Inhibiting eukaryotic initiation factor 5A (eIF5A) hypusination attenuated activation of the SIK2 (salt-inducible kinase 2)-p4E-BP1 pathway involved in ovarian cancer cell proliferation and migration.

BACKGROUND: Eukaryotic initiation factor 5A hypusine (eIF5AHyp) stimulates the translation of proline repeat motifs. Salt inducible kinase 2 (SIK2) containing a proline repeat motif is overexpressed in ovarian cancers, in which it promotes cell proliferation, migration, and invasion. METHODS AND RESULTS: Western blotting and dual luciferase analyses showed that depletion of eIF5AHyp by GC7 or eIF5A-targeting siRNA downregulated SIK2 level and decreased luciferase activity in cells transfected with a luciferase-based reporter construct containing consecutive proline residues, whereas the activity of the mutant control reporter construct (replacing P825L, P828H, and P831Q) did not change. According to the MTT assay, GC7, which has a potential antiproliferative effect, reduced the viability of several ovarian cancer cell lines by 20-35% at high concentrations (ES2 > CAOV-3 > OVCAR-3 > TOV-112D) but not at low concentrations. In a pull-down assay, we identified eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP1 (p4E-BP1) phosphorylated at Ser 65 as downstream binding partners of SIK2, and we validated that the level of p4E-BP1(Ser 65) was downregulated by SIK2-targeting siRNA. Conversely, in ES2 cells overexpressing SIK2, the p4E-BP1(Ser 65) level was increased but decreased in the presence of GC7 or eIF5A-targeting siRNA. Finally, the migration, clonogenicity, and viability of ES2 ovarian cancer cells were reduced by GC7 treatment as well as by siRNA for eIF5A gene silencing and siRNA for SIK2 and 4E-BP1 gene silencing. Conversely, those activities were increased in cells overexpressing SIK2 or 4E-BP1 and decreased again in the presence of GC7. CONCLUSION: The depletion of eIF5AHyp by GC7 or eIF5A-targeting siRNA attenuated activation of the SIK2-p4EBP1 pathway. In that way, eIF5AHyp depletion reduces the migration, clonogenicity, and viability of ES2 ovarian cancer cells.

Eukaryotic initiation factor 5A hypusine as a negative regulator of adenosine 2B receptor (A2bAR) through interaction with stem loop sequences within the A2bAR 3'-untranslated region.

PURPOSE: The aim of this study was to investigate whether eIF5A hypusine (eIF5AHyp) reduces adenosine 2b receptor (A2bAR) gene expression through interaction with highly structured stem-loop sequences within the A2bAR 3'UTR. METHODS AND RESULTS: Based on real-time PCR and western blotting, expression of A2bAR mRNA was significantly decreased upon treatment with eIF5AHyp in mouse embryonic fibroblasts of eIF5A (eIF5A-MEF) and 3T3-L1 cells. Target Scan software and RNAfold web server predicted two different structures formed by stem-loop sequences with overlapping microRNA 27 seed sequences and mutations. The EMSA results showed significantly impaired formation of the wild type (WT) biotin-labeled A2bAR probe (27 base) containing stem loop sequences-eIF5AHyp complex by mutation of stem-loop sequences or by eIF5A non-hypusine (eIF5ALys). The luciferase reporter assay showed that GC7-induced eIF5ALys accumulation increased the activity of pMIR-A2bAR WT containing the same stem-loop sequence in 3T3-L1 cells, whereas the activity with pMIR-A2bAR Mut was increased compared to WT control without dependence on GC7. Oil Red O staining showed that suppression of A2bAR expression (A2bAR siRNA and eIF5AHyp) increased the amount of lipid droplet formation and the mRNA levels of lipid droplet-related genes (C/EBP-ß, PPAR-γ, FABP4, SREBP-1, and Perilipin). In contrast, overexpression of A2bAR (A2bAR vector, eIF5ALys vector, and GC7) significantly decreased the expression of lipid droplet-associated genes and lipid droplet formation. CONCLUSIONS: eIF5AHyp acts as a negative regulator of A2bAR gene expression through stem loop sequences in A2bAR 3'UTR, allowing differentiation of adipocytes.