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Christopher G Proud - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic Elongation Factor 2 kinase promotes angiogenesis in hepatocellular carcinoma via pi3k akt and stat3
International Journal of Cancer, 2020Co-Authors: Christopher G Proud, Ying Zhou, Ziyi Zhu, Shaoli Chen, Yuan Tan, Jianling Xie, Kaikai ShenAbstract:Hepatocellular carcinoma (HCC) is an aggressive malignancy with increasing mortality in China. Angiogenesis is crucial for tumor formation, development and metastasis in HCC. Previous studies indicated that high expression levels of Elongation Factor 2 kinase (eEF2K), a protein kinase that negatively regulates the Elongation stage of translation, were associated with poor prognosis of HCC. Here, we show that pharmacological inhibition or knockdown of eEF2K in highly metastatic liver cancer cells inhibits their colony forming and migratory capacities, as well as reducing their invasiveness. Importantly, knocking down eEF2K by lentiviral directed shRNA prevented tumor growth and angiogenesis of HCC in mice. Silencing of eEF2K in endothelial cells (HUVECs) led to a reduction in vascularization, evidenced by a decrease in capillary-like structures in the matrigel. Notably, knocking down eEF2K reduced the expression of angiogenesis-related growth Factors in liver cancer cells and the expression of growth Factor receptors on HUVECs, and thus restricted signaling crosstalk that promotes angiogenesis between HCC cells and endothelial cells. We also showed that silencing of eEF2K effectively reduced protein levels of SP1/KLF5 transcription Factors and hence decreased the levels of bound SP1/KLF5 to the VEGF promoter, resulted in a decrease in VEGF mRNA expression. Knocking down eEF2K also led to a striking decrease in the phosphorylation of PI3K/Akt and STAT3, indicating inactivation of these tumorigenic pathways. Taken together, our data suggest that eEF2K contributes to angiogenesis and tumor progression in HCC via SP1/KLF5-mediated VEGF expression, as well as the subsequent stimulation of PI3K/Akt and STAT3 signaling.
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a high throughput screening assay for eukaryotic Elongation Factor 2 kinase inhibitors
Acta Pharmaceutica Sinica B, 2016Co-Authors: Ting Xiao, Christopher G Proud, Rui Liu, Mingwei WangAbstract:Eukaryotic Elongation Factor 2 kinase (eEF2K) inhibitors may aid in the development of new therapeutic agents to combat cancer. Purified human eEF2K was obtained from an Escherichia coli expression system and a luminescence-based high-throughput screening (HTS) assay was developed using MH-1 peptide as the substrate. The luminescent readouts correlated with the amount of adenosine triphosphate remaining in the kinase reaction. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Nine initial hits showing inhibitory activities on eEF2K were identified from 56,000 synthetic compounds during the HTS campaign, of which, five were chosen to test their effects in cancer cell lines.
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eukaryotic Elongation Factor 2 kinase an unusual enzyme with multiple roles
Advances in biological regulation, 2014Co-Authors: Justin W Kenney, Claire E Moore, Xuemin Wang, Christopher G ProudAbstract:Eukaryotic Elongation Factor 2 kinase (eEF2K) is a member of the small group of atypical 'α-kinases'. It phosphorylates and inhibits eukaryotic Elongation Factor 2, to slow down the Elongation stage of protein synthesis, which normally consumes a great deal of energy and amino acids. The activity of eEF2K is normally dependent on calcium ions and calmodulin. eEF2K is also regulated by a plethora of other inputs, including inhibition by signalling downstream of anabolic signalling pathways such as the mammalian target of rapamycin complex 1. Recent data show that eEF2K helps to protect cancer cells against nutrient starvation and is also cytoprotective in other settings, including hypoxia. Growing evidence points to roles for eEF2K in neurological processes such as learning and memory and perhaps in depression.
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The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses
Journal of Biological Chemistry, 2005Co-Authors: Ewan M. Smith, Andrew R. Tee, Stephen G. Finn, Gareth J. Brownei, Christopher G ProudAbstract:Amino acids positively regulate signaling through the mammalian target of rapamycin (mTOR). Recent work demonstrated the importance of the tuberous sclerosis protein TSC2 for regulation of mTOR by insulin. TSC2 contains a GTPase-activator domain that promotes hydrolysis of GTP bound to Rheb, which positively regulates mTOR signaling. Some studies have suggested that TSC2 also mediates the control of mTOR by amino acids. In cells lacking TSC2, amino acid withdrawal still results in dephosphorylation of S6K1, ribosomal protein S6, the eukaryotic initiation Factor 4E-binding protein, and Elongation Factor-2 kinase. The effects of amino acid withdrawal are diminished by inhibiting protein synthesis or adding back amino acids. These studies demonstrate that amino acid signaling to mTOR occurs independently of TSC2 and involves additional unidentified inputs. Although TSC2 is not required for amino acid control of mTOR, amino acid withdrawal does decrease the proportion of Rheb in the active GTP-bound state. Here we also show that Rheb and mTOR form stable complexes, which are not, however, disrupted by amino acid withdrawal. Mutants of Rheb that cannot bind GTP or GDP can interact with mTOR complexes. We also show that the effects of hydrogen peroxide and sorbitol, cell stresses that impair mTOR signaling, are independent of TSC2. Finally, we show that the ability of energy depletion (which impairs mTOR signaling in TSC2+/+ cells) to increase the phosphorylation of eukaryotic Elongation Factor 2 is also independent of TSC2. This likely involves the phosphorylation of the Elongation Factor-2 kinase by the AMP-activated protein kinase.
