. Autophagy was induced by nutrient/growth factor deprivation as manifested by autophagosome formation in GFP-LC3- transfected MCF-7 cells. Treatment with a potent and specific inhibitor of eEF-2 kinase, NH125 (Arora et al, Cancer Res, 2003), inhibited autophagy as indicated by a reduction in autophagosome formation. In either transient or stable MCF-7 transfectants, NH125 was 10-times more potent and more effective than 3-methyladenine, a known autophagy inhibitor. To determine the effects of blocking autophagy via inhibition of eEF-2 kinase on cellular energetics, we studied the rate and amount of ATP depletion in NH125- and vehicle-treated MCF-7 transfectants. Following nutrient deprivation, inhibition of eEF-2 kinase by NH125 resulted in a greater and more rapid reduction of cellular ATP as compared to vehicle treatment. These results provide additional evidence that eEF-2 kinase is activated in response to metabolic stress, and that inhibiting eEF-2 kinase may overcome the cellular attempts to survive via autophagic regeneration of ATP.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA