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Akikazu Kawase



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    P2.03 - Biology (ID 162)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Biology
    • Presentations: 1
    • Now Available
    • Moderators:
    • Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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      P2.03-43 - WTAP Activates Oncogenes and Accelerates Tumor Aggressiveness Through Adding m6A RNA Modification in Non-Small-Cell Lung Cancer (Now Available) (ID 2031)

      10:15 - 18:15  |  Author(s): Akikazu Kawase

      • Abstract
      • Slides

      Background

      N6-methyladenosine (m6A) is one of the RNA modifications affecting multiple cellular processes such as RNA metabolism, stem cell self-renewal, and cell differentiation in eukaryotes. m6A is dynamically regulated by three types of enzymes called writer, eraser, and reader. WT1-associated protein (WTAP) is an important component of the m6A writer complex to which the majority of m6A is attributed. Despite having been revealed in several types of cancers, the relevance and roles of m6A and its modifiers are unclear in non-small cell lung cancer (NSCLC). We here quantified the global m6A levels in NSCLC tissues and associated them with clinical data. In addition, the roles of WTAP in the context of m6A regulation were explored in vitro.

      Method

      Amounts of global m6A in the purified RNAs extracted from 126 paired tumor and adjacent normal tissues were evaluated using liquid chromatography mass spectrometry/mass spectrometry. The clinicopathological data were retrospectively retrieved. The expression levels of the three major m6A writers (WTAP, METTL3, and METTL14) in lung cancer cell lines (H1299, PC3, and PC9) were assessed using RT-qPCR and western blot. WTAP knockdown was carried out using siRNAs and its effects on tumoral activities. Also the changes of global and specific transcripts in WTAP-deprived PC9 cells were evaluated using a microarray analysis and m6A specific RNA immunoprecipitation (RIP) assay. WTAP protein expression levels in 641 resected NSCLC were assessed using immunohistochemistry and their prognostic role was determined.

      Result

      The m6A/A was significantly higher in tumor tissues than adjacent normal tissues (P <0.001), and higher m6A/A was significantly associated with worse overall survival (Log-rank, P=0.041). Among the three major writer enzymes assessed, WTAP was identified as the most significant modifier of global m6A. Phenotypically, WTAP knockdown reduced proliferation, invasion, and migration. The microarray analysis showed that WTAP knockdown was significantly associated with reduction of activities in several important signaling pathways such as EGFR, KRAS, and MYC. Furthermore, the significant decrease of m6A in individual transcripts such as EGFR, MAPKAPK2, and MYC was confirmed in WTAP-deprived PC9 cells. Reporter assays containing wild type or mutant EGFR-3’UTR at m6A modification sites indicated that EGFR expression change was mediated by WTAP through its regulation of m6A abundance. Immunohistochemical analysis demonstrated that increased WTAP expression was an independent worse prognostic factor in patients with NSCLC (hazard ratio 1.892; 95% confidence interval 1.356-2.64; P<0.001).

      Conclusion

      WTAP accelerates tumor aggressiveness through, in part, adding m6A in several important oncogenic transcripts such as EGFR and therefore increasing their expression. Both global m6A abundance and WTAP expression levels are prognostic in patients with NSCLC.

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