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a novel mtor regulated phosphorylation site in Elongation Factor 2 kinase modulates the activity of the kinase and its binding to calmodulin
Molecular and Cellular Biology, 2004Co-Authors: Gareth J. Browne, Christopher G ProudAbstract:Eukaryotic Elongation Factor 2 (eEF2) kinase is an unusual calcium- and calmodulin-dependent protein kinase that is regulated by insulin through the rapamycin-sensitive mTOR pathway. Here we show that insulin decreases the ability of eEF2 kinase to bind calmodulin in a rapamycin-sensitive manner. We identify a novel phosphorylation site in eEF2 kinase (Ser78) that is located immediately next to its calmodulin-binding motif. Phosphorylation of this site is increased by insulin in a rapamycin-sensitive fashion. Regulation of the phosphorylation of Ser78 also requires amino acids and the protein kinase phosphoinositide-dependent kinase 1. Mutation of this site to alanine strongly attenuates the effects of insulin and rapamycin both on the binding of calmodulin to eEF2 kinase and on eEF2 kinase activity. Phosphorylation of Ser78 is thus likely to link insulin and mTOR signaling to the control of eEF2 phosphorylation and chain Elongation. This site is not a target for known kinases in the mTOR pathway, e.g., the S6 kinases, implying that it is phosphorylated by a novel mTOR-linked protein kinase that serves to couple hormones and amino acids to the control of translation Elongation. eEF2 kinase is thus a target for mTOR signaling independently of previously known downstream components of the pathway.
Hideyuki Yamawaki - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic Elongation Factor 2 kinase inhibitor a484954 lowered blood pressure in spontaneously hypertensive rats via inducing vasorelaxation
Journal of Pharmacological Sciences, 2020Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Abstract Eukaryotic Elongation Factor 2 (eEF2) kinase (eEF2K) suppresses protein translation. We previously reported eEF2K expression was upregulated in mesenteric arteries (MA) from spontaneously hypertensive rats (SHR). We have recently revealed A484954, an eEF2K inhibitor, acutely suppressed vasopressor agonists-induced increase of blood pressure (BP) in normal Wistar rats. In this study, we examined the acute effects of A484954 on BP in SHR and explored underlying mechanisms. BP was measured by a carotid cannulation method in SHR. Isometric contraction in MA from SHR was measured. Endothelial nitric oxide synthase (eNOS) dimerization was measured by low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A484954 lowered BP in 15-week-old SHR. A484954 induced relaxation in MA from both 4- and 7-9-week-old SHR. In MA from 4-week-old SHR, A484954-induced relaxation was inhibited almost completely by a NOS inhibitor, NG-nitro- l -arginine methyl ester ( l -NAME) and significantly by a β blocker, propranolol. In MA from 7-9-week-old SHR, on the other hand, A484954-induced relaxation was inhibited partly either by l -NAME, indomethacin, a cyclooxygenase inhibitor, or l -NAME + indomethacin. A484954 promoted the dimerization of eNOS in human endothelial cells. In summary, we have revealed A484954 lowers BP in SHR perhaps through the vasorelaxation via the production of endothelium-derived relaxing Factors.
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eukaryotic Elongation Factor 2 kinase inhibitor a484954 potentiates β adrenergic receptor agonist induced acute decrease in diastolic blood pressure in rats
Journal of Veterinary Medical Science, 2019Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Eukaryotic Elongation Factor 2 (eEF2) kinase (eEF2K) acts to inhibit protein translation through phosphorylating a specific substrate, eEF2. We previously found that the increased eEF2K expression in mesenteric artery mediates hypertension development in spontaneously hypertensive rats. More recently, we have revealed that a selective eEF2K inhibitor, A484954 induced vasorelaxation via opening inward rectifier K+ channel and activating β2-adrenergic receptor in smooth muscle of rat isolated mesenteric artery, which contributes to prevent noradrenaline-induced acute increase in blood pressure (BP). In this study, we further explored acute effects of A484954 on BP in rats, especially focusing the action on β-adrenergic receptor. We also examined whether A484954 affects contraction and heart rate (HR) of isolated heart. BP and HR were measured by a carotid cannulation method in rats. Isometric contraction and HR in rat isolated atria were also measured pharmacologically. A484954 potentiated adrenaline-induced decrease in diastolic BP (DBP) but not increase in systolic BP (SBP). A484954 potentiated isoproterenol-induced decrease in DBP but not SBP. Contrastingly, A484954 prevented a non-β-adrenergic receptor agonist, angiotensin II-induced increase in both SBP and DBP. In isolated left atria, A484954 caused contraction, which was prevented by a β-adrenergic receptor antagonist, propranolol. In isolated right atria, A484954 increased HR. In conclusion, we for the first time demonstrated that A484954 potentiates β-adrenergic receptor agonist-induced decrease in DBP possibly through vasorelaxation mediated via activating β2-adrenergic receptor. It was also demonstrated that A484954 causes contraction of rat isolated heart via activating β1-adrenergic receptor.
