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Signaling from mTOR to eIF2α mediates cell migration in response to the chemotherapeutic doxorubicin

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Science Signaling  17 Dec 2019:
Vol. 12, Issue 612, eaaw6763
DOI: 10.1126/scisignal.aaw6763
  • Fig. 1 Doxorubicin-induced DNA damage inhibited global protein synthesis rates.

    (A) MCF10A cells were treated with doxorubicin (500 nM) for the indicated times and pulse-labeled with [35S]-methionine for 30 min. Total counts per minute were normalized to total protein, and values are shown as a fold change relative to untreated control samples for each time point. Error bars represent means ± SD (n = 3 independent experiments); **P ≤ 0.01 and *P ≤ 0.05, by unpaired Student’s t test (ns, not significant). (B) Comparison of polysome profiles from MCF10A cells untreated or treated continuously with doxorubicin (500 nM) for 24 hours. Cytoplasmic lysates were centrifuged at 38,000 rpm through 10 to 50% sucrose gradients at 4°C for 2 hours, and absorbance was measured at 254 nm (A254) using a flow rate of 1 ml/min. Profiles shown were representative of three independent experiments. (C) Schematic representation of the regulation of translation initiation through mTOR-dependent regulation of eIF4F complex formation and eIF2K-dependnent regulation of ternary complex (TC) formation. (D) MCF10A cells were treated with doxorubicin (500 nM) for the indicated times, lysed, and analyzed by immunoblotting with the indicated antibodies. Blots shown were representative of three independent experiments. (E) MCF10A cells were treated with thapsigargin (Tg) (250 nM for 1 hour), with or without ISRIB (200 nM), and pulse-labeled with [35S]-methionine for 30 min. Error bars represent means ± SD (n = 3 independent experiments); *P ≤ 0.05, by unpaired Student’s t test. (F) MCF10A cells were treated with doxorubicin (Dox) (500 nM for 16 hours), with or without ISRIB (200 nM), and pulse-labeled with [35S]-methionine for 30 min. Total counts per minute were normalized to total protein, and values are shown as a fold change relative to untreated control samples for each time point. Error bars represent means ± SD (n = 3 independent experiments); ***P ≤ 0.001 and **P ≤ 0.01, by unpaired Student’s t test. (G) Samples prepared in parallel to (E) and (F) were lysed and analyzed by immunoblotting with the indicated antibodies. Blots shown were representative of three independent experiments.

  • Fig. 2 Doxorubicin-induced mTOR inhibition depended on p53 activity.

    (A) Schematic representation depicting the regulation of mTOR signaling. Blocked arrows indicate the inhibition of the downstream protein, whereas arrows indicate the phosphorylation or activation of the downstream protein. (B) Wild-type p53 MCF10A cells (p53+/+) and p53 knockout (KO) MCF10A cells (p53−/−) were treated with doxorubicin (500 nM) or AZD8055 (100 nM) for 16 hours. Cells were lysed and analyzed by immunoblotting with the indicated antibodies. Blots shown were representative of three independent experiments. (C and D) Quantification of p70 S6K phosphorylation at Thr389 (C) and 4E-BP1 phosphorylation at Ser65 (D) from blots shown in (B). Error bars represent means ± SD (n = 3 independent experiments); ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05, by unpaired Student’s t test. (E) p53+/+ MCF10A cells were transfected with an siRNA directed against p53 (sip53) or a nonspecific scrambled control siRNA (siCt). After two consecutive 24-hour transfections, cells were treated with doxorubicin (500 nM) or AZD8055 (100 nM) for 16 hours. Cells were lysed and analyzed by immunoblotting with the indicated antibodies. Blots shown were representative of three independent experiments. (F and G) Quantification of p70 S6K phosphorylation at Thr389 (F) and 4E-BP1 phosphorylation at Ser65 (G) from blots shown in (E). Error bars represent means ± SD (n = 3 independent experiments); **P ≤ 0.01 and *P ≤ 0.05, by unpaired Student’s t test. (H) Comparison of polysome profiles from MCF10A p53−/− cells untreated or treated continuously with doxorubicin (500 nM) for 24 hours. Cytoplasmic lysates were centrifuged at 38,000 rpm through 10 to 50% sucrose gradients at 4°C for 2 hours, and absorbance was measured at 254 nm using a flow rate of 1 ml/min. Profiles shown were representative of three independent experiments. (I) Polysome/subpolysomal ratio from p53+/+ and p53−/− cells treated with doxorubicin for 24 hours. Ratio was calculated from the area under the curve of polysome profiles separated into polysomal and subpolysomal regions. Error bars represent means ± SD (n = 3 independent experiments); **P ≤ 0.01 and *P ≤ 0.05, by unpaired Student’s t test. (J) MCF10A p53+/+ and p53−/− cells and p53+/+ cells transfected with siRNA specific for p53 or a control nontargeting siRNA were pulse-labeled with [35S]-methionine for 30 min. Total counts per minute were normalized to total protein, and values are shown as a fold change relative to untreated control samples for each time point. Error bars represent means ± SD (n = 3 independent experiments); **P ≤ 0.01 and *P ≤ 0.05, by unpaired Student’s t test.

