Research ArticleCancer

Afatinib restrains K-RAS–driven lung tumorigenesis

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Science Translational Medicine  20 Jun 2018:
Vol. 10, Issue 446, eaao2301
DOI: 10.1126/scitranslmed.aao2301
  • Fig. 1 K-RAS–mutated lung ACs display increased EGFR activity.

    (A) Heat map for mRNA expression in K-RAS–mutated tumor biopsies (T1 to T35) and adjacent nonmalignant, healthy lung parenchyma (N1 to N35) of the same patients. Displayed are the top 50 differentially regulated genes within the Gene Ontology (GO) ERBB signaling pathway (GO: 0038127). Hierarchical clustering was performed using heatmapper.ca online tool. (B) GSEA for GO and KEGG ERBB pathway signatures in K-RAS–mutant tumors versus nonmalignant tissue and (C) in K-RAS–mutated tumors of stage II and higher versus stage I. FDR, false discovery rate; FWER, familywise-error rate. (D) Relative mRNA expression of indicated genes in healthy lung tissue and K-RAS tumors. n = 35 per group, data shown as means ± SD. Data in (A) to (D) were retrieved from the Gene Expression Omnibus (GSE75037). A.U., arbitrary units. (E) Images of representative immunohistochemical staining for indicated EGFR phosphorylation sites in human nonmalignant lung parenchyma and K-RAS–mutated lung AC. Boxplot (min to max) of scoring values shows EGFR phosphorylation specifically in tumor cells versus healthy tissue. n ≥ 30 per group. (F) Relative mRNA expression in WT (K-ras+/+, n = 5 to 6) and tumor-bearing mouse lungs (K-rasG12D/+, n = 6) at 10 weeks after tumor induction via Ad.Cre treatment. Actb was used for normalization. Data are presented as means ± SD. (D to F) *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 Genetic EGFR ablation in K-RAS–mutated lung AC reduces tumor growth.

    (A) Kaplan-Meier analysis of K (K-rasG12D, n = 24) and KE (K-rasG12D:EgfrΔLep/ΔLep, n = 28) mice and (B) of KP (K-rasG12D:p53ΔLep/ΔLep, n = 20) and KPE (K-rasG12D:p53ΔLep/ΔLep:EgfrΔLep/ΔLep, n = 27) mice after intranasal infection with Ad.Cre. (C) Survival analysis of immunocompetent recipient mice after orthotopic transplantation of syngeneic K-rasG12D–mutated and p53-deficient KP cells, with and without Egfr deletion (n = 7 per group). (A to C) The median survival times of the groups are indicated. Differences in survival of groups were tested for significance using the log-rank test, and respective P values are shown. (D) Representative images of hematoxylin and eosin (H&E) staining, including higher magnification of the indicated areas (bottom) of tumor-bearing lungs 10 weeks after Ad.Cre inhalation of mice with specified genotypes. For quantitation, the mean values of two sections per mouse were used. Graphs represent means of ratios ± SD of tumor area versus healthy lung area and mean tumor numbers ± SD per section (n = 13 mice for K-rasG12D and n = 14 mice for K-rasG12D:EgfrΔLep/ΔLep). Scale bars, 1 mm (top) and 250 μm (bottom). (E) Representative images of immunohistochemical staining of mouse lungs 10 weeks after tumor induction using antibodies specific for Ki67 and pERK. Tumor cell intrinsic expression of the respective proteins in at least five individual tumors per mouse was evaluated using TissueGnostics software. Graphs represent means ± SD of Ki67- and pERK-positive tumor cells normalized to all tumor cells (n = 5 to 7 mice per group). Scale bars, 50 μm. (F) Cell count of p53-deficient versus p53/EGFR double-knockout A549 cells after in vitro cultivation. Graph represents means ± SD of three individual clones per group. (G) Mean volumes ± SD of xenografted tumors comparing EGFR-expressing versus EGFR-deficient p53 knockout A549 cells, monitored over the course of the experiment. The graph in the middle presents the endpoint tumor weight ± SD (n = 6 per group). The image on the right shows the tumors at the end of the experiment. Scale bar, 2 cm. (D to G) ***P < 0.001.

  • Fig. 3 Inhibition of EGFR signaling down-regulates activity of mutant K-RAS.

