Eukaryotic elongation factor 2 kinase (eEF2K): its relation to cancer cell survival, autophagy and other cell signalling pathways

Abstract

Eukaryotic elongation factor 2 kinase (eEF2K) is a calcium/calmodulin dependent alpha kinase that plays a central role in the regulation of one of the most important processes in cells – protein synthesis, specifically the elongation stage which consumes large amounts of energy (ATP/GTP) and amino acids. Disruptions in protein synthesis or in its regulation have been linked to various diseases and cancers. This makes eEF2K a prime target for study. eEF2K has been shown to assist cancer cell survival under nutrient-deprived conditions, allowing cells to reduce their rates of protein synthesis and thereby save energy. However, eEF2K’s role in cancer cell survival appears to depend on the cancer cell type, with some cancer cells surviving for shorter times when eEF2K is disrupted, and vice versa for others. Therefore, it becomes important to test eEF2K’s role in cancer cell survival in a variety of cell lines. eEF2K has also been linked to autophagy, a process that allows cells to scavenge their own proteins for amino acids and energy. In this thesis, I have investigated these claims in breast, lung and prostate cancer cell lines. To more accurately assess the effects of eEF2K disruption, I created an eEF2K knock-out model via CRISPR-Cas9. Using this model, I found that autophagy and eEF2K act independently of each other in all the cell lines tested. In the case of lung and breast cancer cells, both processes act to help cells survive nutrient deprivation. In the case of prostate cancer cells, it was found that a lack of eEF2K assisted their survival, highlighting the need to study eEF2K in a range of cell lines. It was also found that eEF2K does not affect glycolysis or mitochondrial respiration. Furthermore, to study how cancer cells gain resistance to nutrient deprivation and autophagy inhibition, I generated ‘resistant’ eEF2K wild-type and knock-out breast cancer cell lines. Importantly, it was found that this resistance is reversible and is lost over time. These cell lines were analysed by proteomics and phospho-proteomics. A variety of proteins and cellular processes were affected in the resistant lines, including aminoacyl tRNA synthesis, translation factors, protein folding, lysosomal proteins, fatty acid metabolism among others. The phosphoproteomic analysis also revealed an overall decrease in phosphorylation of sites downstream of mTORC1 signalling in resistant cells, as well as potential new substrates for eEF2K. This project also investigated the links that eEF2K has, or had been reported to have, to a variety of other signalling pathways, including cell migration/invasion, hypoxia, Nrf2 (nuclear factor erythroid 2-related factor), PD-L1 (programmed cell death 1 ligand 1) and glucose uptake. Overall, this project helped to unravel the links between eEF2K and several other cellular processes. The finding that eEF2K and autophagy act together to assist cell survival under nutrient-deprived conditions could be important for potential therapies against certain cancers. Further supporting this finding, the analysis of resistant cell lines gives more insight into how cancer cells can gain resistance to certain adverse conditions and potential therapeutic agents.