Research ArticleCancer

ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism

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Science Translational Medicine  13 Mar 2019:
Vol. 11, Issue 483, eaaq1238
DOI: 10.1126/scitranslmed.aaq1238
  • Fig. 1 Epithelial tumors have greater MAPK signaling dependency for growth.

    (A) Experimental design for FDAome shRNA dropout screens in epithelial (E) (393P) and mesenchymal (M) (344P) murine lung cancer cell lines implanted subcutaneously in vivo (nude mice) and grown in parallel in vitro (20 doublings). (B) Gene rank analysis highlighting the behavior of RAF1 and MAPK1 genes in the FDAome in vivo screens executed in epithelial (393P) and mesenchymal (344P) murine lung cancer cell lines. shRNA dropout score was calculated as the log of the RSA value shown in table S1. (C) Heatmap of RPPA profile showing statistically significant (P < 0.05) differentially regulated proteins in isogenically matched epithelial and mesenchymal human lung cancer cell lines. Cell lines expressed doxycycline (Dox)–inducible miR-200 or ZEB1, as indicated. ZEB1 was constitutively expressed in HCC827 cells, rather than doxycycline inducible. (D) Dot plot of extracellular signal–regulated kinase (ERK) phosphorylation (T202/Y204) from RPPA dataset in a panel of mesenchymal and epithelial murine (epithelial: 393P, 393P vector control, 393P–miR-200, 393LN, 307P, 412P, 713P, 531P1, 531P2, and 344SQ–miR-200; mesenchymal: 531LN1, 531LN2, 531LN3, 344P, 344LN, 344SQ, and 393P-Zeb1) and human lung cancer cells. (E) Cluster plot analysis of Spearman’s rank correlation between EMT score and the activated downstream MAPK signaling molecule p-p90RSK (T359/S363) in KRAS mutant lung and colorectal cancer patient samples from TCGA RPPA datasets. (F) Western blot of the EMT markers ZEB1, E-cadherin, and vimentin and MAPK signaling molecules in a panel of human KRAS mutant lung cancer cell lines. Numbers at the bottom indicate relative quantification of Western blot signal for phosphorylated ERK (p-ERK), relative to β-actin. (G and H) Western blot of indicated proteins in (G) H157 cells after doxycycline-induced miR-200 expression for 7 days and (H) H441 cells after doxycycline-induced ZEB1 expression at the indicated time points. Numbers at the bottom indicate relative quantification of Western blot signal for p-ERK, relative to β-actin.

  • Fig. 2 RAS-RAF-MEK-ERK pathway regulates MAPK signaling in epithelial cancer cells.

    (A) Schematic of RAS-RAF-MEK-ERK MAPK pathway signaling. (B) Top: Western blot of KRAS from RAS–guanosine 5′-triphosphate pulldown with the RBD of RAF1 after miR-200ab induction in mesenchymal H157 lung cancer cell line. Bottom: Western blots of whole-cell lysates of MAPK signaling molecules in H157 cells with induced miR-200 expression. (C) Western blots of MAPK signaling proteins in HCC827 [epidermal growth factor receptor (EGFR) mutant], H1299 [Neuroblastoma RAS (NRAS) mutant], and H2882 (EGFR and RAS WT) human lung cancer cell lines after 96-hour transient expression of miR-200abc. (D) Western blot of ZEB1, E-cadherin, and vimentin and MAPK signaling proteins in epithelial and mesenchymal KRAS mutant pancreatic and colorectal cancer cell lines after 6-day transient expression of miR-200abc. (E) Western blots of MAPK signaling proteins in nontransformed BEAS2B epithelial human lung cell lines expressing mutant KRAS or mutant EGFR in conjunction with transient expression of miR-200abc. (F to H) Western blots of MAPK signaling proteins in H157 cells with induced miR-200ab expression after (F) KRAS knockdown by shRNA, (G) inhibition of pan-RAF enzymatic activity for 24 hours with TAK-632, and (H) 48-hour siRNA-mediated knockdown of RAF isoforms individually or in combination.

  • Fig. 3 ZEB1 regulates MAPK signaling by suppressing the scaffold protein IL17RD.

