eEF2K Expression Research Articles

FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation, invasion and tumorgenesis of human triple negative breast cancer cells.

Eukaryotic elongation factor 2 kinase (eEF2K), an emerging molecular target for cancer therapy, contributes to cancer proliferation, cell survival, tumorigenesis, and invasion, disease progression and drug resistance. Although eEF2K is highly up-regulated in various cancers, the mechanism of gene regulation has not been elucidated. In this study, we examined the role of Forkhead Box M1 (FOXM1) proto-oncogenic transcription factor in triple negative breast cancer (TNBC) cells and the regulation of eEF2K. We found that FOXM1 is highly upregulated in TNBC and its knockdown by RNA interference (siRNA) significantly inhibited eEF2K expression and suppressed cell proliferation, colony formation, migration, invasion and induced apoptotic cell death, recapitulating the effects of eEF2K inhibition. Knockdown of FOXM1 inhibited regulators of cell cycle, migration/invasion and survival, including cyclin D1, Src and MAPK-ERK signaling pathways, respectively. We also demonstrated that FOXM1 (1B and 1C isoforms) directly binds to and transcriptionally regulates eEF2K gene expression by chromatin immunoprecipitation (ChIP) and luciferase gene reporter assays. Furthermore, in vivo inhibition of FOXM1 by liposomal siRNA-nanoparticles suppressed growth of MDA-MB-231 TNBC tumor xenografts in orthotopic models. In conclusion, our study provides the first evidence about the transcriptional regulation of eEF2K in TNBC and the role of FOXM1 in mediating breast cancer cell proliferation, survival, migration/invasion, progression and tumorgenesis and highlighting the potential of FOXM1/eEF2K axis as a molecular target in breast and other cancers.

SG-04 * THE eEF2 KINASE, A MEDIATOR OF MEDULLOBLASTOMA ADAPTATION TO METABOLIC STRESS, SUPPORTS FATTY ACID OXIDATION

During tumour progression, brain tumour cells are exposed to nutrient deprivation due to defective tumour vasculature. The ability of brain tumour cells to adapt to reduced nutrient availability favours selection for aggressive clones. The molecular pathways supporting such metabolic adaptation are still poorly defined. We previously reported that the translation elongation factor 2 kinase (eEF2K), which controls mRNA translation at the step of elongation, is an evolutionarily conserved mediator of the response to nutrient deprivation. Gene expression analysis show that eEF2K expression is upregulated in the most aggressive subgroup of medulloblastoma (MB), namely group 3, and that high eEF2K expression is strongly associated with poor survival in both MB. In addition, genetic targeting of eEF2K significantly increases sensitivity of MB cells to nutrient deprivation, supporting a role for eEF2K in facilitating adaptation of MB to nutrient stress. To decipher the basis for eEF2K protective functions under nutrient deprivation, we performed gene expression analysis of eEF2K proficient versus deficient cells under nutrient-deprived conditions. Gene set enrichment analysis (GSEA) highlighted fatty acid oxidation (FAO) as one of the biological pathways most dramatically deregulated upon eEF2K loss. Remarkably, many genes involved in FAO are downregulated in eEF2K deficient cells under starvation, including genes encoding acyl-coA synthetases and acyl-coA dehydrogenases. Furthermore, cells lacking eEF2K are deficient in metabolizing exogenous palmitate to produce ATP, strongly supporting a role for eEF2K in stimulating FAO activity. Finally, our data show that pharmacological induction of FAO in eEF2K deficient cells using bezafibrate was sufficient to rescue cell death occurring under nutrient depleted conditions. The molecular basis for eEF2K-mediated regulation of the FAO transcriptional program is under investigation. Together, our work supports a critical role for eEF2K in driving FAO activity under nutrient deprivation, which could profoundly impact medulloblastoma adaptation to nutrient stress and therefore emergence of aggressive phenotypes.

CS-16 * THE eEF2 KINASE IS CRITICAL FOR BRAIN TUMOURS ADAPTATION TO METABOLIC STRESS

During tumour progression, brain tumour cells are exposed to metabolic stress, such as nutrient deprivation, due to abnormal tumour vasculature. The ability of tumour cells to respond and manage reduced nutrient availability has a strong impact on tumour outcome. The molecular pathways supporting metabolic adaptation of brain tumour cells to nutrient stress represent potential therapeutic targets which are still not well defined. We report that the translation elongation factor 2 (eEF2) kinase mediates a protective response under nutrient starvation by restraining mRNA translation at the step of elongation. In aggressive human tumour cells, such as medulloblastoma (MB) cells, ablation of eEF2K expression increases sensitivity to nutrient removal. In addition, gene expression analysis in patient samples show that eEF2K expression is upregulated in the most aggressive subgroup of MB, namely group 3, and that high eEF2K expression is strongly associated with poor survival in both MB and glioblastoma (GBM). In vivo, eEF2K overexpression confers resistance of tumour xenografts to calorie restriction. Finally, our data reveal that eEF2K is an evolutionarily conserved mediator of the physiological response to nutrient starvation, as genetic removal of eEF2K compromises survival of C. elegans in absence of nutrients. Overall, our works highlight a novel pro-survival factor which is hijacked by brain tumour cells to support their adaptation to nutrient stress. The potential for therapeutic targeting of eEF2 kinase in brain tumors will be discussed.

