Research ArticlePharmacology

ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases

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Science Signaling  16 Feb 2016:
Vol. 9, Issue 415, pp. ra18
DOI: 10.1126/scisignal.aac4374
  • Fig. 1 ONC201’s broad-spectrum activity can be attributed to its antiproliferative and proapoptotic effects.

    (A) The EC50 of ONC201 was assessed by CellTiter-Glo (CTG) assay in 23 cell lines treated with ONC201 for 72 hours. (B) Apoptosis measured by sub-G1 analysis in cells treated with ONC201 for 72 hours. (C) Cell cycle profile analyses in HCT116 and A549 cells treated with ONC201 for 24 hours (H). Data are representative of two biological replicates. PI, propidium iodide. (D) BrdU incorporation assays in HCT116 and A549 cells treated with ONC201 for 48 hours. V, vehicle; O, ONC201. (E) Viable cell counts assessed by trypan blue exclusion in cells treated with ONC201 for up to 72 hours. *P < 0.05, Student’s t test with Holm-Sidak correction. (F) Proliferative capacity of cells after 72 hours in the presence of ONC201, assessed by clonogenic assays. Bars that are not visible indicate a proliferative fraction of 0. The concentrations of ONC201 used for each cell line are listed in table S1. Data in (A), (B), (D), (E), and (F) are means ± SEM from three biological replicates.

  • Fig. 2 ONC201 activates the ISR.

    (A) Western blotting analysis for ATF4, CHOP, ATF3, and TRB3 on lysates from HCT116 cells cultured with ONC201 (0 to 10 μM) for 3 to 24 hours. BFA (an ER stressor) served as a positive control. Blots are representative of two experiments. (B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) for the expression of CHOP and DR5 relative to GAPDH in HCT116 cells treated with ONC201 (10 μM) or vehicle for 24 or 48 hours. (C) Immunohistochemical analysis for CHOP in HCT116-derived subcutaneous xenografts from mice that received either vehicle (−) or ONC201 (+; 25 mg/kg). Results are representative of tissues from two vehicle-treated mice and six mice intraperitoneally or intravenously injected with ONC201 (three mice each). Scale bars, 10 μm. (D) Western blotting analysis for CHOP in lysates from colorectal cancer stem cell–like cell-derived xenografts (45) in athymic nude mice extracted 72 hours after treatment [vehicle or ONC201 (50 mg/kg, intraperitoneally)]. Tumor lysates from untreated mice (−) served as controls. (E) Densitometric analyses of the bands in the Western blots in (D). (F) qRT-PCR for the fold change in expression of CHOP and DR5 relative to GAPDH in different cell types cultured with ONC201 (compared to vehicle) for 72 hours. Data in (B), (E), and (F) are means ± SEM of three biological replicates. *P < 0.05, Student’s t test with Holm-Sidak correction, comparing ONC201-treated versus vehicle-treated cells.

  • Fig. 3 ATF4 is required for ONC201-induced cytotoxicity.

    (A) Western blotting analysis for the indicated proteins in wild-type (WT) RKO (RKOp) and ONC201-resistant RKO (RKOr1) cells treated with 10 μM BFA (an ER stressor, positive control) or 5 μM ONC201. (B) qRT-PCR for the fold change in expression of CHOP relative to GAPDH in proteins in WT RKO and ONC201-resistant RKO cells treated with ONC201 (5 μM, 24 to 48 hours) compared with the 0 time point. (C) Viability of HCT116 cells assessed by trypan blue exclusion assay after ATF4 and CHOP knockdown and treatment with vehicle or ONC201 (10 μM, 24 and 48 hours). Scr, scrambled. (D) Western blotting analysis for ATF4, CHOP, and uncleaved (unclvd) and cleaved (clvd) PARP in HCT116 cells transfected with ATF4 or CHOP siRNA. (E) Quantitative PCR of DR5 expression relative to GAPDH in HCT116 cells transfected with ATF4 or CHOP siRNA and subsequently treated with vehicle or ONC201 (10 μM, 48 hours). (F) Western blotting for the indicated proteins in HCT116 cells transfected with ATF4 or TRB3 siRNA and subsequently treated with ONC201 (10 μM, 72 hours for the left set and 48 hours for the right set). Blots in (A), (D), and (F) are representative of two experiments. Data in (B), (C), and (E) are means ± SEM of three biological replicates. *P < 0.05, Student’s t test with Holm-Sidak correction.

  • Fig. 4 ONC201 induces a decrease in cyclin D1 abundance.

    Western blotting analyses for the indicated proteins were performed in HCT116 or A549 cells treated with ONC201 (10 or 5 μM, respectively) for up to 48 hours. Blots are representative of two experiments.

  • Fig. 5 ONC201 activates the ATF4 pathway through the eIF2α kinases HRI and PKR.

    (A) Western blotting analysis for phosphorylated and total eIF2α was performed in the indicated cell types treated with tunicamycin (Tun) or ONC201. (B to D) Western blotting analysis for the indicated proteins in lysates from HCT116 cells that were transfected with siRNA against one (B) or two eIF2α kinases (C) as indicated for 24 hours, and subsequently treated with ONC201 (10 μM) for 12 hours. (D) Western blotting analysis for indicated proteins in lysates from HCT116 cells that were transfected with plasmid constructs for WT and a nonphosphorylatable eIF2α mutant (S51A) for 48 hours and subsequently treated with ONC201 for 12 hours. (E) Western blotting analysis for cyclin D1 in HCT116 cells transfected with the indicated siRNA(s) and treated with ONC201 for 24 hours. Blots in (A) to (E) are representative of at least two independent experiments.

  • Fig. 6 The effect of ONC201 on XIAP amount correlates with fate of cells treated with ONC201.