    (A) Western blot of MAPK signaling molecules in H157 cells ± miR-200 expression after 24 hours of serum-free starvation, followed by stimulation with complete serum medium, serum-free medium, or EGF for 4 hours. (B) qPCR analysis for relative expression of SPRY1, KSR2, 14-3-3σ/SFN, CNK2, SHOC2, and IL17RD in H157 cells with inducible miR-200 expression. *P < 0.05. (C) Cluster plot analysis of Spearman’s rank correlation between IL17RD gene expression and EMT scores of 77 human lung cancer cell lines. (D and E) Western blots of IL17RD in (D) a panel of human epithelial and mesenchymal KRAS mutant lung cancer cell lines and (E) H157 or H441 cells with induced miR-200 or ZEB1 expression, respectively. (F) Western blot of IL17RD and MAPK signaling molecules after transient shRNA knockdown of IL17RD in H157 cells with induced miR-200 expression for 72 hours. (G and H) Western blots of IL17RD and MAPK signaling molecules after 48-hour constitutive expression of IL17RD in (G) H157 and (H) A549 cells. (I) Schematic of human IL17RD promoter region containing predicted ZEB1 binding sites represented by color-coded ellipses. Black arrows indicate location of genomic region used for qPCR amplification, containing potential ZEB1 binding sites in the IL17RD promoter after ZEB1 chromatin immunoprecipitation (ChIP). The IL17RD promoter was cloned 1066 base pairs (bp) upstream of the transcriptional start site and inserted into a luciferase reporter vector. Mutations of potential ZEB1 binding sites indicated with yellow ×. (J) Fold enrichment by qPCR analysis of IL17RD promoter segments containing potential ZEB1 binding sites after endogenous ZEB1 ChIP in H157 cells with inducible vector control or miR-200 expression, using ZEB1 antibody or immunoglobulin G (IgG) control antibody. Promoter regions analyzed by qPCR labeled with black arrows in (I). (K) Relative luciferase activity of IL17RD promoter reporter constructs in (I) transfected into epithelial H441 cells with induced green fluorescent protein (GFP) control or ZEB1 expression. Relative luciferin signal was normalized to promoter-less vector control signal.

  • Fig. 4 Lung cancer cells and tumors with high ZEB1 are resistant to MEK inhibition.

    (A and B) In vitro cell survival response after 72-hour selumetinib (AZD6244) treatment in a panel of epithelial and mesenchymal (A) human and (B) murine RAS mutant lung cancer cell lines. (C) Hematoxylin and eosin (H&E), IL17RD, p-ERK, ZEB1, E-cadherin, and vimentin IHC stains of primary tumor tissues generated by subcutaneous injection of epithelial 393P and mesenchymal 344SQ murine lung cancer cell lines in syngeneic WT mice (n = 8 tumors per group). Scale bars, 100 μm. Inset scale bars, 20 μm. (D) Left: In vivo volume measurements at the indicated time points for 393P and 344SQ subcutaneous tumors in syngeneic WT mice after daily treatment with AZD6244 (25 mg/kg) MEK inhibitor or vehicle control. Treatment start time denoted by a green arrow, end point denoted by a red arrow, and resistant tumors denoted by a purple arrow. Treatment performed in experimental duplicate with four to five mice per replicate in each treatment group. Sample size is as indicated in the middle graph. Tumor volume data plotted as means and SD. Purple data points represent 393P tumors that were initially responsive to MEK inhibition and developed resistance over time. Middle: Tumor volume measurements at week 7 (at the end point, after 4 weeks of AZD6244 treatment). Right: Quantification of lung metastatic surface nodules in the indicated experimental groups at week 7 of the experiment or at week 12 in 393P tumors that developed resistance to AZD6244 (393P AZD-R). *P < 0.05, **P < 0.01. (E) Stains for the indicated markers in 393P tumors in mice that received vehicle or AZD6244 until week 7 of the experiment in (D) (top and middle) or in tumors that developed 393P AZD-R at week 12 of the experiment in (D) (bottom). Scale bars, 50 μm. Inset scale bars, 20 μm. Images are representative of n = 5 tissues per group.

  • Fig. 5 MiR-200 or IL17RD sensitizes mesenchymal lung tumors to MEK inhibition.