Abstract 4054: Elongation factor-2 kinase (eEF-2K) promotes cell invasion and epithelial mesenchymal transition through regulation of TG2-mediated signaling in human pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for the majority of pancreatic cancer (PaCa) cases and is currently uncurable with poor prognosis/survival rates (∼4% 5-year survival). The extensive local tumor invasion, early metastasis/systemic dissemination and resistance to existing cancer therapies are the major characteristics of PaCa and the impediment to effective cure of this disease. Although PaCa has a well-defined spectrum of highly oncogenic lesions (e.g, mutations in K-RAS, p53, p16ink4a, and SMAD4/DPC4), effective therapies have not yet been developed. Therefore, novel molecular targets-based therapeutic strategies are urgently needed. Recently, we discovered that eukaryotic elongation factor-2 kinase (eEF-2K) is dramatically up-regulated in different PaCa cells, promoting their survival, and that targeted-silencing of eEF-2K led to significant cell death (Ashour et al, 2013). However, the role of eEF-2K in the pathways regulating invasion/metastasis and drug-resistance remains largely unknown. Here, we show that siRNA-mediated knockdown of eEF-2K markedly inhibits the invasion of PANC-1and MIAPaCa-2 cells. Furthermore, rottlerin, which we found to down-regulate eEF-2K expression, also significantly reduced the invasion of both cell lines. We later investigated the involvement of tissue transglutaminase (TG2), a multifunctional enzyme implicated in regulation of cell survival, migration and invasion. We found that eEF-2K regulates TG2 at the transcription level, as evidenced by eEF-2K knockdown-mediated down-regulation of TG2 protein and mRNA levels by Western blot and RT-PCR, respectively. Importantly, siRNA-mediated knock-down of eEF-2K or TG2 separately, suppresses the downstream key cellular pathways supporting PaCa invasion, and recapitulates rottlerin-induced invasion inhibition and correlated events. We also found that inhibition of eEF-2K/TG2 axis suppresses the epithelial-mesenchymal transition (EMT) through the modulation of ZEB1 zinc finger transcription factors, and tight junction proteins, claudins. Moreover, eEF-2K overexpression, by lentivirus-based expression system, led to increased TG2 expression, with concomitant induction in the invasion capability of PaCa cells. eEF-2K overexpression also rescued the cells from rottlerin-induced suppression of invasion and invasion-supporting machineries, suggesting that eEF-2K is a prominent target of rottlerin, and is a central regulator of PaCa cell invasion. We are currently treating PANC-1 tumor xenografts in nude mice with eEF-2K siRNA-nanoparticles to demonstrate the role of eEF-2K in invasion and tumor progression, in order to establish eEF-2K as a potential novel therapeutic target in PaCa. Collectively, our data suggest, for the first time, the pivotal role of eEF-2K in PaCa invasion/progression and possibly tumor growth. Citation Format: Ahmed A. Ashour, Sultan N. Alpay, Nilgun Gurbuz, Abdel-Aziz H. Abdel-Aziz, Ahmed M. Mansour, Bulent Ozpolat. Elongation factor-2 kinase (eEF-2K) promotes cell invasion and epithelial mesenchymal transition through regulation of TG2-mediated signaling in human pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4054. doi:10.1158/1538-7445.AM2014-4054

Targeted Silencing of Elongation Factor 2 Kinase Suppresses Growth and Sensitizes Tumors to Doxorubicin in an Orthotopic Model of Breast Cancer

Eukaryotic elongation factor 2 kinase (eEF-2K), through its phosphorylation of elongation factor 2 (eEF2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. The purpose of this study was to examine the therapeutic potential of targeting eEF-2K in breast cancer tumors. Through the systemic administration of liposomal eEF-2K siRNA (twice a week, i.v. 150 µg/kg), the expression of eEF-2K was down-regulated in vivo in an orthotopic xenograft mouse model of a highly aggressive triple negative MDA-MB-231 tumor. This targeting resulted in a substantial decrease in eEF2 phosphorylation in the tumors, and led to the inhibition of tumor growth, the induction of apoptosis and the sensitization of tumors to the chemotherapy agent doxorubicin. eEF-2K down-modulation in vitro resulted in a decrease in the expression of c-Myc and cyclin D1 with a concomitant increase in the expression of p27Kip1. A decrease in the basal activity of c-Src (phospho-Tyr-416), focal adhesion kinase (phospho-Tyr-397), and Akt (phospho-Ser-473) was also detected following eEF-2K down-regulation in MDA-MB-231 cells, as determined by Western blotting. Where tested, similar results were seen in ER-positive MCF-7 cells. These effects were also accompanied by a decrease in the observed invasive phenotype of the MDA-MB-231 cells. These data support the notion that the disruption of eEF-2K expression in breast cancer cells results in the down-regulation of signaling pathways affecting growth, survival and resistance and has potential as a therapeutic approach for the treatment of breast cancer.

