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P. Feingold



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    MINI 38 - Biology and Prognosis (ID 167)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
    • Presentations: 1
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      MINI38.07 - RITA Enhances Mithramycin-Mediated Growth Arrest and Apoptosis of Malignant Pleural Mesothelioma Cells In-Vitro and In-Vivo (ID 2996)

      19:05 - 19:10  |  Author(s): P. Feingold

      • Abstract
      • Presentation
      • Slides

      Background:
      Malignant pleural mesotheliomas (MPM) are relatively rare tumors for which there are no effective treatment options. Previously we reported that mithramycin (MM) dramatically inhibits growth and tumorigenicity of MPM cells in part via depletion of Specificity Protein 1 (SP1) and activation of p53 signaling. We also demonstrated that 24h MM treatment induces G0/G1arrest and senescence with subsequent apoptosis of MPM cells. The present study was undertaken to examine the effects of RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis- a p53 activator and MDM2 inhibitor) with or without MM in cultured MPM cells in vitro and in vivo.

      Methods:
      NCI-SB-MES1 and NCI-SB-MES7 (MES1 and MES7, respectively) with wild-type p53 were cultured in the presence of mithramycin (24h) and/or RITA (48h). DNA damage, senescence and autophagy were assessed by immunoblot/immunofluorescence analysis of g-H2A-X phosphorylation and foci formation, ß-gal staining, and immunoblot/immunofluorescence analysis of LC3 proteins. Propidium iodide and APO-BrdU techniques were used to determine cell cycle kinetics and quantify apoptosis. qRT-PCR and immunoblot techniques were used to examine signal transduction, cell cycle-related and apoptosis-related protein levels in MPM cells. Murine subcutaneous xenograft models were used to evaluate the combinatorial antitumor effects of RITA and MM in-vivo.

      Results:
      MM treatment (10-100nM x 24h) mediated dose-dependent depletion of SP1 and markedly increased p53 levels in MPM cells; these effects coincided with DNA damage, G0/G1 arrest, senescence and an autophagy phenotype as evidenced by induction of LC3 puncta/proteins and p-AMPK and inhibition of p-S6 kinase. Senescence or autophagy phenotype coincided with up-regulation of CDKN1A, MDM2/TP53INP1, MAPLC3B, and down-regulation of EZH2, SP1/MTOR. RITA (100-1000nM x48h) alone mediated low-level, dose-dependent growth inhibition in MPM cells. However treatment with subtherapeutic doses of MM for 24h followed by RITA for 48h resulted in synergistic growth inhibition and apoptosis in MPM cells, detected by flow cytometry, as well as immunoblot analysis of cleaved PARP and cleaved caspase 3. Sequential intraperitoneal treatment with MM (1mg/kg/week) followed by RITA (2 mg/kg/3d/week) significantly reduced volumes/masses of subcutaneous MES1 xenografts in athymic nude mice.

      Conclusion:
      Sequential mithramycin/RITA treatment significantly reduces mesothelioma tumor burden via induction of apoptosis. These findings provide preclinical rationale for evaluation of this drug regimen in MPM patients.

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