Author of

• 14490

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  P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)

  • Event: WCLC 2015
  • Type: Poster
  • Track: Biology, Pathology, and Molecular Testing
  • Presentations: 1
  • Moderators:
  • Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)

  • 14493

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    P2.04-003 - Two Methods for Developing in Vitro Erlotinib-Resistant Cell Lines Lead to Distinct RTK Shifts, but Both Result in EMT (ID 928)

    09:30 - 09:30  |  Author(s): A.T. Madsen
    • Abstract

    Background:
    Several studies have investigated resistance mechanisms underlying acquired erlotinib-resistance in vitro. To mimic the in vivo distribution of the drugs, different approaches such as applying gradually increasing doses of erlotinib to the cells or exposing them to a high fixed concentration of the drug have been used. We demonstrate that two different approaches of developing erlotinib-resistant HCC827 cells results in activation of two distinct RTK bypass-signalling pathways. However, despite these differences both cell lines undergo EMT. Our finding suggests that EMT is a common marker of erlotinib-resistance.
    
    Methods:
    Two HCC827 erlotinib-resistant cell lines were established using either gradually increasing doses of erlotinib (0.01 μM – 5 μM) resulting in erlotinib-resistant HCC827ER cells. Alternatively a fixed concentration of 5 μM generated HCC827HD with erlotinib resistance. Growth of the resistant cell lines was investigated using MTS assay in combination with erlotinib, linsitinib and crizotinib. Phospho-RTK arrays (R&D Systems), qPCR and immunofluorescence were used to characterize the cells.
    
    Results:
    Phospho-RTK array analysis revealed that the erlotinib-resistant HCC827ER cells had an increased activation of MET, and copy number analysis demonstrated the activation to be caused by a MET amplification. Furthermore, HCC827ER showed growth inhibition when treated with the MET-inhibitor crizotinib. The other type of erlotinib-resistant cells, HCC827HD, had increased activation of IGF1R and also responded to the IGF1R-inhibitor linsitinib. However, a common feature is that both HCC827ER and HCC827HD gained EMT features. HCC827ER showed increased expression SLUG, SNAIL and ZEB1, whereas HCC827HD showed increased SLUG and TWIST expression. To detect the relevance of MET and IGF1R signalling in accordance to EMT in the two cell lines, we treated the HCC827ER cells with the tyrosine kinase inhibitor crizotinib (MET) and the HCC827HD cells with linsitinib (IGF1R). In both cases, we saw a decrease in EMT-marker transcription after the treatment.
    
    Conclusion:
    Our study demonstrates that different approaches to developing erlotinib-resistant cell lines can lead to distinct activation of bypass receptor tyrosine kinase signalling pathways. EMT, however, is induced in both types of erlotinib-resistance. This finding indicates that EMT is a common trait of the phenotype of erlotinib-resistant cells. More research needs to be done to establish the functional role of EMT in erlotinib resistance.