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E. Reardon



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    ORAL 07 - Lung Cancer Pathogenesis (ID 91)

    • Event: WCLC 2015
    • Type: Oral Session
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      ORAL07.01 - Evaluation of Epigenetic Mechanisms of Pluripotency in Human Respiratory Epithelia (ID 3041)

      10:45 - 12:15  |  Author(s): E. Reardon

      • Abstract
      • Presentation
      • Slides

      Background:
      Smoking is the number one risk factor for lung cancer worldwide. Recent data indicate that stem cells situated throughout the small airway epithelium may initiate cancer formation following direct exposure to inhaled carcinogens. In the present study we sought to generate induced pluripotent stem cells (iPSCs) from normal human small airway epithelial cells (SAECs) in order to investigate epigenetic mechanisms contributing to the cancer stem cell initiation process, and possibly identify novel targets for lung cancer therapy.

      Methods:
      Several different stocks of SAEC were transduced with Stemcca virus containing OKSM (Yamanaka factors); multiple randomly selected clones were expanded for further analysis. Spectral karyotyping was performed to confirm the purity of pluripotent cells. iPSC cells were injected in SCID mice to study teratoma formation. RNA and DNA were extracted from iPSC and parental SAEC for qRT-PCR and RNA-Seq analyses, as well as pyrosequencing of LINE-1, NBL2 and D4Z4 DNA repetitive elements, and promoter regions of several differentially regulated genes.

      Results:
      SAEC were reprogrammed to a pluripotent state. Generated iPSCs demonstrated hallmarks of pluripotency including morphology, proliferation, expression of surface antigens, stemness gene expression, and in vivo teratoma formation. Interestingly, no chromosomal aberrations were observed in iPSCs. Pyrosequencing did not demonstrate any significant changes in LINE-1, NBL2 and D4Z4 DNA methylation levels in iPSC compared to parental SAEC, suggesting relatively limited global hypomethylation following reprogramming. Consistent with these observations, cancer-testis genes such as NY-ESO-1, MAGE-A1 and MAGE-A3, which are frequently upregulated by DNA demethylation in lung cancer cells, remained transcriptionally repressed in the iPSC. On the other hand, NANOG and POU5F1 genes were hypomethylated in iPSCs relative to SAEC, correlating with their over-expression in iPSCs. RNA-Seq analysis revealed up-regulation of genes encoding components of Polycomb-Repressive Complex 2 (PRC2), and down-regulation of several tumor suppressor genes such as DKK1, p16 and p21 in iPSC relative to parental SAEC. Several novel pluripotency associated genes were also noted to be up-regulated in pulmonary iPSC, which are the focus of ongoing mechanistic studies.

      Conclusion:
      This is the first report demonstrating successful reprogramming of human respiratory epithelia to pluripotency. This model may prove useful for elucidating fundamental epigenomic mechanisms of pulmonary carcinogenesis and identification of novel targets for lung cancer therapy.

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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
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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): E. Reardon

      • Abstract
      • Slides

      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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