Research ArticleCancer

Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy

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Science Translational Medicine  25 Jul 2018:
Vol. 10, Issue 451, eaat3504
DOI: 10.1126/scitranslmed.aat3504
  • Fig. 1 An in vitro siRNA screen for carboplatin synthetic lethality in lung adenocarcinoma cells.

    (A) Carboplatin median inhibitory concentration (IC50) values in lung cancer cell lines, classified as platinum-resistant or sensitive in vitro. n = 4, mean ± SEM. Dotted line indicates peak plasma concentration for carboplatin in humans. (B) Viability of A549 and LX22CL cells treated with a range of carboplatin concentrations in vitro. n = 4, mean ± SEM. Red dashed line indicates peak plasma concentrations for carboplatin in humans; black dashed line, concentration chosen for screen. (C) γH2AX immunofluorescence images in A549 cells treated with carboplatin for 24 hours, counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. (D) Overview of the synthetic lethal screen methodology. (E) Results of a whole-genome siRNA screen for carboplatin synthetic lethality for each gene in order of sensitization index. The top 5% of hits are shown in red. Potentially targetable genes are listed. (F) Secondary screening of the top 230 hits, depicted as a sensitization index heat map. Deconvolution of the primary screen was performed using individual siRNAs (1 to 4) and pooled siRNAs targeting the same genes in HEK293 cells. (G) Western blot analysis of knockdown efficiency of ACVR1B, FKBP3, and MCL1 siRNAs orthogonal to the siRNAs used in the screen. (H) Viability of A549 cells treated with nontargeting (NT) or specific siRNAs in combination with a range of carboplatin concentrations in vitro. IC50 carboplatin concentrations are shown. n = 4, mean ± SEM.

  • Fig. 2 Network analysis of the top 5% of hits from the primary screen in A549 cells.

    Each node represents an enriched pathway, and the size of each node reflects the statistical significance of the term. The thickness of the interconnecting lines reflects the number of interactions between each term. Colors represent broader categories, where all nodes that share the same color represent interrelated terms. IFN, interferon; TCR, T cell receptor; GM-CSF, granulocyte-macrophage colony-stimulating factor; JNK, c-Jun N-terminal kinase; TRAF1, TNF receptor–associated factor 1; NFκB, nuclear factor κB; cGMP, guanosine 3′,5′-monophosphate; DCC, dicyclohexylcarbodiimide; LDL, low-density lipoprotein; GPCR, G protein–coupled receptor; RIG-I, retinoic acid–inducible gene-1; MDA5, melanoma differentiation–associated gene 5; ISG15, interferon-stimulated protein, 15 KDa; FGFR, fibroblast growth factor receptor; CaM, calmodulin; IRS, insulin receptor substrate; ATM, ataxia telangiectasia–mutated.

  • Fig. 3 Activin A and GDF11 signaling and platinum resistance in A549 cells in vitro.

    (A) Flowchart depicting the sensitization index (change in viability comparing carboplatin alone, siRNA alone, and carboplatin + siRNA) of genes in the activin/TGFβ pathway from the primary screen. ROS, reactive oxygen species. (B) Western blot analysis for phosphorylated TAK1 (p-TAK1), activin A (precursor, 55 kDa; active fragment, 25 kDa), GDF8/11, TGFβ (precursor, 67 kDa; active fragment, 12 kDa), ACVR1B, TGFBR1, phospho-SMAD2/3 (p-SMAD2/3), SMAD2/3, and actin in A549 cells treated with carboplatin (25 μg/ml) for 0 to 48 hours (hr). Positive controls for p-SMAD2/3 and SMAD2/3 are shown in fig. S4A. (C) Quantitative analysis of the data shown in Fig. 3B. n = 4, mean ± SEM. **P < 0.01, *P < 0.05, one-way analysis of variance (ANOVA)/Bonferroni correction. (D) Viability of A549 cells after treatment with vehicle (DMSO) or the TAK1 inhibitor LLZ-1640-2 (10 μM) in combination with a range of carboplatin concentrations. Carboplatin IC50 concentrations are shown. n = 4, mean ± SEM. (E) Western blot analysis of A549 cells treated with vehicle (DMSO) or LLZ-1640-2 (10 μM), in combination with carboplatin (Carbo) at 25 mg/ml for 0 to 48 hours. Blots were probed with antibodies specific for phosphorylated TAK1 (p-TAK1, T184/187), TAK1, phosphorylated MKK4 (p-MKK4, S257/T261), MKK4, phosphorylated P38 (p-P38, T180/Y182), P38, phosphorylated MCL1 (p-MCL1, T163), MCL1, and actin. (F) Orthogonal siRNA validation of TAK1. Top: Western blot analysis of TAK1 expression in A549 cells treated with a nontargeting (NT) siRNA, siRNA directed at MAP3K7 (the gene coding for TAK1), orthogonal to the siRNA used in the screen, the gene encoding TAK1. Bottom: Viability of A549 cells after treatment with a NT siRNA or orthogonal siRNA directed at MAP3K7, in combination with a range of carboplatin concentrations in vitro. Carboplatin IC50 concentrations for each experimental group are shown. n = 4, mean ± SEM. (G) CRISPR/Cas9 knockout of TAK1. Top: Western blot analysis of TAK1 expression in control cells and in cells carrying a MAP3K7 mutation induced by CRISPR/Cas9. Bottom: Viability of A549 parental cells and cells carrying a MAP3K7 mutation induced by CRISPR/Cas9. n = 4, mean ± SEM. Carboplatin IC50 concentrations for each group are shown. WT, wild type.

