Abstract
BackgroundAutophagy is a highly conserved and regulated cellular process employed by living cells to degrade proteins and organelles as a response to metabolic stress. We have previously reported that eukaryotic elongation factor-2 kinase (eEF-2 kinase, also known as Ca2+/calmodulin-dependent protein kinase III) can positively modulate autophagy and negatively regulate protein synthesis. The purpose of the current study was to determine the role of the eEF-2 kinase-regulated autophagy in the response of breast cancer cells to inhibitors of growth factor signaling.Methodology/Principal FindingsWe found that nutrient depletion or growth factor inhibitors activated autophagy in human breast cancer cells, and the increased activity of autophagy was associated with a decrease in cellular ATP and an increase in activities of AMP kinase and eEF-2 kinase. Silencing of eEF-2 kinase relieved the inhibition of protein synthesis, led to a greater reduction of cellular ATP, and blunted autophagic response. We further showed that suppression of eEF-2 kinase-regulated autophagy impeded cell growth in serum/nutrient-deprived cultures and handicapped cell survival, and enhanced the efficacy of the growth factor inhibitors such as trastuzumab, gefitinib, and lapatinib.Conclusion/SignificanceThe results of this study provide new evidence that activation of eEF-2 kinase-mediated autophagy plays a protective role for cancer cells under metabolic stress conditions, and that targeting autophagic survival may represent a novel approach to enhancing the effectiveness of growth factor inhibitors.
Highlights
Autophagy is a highly conserved process by which cytoplasm and organelles are digested via autophagosomes and autolysosomes and cellular components are recycled for energy utilization [1,2]
Autophagy can optimize nutrient utilization in rapidly growing cells when faced with hypoxic or metabolic stresses, contributing to cancer cell survival [3,4,5]. eEF-2 kinase, a Ca2+/ calmodulin-dependent protein kinase, acts as a negative regulator of protein synthesis: this kinase phosphorylates eEF-2, a 100 kDa protein that mediates the translocation step in peptide-chain elongation by inducing the transfer of peptidyl-tRNA from the ribosomal A to P site; phosphorylation of eEF-2 at Thr56 by eEF-2 kinase decreases the affinity of the elongation factor for ribosome and terminates elongation [6]
We further found that MCF-7 and MDA-MB-468 breast cancer cells treated with the growth factor inhibitors, gefitinib and lapatinib, showed an increased in LC3-II amount in a dose-dependent manner, as determined by Western blot (Fig. 1C and D)
Summary
Autophagy is a highly conserved process by which cytoplasm and organelles are digested via autophagosomes and autolysosomes and cellular components are recycled for energy utilization [1,2]. The role of eEF-2 kinase in the regulation of stress-induced autophagy has further been confirmed by others [9]. Since protein synthesis is a major energy-consuming process, termination of protein synthesis and induction of autophagy via activation of eEF-2 kinase should conserve energy and support cell survival during time of metabolic stress. Autophagy is a highly conserved and regulated cellular process employed by living cells to degrade proteins and organelles as a response to metabolic stress. The purpose of the current study was to determine the role of the eEF-2 kinaseregulated autophagy in the response of breast cancer cells to inhibitors of growth factor signaling
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