Abstract 4985: The essential role of eEF-2 kinase in determining fate of glioma cells under ER stress

Abstract Endoplasmic reticulum (ER), an organelle that plays an essential role in protein folding and post-translational modification, can be stressed a variety of stimuli and pathological conditions. To recover normal ER function, an evolutionarily conserved response, termed unfolded protein response, is triggered. When ER homeostasis fails to be re-established, persistent ER stress can induce apoptosis. Recently, it has been demonstrated that ER stress can also induce autophagy, another mechanism for removing unfolded proteins that cannot be eliminated via the ubiquitin/proteasome system, thereby reducing ER stress. However, the precise mechanisms by which the ER stress-induced autophagy is regulated and the functional association between apoptosis and autophagy in tumor cells under ER stress remains unclear. We report here that eukaryotic elongation factor-2 kinase (eEF-2K) is a critical mediator of the ER stress-induced autophagy. We found in the human glioma cells (T98G and LN229) that eEF-2K and autophagy were simultaneously activated following treatment with the ER stress inducers, thapsigargin and tunicamycin; silencing of eEF-2K expression blunted the autophagic response to ER stress. To explore the pathway linking eEF-2K activation and ER stress, we tested the role of REDD1, a stress-induced protein involved in repression of the mTOR signaling. We demonstrated that under ER stress, silencing of REDD1 blocked the phosphorylation of eEF-2, the only known substrate of eEF-2K, indicating that REDD1 is required for transducing the signal for eEF-2K activation. As phosphorylation of eEF-2K at different sites can either activate or inhibit the activity of the enzyme, we next determined the roles of phosphorylation of the kinase in regulating autophagy. We showed that phosphorylation of eEF-2K at Ser398 was essential for the induction of autophagy, while phosphorylation at Ser 366 and Ser78 exerted an inhibitory effect on the kinase activity and autophagy. We further observed that suppression of the ER stress-activated autophagy via silencing of eEF-2K expression aggravated ER stress and promoted apoptotic cell death. Moreover, inhibiting eEF-2K by either RNAi or NH125, a small molecule inhibitor of the enzyme, rendered the glioma cells 2∼3-fold more sensitive to curcumin and velcade, two cytotoxic agents that trigger anti-tumor activities by inducing ER stress. Our study indicates that REDD1-eEF-2K pathway is essential for inducing autophagy and for determining fate of tumor cells under ER stress, and suggests that inhibiting the eEF-2K-mediated autophagy can enhance ER stress and lead to greater apoptotic response, thereby potentiating the efficacy of anticancer agents. These results underscore the potential of eEF-2K as a novel target for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4985. doi:1538-7445.AM2012-4985

Eukaryotic elongation factor 2 kinase (eEF2K) downregulation correlated with de novo DNA methylation and decreased DNA accessibility in neonatal IUGR rat liver

Uteroplacental insufficiency (UPI) alters protein synthesis in intrauterine growth restriction (IUGR). A key regulator of protein synthesis is eEF2K, which regulates protein synthesis by phosphorylating and inactivating eEF2. eEF2K is a classic CpG island gene. We hypothesized that UPI would affect hepatic eEF2K expression by altering eEF2K DNA methylation and possibly DNA accessibility in neonatal IUGR rats.MethodsIUGR rats were generated by bilateral uterine artery ligation. Nucleosome position and DNA accessibility was established by M.SssI methyltransferase (MMT) footprinting. EEF2K CpG methylation, mRNA and protein levels were determined.ResultsIUGR animals had decreased hepatic eEF2K mRNA and protein levels. 45 CpG sites around the transcription start site (TSS) from ‐282 through +217 on the eEF2K gene were analyzed. Increased CpG methylation between ‐206 and ‐282 occurred in IUGR relative to control. The position of 2.5 nucleosomes around the TSS was mapped by MMT footprinting. The ‐1 nucleosome was highly accessible in both groups, while ‐2 and +1 nucleosomes had significantly decreased accessibility in IUGR relative to control.ConclusionsUPI downregulates hepatic eEF2K expression in neonatal IUGR rats. This downregulation is correlated with de novo DNA methylation and reduced DNA accessibility. These results demonstrate IUGR induced alterations to nucleosome positioning.