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

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.

Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations

“…Plating for colony survival assay, colony counting, and calculation of percent clonogenic death (CD) was performed in triplicates as described before [ 19 – 21 ]. Colonies were fixed, stained and counted 10–14 days following plating, when 90–95% of the colonies in the control possessed more than 50 cells.…”

“…Preparation of cell lysates and analysis of treatment-induced changes in protein level and phosphorylation were done as we described before [ 19 ]. Protein content was determined with a bicinchoninic acid reagent (Bio-Rad, Hercules, CA), and equal loading was verified by measuring the absorbance at 520 nm of Ponceau S (Sigma, St-Louis, MO) extracted with phosphate buffer (2.67 mM KCl, 1.47 mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 and 1.125 M NaCl) from individual strips of a twin run [ 19 , 20 ]. Blots were exposed to x-ray film for chemiluminescence following treatment with West Pico ECL reagent (Thermo Scientific Rockford, IL).…”

“…Also, neither vorinostat nor 6-TG are multiple drug resistant (MDR) 1 substrates [ 7 , 15 ] and therefore their combined action should not be affected by a possible increase in the level of MDR1. Lastly, vorinostat-induced increase in phosphorylation of eIF2α is also expected to contribute to inhibition of HRR [ 19 ] thus leading to enhanced cellular response to both PARPis and 6-TG.…”

Activation of endoplasmic reticulum stress promotes autophagy and apoptosis and reverses chemoresistance of human small cell lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway

“…Excessive phosphorylation of eIF2α decreases cancer cell survivals. Thus, eIF2α is a worthy target for drug development to enhance the cytotoxic effects of established anti-neoplastic therapies by inhibiting upstream components of the mTOR signaling pathway [28]. CHOP can regulate cell apoptosis effectors such as Bcl-2 and Bim, and eventually lead to apoptosis activation of caspase-3 [29].…”

mTORC2 Balances AKT Activation and eIF2α Serine 51 Phosphorylation to Promote Survival under Stress

“…However, the role of mTOR in eIF2aS51P and translational control in mammalian cells under stress conditions is not well understood. For example, eIF2aS51P was shown to be induced by either the inhibition or the activation of mTORC1 in response to rapamycin or TSC inactivation, respectively, rendering the interpretation of the findings difficult (25,26). We have investigated the regulation of eIF2aS51P in mouse and human cells rendered defective in mTORC1 and/or mTORC2 activity by genetic or pharmacological means.…”