Research ArticleCancer

Phosphorylation of FADD by the kinase CK1α promotes KRASG12D-induced lung cancer

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Science Signaling  27 Jan 2015:
Vol. 8, Issue 361, pp. ra9
DOI: 10.1126/scisignal.2005607
  • Fig. 1 A requirement for FADD in Kras-driven lung cancer.

    (A) Genetic strategy used to activate KrasLSL-G12D and Rosa26LSL-Luciferase and deplete Fadd:GFP expression in a lung-specific manner. EGFP, enhanced green fluorescent protein. BLI, bioluminescence imaging. (B) Representative bioluminescent images of KLuc and KFLuc mice at 7 to 22 weeks after intranasal AdCre administration. Pseudocolor scale is radiance as photons/second/cm2/steradian (photons/sec/cm2/sr). (C) Average bioluminescence in Luc (n = 7), FLuc (n = 7), KLuc (n = 25), and KFLuc (n = 34) mice at specified times. (D) Representative images of CT scans of lungs from Luc, FLuc, KLuc, and KFLuc animals at shown times, with hematoxylin and eosin (H&E) slide corresponding to that animal from week 18 or 22 as indicated. Arrows, lesions. H, heart. (E) Tumor and vascular volumes in KLuc (n = 10) and KFLuc (n = 17) mice analyzed by CT at indicated times. Data are means + SEM. *P = 0.009 and **P = 0.018, unpaired Student’s t test. (F) Colocalization of bioluminescence and CT imaging for lung tumors in Luc, KFLuc, and KLuc mice. Inset, H&E of lesion in KLuc mouse. (G) Survival plot of KLuc (n = 19) and KFLuc (n = 15) mice. Median survival time: KFLuc mice, 51.4 weeks; KLuc mice, 34 weeks. ***P = 0.005, Wilcoxon log-rank test (95% confidence interval).

  • Fig. 2 Fadd-null lung tumors are less proliferative.

    (A) Representative histology images of lungs removed from mice 18 weeks after AdCre administration. Tissues were stained with H&E or antibodies against cyclin D1, Ki-67, and FADD. Scale bars, 10×, 500 μm; 40×, 200 μm; and 100×, 50 μm. (B) Average tumor area quantified from H&E-stained lung tissue sections from KLuc (n = 6) and KFLuc (n = 10) mice. *P = 0.04, unpaired Student’s t test. (C) Average percentage of positive Ki-67–stained cells in lung tissue sections from KLuc and KFLuc mice, four fields per 10 mice each. **P = 2 × 10−5, unpaired Student’s t test. (D) Representative Western blot for endogenous FADD, GFP (Fadd transgene), phosphorylated ERK1/2 (pERK1/2) and total ERK1/2, phosphorylated RB (pRB), cyclin D1, cyclin B1, and β-actin in lung tissue from the indicated mice. Blots are representative of three independent experiments. pFADD, phosphorylated FADD.

  • Fig. 3 FADD and FADD phosphorylation are required for Kras-driven cell proliferation.

    (A) Western blotting for FADD, cyclin D1, and β-actin in lysates from Luc, KLuc, and KFLuc MEFs. (B and C) AlamarBlue proliferation assay in cultures of Luc, KLuc, and KFLuc MEFs that were either (B) untreated or (C) treated with DMSO (dimethyl sulfoxide), 250 μM CKI-7, 10 μM lonafarnib, or 200 nM PD0325901. (D) Agar colony formation of KLuc, KFLuc, and Fadd-reconstituted KFLuc MEF cells treated as indicated for 2 weeks, concentrations as in (C). (E) Fluorescence-activated cell sorting (FACS) analysis of cell cycle distribution in KLuc and KFLuc MEFs 24 hours after the indicated treatment, concentrations as in (C). (F) Western blotting for the indicated proteins in KLuc MEFs treated for 6 hours as in (C). Blots (A and F) and FACS data (E) are representative of three independent experiments. Data are means + SEM from three independent experiments. pCDC20, phosphorylated cell division cycle 20.

  • Fig. 4 FADD interacts with key mediators of G2/M transition.

    (A) Western blotting for the indicated proteins in H1975 cells after double thymidine block and release (G2/M). Cells were harvested at 6, 7, 8, and 9 hours after synchronization. AS, asynchronous cells. pHistone H3, phosphorylated histone H3. (B and C) Western blotting (WB) after FADD immunoprecipitation (IP) from A549 cells (B) and quantification of the BUB1-FADD interaction (C). Cells were asynchronous (AS) or treated with CKI-7, nocodozole (G2/M), or hydroxyurea (G1/S). BUB1 abundance was normalized to that in asynchronous cells. Data are means ± SEM from three independent experiments. *P = 0.005, unpaired Student’s t test.

  • Fig. 5 CK1α mediates Kras-driven lung cancer.

