Eukaryotic elongation factor 2 is a prognostic marker and its kinase a potential therapeutic target in HCC

Abstract

Hepatocellular carcinoma is a cancer with increasing incidence and largely refractory to current anticancer drugs. Since Sorafenib, a multikinase inhibitor has shown modest efficacy in advanced hepatocellular carcinoma additional treatments are highly needed. Protein phosphorylation via kinases is an important post-translational modification to regulate cell homeostasis including proliferation and apoptosis. Therefore kinases are valuable targets in cancer therapy. To this end we performed 2D differential gel electrophoresis and mass spectrometry analysis of phosphoprotein-enriched lysates of tumor and corresponding non-tumorous liver samples to detect differentially abundant phosphoproteins to screen for novel kinases as potential drug targets. We identified 34 differentially abundant proteins in phosphoprotein enriched lysates. Expression and distribution of the candidate protein eEF2 and its phosphorylated isoform was validated immunohistochemically on 78 hepatocellular carcinoma and non-tumorous tissue samples. Validation showed that total eEF2 and phosphorylated eEF2 at threonine 56 are prognostic markers for overall survival of HCC-patients. The activity of the regulating eEF2 kinase, compared between tumor and non-tumorous tissue lysates by in vitro kinase assays, is more than four times higher in tumor tissues. Functional analyzes regarding eEF2 kinase were performed in JHH5 cells with CRISPR/Cas9 mediated eEF2 kinase knock out. Proliferation and growth is decreased in eEF2 kinase knock out cells.ConclusioneEF2 and phosphorylated eEF2 are prognostic markers for survival of hepatocellular carcinoma patients and the regulating eEF2 kinase is a potential drug target for tumor therapy.

Eukaryotic elongation factor 2 kinase promotes angiogenesis in hepatocellular carcinoma via PI3K/Akt and STAT3

“…A recent study showed that high levels of total eEF2 and P-eEF2 are associated with poor prognosis of HCC patients. 9 Here, we demonstrate for the first time that inhibition of eEF2K prevents HCC angiogenesis and progression in an HCC xenograft tumor model, indicating that pharmacologically targeting eEF2K may be a promising therapeutic strategy against HCC, and as well other types of angiogenic solid tumors.…”

“…Previously, it has been reported that the relative abundances of total and phosphorylated (at Thr56) eEF2 are higher in HCC tissues compared to adjacent nontumor tissues or normal liver tissues. 9 We first compared the levels of eEF2K and phospho (p-) eEF2 Thr56 among different HCC cell lines. MHCC97H cells exhibited highest levels of both eEF2K and p-eEF2 Thr56 (Zhou Y and Shen K, unpublished data).…”

“…Eukaryotic elongation factor 2 kinase (eEF2K), an atypical Ca 2+ /calmodulin‐dependent protein kinase, is a negative regulator of the elongation stage of mRNA translation, which phosphorylates and hence inactivates eEF2 . It is highly expressed in various metastatic tumors and is associated with poor prognosis in different types of human cancers . Several recent studies suggest that eEF2K plays an important role in promoting tumor cell migration and invasion .…”

“…It is highly expressed in various metastatic tumors and is associated with poor prognosis in different types of human cancers . Several recent studies suggest that eEF2K plays an important role in promoting tumor cell migration and invasion . siRNA‐mediated knockdown of eEF2K in A549 cells (human lung carcinoma) suppresses tumor development and metastasis .…”

CRISPR/Cas9: at the cutting edge of hepatology

“…The most common method is to use a single sgRNA targeted to a critical exon in order to introduce indels, likely causing a frameshift mutation and/or NMD (figure 2A). This approach has been used to validate, in vitro, potential oncogenes discovered in human liver cancers, such as acid sensing ion channel 1a43 and eukaryotic elongation factor 2 kinase 44. Similarly, isocitrate dehydrogenase 1, commonly mutated in intrahepatic cholangiocarcinoma,45 has been deleted in the HepG2 cell lines to dissect its biological and metabolic functions 46…”