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

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) 2 alpha in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2 alpha phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal - an inhibitor of eIF2 alpha 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 eIF2 alpha is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2 alpha phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2 alpha phosphorylation than non-irradiated ones. Similar to salubrinal - the phosphomimetic eIF2 alpha variant - S51D - increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2 alpha 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 eIF2 alpha phosphorylation and clonogenic death in response to the histone deacetylase inhibitor - vorinostat. Finally, the catalytic competitive inhibitor of mTOR - Ku-0063794 - increased phosphorylation of eIF2 alpha demonstrating further the involvement of mTOR activity in modulating eIF2 alpha phosphorylation. These experiments suggest that excessive phosphorylation of eIF2 alpha decreases survival of cancer cells; making eIF2 alpha 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.