    (A to C) In vitro cell survival response after 72 hours of AZD6244 treatment in (A) mesenchymal human H157 (top) and murine 344SQ (bottom) cells with or without inducible miR-200 expression. *P < 0.05, **P < 0.01. IC50, median inhibitory concentration. (B) Epithelial human H441 (top) and murine 393P (bottom) cells with or without inducible ZEB1 expression. *P < 0.05, **P < 0.01. (C) Mesenchymal human H157 (top) and murine 344SQ (bottom) cells with stable vector control or IL17RD expression. *P < 0.05, **P < 0.01. (D) Left: In vivo subcutaneous tumor volume measurements at the indicated time points for 344SQ tumors ± doxycycline-inducible miR-200 expression, treated daily with AZD6244 or vehicle control. Starting time of treatment denoted by red arrow. Treatment performed in experimental duplicate with three to five mice per replicate, and total sample size is denoted in the middle graph. Tumor volume data plotted as means and SD. Middle: Final tumor volume measurements at week 9 of experiment (5 weeks of treatment). Right: Quantification of lung metastatic surface nodules in the indicated experimental groups at week 9 of experiment. *P < 0.05, **P < 0.01. (E) Left: In vivo subcutaneous tumor volume measurements at the indicated time points for 344SQ tumors ± IL17RD expression, treated daily with AZD6244 or vehicle control. Starting time of treatment is denoted by a red arrow; n = 4 to 5 mice per treatment group. Tumor volume data plotted as means and SD. *P < 0.05, **P < 0.01. Middle: Final tumor weight measurements at week 7 of experiment (4 weeks of treatment). Right: Quantification of lung metastatic surface nodules in the indicated experimental groups at week 7 of experiment. (F) Left: In vivo subcutaneous tumor volume measurements at the indicated time points for 393P tumors ± IL17RD knockdown, treated daily with AZD6244 or vehicle control starting at the time indicated by the red arrow; n = 5 mice per treatment group. Tumor volume data plotted as means and SD. Middle: Final tumor weight measurements at week 8 of experiment (5 weeks of treatment). Right: Images of three representative primary subcutaneous tumors for each of the indicated treatment groups. Scale bar, 1 cm. **P < 0.01.

  • Fig. 6 ZEB1 expression correlates with decreased IL17RD, decreased MAPK signaling, and MEK inhibitor resistance in KRAS mutant mouse lung tumors.

    (A) H&E, IL17RD, p-ERK, ZEB1, and TTF1 IHC stains of lung tumor tissue from KrasG12D, KP−/−, and KM−/− mice (n = 6 tissues per group). KP and KM lung tumor images show regions from two different mice. Scale bars, 100 μm. Inset scale bars, 20 μm. (B) Top: Percent change in overall lung tumor area of age-matched KrasG12D, KP−/+, KP−/−, KM−/+, and KM−/− mice after 4 weeks of daily treatment with AZD6244 (25 mg/kg), as assessed by micro-CT imaging of mouse lungs. Bottom: Trendlines of percent change in overall tumor area at indicated time points for each cohort over 4 weeks of daily treatment with AZD6244. Mouse sample sizes are indicated in the top graph. Percent change in tumor volume data plotted as the mean. (C) Representative cross-sectional micro-CT images of KrasG12D, KP−/−, and KM−/− mouse lungs before AZD6244 administration (week 0) and at the treatment endpoint (week 4). Yellow circles outline representative target lesions. White “H” indicates mouse heart.

  • Fig. 7 Mocetinostat increases miR-200 and IL17RD expression and sensitizes resistant tumor cells to MEK inhibition.

