Author of

• 11481

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  P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Biology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 11497

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    P2.02-016 - PRMT5, a novel epigenetic target in lung cancers (ID 2886)

    13:45 - 14:02  |  Author(s): G.A. Otterson
    • Abstract

    Background
    PRMT5 is an arginine methyltransferase that regulates cellular events by methylation of Arginine residues on histone and non-histone proteins. PRMT5 cooperates with chromatin remodelers and co-repressors to induce epigenetic silencing, and is overexpressed in several human cancers. It also has impact on cell growth and transformation pathways by modulation of E2F1, p53, EGFR and CRAF. We investigated the role of PRMT5 in lung cancer.

    Methods
    The expression pattern of PRMT5 using immunohistochemistry from resection specimens obtained with an IRB approved protocol. Immortalized lung cancer cells (A549, H719, H520 and H1299) were obtained from ATCC and normal bronchial airway cells (HPAEpiC, HBEpiC) were obtained from ScienCell. Western immunoblot and cell cycle analysis by flow cytometry was performed using standard techniques. A novel and specific inhibitor of PRMT5 developed by colleagues at OSU was applied to in vitro culture systems.

    Results
    The expression of PRMT5 was analyzed in 9 lung cancer resection specimens (3 adenocarcinoma, 3 squamous, 2 small cell and 1 large cell neuroendocrine cancer). All 9 showed diffuse cytoplasmic and variable nuclear PRMT5 expression. PRMT5 was also seen in reactive type 2 pneumocytes and respiratory epithelium adjacent to the tumors but not in alveolar parenchyma, fibroblasts or endothelial cells. Using Western immunoblot, PRMT5 is highly expressed in A549, H719, H1299 and H520 cells compared with normal cells such as HPAEpiC and HBEpiC. We knockdowned the expression of PRMT5 by lentiviral shRNAs and identified several clones with effective PRMT5 inhibition. Inhibition of PRMT5 was associated with slow cell growth. The cell proliferation decreased 58.4% and 62.3% in H1299 and A549 knockdown cells respectively. Interestingly, while H4R3 methylation was decreased with PRMT5 knockdown in A549 it was not in H1299 cells. We further analyzed the role of PRMT5 by using a specific inhibitor developed by researchers at OSU, CPD5. One of the PRMT5 specific marks of histone H4R3 methylation was inhibited and significant cell cycle changes were observed in A549 and H1299 cells treated for 24 hr and 48 hr. At 24 hours, the percentage of cells in G0/G1 was 57.1% (control) compared with 66.4% in CPD5 treated A549 cells, and 52.5% (control) compared with 70.9% in CPD5 treated H1299 cells. The expression of p21 was increased while cyclin E1 was decreased in A549 cells treated with CPD5. In contrast, the expression of cell cycle related proteins were not found in PRMT5 knock-down cells. Immunoprecipitation and other protein interaction techniques are in process to identify new PRMT5 targets.

    Conclusion
    In summary, PRMT5 is highly expressed in lung cancer cells compared to normal lung. A novel PRMT5 inhibitor and shRNAs can inhibit cell proliferation. Although PRMT5 inhibitor could induce cell death by cell cycle regulation and apoptosis, different pathways may be involved in PRMT5 knock-down cells and speaks to the complexity regulating mechanisms in different histological lung cancer patients. The differences in H4R3 methylation suggest that epigenetic repression may be dominant in some cancers, but that non-epigenetic mechanisms may be relevant in others. Further exploring of PRMT5 targets are ongoing.