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eukaryotic Elongation Factor 2 kinase inhibitor a484954 inhibits noradrenaline induced acute increase of blood pressure in rats
Journal of Veterinary Medical Science, 2019Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Eukaryotic Elongation Factor 2 (eEF2) kinase (eEF2K) inhibits protein translation through the phosphorylation of its specific substrate, eEF2. We previously demonstrated that eEF2K expression increases in superior mesenteric artery from spontaneously hypertensive rats (SHR) and that eEF2K mediates development of hypertension in SHR. In addition, we recently revealed that A484954, a selective eEF2K inhibitor induced relaxation via opening smooth muscle inward rectifier K+ (Kir) channel in rat isolated superior mesenteric artery. Here, we further examined the effects of A484954 on contractility and blood pressure (BP) in rats. Isometric contraction of rat isolated superior mesenteric artery was measured. BP was measured by a carotid cannulation method. A484954 (10 µM) inhibited noradrenaline (NA)-induced contraction in a biphasic manner (magnitude of inhibition higher at high dose NA). A484954 also inhibited an α1-receptor agonist, phenylephrine-induced contraction, while it was not biphasic. Specifically, a β-receptor antagonist, propranolol (1 µM) prevented the A484954-mediated inhibition of NA (high-dose)-induced contraction. A484954 (10 µM) potentiated a β-receptor agonist, isoproterenol-induced relaxation, which was completely prevented by BaCl2 (1 mM), a Kir channel blocker. In vivo, A484954 (122 µg/kg) inhibited NA-induced increase of BP in rats. Another eEF2K inhibitor, NH125 (22 µg/kg) also inhibited the NA-induced BP increase in rats. In summary, it was concluded that A484954 lowers NA-induced BP rise perhaps through activation of β2-receptor-Kir channel and subsequent vasorelaxation via inhibiting eEF2K activity.
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mechanisms underlying the relaxation by a484954 a eukaryotic Elongation Factor 2 kinase inhibitor in rat isolated mesenteric artery
Journal of Pharmacological Sciences, 2018Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Abstract Eukaryotic Elongation Factor 2 kinase (eEF2K) is a calmodulin-related protein kinase which regulates protein translation. A484954 is an inhibitor of eEF2K. In the present study, we investigated the acute effects of A484954 on contractility of isolated blood vessels. Isometric contraction of rat isolated aorta and main branch of superior mesenteric artery (MA) was measured. Expression of an inward rectifier K+ (Kir) channel subtype mRNA and protein was examined. A484954 caused relaxation in endothelium-intact [E (+)] and -denuded [E (−)] aorta or MA precontracted with noradrenaline (NA). The relaxation was higher in MA than aorta. The relaxation was partially inhibited by a nitric oxide (NO) synthase inhibitor, NG-nitro- l -arginine methyl ester (300 μM) in E (+) MA. The relaxation was significantly smaller in MA precontracted with high K+ than NA. The A484954-induced relaxation was significantly inhibited by a Kir channel blocker, BaCl2 (1 mM) compared with vehicle control in E (−) MA. Expression of Kir2.2 mRNA and protein was significantly higher in MA than aorta. We for the first time revealed that A484954 induces relaxation through opening smooth muscle Kir (Kir2.2) channel and through endothelium-derived NO in MA.
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Mechanisms underlying the relaxation by A484954, a eukaryotic Elongation Factor 2 kinase inhibitor, in rat isolated mesenteric artery
Elsevier, 2018Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Eukaryotic Elongation Factor 2 kinase (eEF2K) is a calmodulin-related protein kinase which regulates protein translation. A484954 is an inhibitor of eEF2K. In the present study, we investigated the acute effects of A484954 on contractility of isolated blood vessels. Isometric contraction of rat isolated aorta and main branch of superior mesenteric artery (MA) was measured. Expression of an inward rectifier K+ (Kir) channel subtype mRNA and protein was examined. A484954 caused relaxation in endothelium-intact [E (+)] and -denuded [E (−)] aorta or MA precontracted with noradrenaline (NA). The relaxation was higher in MA than aorta. The relaxation was partially inhibited by a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (300 μM) in E (+) MA. The relaxation was significantly smaller in MA precontracted with high K+ than NA. The A484954-induced relaxation was significantly inhibited by a Kir channel blocker, BaCl2 (1 mM) compared with vehicle control in E (−) MA. Expression of Kir2.2 mRNA and protein was significantly higher in MA than aorta. We for the first time revealed that A484954 induces relaxation through opening smooth muscle Kir (Kir2.2) channel and through endothelium-derived NO in MA. Keywords: Blood vessel, K+ channel, Vascular smooth muscle, Vasorelaxatio
Bulent Ozpolat - One of the best experts on this subject based on the ideXlab platform.