    (A) qPCR analysis for relative expression of miR-200a, miR-200b, and miR-200c in H157 cells after 72 hours of treatment with mocetinostat. **P < 0.01. DMSO, dimethyl sulfoxide. (B) Western blots of EMT markers in H157 cells after treatment with mocetinostat for the indicated time course. (C) Western blots of IL17RD and MAPK signaling proteins in H157 cells after treatment with mocetinostat for the indicated time course. Acetylated histone (H3K9) is included to confirm HDAC inhibition by mocetinostat. (D) H&E, IL17RD, p-ERK, and E-cadherin IHC stains of primary 344SQ tumor tissues in syngeneic WT mice after 3-week treatment with mocetinostat (80 mg/kg) or vehicle control daily (n = 5 tumors per group). Scale bars, 100 μm. Inset scale bars, 20 μm. (E) qPCR analysis of miR-200c and IL17RD expression in 344SQ tumor tissues treated with mocetinostat or vehicle control from (D). Technical triplicates of five tumor tissue samples from each treatment group were analyzed for each qPCR. (F) Left: Tumor volume measurements at the indicated time points for 344SQ subcutaneous syngeneic tumors treated daily with single-agent AZD6244 (AZD; 25 mg/kg), single-agent mocetinostat (Moc; 80 mg/kg), or both drugs in combination (Combo). Starting time of treatment is denoted by a red arrow; n = 5 mice per treatment group. Tumor volume data plotted as means and SD. Middle: Final tumor volume measurements at week 6 of experiment (3 weeks of treatment). Right: Quantification of lung metastatic surface nodules in the indicated treatment groups at week 6 of experiment. (G) Top: Percent change in overall tumor area of Kras, KP, and KM−/− mice after 4 to 5 weeks of daily treatment with single-agent AZD6244 (25 mg/kg) or daily combinatorial treatment with AZD6244 and mocetinostat (80 mg/kg) (Combo). Bottom: Trendline of percent change in overall tumor area at the indicated time points for Kras, KP, and KM mice with single-agent AZD6244 treatment or combinatorial treatment with AZD6244 and mocetinostat; n = 5 mice per treatment group. Percent change in tumor volume data plotted as means. (H) Representative micro-CT images of Kras, KP, and KM−/− mice before treatment (week 0) and at treatment end point (week 4 or 5). White “H” indicates location of mouse heart.

  • Fig. 8 Patients with lung ADC with high ZEB1 have lower phosphorylation of ERK.

    (A) Examples of well-differentiated and poorly differentiated human lung ADC tissue sections stained by IHC for ZEB1 and p-ERK. Scale bars, 200 μm. (B) Top: Nuclear H scores of p-ERK in ADC specimens of different differentiation grades. Bottom: Cluster plot analysis of Spearman’s rank correlation between ZEB1 and nuclear p-ERK H scores in ADC specimens. Solid line indicates the linear correlation, and dotted lines represent 90% confidence bands. (C) Cluster plot analysis of Spearman’s rank correlation between ZEB1 and IL17RD mRNA expression in human lung cancer patient tumor samples from PROSPECT dataset. (D) Proposed model demonstrating differential MAPK signaling pathway activation and sensitivity to MEK inhibitor treatment between epithelial and mesenchymal lung cancer cells due to ZEB1 regulation of IL17RD expression.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/483/eaaq1238/DC1

    Materials and Methods

    Fig. S1. Epithelial tumor cells exhibit greater MAPK signaling.

    Fig. S2. MAPK signaling in epithelial cells is only partially dependent on FAK-PAK signaling.

    Fig. S3. IL17RD expression in epithelial cancer cells correlates with MAPK signaling and inversely correlates with FGFR1.

    Fig. S4. ZEB1-expressing lung cancer cells are resistant to MEK inhibition.

    Fig. S5. MiR-200 sensitizes mesenchymal lung tumors to MEK inhibition through IL17RD up-regulation.

    Fig. S6. ZEB1 expression correlates with decreased IL17RD and decreased MAPK signaling in Kras mutant mouse lung tumors.

    Fig. S7. KrasG12D;miR-141/200c−/+ (KM) mouse lung tumors display high ZEB1, low MAPK signaling, and heterogeneity.

    Fig. S8. ZEB1 expression in MEK inhibitor–treated lung tumors of Kras mutant mice correlates with resistance.

    Fig. S9. Mocetinostat recapitulates miR-200 expression, sensitizing resistant tumor cells to MEK inhibition.

    Fig. S10. ZEB1 inversely correlates with ERK phosphorylation in patients with lung ADC.

    Data file S1 contains the following supplementary tables:

    Table S1. Quality control metrics for the shRNA screens.

    Table S2. Gene-level dropout scores for each of the four screens described in Fig. 1A.

    Table S3. RPPA dataset of protein expression fold change between epithelial and mesenchymal human lung cancer cell lines.

