Author of

• 11481

  +

  P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Biology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 11487

    +

    P2.02-006 - The selective regulation of EGFR mutants by C-terminus of Hsc70-interacting-protein(CHIP) in lung adenocarcinoma (ID 1116)

    10:55 - 11:12  |  Author(s): H.R. Cha
    • Abstract
    • Slides

    Background
    Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) are response predictive markers for the treatment of lung adenocarcinoma by using EGFR tyrosine kinase inhibitors. The stability of EGFR is dependent on its interaction with heat shock protein 90 (Hsp90), and Hsp90 inhibition may induce ubiquitin-mediated degradation of EGFR proteins. However, the mechanism of EGFR downregulation by HSP90 inhibition remains unclear. Here, we report a novel interaction between EGFR mutants and the E3 ubiquitin ligase C-terminus of Hsp70-interacting protein (CHIP), which can ubiquitinate and destabilize EGFR mutants

    Methods
    Cell culture, transfection and drug treatments NSCLC (A549, H358, H827, and PC9 cells) and Chinese hamster ovarian (CHO) cell lines used in the study were obtained from ATCC. Transfections of different DNA constructs were performed using Lipofectamine 2000 or PolyJet Western blot and immunoprecipitation Cells were then chilled on ice, washed twice with ice-cold PBS, and lysed in a buffer. Protein concentrations were determined using a Bradford assay kit. Equal amounts of protein (20 μg) in cell lysates were separated by SDS-PAGE, transferred to membranes, immunoblotted with specific primary and secondary antibodies, and detected by chemiluminescence with the enhanced chemiluminescence detection reagents. Cell viability assay After trasnfection with or without CHIP, cell metabolic activity was determined every 24 hours by using the CCK-8 (Dojindo, Gaithersburg, MD) colorimetric assay according to the manufacturer’s instructions. Cell cycle analysis by FACS Cells were seeded in 6-well plate and incubated overnight before transfection. After harvest at 48 h following transfection, cells were washed twice with pre-chilled PBS and resuspended in PBS (100 μL) at a concentration of 1 × 106 cells/mL. Cell cycle analysis was performed using the Coulter DNA PrepTM Reagents Kit. Xenograft studies A549 and PC9 cells were cultured as monolayers, trypsinized and resuspended in an equal volume of PBS at 5 × 107 per ml, respectively. BALB/c female nu/nu mice (5 weeks of age) were given bilateral subcutaneous injections of 5 × 106 (0.1 ml). Tumor growth was monitored twice each week by measuring the tumor size using calipers

    Results
    In CHO cells expressing either WT or mutant EGFR, the expression of the EGFR mutants L858R, G719S, and L747_E749del A750P was inhibited following overexpression of CHIP, whereas WT EGFR expression was not diminished in CHIP transfected cells. In a pull-down assay, CHIP interacted with G719S and del L747_E749del A750P, but not L858R, and simultaneously induced the ubiquitination and proteasomal degradation of its proteins. Similarly, the expression of mutant EGFR proteins in the non-small cell lung cancer cell line PC9 was also diminished by CHIP-mediated ubiquitination and degradation relative to WT EGFR. In EGFR mutant cell lines, CHIP inhibited cell proliferation as well as the depletion of phosphorylated Akt. In addition, CHIP inhibited the tumor growth of PC9 cells in xenografts of CHIP-overexpressing stable cell lines.

    Conclusion
    These data suggest that CHIP-induced ubiquitination of EGFR mutants may be responsible for EGFR regulation in lung adenocarcinoma and provide evidence that CHIP could be a novel E3 ligase for EGFR mutants.

    Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.