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

<div><p>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.</p> </div

Catalytic inhibitor of mTOR increases eIF2alpha phosphorylation:

<div><p><b>a</b>. Cells were treated with the noted concentrations of Ku-0063794 for 48 hours, then harvested and processed for Western blot analysis of eIF2α phosphorylation. <b>b</b>. Ku-0063794 induced changes in eIF2α (e), p-eIF2α (p) and the ratio p-eIF2α/eIF2α (p/e) represent the analysis of 3 determinations for 100 and 1000 nM and of 2 determinations for 300 nM.</p> <p>*Denotes significant change relative to control - <i>p</i><0.05. Values of e, p and p/e for 300 nM Ku-0063794 from the two determinations are presented in Table S6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077260#pone.0077260.s001" target="_blank">File S1</a>).</p></div

Salubrinal enhances radiation induced eIF2α phosphorylation:

<div><p><b>a</b>. Cells were processed for analysis of eIF2α phosphorylation 48 hours post-addition of salubrinal (Sal). b. Mean ± SEM fold change relative to control of eIF2α (e), p-eIF2α (p) and of the ratio p-eIF2α/eIF2α (p/e) in salubrinal (Sal) treated cells. The experiment was reproduced 3 times for 4.5 µM and twice for 16 µM. * Denotes significant change relative to control - P<0.05.</p> <p><b>c</b>. Cells were irradiated with the noted radiation dose prior to addition of 4.5 µM Salubrinal (Sal). <b>d</b>. Mean fold change relative to control of eIF2α (e), p-eIF2α (p) and the ratio p eIF2α/eIF2α (p/e).The experiment was reproduced once with similar results (Values for e, p, and p/e from both experiments are presented in Table S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077260#pone.0077260.s001" target="_blank">File S1</a>). </p></div

Increased eIF2α phosphorylation in response to radiation:

<p><b>a</b>. MCF-7 and <b>b</b>. MDA-MB-231 cells were irradiated with the indicated radiation dose and processed for analysis of eIF2α phosphorylation at the noted time post-irradiation. <b>c</b>. Mean ± SEM of fold change relative to control of eIF2alpha (e), p-eIF2α (p) and the ratio of p-eIF2α/eIF2α (p/e) in irradiated MCF-7 and MDA-MB-231 cells. Analysis for MCF-7 at 24 hours post-irradiation with 1.5 and 9.5 Gy represents 2 and 3 separate determinations respectively and at 48 hours 3 separate determinations for each dose. Analysis for MDA-MB-231 cells at 24 hours represents 3 determinations for each dose and at 48 hours 5 determination for 1.5 Gy and 6 for 9.5 Gy. *Denotes significant change relative to control - <i>p</i>< 0.05.</p

Salubrinal reduced expression of surface ESA:

<p>Cells were incubated with 24 µM salubrinal for 96 hours prior to harvesting, Cell staining with FITC-anti-ESA or with isotype-matched controls was detected with FACSCalibur. The experiment was reproduced twice with similar results. 7AAD - viability dye, FITC - anti-ESA.</p

The phosphomimetic eIF2α variant increases clonogenic death in control and irradiated cells:

<p><b>a</b>. Cells transfected with 2 µg/2ml eIF2α S51A (SA) or with eIF2α S51D (SD) were processed for analysis 17 days post-plating. At this concentration SD decreased clonogenic survival. <b>b</b>,<b>c</b>. Cells transfected with 1.5µg/2ml SA or with SD. Cells in <b>b</b> were not irradiated whereas cells in <b>c</b> were irradiated with 1.5 Gy 24 hours post-transfection. Cells in b and c were processed for analysis 10 days post-irradiation. At this concentration SD by itself did not affect clonogenic survival but did increase sensitivity to radiation. The experiment was reproduced once with similar results. </p

Salubrinal inhibits NHEJ and HR repair:

<p><b>a</b>. Measurement of NHEJ activity was conducted 24 hours post-transfection with I-SceI. The experiment was reproduced once with similar results i.e. 4% repair activity for control and 3% for salubrinal treated cells. <b>b</b>. Measurement of HR activity was conducted 36 hours post-transfection with I-SceI. The experiment was reproduced once with similar results i.e. 2.35% activity for control and 1.6% activity for salubrinal treated cells at 24 hours post-transfection with I-SceI. Sal - 4.5 µM salubrinal.</p

Increased eIF2α phosphorylation in irradiated cells abrogated radiation-induced increase in cellular BRCA1

<div><p>Upper panel: Control and irradiated cells (1.5 Gy) were treated with 4.5 μM salubrinal or the vehicle. Cells were harvested 48 hours post-irradiation and processed for western blot analysis of changes in the level of BRCA1. </p> <p>Lower panels: Cells transfected with 10 µg/ml of the S51A and S51D eIF2α variants or with transfection reagents alone (SHAM). Cells were irradiated with 9.5 Gy 24 hours post-transfection and processed for BRCA1 analysis 24 hours post- radiation. CD2 was expressed in transfected cells indicating a successful transfection. Sal - 4.5 µM salubrinal. </p></div

Salubrinal increases activity of senescence-associated β-Galactosidase:

<p>Cells were plated for colony survival assay. Salubrinal (4.5 µM) or the vehicle was added immediately following radiation with 1 Gy. Acidic β-Galactosidase activity is reflected in the blue stain. Numbers are average of senescent looking cells/colony ± SD in triplicate plates and differences between the combined treatments and each one of the sole treatments as well as between each treatment and control were statistically significant <i>p</i>< 0.05. Bar,100 µM.</p

Increased eIF2α phosphorylation in response to rapamycin:

<p><b>a</b>. Cells were incubated for 24 hours with 50 nM rapamycin, harvested and processed for determination of changes in eIF2α phosphorylation <b>b</b>. Mean ± SEM fold change relative to control of eIF2α (e), p-eIF2α (p) and the ratio p-eIF2α/eIF2α (p/e) in rapamycin treated cells. The analysis represents 6 separate determinations for MCF-7 cells and 3 for MDA-MB-231 cells. *Denotes significant change relative to control - <i>p</i>< 0.05.</p