  • Fig. 3 mTOR inhibition enhanced the phosphorylation of eIF2α through a cross-talk signaling cascade.

    (A) Wild-type p53 MCF10A cells (p53+/+), p53 knockout MCF10A cells (p53−/−), and p53+/+ cells transfected with either control nontargeting siRNA (siCt) or siRNA specific for p53 (sip53) were treated with doxorubicin (500 nM) or AZD8055 (100 nM) for 16 hours. Cells were lysed and analyzed by immunoblotting with the indicated antibodies. Blots shown were representative of three independent experiments. (B) Quantification of eIF2α phosphorylation at Ser51 from blots shown in (A). Error bars represent means ± SD (n = 3 independent experiments); **P ≤ 0.01 and *P ≤ 0.05, by unpaired Student’s t test. (C) MCF10A p53+/+ cells were treated with Torin 1 (100 nM), AZD8055 (100 nM), or rapamycin (100 nM) for the indicated times. Cells were lysed and analyzed by immunoblotting with the indicated antibodies. Blots shown were representative of three independent experiments. (D and E) A549 cells were treated with doxorubicin (500 nM) or AZD8055 (100 nM) for 16 hours and analyzed by immunoblotting with the indicated antibodies (D). Blots shown were representative of three independent experiments. Quantification of p70 S6K phosphorylation at Thr389 and eIF2α phosphorylation at Ser51 (E) from blots shown in (D). Error bars represent means ± SD (n = 3 independent experiments); ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05, by unpaired Student’s t test. (F and G) A549 cells that were transfected with either control nontargeting siRNA (siCt) or siRNA specific for p53 (sip53) were treated with doxorubicin (500 nM) or AZD8055 (100 nM) for 16 hours. Cells were lysed and analyzed by immunoblotting with the indicated antibodies (F). Blots shown were representative of three independent experiments. Quantification of p70 S6K phosphorylation at Thr389 and eIF2α phosphorylation at Ser51 (G) from blots shown in (F). Error bars represent means ± SD (n = 3 independent experiments); ***P ≤ 0.001 and *P ≤ 0.05, by unpaired Student’s t test. SESN2, Sestrin2. (H) BT474 cells were grown at either 37°C (leading to loss of p53 function) or 32°C (restoring p53 function) and treated with doxorubicin (500 nM) or AZD8055 (100 nM) for 16 hours. Cells were lysed and analyzed by immunoblotting with the indicated antibodies. Blots shown were representative of two independent experiments.

  • Fig. 4 Doxorubicin-induced eIF2α phosphorylation was mediated by the eIF2Ks GCN2 and PERK.

    (A) Schematic representation of the phosphorylation of eIF2α in response to the activation of the eIF2Ks PERK, GCN2, and PKR in response to the indicated stimulus. (B and C) p53+/+ MCF10A cells were transfected with individual siRNAs directed against the eIF2Ks PERK (1 nM), PKR (1 nM), or GCN2 (2 nM). Forty-eight hours after transfection, cells were treated with AZD8055 (100 nM) for 16 hours. Cells were lysed and analyzed by immunoblotting with the indicated antibodies (B). Blots shown were representative of three independent experiments. Quantification of eIF2α phosphorylation (Ser51) (C) from (B). Error bars represent means ± SD (n = 3 independent experiments); *P ≤ 0.05, by unpaired Student’s t test. (D and E) p53+/+ MCF10A cells were transfected with combinations of siRNAs directed against the eIF2Ks PERK (1 nM), PKR (1 nM), or GCN2 (2 nM). Forty-eight hours after transfection, cells were treated with AZD8055 (100 nM) for 16 hours. Cells were lysed and analyzed by immunoblotting with the indicated antibodies (D). Blots shown were representative of three independent experiments. Quantification of eIF2α phosphorylation (Ser51) (E) from (D). Error bars represent means ± SD (n = 3 independent experiments); *P ≤ 0.05 and **P ≤ 0.01, by unpaired Student’s t test. (F and G) p53+/+ MCF10A cells were transfected with individual siRNAs directed against the eIF2Ks PERK (1 nM), PKR (1 nM), or GCN2 (2 nM). Forty-eight hours after transfection, cells were treated with doxorubicin (500 nM) for 16 hours. Cells were lysed and analyzed by immunoblotting with the indicated antibodies (F). Blots shown were representative of three independent experiments. Quantification of eIF2α phosphorylation (Ser51) (G) from (F). Error bars represent means ± SD (n = 3 independent experiments), and significance was calculated by unpaired Student’s t test. (H and I) p53+/+ MCF10A cells were transfected with combinations of siRNAs directed against the eIF2Ks PERK (1 nM), PKR (1 nM), or GCN2 (2 nM). Forty-eight hours after transfection, cells were treated with doxorubicin (500 nM) for 16 hours. Cells were lysed and analyzed by immunoblotting with the indicated antibodies (H). Blots shown were representative of three independent experiments. Quantification of eIF2α phosphorylation (Ser51) (I) from (H). Error bars represent means ± SD (n = 3 independent experiments); *P ≤ 0.05 and **P ≤ 0.01, by unpaired Student’s t test.