  • Fig. 4 Activation of TAK1 by ACVR1B and TGFBR1 after treatment with carboplatin in vitro.

    (A) Viability of A549, NCI-H358, NCI-H727, and NCI-H2009 lung adenocarcinoma cells after treatment with vehicle (DMSO) or the ACVR1B/TGFBR1 inhibitor SB-505124 (5 μM) in combination with a range of carboplatin concentrations. Carboplatin IC50 concentrations for each experimental group are shown. n = 4, mean ± SEM. Genotypes and treatment histories are listed. Del, homozygous deletion; Ins, in-frame insertion. (B) Western blot analysis of phospho-TAK1 (p-TAK1), TAK1, phospho-SMAD2/3 (p-SMAD2/3), SMAD2/3, MCL1, activin A, GDF8/11, and actin expression in A549, NCI-H358, NCI-H727, and NCI-H2009 cells treated with vehicle (DMSO) or SB-505124 (5 μM) in combination with carboplatin at 25 mg/ml for 0 to 48 hours. Positive controls for p-SMAD2/3 and SMAD2/3 are shown in fig. S4.

  • Fig. 5 Expression of TGFβ superfamily components in lung adenocarcinoma.

    (A) Heat map depicting expression of genes belonging to the TGFβ superfamily in the TCGA lung adenocarcinoma data set. (B) Association between GDF11, TGFB1, INHBA, INHBB, ACVR1B, and TGFBR1 expression and overall survival in the KMPlot lung adenocarcinoma data set. (C) Activin A and GDF8/11 expression detected by immunohistochemistry (IHC) in stage IV lung adenocarcinoma. Immunoperoxidase signal is shown in brown, counterstained with hematoxylin. Scale bars, 50 μm. (D) Kaplan-Meier analysis of progression-free survival (PFS) in a cohort of patients with stage IV lung adenocarcinoma (n = 59) treated with platinum-based chemotherapy. Survival outcomes are shown with respect to quantitative immunohistochemical analysis of activin A and GDF8/11 staining. Statistical significance was calculated using log-rank analysis. CI, confidence interval; HR, hazard ratio.

  • Fig. 6 Inhibition of ACVR1B/TGFR1 signaling in vivo.

    (A) ACVR1B and TGFBR1 expression detected by immunohistochemistry in A549 xenografts. Immunoperoxidase signal is shown in brown, counterstained with hematoxylin. Scale bars, 100 μm. (B) Schema describing an experiment to determine the effect of combination carboplatin and SB-505124 treatment on the growth of A549 flank xenograft tumors. (C) Growth of A549 xenografts in mice treated with vehicle control, SB-505124, and/or Carbo. Data are presented as the growth of tumors in independent mice. (D) Kaplan-Meier analysis of survival to ethical endpoint of mice from the same experiment. n for each treatment group is shown. ***P < 0.001 compared to mice treated with vehicle + cisplatin (CisPt), log-rank test. (E) Schema describing an experiment to determine the effect of treatment with SB-505124 on acute kidney injury induced by CisPt. (F) Serum urea and creatinine concentrations in mice treated with vehicle, SB-505124, and/or CisPt. Blood samples were taken on day 5. n = 7, mean ± SEM. **P < 0.01, ***P < 0.001, one-way ANOVA with Bonferroni correction. (G) Top: Macroscopic appearance of kidneys from mice from the experiment in (E) and (F). Scale bar, 2 mm. Bottom: Photomicrographs of periodic acid-Schiff (PAS)–stained sections of whole kidneys. Scale bar, 2 mm.

