Eukaryotic Initiation Factor 2α - a Downstream Effector of Mammalian Target of Rapamycin - Modulates DNA Repair and Cancer Response to Treatment

Liron Tuval-Kochen,Amos Toren,Shoshana Paglin,Yaacov Lawrence,Joachim Yahalom,Raphael Pfeffer,Gilmor Keshet,Tamar Golani,Charles Nakar,Yaniv Lerenthal,Vladislav V Glinskii

doi:10.1371/journal.pone.0077260

Abstract

In an effort to circumvent resistance to rapamycin – an mTOR inhibitor - we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF) 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal – an inhibitor of eIF2α dephosphorylation – decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal - the phosphomimetic eIF2α variant - S51D - increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor – vorinostat. Finally, the catalytic competitive inhibitor of mTOR - Ku-0063794 - increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for drug development, with the potential to enhance the cytotoxic effects of established anti-neoplastic therapies and circumvent resistance to rapalogues and possibly to other drugs that inhibit upstream components of the mTOR pathway.

Highlights

The phosphatidylinositol 3-kinase - protein kinase B mammalian target of rapamycin (PI3K-Akt-mTOR) pathway regulates cell growth and proliferation
While this work was in progress, later studies showed that temsirolimous at concentrations higher than 10 μM can induce eIF2α phosphorylation in temsirolimous-resistant cells [14], and nM concentrations of rapamycin induced eIF2α phosphorylation in acute myeloid leukemia cells [13]
Very recently Mounir et al demonstrated that inhibition of PI3K in mouse embryonic fibroblasts (MEF) leads to inhibition of Akt with the consequent activation of PKR-like ER-localized eIF2α kinase (PERK) and phosphorylation of eIF2α [12]

Summary

Introduction

The phosphatidylinositol 3-kinase - protein kinase B mammalian target of rapamycin (PI3K-Akt-mTOR) pathway regulates cell growth and proliferation. The mTOR's inhibitors - rapamycin, and its derivatives decrease cancer cell proliferation and have been tested as anti-cancer agents in clinical trials [1,2,3]. Its derivatives - temsirolimous and everolimous have been approved for the treatment of various types of cancer [5,6]. Rapalogues bind their intracellular receptor FK506 binding protein 12 (FKBP12), forming a complex that inhibits mTOR complex 1 (mTORC1) by binding mTOR's FKBP12 rapamycinbinding domain [7].

Methods

Results

Conclusion