    (A) Heat map of top 100 up-regulated genes in K-rasG12D versus WT AT2 cells and hierarchical clustering of WT (wt_0-2), K-rasG12D (Khet_0 – 2), and K-rasG12D:EgfrΔ/Δ (KhetEko_0-2) mouse pneumocytes. (B) GSEA of K-rasG12D (K) versus K-rasG12D:EgfrΔ/Δ (KE) mouse pneumocytes for the indicated gene sets. (C) Representative image of antibody array of cell lysates of A549 and A549ΔEGFR cells (n = 2 clones per group with two spots each). Antibody probes are decoded at the bottom panel, black color indicates proteins that were not detected, red color marks proteins that were down-regulated, and blue color highlights proteins that were not differentially expressed in the A549ΔEGFR clones. (D) Densitometric quantitation of microarray films (n = 2 clones per group). (E) Western blot (WB) probing for RAS after GST-RAF1-RBD–mediated pulldown in A549 and A549ΔEGFR cell lysates and respective input samples (n = 3 per group). IP, immunoprecipitation.

  • Fig. 4 Afatinib reduces growth of K-RAS–mutant lung AC in vivo.

    (A) Graphs display tumor volumes of (xeno-)grafts using indicated cell lines monitored over the experimental period and tumor weights at the end of the experiment. Mice were treated with vehicle alone or afatinib at 5 mg/kg body weight via oral gavage, five times per week, and the start of treatment is indicated. Means ± SD are shown. n = 4 per group in the A549 experiment and n ≥ 5 per group in A427 and 368T1 experiments. (B) Representative images of Ki67 and cleaved caspase-3 staining of 368T1 cell line–derived grafts upon vehicle and afatinib treatment. Scale bars, 100 μm. (C) Quantitation of positive cells in (B) (n = 5). (D) Mean tumor volumes ± SD of PDXs of lung AC tissue with K-RASG12C mutation. Mice were treated with vehicle, afatinib (15 mg/kg body weight, daily), paclitaxel (15 mg/kg body weight, once per week), or a combination of both treatments (n = 8 per group). (E) Representative Ki67 and cleaved caspase-3 staining for sections of vehicle-treated versus afatinib-treated PDXs. Scale bars, 200 μm. (A, C, and D) **P < 0.01, ***P < 0.001, unpaired two-tailed t test.

  • Fig. 5 Afatinib, but not first-generation EGFR TKIs, inhibits growth of autochthonous K-ras tumors.

    (A) Representative images of H&E-stained lung sections of K-rasG12D/+ mice 10 weeks after Ad.Cre inhalation (left) or 20 weeks after Ad.Cre inhalation, with treatment over the last 10 weeks with vehicle, afatinib, erlotinib, or gefitinib (5 mg/kg body weight, five times per week via oral gavage). Bottom: Magnifications of the indicated sections at the top panel. n ≥ 4 per group. Scale bars, 1 mm (top) and 500 μm (bottom). (B and C) Graphs represent means ± SD of tumor area versus total lung area ratios (B) and mean tumor numbers ± SD per section of lung (C) in mice. Each data point represents the mean value of two sections derived from one mouse. One-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. (D) Representative images of Ki67 staining of lung tumors 20 weeks after Ad.Cre induction and treated for 10 weeks with vehicle or afatinib. Ki67-positive tumor cells in at least three tumors per mouse were quantitated, and plot shows means ± SD of Ki67-positive tumor cells. Student’s t test, n = 4 mice per group. Scale bars, 50 μm. (E) Survival analysis of immunocompetent mice after orthotopic transplantation of syngeneic 368T1 lung AC cells. Three weeks after injection, treatment with vehicle, afatinib, or erlotinib (5 mg/kg body weight, five times per week via oral gavage) was started. Median survival times were 42 days for vehicle group, 49 days for afatinib group, and 44 days for erlotinib group. Log-rank test, n = 5. ns, not significant. (B to E) *P < 0.05, ***P < 0.001.

  • Fig. 6 ERBB family members mediate resistance to EGFR inhibition, which can be blocked by afatinib.

