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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Antineoplastic Agents / pharmacology*
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Cell Line, Tumor
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Cellular Senescence / drug effects
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Cinnamates / pharmacology
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DNA Repair / drug effects*
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DNA, Neoplasm / genetics*
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DNA, Neoplasm / metabolism
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Deoxyribonucleases, Type II Site-Specific / genetics
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Deoxyribonucleases, Type II Site-Specific / metabolism
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Drug Resistance, Neoplasm / drug effects
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Drug Resistance, Neoplasm / genetics*
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Drug Resistance, Neoplasm / radiation effects
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Eukaryotic Initiation Factor-2 / antagonists & inhibitors
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Eukaryotic Initiation Factor-2 / genetics*
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Eukaryotic Initiation Factor-2 / metabolism
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Female
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Gamma Rays
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Gene Expression Regulation, Neoplastic / drug effects*
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Gene Expression Regulation, Neoplastic / radiation effects
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Humans
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Hydroxamic Acids / pharmacology
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Morpholines / pharmacology
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Peptidomimetics / pharmacology
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Phosphorylation / drug effects
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Phosphorylation / radiation effects
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Pyrimidines / pharmacology
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Signal Transduction
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Sirolimus / pharmacology
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TOR Serine-Threonine Kinases / antagonists & inhibitors
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TOR Serine-Threonine Kinases / genetics*
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TOR Serine-Threonine Kinases / metabolism
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Thiourea / analogs & derivatives
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Thiourea / pharmacology
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Transgenes
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Vorinostat
Substances
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Antineoplastic Agents
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Cinnamates
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DNA, Neoplasm
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Eukaryotic Initiation Factor-2
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Hydroxamic Acids
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Membrane Proteins
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Morpholines
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Peptidomimetics
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Pyrimidines
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SFXN4 protein, human
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Saccharomyces cerevisiae Proteins
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salubrinal
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Green Fluorescent Proteins
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Vorinostat
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Ku 0063794
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MTOR protein, human
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TOR Serine-Threonine Kinases
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SCEI protein, S cerevisiae
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Deoxyribonucleases, Type II Site-Specific
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Thiourea
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Sirolimus
Grants and funding
The research was supported by a generous donation from the estate of Miss HARAN ESTER of blessed memory through Keren Izvonot, Israel, Ministry of health (S.P) and by the Medical Research Infrastructure Development Fund – Sheba Medical Center (Y.L). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.