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P. Singleton

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    P1.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 206)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Treatment of Advanced Diseases - NSCLC
    • Presentations: 1
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      P1.01-073 - MET and Invasive Function in NSCLC (ID 2951)

      09:30 - 17:00  |  Author(s): P. Singleton

      • Abstract
      • Slides

      There is molecular heterogeneity of lung cancers, especially non-small cell lung cancer (NSCLC). Even though pathways such as EGFR and ALK are understood much more, we are beginning to understand the role of MET receptor tyrosine kinase (RTK). The initial trial of EGFR with MET inhibitor was no better than single agent, we strongly believe that it is a subset of MET abnormalities that lead to the pathogenesis of lung cancer. In order to better define this, we are studying the MET function in invasion of NSCLC. We are utilizing the novel ECIS method to study biological behavior.

      Immunofluorescence of Met/pMet staining was performed with the following antibodies: c-MET (NovusBio) and p-METet (Tyr1230,1234,1235, Invitrogen). Immunoblot analyses of MET/pMET and related signal transduction molecules were performed on H1993 (MET amplified cell line) and A549 (KRAS mutated cell line, with activated MET) cell lysates. ECIS instrument (Applied Biophysics Inc) was used for motility assay and proliferation assay. Confluent cell monolayer was electrically abrased at 6V for 30 seconds. Impedance and resistance of the cell layer were immediately recorded for a period of up to 20 hours. For proliferation assay cells were seeded in 8W10E plates without or with inhibitors. Impedance and resistance were measured for 48 hours at 15 kHz. MET inhibitors were also utilized in the study.

      In our study we investigated the inhibition potential of MET inhibitors. Staining with MET antibody resulted in nuclear and perinuclear staining of both of the cell lines. HGF treatment (100 nM, 20 min) increased nuclear staining. p-MET also increased in the presence of HGF and had plasma membrane, perinuclear and nuclear pattern. 48 hours pre-incubation with MET inhibitors reduced cellular MET staining and abolished MET phosphorylation. Moreover immunoblotting assay demonstrated significant reduction of MET phosphorylation in untreated and HGF treated cells. We measured biological activity of the cells in the presence of inhibitors. MET inhibitors significantly reduced growth rate compared with untreated cells as assessed by electrical resistance measurement. Next we did ECIS based wound-healing assay for a quantitative determination of MET inhibitors on migration potential of NSCLC cells. Voltage pulse led to drastic decrease of cell resistance. MET inhibitors delayed for 35% return to resistance value of the resting cells.

      MET inhibitors reduced NSCLC cell motility, migration and invasion. Novel MET inhibitors can be used for therapeutic intervention against NSCLC. The nuclear localization of MET is a novle function and needs to be explored further. ECIS also is a novel technique to study the biology of epithelial cancers.

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