In an effort to circumvent rapamycin-resistance in cancer cells 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)2alpha 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 eIF2(alpha) dephosphorylation– decreased the size of the sub-population with surface epithelial specific antigen, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2(alpha) can hinder the cells' capacity for self-renewal and is detrimental to their survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and demonstrated that levels of eIF2α phosphorylation that are tolerated by non-irradiated cells, are harmful to irradiated ones. Similar to salubrinal - the phosphomimetic eIF2(alpha) variant - S51D - increased sensitivity to radiation. Both salubrinal and the phosphomimetic eIF2alphaS51D abrogated radiation-induced increase in BRCA1, thus implicating enhanced phosphorylation of eIF2alpha in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks (DSB) that were induced by I-SceI in GFP reporter plasmids. In addition to radiation, salubrinal enhanced eIF2alpha phosphorylation and clonogenic cell death in response to the histone deacetylase inhibitor – Vorinostat.Finally, the ATP-competitive catalytic 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 negatively affects survival of cancer cells, making eIF2α a worthy target for drug development, with the potential to both increase the cytotoxic effects of established anti-neoplastic therapy and circumvent resistance to rapalogues and possibly to other drugs that inhibit upstream components of the PI3K/Akt/mTOR pathway.
