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  P3.01 - Poster Session 3 - Cancer Biology (ID 147)

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

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    P3.01-004 - Pre-clinical evaluation of Death Receptor-5 activation as a feasible option for K-ras mutant NSCLC therapy (ID 3399)

    09:30 - 09:30  |  Author(s): M.D.S. Caetano
    • Abstract

    Background
    Activating mutations of K-ras are one of the most common alterations associated with tobacco exposure-related lung cancer(LC). There are two major types of LC - small-cell LC(SCLC) and non-small cell LC(NSCLC) - and the later accounts for 80% of the cases. NSCLC can be divided into three histological subtypes: Adenocarcinoma, large cells and squamous cell carcinoma. First-line treatment comprises surgical resection of tumor, followed or not by chemo/radiotherapy. In non-surgical cases, platinum compounds remain the cornerstone for both early and advanced NSCLC stages management, in spite of its toxicity, high rate of chemo-resistance and poor long term results. Several attempts to develop therapies based on molecular targets, such as K-ras, have been developed and thus far failed, clearly stating the need for new approaches to bring clinical benefits to patients. Among its several aberrations, NSCLC harbors alterations in the apoptotic pathways, leading to impaired pro-apoptotic signaling and positive modulation of anti-apoptotic pathways. Therapeutic strategies targeting such pathways can emerge as an alternative to the cytotoxic therapies selected to wild-type EGFR-patients – especially for K-ras mutated patients, comprising about 20% of this population.

    Methods
    Here we have analyzed a representative panel of NSCLC cell lines (A549, H460, Calu-1 and LC319), that display distinct sensitivities towards cisplatin, mutational profiles and histological subtypes, to test a pre-clinical therapeutic strategy engaging the TNF-related apoptosis-inducing ligand (TRAIL) receptor (Death receptor 4 and 5).

    Results
    TRAIL is a member of the TNF family of cytokines that induces apoptotic cell death in a variety of tumor cells by means of activation of its specific receptors, DR4 and DR5, while displaying low toxicity towards normal cells. We sought to investigate if DRs activation could subvert the relative resistance to cisplatin intrinsically presented by NSCLC cell lines. NSCLC cell lines treated with suboptimal concentrations of cisplatin (IC30) for 48h had their gene expression analyzed by qPCR and the results showed an increased expression of DR4 and DR5 in these cells. These results were confirmed at protein level by Western blot analysis. NSCLC cells are naturally regarded as resistant to TRAIL-induced cell death. Such resistance can rise, among other reasons, from low expression of DRs or increased expression of decoy receptors and/or anti-apoptotic proteins. LBY135 (Novartis) is a DR5 agonist monoclonal antibody that mimics TRAIL and induces cell death in DR5 expressing cells. As cisplatin modulated DR5 protein expression, we have combined it with LBY135 as a strategy to improve cell death induction upon NSCLC cells. LBY135 monotherapy did show some effects when analyzed by MTT assay, although it did not induce cell death accessed by Annexin/PI FACS analysis. However, when cisplatin IC30 and LBY135 were combined, we observed a significant decrease in MTT measurements and increased cell death incidence, suggesting a synergistic effect of these drugs. Such pro-apoptotic effect was blocked by zVAD-fmk, a pan-caspase inhibitor.

    Conclusion
    The synergistic effect observed was more pronounced in cell lines bearing the classical G12K-ras mutation, suggesting an alternative way to subvert chemo-resistance of K-ras mutated NSCLC and restore cisplatin-induced apoptotic signaling leading to cell death.