MNK1 pathway activity maintains protein synthesis in rapalog-treated gliomas
Michal Grzmil, … , Adrian Merlo, Brian A. Hemmings
Michal Grzmil, … , Adrian Merlo, Brian A. Hemmings
Published January 9, 2014
Citation Information: J Clin Invest. 2014;124(2):742-754. https://doi.org/10.1172/JCI70198.
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Research Article Oncology

MNK1 pathway activity maintains protein synthesis in rapalog-treated gliomas

Abstract

High levels of mammalian target of rapamycin complex 1 (mTORC1) activity in malignant gliomas promote tumor progression, suggesting that targeting mTORC1 has potential as a therapeutic strategy. Remarkably, clinical trials in patients with glioma revealed that rapamycin analogs (rapalogs) have limited efficacy, indicating activation of resistance mechanisms. Targeted depletion of MAPK-interacting Ser/Thr kinase 1 (MNK1) sensitizes glioma cells to the mTORC1 inhibitor rapamycin through an indistinct mechanism. Here, we analyzed how MNK1 and mTORC1 signaling pathways regulate the assembly of translation initiation complexes, using the cap analog m7GTP to enrich for initiation complexes in glioma cells followed by mass spectrometry–based quantitative proteomics. Association of eukaryotic translation initiation factor 4E (eIF4E) with eIF4E-binding protein 1 (4EBP1) was regulated by the mTORC1 pathway, whereas pharmacological blocking of MNK activity by CGP57380 or MNK1 knockdown, along with mTORC1 inhibition by RAD001, increased 4EBP1 binding to eIF4E. Furthermore, combined MNK1 and mTORC1 inhibition profoundly inhibited 4EBP1 phosphorylation at Ser65, protein synthesis and proliferation in glioma cells, and reduced tumor growth in an orthotopic glioblastoma (GBM) mouse model. Immunohistochemical analysis of GBM samples revealed increased 4EBP1 phosphorylation. Taken together, our data indicate that rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1 and indicate a molecular cross-talk between the mTORC1 and MNK1 pathways that has potential to be exploited therapeutically.

Authors

Michal Grzmil, Roland M. Huber, Daniel Hess, Stephan Frank, Debby Hynx, Gerald Moncayo, Dominique Klein, Adrian Merlo, Brian A. Hemmings

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Figure 2

MNK1-dependent 4EBP1 phosphorylation at Ser65 in RAD001-treated glioma cells.

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MNK1-dependent 4EBP1 phosphorylation at Ser65 in RAD001-treated glioma c...
(A) LN229 cells were treated with 10 μM CGP5730 and/or 10 nM RAD001 for 2 hours, and 4EBP1 immunoprecipitated using a 4EBP1-specific antibody was analyzed by LC-MS/MS. Bars represent relative quantification of phosphopeptides. The abundance of each identified phosphopeptide in control DMSO-treated cells was set to 1. (B) Representation of the 4EBP1 protein showing phosphorylation sites affected by inhibitor treatment. BD, eIF4G binding domain. (C) Relative abundance and (D) MS2 spectra for peptides covering 4EBP1 phosphorylation sites at Ser65/Thr70. Detected y- and b-ions are indicated in the sequence and H3PO4 loss is marked with asterisks. (E) Phosphorylation of 4EBP1 at Ser65 was analyzed by immunoblotting using a phospho-specific antibody in whole protein lysates prepared from U373 and LN229 cells after 20 hours of treatment. Blots were stripped and reprobed with 4EBP1 and/or tubulin-specific antibodies. Twenty-four hours after transfection with duplex siRNA oligonucleotides against MNK1 or control duplex against luciferase, cells were treated with 10 nM RAD001 for a further 20 hours and lysates were subjected to Western blot analysis. (F) 4EBP1 phosphorylation in MNK1-overexpressing U373 cells 24 hours after transfection and treatment with 10 nM RAD001 was analyzed by immunoblotting as described above.