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)

  • 14517

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    P2.04-027 - Erlotinib Induce miRNA Alterations in T790M EGFR Mutated NSCLC: Preclinical Study (ID 2483)

    09:30 - 09:30  |  Author(s): N. Van Der Steen
    • Abstract
    • Slides

    Background:
    Lung cancer is one of the leading causes of cancer-related deaths worldwide. In the most common type, non-small cell lung cancer (NSCLC), an array of oncogenic driver mutations affecting growth factor receptors and signaling pathways, such as EGFR, KRAS, c-Met or ALK translocation has driven the development of directed-drug therapies with tyrosine kinase inhibitors (TKIs) and monoclonal antibodies. Nevertheless, the appearance of resistance mechanisms, such as c-Met amplification or de novo mutations in EGFR, decreases the effectiveness of these therapies. Therefore, we analyzed the levels of miRNA which have proved to be involved in disease progression (miR-30b-5p,195-5p,221-3p) in NSCLC cells that are resistant (H1975) or sensitive (HCC827) against first generation TKI (erlotinib) treatment, to assess whether they are markers of response to the treatment.
    
    Methods:
    The H1975 cell line (EGFR mutations T790M/L858R) was cultured in RPMI 1640 medium, supplied with 10% FBS, 2 mM L-glutamine and 1% penicillin/streptomycin (Gibco). The sulforhodamine B assay was performed to assess cell proliferation. The cells were treated during 72h with 10µM erlotinib (SelleckChem) or DMSO. After collection, cells were lysed and RNA was extracted through commercial kit (RNA Mini Spin, GE Healthcare). The miRNAs profile analysis was performed through TaqMan Real-Time PCR and miRNA RNU-48 was used as endogenous control. Data was processed according to the formula 2[-ΔΔct]. Control values (H1975 + vehicle) are used as baseline and results are shown in logarithmic scale (Image).
    
    Results:
    Cells treated with 10µM erlotinib show an increased expression of miR-30b-5p, miR-195-5p compared to control values. On the other hand, we detected that the expression of the miR-221-3p was strongly down-regulated in respect the control values.
    
    Conclusion:
    H1975 carries the T790M mutation in EGFR, associated in NSCLC with appearance of resistance to TKIs treatment. However, we observed after treatment an increase of onco-suppressor miR-30b-5p and decrease of the oncogenic mir-221-3p, which are reported to correlate with good prognosis (Garofalo 2013, Zhong 2014). Moreover, mir-195-5p, another miRNA related with onco-suppression, is also upregulated, which has been reported to correlate with good response (Liu, 2015). Overall, our data suggests that erlotinib treatment alters miRNA in an EGFR mutated cell line. Further analysis of miRNA in exosomes produced by H1975, and comparison with HCC827 exosomal and cellular miRNA levels is currently undergoing in our group to confirm their value as response to treatment biomarkers.Figure 1
    
    
    
    

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