Targeting elongation factor-2 kinase (eEF-2K) induces apoptosis in human pancreatic cancer cells

Abstract

Pancreatic cancer (PaCa) is one of the most aggressive, apoptosis-resistant and currently incurable cancers with a poor survival rate. Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase, whose role in PaCa survival is not yet known. Here, we show that eEF-2K is overexpressed in PaCa cells and its down-regulation induces apoptotic cell death. Rottlerin (ROT), a polyphenolic compound initially identified as a PKC-δ inhibitor, induces apoptosis and autophagy in a variety of cancer cells including PaCa cells. We demonstrated that ROT induces intrinsic apoptosis, with dissipation of mitochondrial membrane potential (ΔΨm), and stimulates extrinsic apoptosis with concomitant induction of TNF-related apoptosis inducing ligand (TRAIL) receptors, DR4 and DR5, with caspase-8 activation, in PANC-1 and MIAPaCa-2 cells. Notably, while none of these effects were dependent on PKC-δ inhibition, ROT down-regulates eEF-2K at mRNA level, and induce eEF-2K protein degradation through ubiquitin-proteasome pathway. Down-regulation of eEF-2K recapitulates the events observed after ROT treatment, while its over-expression suppressed the ROT-induced apoptosis. Furthermore, eEF-2K regulates the expression of tissue transglutaminase (TG2), an enzyme previously implicated in proliferation, drug resistance and survival of cancer cells. Inhibition of eEF-2K/TG2 axis leads to caspase-independent apoptosis which is associated with induction of apoptosis-inducing factor (AIF). Collectively, these results indicate, for the first time, that the down-regulation of eEF-2K leads to induction of intrinsic, extrinsic as well as AIF-dependent apoptosis in PaCa cells, suggesting that eEF-2K may represent an attractive therapeutic target for the future anticancer agents in PaCa.

Down-Regulation of 5-HT1B and 5-HT1D Receptors Inhibits Proliferation, Clonogenicity and Invasion of Human Pancreatic Cancer Cells

“…Plated cells were transfected with double-stranded siRNA targeting the mRNA of the serotonergic receptors (5-HT-1) subtype –B or –D (Sigma-Aldrich, St. Louis, MO), or transfected with control (non-silencing) siRNA; (5′-AAUUCUCCGAACGUGUCACGU-3′) [34], [35] (Sigma-Aldrich, St. Louis, MO). siRNA targeting tissue transglutaminase (TG2) (Qiagen, Valencia, CA) were also employed [35]. Cells were transfected with either siRNA, at a final concentration of 25–50 nM for 72 h, using HiPerFect Transfection Reagent (Qiagen, Valencia, CA) according to the manufacturer's protocol.…”

“…pyrophosphate, 1 mM Na 3 VO 4 and 10% glycerol, containing proteinase and phosphatase inhibitors (Roche Applied Science, Indianapolis)] were added to each well. The cell lysates were collected, and RPPA was processed as described before [35].…”

MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase

“…Recently, emerging evidence has revealed that eukaryotic elongation factor 2 kinase (eEF2K) is a potential molecular driver in several cancers, including pancreatic, brain and breast cancer [11–16]. eEF2K is the only calcium/calmodulin activated member of the α-kinase family and is considered an atypical kinase since its catalytic domain is not structurally similar to those of conventional protein kinases [17, 18].…”

“…eEF2K promotes cell survival in conditions of nutrient deprivation, hypoxia and metabolic stress by regulating the rate of translation [24]. Recently, eEF2K was shown to promote cell proliferation, cell migration, invasion, epithelial-mesenchymal transition (EMT) and TNBC tumorigenesis and progression through modulating the cell cycle (G1/S transition) by regulating cyclin D1, c-myc, PI3K/Akt, Src/Fak and insulin-like growth factor receptor (IGFR) signaling [11, 13, 14, 16]. Therapeutic targeting of eEF2K triggers apoptosis and suppresses TNBC tumor growth, in addition to and increased doxorubicin and paclitaxel efficacy [16].…”

Oligonucleotide-Based Theranostic Nanoparticles in Cancer Therapy

“…siRNAs have been used with breast cancer therapeutics to specifically target C-X-C chemokine receptor 4 [61,62], elongation factor 2 kinase [63], Aurora kinase A [64], beta 3 integrin [65], B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11A gene [66], HER2 [67], tumor p53 gene [68], adenosine triphosphate-binding cassette E1 and eukaryotic release factor 3 [69].…”