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S. Azoury
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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
- Moderators:R. Tsuchiya, M. Wynes
- Coordinates: 9/09/2015, 18:30 - 20:00, 702+704+706
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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)
18:30 - 20:00 | Author(s): S. Azoury
- Abstract
- Presentation
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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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)
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P2.04-051 - The Pluripotency Factor Musashi-2 Is a Potential Target for Lung Cancer Therapy (ID 2973)
09:30 - 17:00 | Author(s): S. Azoury
- Abstract
Background:
Recent studies have demonstrated that mithramycin represses multiple pathways critical for stem cell signaling and pluripotency in lung cancer cells. This phenomenon coincides with decreased side population (SP) fraction, and dramatic dose-dependent growth arrest of lung cancer cells in-vitro and in-vivo. The present study was performed to further examine the effects of mithramycin on stem cell signaling in an attempt to identify novel targets for lung cancer therapy.
Methods:
Microarray, quantitative RT-PCR (qRT-PCR) and immunoblot techniques were used to examine stem cell gene expression and proliferation of human lung cancer cells and normal/immortalized human respiratory epithelial cells (SAEC/NHBE/HBEC) cultured in the presence or absence of mithramycin, or lung cancer cells following stem cell gene knockdown. Micro-array and qRT-PCR techniques were used to assess effects of systemic mithramycin exposure on stem cell gene expression in subcutaneous lung cancer xenografts in athymic nude mice. qRT-PCR and immunoblot techniques were used to examine endogenous levels of selected stem cell genes in induced pluripotent stem cells (iPSC) derived from SAEC, as well as primary lung cancers and paired normal respiratory tissues. siRNA techniques were used to knockdown Msi-2 to confirm potential mechanisms of action of mithramycin-mediated cytotoxicity in lung cancer cells.
Results:
Preliminary microarray analysis of cultured lung cancer cells and xenografts demonstrated that mithramycin decreased expression of musashi-2 (Msi-2), a RNA binding protein which mediates self-renewal in normal stem cells and aggressive phenotype of several human cancers. Subsequent qRT-PCR and immunoblot experiments confirmed that mithramycin depletes Msi-2 in lung cancer cells in a time and dose-dependent manner. Expression levels of Msi-2 were significantly elevated in non-small cell as well as small-cell lung cancer lines relative to normal/immortalized human respiratory epithelial cells (p < 0.001). Consistent with these findings, Msi-2 mRNA levels in primary lung cancers were significantly higher than those detected in adjacent paired normal lung parenchyma (p< 0.0003). Msi-2 expression was enriched in SP fractions of cultured lung cancer cells, and was significantly increased in SAEC following reprogramming to pluripotency. si-RNA-mediated knock-down of Msi-2 decreased expression of Oct4, Nanog and Myc, and transiently inhibited proliferation of lung cancer cells. Attempts to permanently knockdown Msi-2 by shRNA techniques thus far have been unsuccessful, suggesting a strong selective pressure to maintain Msi-2 expression in these cells.
Conclusion:
Mithramycin depletes Msi-2 in lung cancer cells. Pharmacologic depletion of this pluripotency factor may be a novel strategy for lung cancer therapy.
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P3.05 - Poster Session/ Prevention and Tobacco Control (ID 217)
- Event: WCLC 2015
- Type: Poster
- Track: Prevention and Tobacco Control
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.05-012 - Hookah Smoke Mediates Cancer-Associated Alterations in Normal Human Respiratory Epithelial Cells (ID 2767)
09:30 - 17:00 | Author(s): S. Azoury
- Abstract
Background:
Cigarette smoking is the leading cause of lung cancers worldwide, and numerous countries have initiated public health programs to curtail cigarette abuse. Although hookah tobacco is perceived to be a safe alternative to cigarettes, the effects of hookah smoke in human respiratory epithelial cells and lung cancer cells are currently unknown. The present study was undertaken to examine if hookah smoke mediates cancer-associated alterations in normal respiratory epithelia and lung cancer cells.
Methods:
Human small airway epithelial cells (SAEC), cdk4/hTERT-immortalized human bronchial epithelial cells (HBEC) and lung cancer cells (Calu-6 and A549) were cultured in normal media in the presence or absence of waterpipe condensates (WPC) or cigarette smoke condensates (CSC) under relevant exposure conditions. MTS, quantitative RT-PCR and western blot techniques were used to examine the effects of hookah smoke on cell proliferation, mRNA/ microRNA expression and the histone code relative to those induced by cigarette smoke.
Results:
Five day WPC exposures mediated variable effects in cultured respiratory epithelia and lung cancer cells. In SAEC and HBEC, WPC mediated dose-dependent growth inhibition. In Calu-6 cells, low dose WPC (0.1mg/ml and 0.5mg/ml) enhanced proliferation, whereas higher concentrations of WPC (1.0mg/ml and 2.0mg/ml) inhibited growth. High concentrations of WPC (2.0mg/ml) inhibited proliferation of A549 cells. Similar to CSC, WPC decreased H4K16Ac and H4K20Me3 levels in SAEC and HBEC. In addition, like CSC, WPC increased miR-31 and decreased miR-487b expression in SAEC, HBEC and lung cancer cells. Furthermore, WPC increased expression of Cyp1b1 in SAEC cells, enhanced expression of ABCG2, Lin28B, Myc, SALL4, CTCF, JARID2 and Cyp1a1 in HBEC cells, up-regulated ABCG2 and Cyp1a1 in Calu-6 lung cancer cells, and induced dose-dependent up-regulation of Cyp1a1 and Cyp1b1 in A549 lung cancer cells. WPC exposure significantly decreased expression of Dkk-1 in HBEC and Calu-6 cells. Cigarette smoke induced similar changes in these cells.
Conclusion:
These preliminary findings demonstrate that hookah smoke mediates cancer-associated alterations in human respiratory epithelial cells, and suggest that waterpipe tobacco is not a safe alternative to cigarettes.