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abstract 4262 mir 873 is the master regulator of autophagy genes through a novel negative feedback mechanism mediated by Elongation Factor 2 kinase eef 2k and suppresses tumor growth and progression of triple negative breast cancer
Cancer Research, 2019Co-Authors: Hamada Ahmed Mokhlis, Nermin Kahraman, Seyda Baydogan, Abdelaziz H Abdelaziz, Ahmed A Ashour, Cristina Ivan, Gabriel Lopezberestein, Bulent OzpolatAbstract:Autophagy is a highly complex lysosomal degradation process. Recently we demonstrated that autophagy genes encoding Beclin1, ATG7, LC3 promotes cell proliferation, survival, and invasion by promoting Cyclin D1 and Integrin β1/ Src signaling in triple negative breast cancer (TNBC). Studies showed that increased basal autophagy is linked to development of chemoresistance, relapses and metastasis and poor prognosis in TNBC. However, the major molecular mechanisms and the integrated global regulators controlling the autophagic machinery still remains unknown. Here, we identified miRNA-873 as a p53-driven tumor suppressor that is associated with favorable patient survival (TCGA database) and the key Factor for regulating the major autophagy genes, including BCN1, LC3, ATG7, ATG16L1 and ATG13 in TNBC. Using in silico prediction algorithms we demonstrated that miR-873 has binding sites on the 3’-untranslated region (3’-UTR) of these genes and directly binds and suppresses their expression using by gene reporter assays. Introduction of mutations to the miR-873 binding sites on 3-UTR of these genes reversed the inhibitory effect of miR-873on these genes. Furthermore, knockdown of Beclin1, LC3 and ATG7 genes significantly suppressed Eukaryotic Elongation Factor‐2 kinase (eEF2K), an unusual alpha kinase, which is highly overexpressed in TNBC patients and associated with poor prognosis. We also found that miR-873 also binds to the 3’-UTR of eEF2K mRNA and regulates its expression and inhibits starvation induced autophagy. Through knockdown and overexpression studies we also demonstrated that eEF2K regulates expression of abovementioned autophagic proteins. Interestingly, we found that BCN1, LC3, and ATG7 also regulates expression of EF2K, suggesting an existence of a novel negative feed-back loop. Lastly, we demonstrated that miR-873 expression is suppressed in TNBC patients and cell lines and restoration of its expression in vivo in MDA-MB-231 and MDA-MB-436 orthotopic xenograft models by systemic injection (I.V, tail vein once a week, 0.15 mg/kg) of miR-873 mimic molecules incorporated in novel single lipid (SLNP)-nanoparticles suppressed tumor growth. Furthermore, in vivo treatment of mice with SLNP-Beclin1, ATG7 or ATG8 siRNAs also completely suppressed TNBC tumor growth. In conclusion, our data suggest that p53/miR‐873/eEF2K axis is a novel post-transcriptional regulator of autophagy and miR-873 functions as a master regulator of the post-transcriptional regulation of the major autophagy genes directly and indirectly through eEF2K dependent dual-suppressor mechanism and modulation of this axis could be used as a potential therapy for TNBC. Citation Format: Hamada Ahmed Mokhlis, Nermin Kahraman, Seyda Baydogan, Abdel-Aziz Hamed Abdel-Aziz, Ahmed Ashour, Cristina Ivan, Gabriel Lopez-Berestein, Bulent Ozpolat. MiR-873 is the master regulator of autophagy genes through a novel negative feedback mechanism mediated by Elongation Factor 2 kinase (eEF-2K) and suppresses tumor growth and progression of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4262.
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thymoquinone inhibits cell proliferation migration and invasion by regulating the Elongation Factor 2 kinase eef 2k signaling axis in triple negative breast cancer
Breast Cancer Research and Treatment, 2018Co-Authors: Nashwa N Kabil, Hamada Ahmed Mokhlis, Nermin Kahraman, Gabriel Lopezberestein, Recep Bayraktar, George A Calin, Bulent OzpolatAbstract:Triple-negative breast cancer (TNBC) is the most aggressive and chemoresistant subtype of breast cancer. Therefore, new molecular targets and treatments need to be developed to improve poor patient prognosis and survival. We have previously shown that eukaryotic Elongation Factor 2 kinase (eEF-2K) is highly expressed in TNBC cells, is associated with poor patient survival and prognosis, and promotes cell proliferation, migration, and invasion. In vivo targeting of eEF-2K significantly reduces the tumor growth of orthotopic TNBC xenograft mouse models, suggesting that eEF-2K may serve as a potential novel therapeutic target. In the current study, we identified thymoquinone (TQ), an active ingredient of Nigella sativa, as a potential safe and effective eEF-2K inhibitor in TNBC. We demonstrated for the first time that TQ inhibits the protein and mRNA expression of eEF-2K, as well as the clinically relevant downstream targets, including Src/FAK and Akt, and induces the tumor suppressor miR-603, in response to NF-kB inhibition. This effect was associated with a significant decrease in the proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, systemic in vivo injection of TQ (20 and 100 mg/kg) significantly reduced the growth of MDA-MB-231 tumors and inhibited the eEF-2K expression in an orthotopic tumor model in mice. Our study provides first evidence that TQ treatment inhibits cell proliferation, migration/invasion, and tumor growth, in part through the inhibition of eEF-2K signaling in TNBC. Thus, our findings suggest that systemic TQ treatment may be used as a targeted therapeutic strategy for the inhibition of eEF-2K in TNBC tumor growth and progression.