    Western blotting analysis for XIAP in cells treated with the indicated concentrations of ONC201 for 72 hours. Blots are representative of at least two independent experiments.

  • Table 1 ONC201 induces the expression of mRNAs encoding aminoacyl tRNA synthetases.

    Fold changes in expression of mRNAs encoding aminoacyl tRNA synthetases in HCT116 and RKO cells in response to 18 and 48 hours treatment of ONC201, as determined by microarray analyses (data files S1 to S7).

    Aminoacyl tRNA synthetaseFold change versus
    untreated
    18 hours of ONC201 incubation
    HCT116
    Cysteinyl-tRNA synthetase (CARS)3.4
    CARS2.5
    Seryl-tRNA synthetase (SARS)2.4
    Alanyl-tRNA synthetase (AARS)2.2
    Glycyl-tRNA synthetase (GARS)2.2
    Tryptophanyl-tRNA synthetase (WARS)2
    Tyrosyl-tRNA synthetase (YARS)2
    Leucyl-tRNA synthetase (LARS)1.8
    Isoleucyl-tRNA synthetase (IARS)1.7
    48 hours of ONC201 incubation
    HCT116
    GARS2.2
    RKO
    SARS1.9
    WARS1.8
    WARS1.6

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/415/ra18/DC1

    Fig. S1. The broad-spectrum anticancer activity of ONC201 can be attributed to its antiproliferative and proapoptotic effects.

    Fig. S2. ONC201 activates ATF4 and CHOP.

    Fig. S3. The differential response of ONC201-sensitive and ONC201-resistant lines to ER stress inducers is correlated with ATF4 pathway activation.

    Fig. S4. ONC201 induces a decrease in cyclin D1 abundance but not in Akt and ERK phosphorylation.

    Fig. S5. ONC201 does not activate the eIF2α-ATF4 pathway by inhibiting the eIF2α phosphatase or by activating GCN2 or PERK.

    Fig. S6. The abundance of cIAP-2 and Bcl-2 family members is similarly altered regardless of whether cells undergo apoptosis.

    Fig. S7. Altering the amount of XIAP does not change ONC201’s effects on PARP cleavage.

    Table S1. ONC201 concentrations used according to cell type.

    Table S2. Top gene expression changes induced by ONC201 in HCT116 cells.

    Table S3. Top gene expression changes induced by ONC201 in RKO cells.

    Data file S1. Genes in HCT116 cells with altered expression after 18 hours of ONC201 treatment.

    Data file S2. Genes in HCT116 cells with altered expression after 48 hours of ONC201 treatment.

    Data file S3. Additional information on the genes in data file S2.

    Data file S4. Genes in HCT116 cells with altered expression after 18 or 48 hours of ONC201 treatment.

    Data file S5. Genes in HCT116 cells with altered expression after 18 hours but not 48 hours of ONC201 treatment.

    Data file S6. Genes in parental RKO cells with altered expression after 48 hours of ONC201 treatment.

    Data file S7. Genes in ONC201-resistant RKO cells with altered expression after 48 hours of ONC201 treatment.

  • Supplementary Materials for:

    ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases

    C. Leah B. Kline, A. Pieter J. Van den Heuvel, Joshua E. Allen, Varun V. Prabhu, David T. Dicker, Wafik S. El-Deiry*

    *Corresponding author. E-mail: wafik.eldeiry{at}fccc.edu

    This PDF file includes:

    • Fig. S1. The broad-spectrum anticancer activity of ONC201 can be attributed to its antiproliferative and proapoptotic effects.
    • Fig. S2. ONC201 activates ATF4 and CHOP.
    • Fig. S3. The differential response of ONC201-sensitive and ONC201-resistant lines to ER stress inducers is correlated with ATF4 pathway activation.
    • Fig. S4. ONC201 induces a decrease in cyclin D1 abundance but not in Akt and ERK phosphorylation.
    • Fig. S5. ONC201 does not activate the eIF2α-ATF4 pathway by inhibiting the eIF2α phosphatase or by activating GCN2 or PERK.
    • Fig. S6. The abundance of cIAP-2 and Bcl-2 family members is similarly altered regardless of whether cells undergo apoptosis.
    • Fig. S7. Altering the amount of XIAP does not change ONC201's effects on PARP cleavage.
    • Table S1. ONC201 concentrations used according to cell type.
    • Table S2. Top gene expression changes induced by ONC201 in HCT116 cells.
    • Table S3. Top gene expression changes induced by ONC201 in RKO cells.
    • Legends for data file S1 to S7

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.77 MB

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Genes in HCT116 cells with altered expression after 18 hours of ONC201 treatment.
    • Data file S2 (Microsoft Excel format). Genes in HCT116 cells with altered expression after 48 hours of ONC201 treatment.
    • Data file S3 (Microsoft Excel format). Additional information on the genes in data file S2.
    • Data file S4 (Microsoft Excel format). Genes in HCT116 cells with altered expression after 18 or 48 hours of ONC201 treatment.
    • Data file S5 (Microsoft Excel format). Genes in HCT116 cells with altered expression after 18 hours but not 48 hours of ONC201 treatment.
    • Data file S6 (Microsoft Excel format). Genes in parental RKO cells with altered expression after 48 hours of ONC201 treatment.
    • Data file S7 (Microsoft Excel format). Genes in ONC201-resistant RKO cells with altered expression after 48 hours of ONC201 treatment.

    [Download Data files S1 to S7]


    Citation: C. L. B. Kline, A. P. J. Van den Heuvel, J. E. Allen, V. V. Prabhu, D. T. Dicker, W. S. El-Deiry, ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases. Sci. Signal. 9, ra18 (2016).

    © 2016 American Association for the Advancement of Science

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