eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA

Emerging advances in cancer therapy have transformed the landscape towards cancer immunotherapy regimens. Recent discoveries have resulted in the development of clinical immune checkpoint inhibitors that are 'game-changers' for cancer immunotherapy. Here we show that eEF2K, an atypical protein kinase that negatively modulates the elongation stage of protein synthesis, promotes the synthesis of PD-L1, an immune checkpoint protein which helps cancer cells to escape from immunosurveillance. Ablation of eEF2K in prostate and lung cancer cells markedly reduced the expression levels of the PD-L1 protein. We show that eEF2K promotes the association of PD-L1 mRNAs with translationally active polyribosomes and that translation of the PD-L1 mRNA is regulated by a uORF (upstream open reading-frame) within its 5'-UTR (5'-untranslated region) which starts with a non-canonical CUG as the initiation codon. This inhibitory effect is attenuated by eEF2K thereby allowing higher levels of translation of the PD-L1 coding region and enhanced expression of the PD-L1 protein. Moreover, eEF2K-depleted cancer cells are more vulnerable to immune attack by natural killer cells. Therefore, control of translation elongation can modulate the translation of this specific mRNA, one which contains an uORF that starts with CUG, and perhaps others that contain a similar feature. Taken together, our data reveal that eEF2K regulates PD-L1 expression at the level of the translation of its mRNA by virtue of a uORF in its 5'-region. This, and other roles of eEF2K in cancer cell biology (e.g., in cell survival and migration), may be exploited for the design of future therapeutic strategies.Yu Wu, Jianling Xie, Xin Jin, Roman V. Lenchine, Xuemin Wang, Danielle M. Fang ... et al

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

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.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

Development of a technique to minimise the wind-induced noise in shielded microphones

S.V. Alamshah, A.C.Zander, V.V. Lenchinehttp://www.acoustics.asn.au/conference_proceedings/AAS2013/papers/AAS2013-Information.pdfhttp://www.acoustics.asn.au/conference_proceedings/AAS2013

Eukaryotic elongation factor 2 kinase upregulates the expression of proteins implicated in cell migration and cancer cell metastasis

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation phase of mRNA translation and hence protein synthesis. Increasing evidence indicates that eEF2K plays an important role in the survival and migration of cancer cells and in tumor progression. As demonstrated by two-dimensional wound-healing and three-dimensional transwell invasion assays, knocking down or inhibiting eEF2K in cancer cells impairs migration and invasion of cancer cells. Conversely, exogenous expression of eEF2K or knocking down eEF2 (the substrate of eEF2K) accelerates wound healing and invasion. Importantly, using LC-HDMSE analysis, we identify 150 proteins whose expression is decreased and 73 proteins which are increased upon knocking down eEF2K in human lung carcinoma cells. Of interest, 34 downregulated proteins are integrins and other proteins implicated in cell migration, suggesting that inhibiting eEF2K may help prevent cancer cell mobility and metastasis. Interestingly, eEF2K promotes the association of integrin mRNAs with polysomes, providing a mechanism by which eEF2K may enhance their cellular levels. Consistent with this, genetic knock down or pharmacological inhibition of eEF2K reduces the protein expression levels of integrins. Notably, pharmacological or genetic inhibition of eEF2K almost completely blocked tumor growth and effectively prevented the spread of tumor cells in vivo. High levels of eEF2K expression were associated with invasive carcinoma and metastatic tumors. These data provide the evidence that eEF2K is a new potential therapeutic target for preventing tumor metastasis.</p