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abstract 3249 thymoquinone inhibits Elongation Factor 2 kinase signaling axis by inducing tumor suppressor mir 603 in triple negative breast cancer cells
Cancer Research, 2017Co-Authors: Nashwa N Kabil, Nermin Kahraman, Recep Bayraktar, Bulent OzpolatAbstract:Triple negative breast cancer (TNBC), is a highly heterogeneous and aggressive subtype of breast cancer (BC), which poses a significant clinical challenge. TNBC constitutes about 15-20 % of BC cases, and is characterized by lack of estrogen (ER), progesterone (PR) and human epidermal growth Factor 2 (HER2) receptors. Thus, patients cannot benefit from targeted therapies such as anti-estrogens (eg.Tamoxifen) and anti-HER2 (eg.trastuzumab) treatments. Therefore, identification of new molecular targets and treatment strategies are highly warranted to improve patient outcome. We previously reported that Elongation Factor 2 kinase (EF-2K) is highly expressed in TNBC cell lines and is associated with poor patient survival and prognosis. Furthermore, in vivo targeting of EF-2K by siRNA nano-therapeutics inhibited cell proliferation, migration/invasion, and significantly decreased tumor growth in 2 different orthotopic TNBC mouse models (MDA MB-231 and MDA MB-436). Collectively, our work suggests that EF-2K is an excellent novel therapeutic target in TNBC. In search of a potential safe and effective EF-2K inhibitor, we identified Thymoquinone (TQ), a dietary natural compound, known to have diverse anti-cancerous properties in several in vitro and in vivo models, including TNBC. However, the mechanism by which TQ mediates its effects are not well elucidated. Our current study is the first to demonstrate that TQ inhibits protein and mRNA expression of EF-2K, as well as its clinically significant downstream targets such as Src/FAK, PI3K/AKT, and CyclinD1; resulting in a significant decrease in proliferation, migration/invasion of TNBC cells. To determine the molecular mechanism by which TQ inhibits EF-2K expression, we investigated if TQ induces tumor suppressor microRNAs that we identified to bind to the 3’-UTR of EF-2K. We found that TQ induces miR-603 expression, which we had reported to directly bind and inhibit EF-2K expression; resulting in decreased TNBC growth and progression, both in vitro and in vivo. Furthermore, we showed that inhibition of nuclear Factor kappa B (NF-κB) (a well established target for TQ), also induces miR-603 and inhibits EF-2K expression in TNBC cells. In conclusion, our study is the first to show that TQ treatment decreases EF-2K expression through modulating the NF-κB/miR-603 axis; ultimately resulting in decreased cell proliferation, migration/invasion of TNBC. Our data suggests a novel molecular mechanism for TQ that represents a potential therapeutic strategy in inhibiting TNBC tumor growth and progression. Citation Format: Nashwa N. Kabil, Recep Bayraktar, Nermin Kahraman, Bulent Ozpolat. Thymoquinone inhibits Elongation Factor 2 kinase signaling axis by inducing tumor suppressor miR-603 in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3249. doi:10.1158/1538-7445.AM2017-3249
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abstract 1092 mir 603 acts as a tumor suppressor in triple negative breast cancer and inhibits cell proliferation invasiveness and tumorigenesis by targeting Elongation Factor 2 kinase eef2k
Cancer Research, 2016Co-Authors: Recep Bayraktar, Cristina Ivan, Martin Pichler, George A Calin, Bulent OzpolatAbstract:Triple Negative Breast Cancer (TNBC) is characterized by heterogeneous disease due to its unique molecular profile, aggressiveness, distinct metastatic patterns and lack of molecular targets such as ER, PR and HER2/neu for effective targeted therapies including tamoxifen and herceptin reduced sensitivity to chemotherapies, representing unmet therapeutic challenge. Eukaryotic Elongation Factor-2 kinase (eEF2K) is an atypical calcium/calmodulin dependent serine/tyrosine kinase that regulates protein synthesis through phosphorylation of EF2. Recently, we reported that eEF2K promotes TNBC cell proliferation, colony formation, migration/invasion, angiogeneis and tumorigenesis and drug resistance by inducing PI3K/Akt, mTOR, IGF1R, Src/FAK, C-myc,, Bcl-2, CyclinD1, and VEGF (Tekedereli 2012). We also demonstrated that in vivo therapeutic targeting of eEF2K by systemically administered liposomal eEF2KsiRNA nanoparticles significantly inhibited growth of highly aggressive MDA-MB-231 tumor xenografts in and enhanced the efficacy of standard chemoagents (ie, doxorubicin, paclitaxel), representing a potential molecular target in TNBC. However, currently the transcriptional regulation of eEF2K is unknown. Here, we report that eEF2K expression is highly upregulated in TNBC cells and associated with poor patient survival and clinical outcome in these patients.. Thorough extensive analysis of databases and predictive algorithms, and in vitro and in vivo correlation studies we identified that miR-603 inversely correlated with eEF2K and demonstrated miR603 as the first microRNA that directly binds to the 3’-UTR of eEF2K gene and suppresses its expression, though gene reporter assays. Introduction of mutations to the miR-603 binding site completely reversed eEF2K expression in gene reporter assays. We also showed that ectopic expression of miR-603 in TNBC