    (A) Genetic strategy used to activate KrasLSL-G12D and Rosa26LSL-Luciferase and delete Csnk1a expression in a lung-specific manner. Bioluminescence and/or CT imaging was performed at the indicated weeks after administration of Cre recombinase. (B) Bioluminescence of Luc (n = 8), CLuc (n = 5), KLuc (n = 38), and KCLuc (n = 25) mice at the specified times. Data are means ± SEM. (C) Representative images of CT scans of lungs from Luc, KLuc, CLuc, and KCLuc mice, with corresponding H&E slides. Arrows, lesions. H, heart. (D) Average tumor and vascular volumes of KLuc (n = 8) and KCLuc (n = 7) mice analyzed by CT 18 weeks after AdCre administration. Data are means ± SEM; *P = 7.47 × 10−6, unpaired Student’s t test. (E) Representative histology for H&E or indicated proteins in lungs from mice 18 weeks after AdCre administration. Scale bars, 10×, 500 μm; 40×, 200 μm; and 100×, 50 μm. (F) Representative Western blot for CK1α, FADD, and β-actin, in lysates from KCLuc MEFs treated with various concentrations of AdCre. PFU, plaque-forming units. (G and H) Representative Western blot (G) and quantification of phosphorylated fractions (H) for the indicated proteins in lysates of KLuc and KCLuc MEFs treated with AdCre or AdLuc (Adenovirus-luciferase). Data are means ± SEM from three independent experiments. **P = 0.003, unpaired Student’s t test. Ctrl, control. p–β-Catenin, phosphorylated β-catenin. pMDM2, phosphorylated MDM2.

  • Fig. 6 Model for the role of FADD in KRAS-mediated cell proliferation.

    FADD and CK1α are necessary in mediating mitogenic KRAS signaling in cancer cells. Inhibiting KRAS, MEK, or CK1α or deleting Csnk1a1 or Fadd decreased the abundance of phosphorylated FADD and decreased cell proliferation. A requirement for phosphorylated FADD in G2/M progression through the interaction with AURKA, PLK1, or BUB1 provides a mechanistic basis for these results.

  • Table 1 FADD interacts with proteins involved in the G2/M transition.

    The top cell cycle–related proteins that were pulled down with Halo-tagged FADD in HEK293T cells. The cutoff that determined FADD-interacting proteins was a spectral count (SpC) of 5 or greater and a control SpC of 0. NSAF, normalized spectral abundance factor.

    Identified proteinsGene
    symbol
    Molecular
    weight (kD)
    FADD
    peptide SpC
    Control
    peptide SpC
    FADD NSAFControl
    NSAF
    FADDFADD2355500.1048768870
    CK1αCSNK1A1392000.0022288460
    Polo-like kinase 1PLK168500.0003195770
    Aurora kinase BAURKB39600.0006686540
    Cell division cycle 20CDC20551700.0013433860
    Centrosomal protein 170CEP1701751100.0002731930
    Structural maintenance of chromosomes 1ASMC1A1431300.0003951140
    Non-SMC condensing II complex, subunit D3NCAPD31691900.0001660990
    Non-SMC condensing II complex, subunit G2NCAPG21311800.0005971950
    Non-SMC condensing II complex, subunit D2NCAPD2157600.0001660990
    Dynactin 2DCTN244500.0004938920
    Cell division cycle 73CDC7361700.00048750
    Fanconi anemia complementation group D2FANCD21651500.0003927330
    Fanconi anemia complementation group IFANCI1493600.0010501010
    Cytoskeleton-associated protein 5CKAP5226700.0001346180

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/361/ra9/DC1

    Fig. S1. Fadd-null lesions have lower abundance of phosphorylated ERK1/2 but no detectable difference for an apoptotic marker.

    Fig. S2. Fadd-null lesions still express Fadd transgene.

    Fig. S3. Increased FADD mRNA correlates with KRAS mutation status in lung cancer patients.

    Fig. S4. FADD interacts with proteins involved in the cell cycle.

    Fig. S5. Immunohistochemistry of Csnk1a1-null lesions reveals residual CK1α protein.

    Data file S1. Mass spectrometry data.

  • Supplementary Materials for:

    Phosphorylation of FADD by the kinase CK1α promotes KRASG12D-induced lung cancer

    Brittany M. Bowman, Katrina A. Sebolt, Benjamin A. Hoff, Jennifer L. Boes, Danette L. Daniels, Kevin A. Heist, Craig J. Galbán, Rajiv M. Patel, Jianke Zhang, David G. Beer, Brian D. Ross, Alnawaz Rehemtulla* Stefanie Galbán

    *Corresponding author. E-mail: alnawaz{at}umich.edu

    This PDF file includes:

    • Fig. S1. Fadd-null lesions have lower abundance of phosphorylated ERK1/2 but no detectable difference for an apoptotic marker.
    • Fig. S2. Fadd-null lesions still express Fadd transgene.
    • Fig. S3. Increased FADD mRNA correlates with KRAS mutation status in lung cancer patients.
    • Fig. S4. FADD interacts with proteins involved in the cell cycle.
    • Fig. S5. Immunohistochemistry of Csnk1a1-null lesions reveals residual CK1α protein.
      Legend for Data file S1

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1. Mass spectrometry data.

    [Download Data File S1]


    Citation: B. M. Bowman, K. A. Sebolt, B. A. Hoff, J. L. Boes, D. L. Daniels, K. A. Heist, C. J. Galbán, R. M. Patel, J. Zhang, D. G. Beer, B. D. Ross, A. Rehemtulla, S. Galbán, Phosphorylation of FADD by the kinase CK1α promotes KRASG12D-induced lung cancer. Sci. Signal. 8, ra9 (2015).

    © 2014 American Association for the Advancement of Science

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