  • Fig. 7 Inhibition of activin/GDF11 signaling in vivo.

    (A) Activin A and GDF8/11 expression detected by immunohistochemistry in A549, NCI-H358, NCI-H727, and PDX1.1 xenografts, and LLC allografts. Immunoperoxidase signal is shown in brown, counterstained with hematoxylin. Scale bars, 100 μm. (B) Quantification of the data shown in (A). n = 4, mean ± SEM. (C) Schema describing an experiment to determine the effect of combination carboplatin and FST treatment on the growth of A549 flank xenograft tumors. (D) Growth of A549 flank xenografts in nude mice treated with vehicle control, FST, and/or Carbo. Data are presented as the growth of tumors in independent mice. (E) Kaplan-Meier analysis of survival to ethical endpoint of mice from the same experiment, n for each experimental group is shown. #P < 0.0001 compared to mice treated with vehicle + cisplatin (Pt), log-rank test. (F) Kaplan-Meier analysis of survival to ethical endpoint of nude mice bearing NCI-H358, NCI-H727, PDX1.1, and LLC flank tumors treated with phosphate-buffered saline (PBS; control), FST, and/or Carbo as shown in (C). n for each group is shown. ***P < 0.001, *P < 0.05 compared to mice treated with vehicle + Pt, log-rank test. (G) Schema describing an experiment to determine the effect of treatment with FST on acute kidney injury induced by CisPt. (H) Serum urea and creatinine concentrations in mice treated with PBS, FST, and/or CisPt. Blood samples were taken on day 5. n = 7, mean ± SEM. **P < 0.01, one-way ANOVA with Bonferroni correction. (I) Top: Macroscopic appearance of kidneys from mice from the experiment in (G) and (H). Scale bar, 2 mm. Bottom: Photomicrographs of PAS-stained sections of whole kidneys from the same experiment. Scale bar, 2 mm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/451/eaat3504/DC1

    Fig. S1. Controls used in the siRNA screen.

    Fig. S2. Controls for SMAD2/3 expression and carboplatin responses in HEK293 cells in vitro.

    Fig. S3. Positive controls for SMAD2/3 expression in vitro.

    Fig. S4. Responses of lung adenocarcinoma cell lines to chemotherapy agents with TGFβ signaling pathway activation in vitro.

    Fig. S5. Evaluation of activin A and GDF8/11 expression in lung adenocarcinoma.

    Fig. S6. Effect of SB-505124 on the platinum response in vivo.

    Fig. S7. Effect of FST on the platinum response in vivo.

    Fig. S8. Mouse weights in xenograft/allograft experiments.

    Fig. S9. Examples of full-length Western blots with image cropping.

    Table S1. Complete data set from the primary siRNA screen (provided as an Excel file).

    Table S2. High-confidence hits targetable with existing small or large molecules.

    Table S3. Characteristics of the patient cohort used to generate immunohistochemical data.

    Table S4. Details of reagents used (provided as an Excel file).

  • The PDF file includes:

    • Fig. S1. Controls used in the siRNA screen.
    • Fig. S2. Controls for SMAD2/3 expression and carboplatin responses in HEK293 cells in vitro.
    • Fig. S3. Positive controls for SMAD2/3 expression in vitro.
    • Fig. S4. Responses of lung adenocarcinoma cell lines to chemotherapy agents with TGFβ signaling pathway activation in vitro.
    • Fig. S5. Evaluation of activin A and GDF8/11 expression in lung adenocarcinoma.
    • Fig. S6. Effect of SB-505124 on the platinum response in vivo.
    • Fig. S7. Effect of FST on the platinum response in vivo.
    • Fig. S8. Mouse weights in xenograft/allograft experiments.
    • Fig. S9. Examples of full-length Western blots with image cropping.
    • Table S2. High-confidence hits targetable with existing small or large molecules.
    • Table S3. Characteristics of the patient cohort used to generate immunohistochemical data.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Complete data set from the primary siRNA screen (provided as an Excel file).
    • Table S4. Details of reagents used (provided as an Excel file).

    [Download Tables S1 and S4]

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