cell lines resulted in the inhibition of cell growth, colony formation, migration and invasion of TNBC cells, recapitulating the effects of eEF2K inhibition by siRNA. Systemic administration of miR-603 mimics incorporated in liposomal nanoparticles (0.3 mg/kg once a week for 4 weeks) in an orthotopic xenograft TNBC mouse model led to significant inhibition of eEF2K protein expression and tumor growth. Analysis of the tumors after the end of study reveled that miR-603-induced downregulation of eEF2K expression was correlated with Src and Akt. In conclusion, our findings suggest for the first time that miR-603 function as a tumor suppressor and loss of its expression contributes to upregulation of eEF2K, tumor growth and progression of TNBC and miR-603 based gene targeting strategy may be used to control eEF2K in TNBC, representing a novel molecularly targeted therapy. Citation Format: Recep Bayraktar, Martin Pichler Pichler, Cristina Ivan, George Calin, Bulent Ozpolat. miR-603 acts as a tumor suppressor in triple-negative breast cancer and inhibits cell proliferation, invasiveness and tumorigenesis by targeting Elongation Factor 2-kinase (eEF2K). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1092.
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foxm1 regulates expression of eukaryotic Elongation Factor 2 kinase and promotes proliferation invasion and tumorgenesis of human triple negative breast cancer cells
Oncotarget, 2016Co-Authors: Nermin Kahraman, Ahmed A Ashour, Zuhal Hamurcu, Bulent OzpolatAbstract: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.
Tomoko Kodama - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic Elongation Factor 2 kinase inhibitor a484954 lowered blood pressure in spontaneously hypertensive rats via inducing vasorelaxation
Journal of Pharmacological Sciences, 2020Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Abstract Eukaryotic Elongation Factor 2 (eEF2) kinase (eEF2K) suppresses protein translation. We previously reported eEF2K expression was upregulated in mesenteric arteries (MA) from spontaneously hypertensive rats (SHR). We have recently revealed A484954, an eEF2K inhibitor, acutely suppressed vasopressor agonists-induced increase of blood pressure (BP) in normal Wistar rats. In this study, we examined the acute effects of A484954 on BP in SHR and explored underlying mechanisms. BP was measured by a carotid cannulation method in SHR. Isometric contraction in MA from SHR was measured. Endothelial nitric oxide synthase (eNOS) dimerization was measured by low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A484954 lowered BP in 15-week-old SHR. A484954 induced relaxation in MA from both 4- and 7-9-week-old SHR. In MA from 4-week-old SHR, A484954-induced relaxation was inhibited almost completely by a NOS inhibitor, NG-nitro- l -arginine methyl ester ( l -NAME) and significantly by a β blocker, propranolol. In MA from 7-9-week-old SHR, on the other hand, A484954-induced relaxation was inhibited partly either by l -NAME, indomethacin, a cyclooxygenase inhibitor, or l -NAME + indomethacin. A484954 promoted the dimerization of eNOS in human endothelial cells. In summary, we have revealed A484954 lowers BP in SHR perhaps through the vasorelaxation via the production of endothelium-derived relaxing Factors.
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eukaryotic Elongation Factor 2 kinase inhibitor a484954 potentiates β adrenergic receptor agonist induced acute decrease in diastolic blood pressure in rats
Journal of Veterinary Medical Science, 2019Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Eukaryotic Elongation Factor 2 (eEF2) kinase (eEF2K) acts to inhibit protein translation through phosphorylating a specific substrate, eEF2. We previously found that the increased eEF2K expression in mesenteric artery mediates hypertension development in spontaneously hypertensive rats. More recently, we have revealed that a selective eEF2K inhibitor, A484954 induced vasorelaxation via opening inward rectifier K+ channel and activating β2-adrenergic receptor in smooth muscle of rat isolated mesenteric artery, which contributes to prevent noradrenaline-induced acute increase in blood pressure (BP). In this study, we further explored acute effects of A484954 on BP in rats, especially focusing the action on β-adrenergic receptor. We also examined whether A484954 affects contraction and heart rate (HR) of isolated heart. BP and HR were measured by a carotid cannulation method in rats. Isometric contraction and HR in rat isolated atria were also measured pharmacologically. A484954 potentiated adrenaline-induced decrease in diastolic BP (DBP) but not increase in systolic BP (SBP). A484954 potentiated isoproterenol-induced decrease in DBP but not SBP. Contrastingly, A484954 prevented a non-β-adrenergic receptor agonist, angiotensin II-induced increase in both SBP and DBP. In isolated left atria, A484954 caused contraction, which was prevented by a β-adrenergic receptor antagonist, propranolol. In isolated right atria, A484954 increased HR. In conclusion, we for the first time demonstrated that A484954 potentiates β-adrenergic receptor agonist-induced decrease in DBP possibly through vasorelaxation mediated via activating β2-adrenergic receptor. It was also demonstrated that A484954 causes contraction of rat isolated heart via activating β1-adrenergic receptor.
