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J. Beers

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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): J. Beers

      • Abstract
      • Presentation
      • Slides

      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.

      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.

      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.

      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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