    (A) Representative images of Ki67 and (B) of pERK in lung tumors of indicated mice 20 weeks after Ad.Cre administration. The percentages of tumor cells expressing each protein were quantitated in at least eight individual tumors per mouse. Graphs represent mean percentages ± SD of Ki67- and pERK-positive tumor cells (n = 6 mice per group). Scale bars, 50 μm. (C) mRNA expression of indicated genes in lungs of K-rasG12D:EgfrΔLep/ΔLep mice 10 and 20 weeks after Ad.Cre inhalation. Actb was used as a housekeeper gene control, and relative expression of each gene was normalized to its expression in K-rasG12D mice at the same time points (dotted line). n ≥ 6 per group. (D) Representative photographs of H&E-stained mouse lung sections, 5 weeks after orthotopic transplantation of A549Δp53 cells by tail vein injection and 3 weeks after the start of treatment with vehicle, afatinib, or erlotinib (5 mg/kg body weight, five times per week via oral gavage). Scale bars, 1 mm. (E) Relative mRNA expression ratios of human versus mouse housekeeping genes (ACTB and 28S) from mice treated as in (D). (F) Relative mRNA expression of human variants of the indicated genes normalized to human housekeeping genes (ACTB and 28S). n = 3. (G) Western blot probing for indicated proteins in A549, SK-LU1, and 368T1 cell lysates after treatment with 1 μM afatinib, erlotinib, or gefitinib for 48 hours. (H) Tumor volumes of A549ΔEGFR xenografts in mice receiving vehicle, afatinib, or erlotinib treatment (5 mg/kg body weight, five times per week via oral gavage), starting 14 days after transplantation, monitored over the experimental period. The graph on the right shows tumor weights at the end of the experiment. n ≥ 5. (A to H) *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/446/eaao2301/DC1

    Materials and Methods

    Fig. S1. K-RAS–mutated lung ACs display increased ERBB expression profile.

    Fig. S2. K-RAS–mutated lung ACs exhibit activated EGFR.

    Fig. S3. Genetic EGFR ablation in K-RAS–mutated lung AC reduces tumor growth.

    Fig. S4. Genetic EGFR ablation in K-RAS–mutated lung AC cells reduces tumor growth.

    Fig. S5. Inhibition of EGFR signaling down-regulates mutated K-RAS activity.

    Fig. S6. Afatinib reduces growth of K-RAS–mutant lung AC in vitro.

    Fig. S7. Afatinib reduces K-RAS–mediated tumorigenesis in vivo.

    Fig. S8. ERBB family members mediate resistance to EGFR inhibition, which can be blocked by afatinib.

    Table S1. Alveolar_KRAS_up gene set (provided as an Excel file).

    Table S2. KRAS_NSCLC_up gene set (provided as an Excel file).

    Table S3. Primary data shown in the figures (provided as an Excel file).

    Table S4. List of genotyping primers.

    Table S5. List of primers for quantitative PCR analysis.

    References (4451)

  • Supplementary Material for:

    Afatinib restrains K-RAS–driven lung tumorigenesis

    Herwig P. Moll, Klemens Pranz, Monica Musteanu, Beatrice Grabner, Natascha Hruschka, Julian Mohrherr, Petra Aigner, Patricia Stiedl, Luka Brcic, Viktoria Laszlo, Daniel Schramek, Richard Moriggl, Robert Eferl, Judit Moldvay, Katalin Dezso, Pedro P. Lopez-Casas, Dagmar Stoiber, Manuel Hidalgo, Josef Penninger, Maria Sibilia, Balázs Győrffy, Mariano Barbacid, Balázs Dome, Helmut Popper, Emilio Casanova*

    *Corresponding author. Email: emilio.casanova{at}meduniwien.ac.at

    Published 20 June 2018, Sci. Transl. Med. 10, eaao2301 (2018)
    DOI: 10.1126/scitranslmed.aao2301

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. K-RAS–mutated lung ACs display increased ERBB expression profile.
    • Fig. S2. K-RAS–mutated lung ACs exhibit activated EGFR.
    • Fig. S3. Genetic EGFR ablation in K-RAS–mutated lung AC reduces tumor growth.
    • Fig. S4. Genetic EGFR ablation in K-RAS–mutated lung AC cells reduces tumor growth.
    • Fig. S5. Inhibition of EGFR signaling down-regulates mutated K-RAS activity.
    • Fig. S6. Afatinib reduces growth of K-RAS–mutant lung AC in vitro.
    • Fig. S7. Afatinib reduces K-RAS–mediated tumorigenesis in vivo.
    • Fig. S8. ERBB family members mediate resistance to EGFR inhibition, which can be blocked by afatinib.
    • Table S4. List of genotyping primers.
    • Table S5. List of primers for quantitative PCR analysis.
    • References (4451)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Alveolar_KRAS_up gene set (provided as an Excel file).
    • Table S2. KRAS_NSCLC_up gene set (provided as an Excel file).
    • Table S3. Primary data shown in the figures (provided as an Excel file).

    [Download Tables S1 to S3]

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