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eukaryotic Elongation Factor 2 kinase inhibitor a484954 inhibits noradrenaline induced acute increase of blood pressure in rats
Journal of Veterinary Medical Science, 2019Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Eukaryotic Elongation Factor 2 (eEF2) kinase (eEF2K) inhibits protein translation through the phosphorylation of its specific substrate, eEF2. We previously demonstrated that eEF2K expression increases in superior mesenteric artery from spontaneously hypertensive rats (SHR) and that eEF2K mediates development of hypertension in SHR. In addition, we recently revealed that A484954, a selective eEF2K inhibitor induced relaxation via opening smooth muscle inward rectifier K+ (Kir) channel in rat isolated superior mesenteric artery. Here, we further examined the effects of A484954 on contractility and blood pressure (BP) in rats. Isometric contraction of rat isolated superior mesenteric artery was measured. BP was measured by a carotid cannulation method. A484954 (10 µM) inhibited noradrenaline (NA)-induced contraction in a biphasic manner (magnitude of inhibition higher at high dose NA). A484954 also inhibited an α1-receptor agonist, phenylephrine-induced contraction, while it was not biphasic. Specifically, a β-receptor antagonist, propranolol (1 µM) prevented the A484954-mediated inhibition of NA (high-dose)-induced contraction. A484954 (10 µM) potentiated a β-receptor agonist, isoproterenol-induced relaxation, which was completely prevented by BaCl2 (1 mM), a Kir channel blocker. In vivo, A484954 (122 µg/kg) inhibited NA-induced increase of BP in rats. Another eEF2K inhibitor, NH125 (22 µg/kg) also inhibited the NA-induced BP increase in rats. In summary, it was concluded that A484954 lowers NA-induced BP rise perhaps through activation of β2-receptor-Kir channel and subsequent vasorelaxation via inhibiting eEF2K activity.
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mechanisms underlying the relaxation by a484954 a eukaryotic Elongation Factor 2 kinase inhibitor in rat isolated mesenteric artery
Journal of Pharmacological Sciences, 2018Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Abstract Eukaryotic Elongation Factor 2 kinase (eEF2K) is a calmodulin-related protein kinase which regulates protein translation. A484954 is an inhibitor of eEF2K. In the present study, we investigated the acute effects of A484954 on contractility of isolated blood vessels. Isometric contraction of rat isolated aorta and main branch of superior mesenteric artery (MA) was measured. Expression of an inward rectifier K+ (Kir) channel subtype mRNA and protein was examined. A484954 caused relaxation in endothelium-intact [E (+)] and -denuded [E (−)] aorta or MA precontracted with noradrenaline (NA). The relaxation was higher in MA than aorta. The relaxation was partially inhibited by a nitric oxide (NO) synthase inhibitor, NG-nitro- l -arginine methyl ester (300 μM) in E (+) MA. The relaxation was significantly smaller in MA precontracted with high K+ than NA. The A484954-induced relaxation was significantly inhibited by a Kir channel blocker, BaCl2 (1 mM) compared with vehicle control in E (−) MA. Expression of Kir2.2 mRNA and protein was significantly higher in MA than aorta. We for the first time revealed that A484954 induces relaxation through opening smooth muscle Kir (Kir2.2) channel and through endothelium-derived NO in MA.
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Mechanisms underlying the relaxation by A484954, a eukaryotic Elongation Factor 2 kinase inhibitor, in rat isolated mesenteric artery
Elsevier, 2018Co-Authors: Tomoko Kodama, Muneyoshi Okada, Hideyuki YamawakiAbstract:Eukaryotic Elongation Factor 2 kinase (eEF2K) is a calmodulin-related protein kinase which regulates protein translation. A484954 is an inhibitor of eEF2K. In the present study, we investigated the acute effects of A484954 on contractility of isolated blood vessels. Isometric contraction of rat isolated aorta and main branch of superior mesenteric artery (MA) was measured. Expression of an inward rectifier K+ (Kir) channel subtype mRNA and protein was examined. A484954 caused relaxation in endothelium-intact [E (+)] and -denuded [E (−)] aorta or MA precontracted with noradrenaline (NA). The relaxation was higher in MA than aorta. The relaxation was partially inhibited by a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (300 μM) in E (+) MA. The relaxation was significantly smaller in MA precontracted with high K+ than NA. The A484954-induced relaxation was significantly inhibited by a Kir channel blocker, BaCl2 (1 mM) compared with vehicle control in E (−) MA. Expression of Kir2.2 mRNA and protein was significantly higher in MA than aorta. We for the first time revealed that A484954 induces relaxation through opening smooth muscle Kir (Kir2.2) channel and through endothelium-derived NO in MA. Keywords: Blood vessel, K+ channel, Vascular smooth muscle, Vasorelaxatio
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Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic Elongation Factor 2.