    Table S4. RPPA dataset of protein expression fold change between epithelial and mesenchymal murine lung cancer cell lines.

    Table S5. RPPA dataset of protein expression fold change in mesenchymal H157 cells after induced miR-200 expression.

    Table S6. RPPA dataset of protein expression fold change in mesenchymal H1299 cells after induced miR-200 expression.

    Table S7. RPPA dataset of protein expression fold change in epithelial HCC827 cells with constitutive ZEB1 expression.

    Table S8. RPPA dataset of protein expression fold change in epithelial H441 cells after induced ZEB1 expression.

    Table S9. Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 4D.

    Table S10. Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 5D.

    Table S11. Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 5E.

    Table S12. Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 5F.

    Table S13. Individual mouse lung tumor area measurements and percent change in lung tumor area from experiment in Fig. 6B.

    Table S14. Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 7F.

    Table S15. Individual mouse lung tumor area measurements and percent change in lung tumor area from experiment in Fig. 7G.

    Table S16. All commercially used antibodies, primers, shRNA, and complementary DNA open reading frames, including catalog numbers and sequences.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Epithelial tumor cells exhibit greater MAPK signaling.
    • Fig. S2. MAPK signaling in epithelial cells is only partially dependent on FAK-PAK signaling.
    • Fig. S3. IL17RD expression in epithelial cancer cells correlates with MAPK signaling and inversely correlates with FGFR1.
    • Fig. S4. ZEB1-expressing lung cancer cells are resistant to MEK inhibition.
    • Fig. S5. MiR-200 sensitizes mesenchymal lung tumors to MEK inhibition through IL17RD up-regulation.
    • Fig. S6. ZEB1 expression correlates with decreased IL17RD and decreased MAPK signaling in Kras mutant mouse lung tumors.
    • Fig. S7. KrasG12D;miR-141/200c−/+ (KM) mouse lung tumors display high ZEB1, low MAPK signaling, and heterogeneity.
    • Fig. S8. ZEB1 expression in MEK inhibitor–treated lung tumors of Kras mutant mice correlates with resistance.
    • Fig. S9. Mocetinostat recapitulates miR-200 expression, sensitizing resistant tumor cells to MEK inhibition.
    • Fig. S10. ZEB1 inversely correlates with ERK phosphorylation in patients with lung ADC.
    • Legends for tables S1 to S16

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 contains the following supplementary tables:
    • Table S1 (Microsoft Excel format). Quality control metrics for the shRNA screens.
    • Table S2 (Microsoft Excel format). Gene-level dropout scores for each of the four screens described in Fig. 1A.
    • Table S3 (Microsoft Excel format). RPPA dataset of protein expression fold change between epithelial and mesenchymal human lung cancer cell lines.
    • Table S4 (Microsoft Excel format). RPPA dataset of protein expression fold change between epithelial and mesenchymal murine lung cancer cell lines.
    • Table S5 (Microsoft Excel format). RPPA dataset of protein expression fold change in mesenchymal H157 cells after induced miR-200 expression.
    • Table S6 (Microsoft Excel format). RPPA dataset of protein expression fold change in mesenchymal H1299 cells after induced miR-200 expression.
    • Table S7 (Microsoft Excel format). RPPA dataset of protein expression fold change in epithelial HCC827 cells with constitutive ZEB1 expression.
    • Table S8 (Microsoft Excel format). RPPA dataset of protein expression fold change in epithelial H441 cells after induced ZEB1 expression.
    • Table S9 (Microsoft Excel format). Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 4D.
    • Table S10 (Microsoft Excel format). Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 5D.
    • Table S11 (Microsoft Excel format). Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 5E.
    • Table S12 (Microsoft Excel format). Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 5F.
    • Table S13 (Microsoft Excel format). Individual mouse lung tumor area measurements and percent change in lung tumor area from experiment in Fig. 6B.
    • Table S14 (Microsoft Excel format). Individual mouse subcutaneous tumor volume measurements from experiment in Fig. 7F.
    • Table S15 (Microsoft Excel format). Individual mouse lung tumor area measurements and percent change in lung tumor area from experiment in Fig. 7G.
    • Table S16 (Microsoft Excel format). All commercially used antibodies, primers, shRNA, and complementary DNA open reading frames, including catalog numbers and sequences.

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