  • Fig. 5 Doxorubicin-induced mTOR-eIF2 cross-talk signaling mediates cell migration.

    (A) Schematic representation of mTOR-eIF2 cross-talk signaling and the alleviation of eIF2α phosphorylation–dependent translational repression. (B) Cell cycle analysis of wild-type p53 MCF10A cells (p53+/+) treated with doxorubicin (500 nM) for the indicated times. Cells were incubated with EdU (10 μM) for the final 1.5 hours of treatment (to quantify S phase cells), stained with FxCycle violet dye (to quantify cells within G1 and G2 phases), and analyzed by flow cytometry. Data values were an average of three independent experiments, and error bars represent means ± SD. (C) Wound-healing assay after treatment with doxorubicin (500 nM), ISRIB (200 nM), or trazodone (TRZ) (50 μM) for 24 hours. The wound was made with a p200 pipette tip (0 hours), and migration into the wound was analyzed by microscopy after 48 hours. Dashed lines represent the area of the initial wound, and the scale bar represents 500 μM. Images are representative from three independent experiments. (D) Quantification of cell migration into the wounded area from (C) using ImageJ. All values are displayed relative to the migration of the untreated sample. Error bars represent means ± SD (n = 3 independent experiments); *P ≤ 0.05 and **P ≤ 0.01, by unpaired Student’s t test. (E) Quantification of cell death using annexin V–FITC and Draq7 staining of MCF10A p53+/+ cells treated with a combination of doxorubicin (500 nM), ISRIB (200 nM), or trazodone (50 μM) for 72 hours. Error bars represent means ± SD (n = 3 independent experiments); ***P ≤ 0.001 and *P ≤ 0.05, by unpaired Student’s t test. (F) Quantification of cell cycle state using FxCycle staining in MCF10A p53+/+ cells treated with a combination of doxorubicin (500 nM), ISRIB (200 nM), or trazodone (50 μM) for 72 hours. Error bars represent means ± SD (n = 3 independent experiments). (G) Cell migration profiles from the xCELLigence RTCA DP instrument. MCF10A cells were treated for 24 hours with doxorubicin (500 nM) or trazodone (50 μM) before seeding in the upper well of the migration CIM-16 plate, and migration was monitored for 48 hours. Medium containing only 0.1% horse serum (HS) was used as a negative control. Data values were an average of three independent experiments, and error bars represent means ± SD. (H) Quantification of cell migration relative to the untreated sample from (G) at 48 hours. Error bars represent means ± SD (n = 3 independent experiments); **P ≤ 0.01 and ***P ≤ 0.001, by unpaired Student’s t test. (I) Cell migration profiles from the xCELLigence RTCA DP instrument. A549 cells were treated for 24 hours with doxorubicin (500 nM) or trazodone (50 μM) before seeding in the upper well of the migration CIM-16 plate, and migration was monitored for 48 hours. Medium containing only 0.1% FBS was used as a negative control. Data values were an average of three independent experiments, and error bars represent means ± SD. (J) Quantification of cell migration relative to the untreated sample from (I) at 48 hours. Error bars represent means ± SD (n = 3 independent experiments); *P ≤ 0.05 and ***P ≤ 0.001, by unpaired Student’s t test.

  • Fig. 6 A model of doxorubicin-induced mTOR-eIF2K cross-talk signaling.

    Doxorubicin-induced DNA damage leads to the activation of p53 and subsequent inhibition of mTOR through TSC-independent mechanisms, most likely resulting in the inhibition of global protein synthesis. Inhibition of mTOR activity directly promotes the activation of both GCN2 and PERK, possibly in a PP6-dependent manner, to enhance the phosphorylation of eIF2α and decrease ternary complex availability. Moreover, cross-talk signaling–mediating eIF2α phosphorylation is required for cell migration. Our data suggest that in a tumor setting, this signaling pathway could regulate cell adaptation to promote invasion and metastasis.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/612/eaaw6763/DC1

    Fig. S1. Doxorubicin inhibited protein synthesis and induced cell death in MCF10A cells.

    Fig. S2. Doxorubicin-induced mTORC1 inhibition was not dependent on TSC activity.

    Fig. S3. Doxorubicin inhibits mTORC1 signaling.

    Fig. S4. Knockdown of PP6 reduced protein synthesis rates.

    Fig. S5. eIF2α-mediated cell migration was not caused by changes in cell death or cell cycle regulation.

  • This PDF file includes:

    • Fig. S1. Doxorubicin inhibited protein synthesis and induced cell death in MCF10A cells.
    • Fig. S2. Doxorubicin-induced mTORC1 inhibition was not dependent on TSC activity.
    • Fig. S3. Doxorubicin inhibits mTORC1 signaling.
    • Fig. S4. Knockdown of PP6 reduced protein synthesis rates.
    • Fig. S5. eIF2α-mediated cell migration was not caused by changes in cell death or cell cycle regulation.

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