Microbiology, 2001Co-Authors: Mythili S. Shastry, Ming-jo Hsu, Dennis M. Schmatz, Jennifer Nielsen, Paul A. Liberator, Jennifer W. Anderson, Michael C. JusticeAbstract:The sordarin class of natural products selectively inhibits fungal protein synthesis by impairing the function of eukaryotic Elongation Factor 2 (eEF2). Mutations in Saccharomyces cerevisiae eEF2 or the ribosomal stalk protein rpP0 can confer resistance to sordarin, although eEF2 is the major determinant of sordarin specificity. It has been shown previously that sordarin specifically binds S. cerevisiae eEF2 while there is no detectable binding to eEF2 from plants or mammals, despite the high level of amino acid sequence conservation among these proteins. In both whole-cell assays and in vitro translation assays, the efficacy of sordarin varies among different species of pathogenic fungi. To investigate the basis of sordarin’s fungal selectivity, eEF2 has been cloned and characterized from several sordarin-sensitive and -insensitive fungal species. Results from in vivo expression of Candida species eEF2s in S. cerevisiae and in vitro translation and growth inhibition assays using hybrid S. cerevisiae eEF2 proteins demonstrate that three amino acid residues within eEF2 account for the selectivity of this class of compounds. It is also shown that the corresponding residues at these positions in human eEF2 are sufficient to confer sordarin insensitivity to S. cerevisiae identical to that observed with mammalian eEF2.
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mutations in ribosomal protein l10e confer resistance to the fungal specific eukaryotic Elongation Factor 2 inhibitor sordarin
Journal of Biological Chemistry, 1999Co-Authors: Michael C. Justice, Ming-jo Hsu, Dennis M. Schmatz, Karen Carniol, Jennifer NielsenAbstract:The natural product sordarin, a tetracyclic diterpene glycoside, selectively inhibits fungal protein synthesis by impairing the function of eukaryotic Elongation Factor 2 (eEF2). Sordarin and its derivatives bind to the eEF2-ribosome-nucleotide complex in sensitive fungi, stabilizing the post-translocational GDP form. We have previously described a class of Saccharomyces cerevisiaemutants that exhibit resistance to varying levels of sordarin and have identified amino acid substitutions in yeast eEF2 that confer sordarin resistance. We now report on a second class of sordarin-resistant mutants. Biochemical and molecular genetic analysis of these mutants demonstrates that sordarin resistance is dependent on the essential large ribosomal subunit protein L10e in S. cerevisiae. Five unique L10e alleles were characterized and sequenced, and several nucleotide changes that differ from the wild-type sequence were identified. Changes that result in the resistance phenotype map to 4 amino acid substitutions and 1 amino acid deletion clustered in a conserved 10-amino acid region of L10e. Like the previously identified eEF2 mutations, the mutant ribosomes show reduced sordarin-conferred stabilization of the eEF2-nucleotide-ribosome complex. To our knowledge, this report provides the first description of ribosomal protein mutations affecting translocation. These results and our previous observations with eEF2 suggest a functional linkage between L10e and eEF2.
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Elongation Factor 2 as a novel target for selective inhibition of fungal protein synthesis.
The Journal of biological chemistry, 1998Co-Authors: Michael C. Justice, Ming-jo Hsu, Bruno Tse, James M. Balkovec, Dennis M. Schmatz, Jennifer NielsenAbstract:Elongation Factor 2 (EF2) is an essential protein catalyzing ribosomal translocation during protein synthesis and is highly conserved in all eukaryotes. It is largely interchangeable in translation systems reconstituted from such divergent organisms as human, wheat, and fungi. We have identified the sordarins as selective inhibitors of fungal protein synthesis acting via a specific interaction with EF2 despite the high degree of amino acid sequence homology exhibited by EF2s from various eukaryotes. In vitroreconstitution assays using purified components from human, yeast, and plant cells demonstrate that sordarin sensitivity is dependent on fungal EF2. Genetic analysis of sordarin-resistant mutants of Saccharomyces cerevisiae shows that resistance to the inhibitor is linked to the genes EFT1 and EFT2that encode EF2. Sordarin blocks ribosomal translocation by stabilizing the fungal EF2-ribosome complex in a manner similar to that of fusidic acid. The fungal specificity of the sordarins, along with a detailed understanding of its mechanism of action, make EF2 an attractive antifungal target. These findings are of particular significance due to the need for new antifungal agents.