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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

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Abstract

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.

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All data generated or analyzed during this study are included in this published article.

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Acknowledgements

GC7 (N1-guanyl-1,7-diamineoheptane) was a gift from Myung Hee Park (NIH/NIDCR, Bethesda, MD).

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2058125).

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Authors and Affiliations

  1. Research Institute of Medical Science, School of Medicine, KonKuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 05029, South Korea

    Jong Hwan Park

  2. Chingchai Wanidworanun, MD PLLC, 4001 9th St N Suite 228, Arlington, VA, 22203, USA

    Grace Kelly Lee

  3. Institute of Life Science and Resources and Department of Food Science and Biotechnology, Kyung Hee University, Yongin, 17104, South Korea

    Hae-Yeong Kim

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Contributions

JHP conceived and designed the study. JHP and GKL performed the experiments and collected the data. JHP and GKL wrote the paper. JHP, GKL, and HYK analyzed the data. JHP and HYK reviewed the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Hae-Yeong Kim or Jong Hwan Park.

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Lee, G.K., Kim, HY. & Park, J.H. 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. Mol Biol Rep 50, 5807–5816 (2023). https://doi.org/10.1007/s11033-023-08510-5

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