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Mayu Koike



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    P2.03 - Biology (ID 162)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Biology
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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      P2.03-23 - UHRF1 as a Potential Therapeutic Target for KRAS Mutated Non-Small Cell Lung Cancer (ID 687)

      10:15 - 18:15  |  Author(s): Mayu Koike

      • Abstract

      Background

      KRAS mutation occurring in approximately 20% of non-small cell lung cancer (NSCLC) functions as a driver oncogene and it serves as a potential therapeutic target. However, development of KRAS-targeted drugs has not been successful primarily because of difficulty in pharmacologically inhibiting a constitutively activated KRAS signaling. Activation of several driver oncogenes including mutant KRAS in normal cells causes cellular senescence, leading to permanent cell cycle arrest, which is termed oncogene-induced senescence (OIS). Thus, KRAS mutated cancers are thought to acquire additional alterations that allow bypassing OIS and such alterations could be good targets for them. With this background, we designed the present study to identify therapeutic targets whose inhibition cause growth suppression in KRAS mutated NSCLC through inducing OIS.

      Method

      We established cdk4/hTERT-immortalized normal human bronchial epithelial cell (HBEC) that expresses mutant KRAS upon tetracycline treatment (designated HBEC-RIN). A semi-genome wide shRNA library (DECIPHER) targeting 5,000 genes was transduced in HBEC- RIN. Each shRNA vector is barcoded with unique sequence for quantification. Two weeks after culturing the cells, we extracted genomic DNA from cells at two points: before (point#1) and after (point #2) tetracycline treatment. The barcodes of DNA were sequenced with NGS at a depth of 20 million reads. The effects of senescence-bypass were determined by dividing the normalized barcode abundance at point #2 by that of point#1. The significance of change of each gene was determined by performing t-test to compare replicates of shRNA with a given gene with those of luciferase. Senescence was evaluated by senescence associated b-gal staining. Three KRAS mutated lung cancer cell lines (A549, H2009, and H460) were used to examine effects of silencing candidates of OIS-bypassing genes. Association between prognosis and expression of candidate genes in NSCLC patients was analyzed with several online datasets.

      Result

      We drew a volcano plot from results of a shRNA screen. We selected genes based on significant average suppressive effects. In the present study, we focused on Ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1) because we were able to confirm OIS-bypassing ability of this gene in HBEC-RIN by its transient silencing with synthetic siRNA. Colorimetric growth and colony formation assays showed that UHRF1 knockdown suppresses cell growth and colony formation in H2009 but not in A549 or H460. Importantly, we found that expression of UHRF1 mRNA correlated with worse prognosis in patients with NSCLC in multiple independent datasets, suggesting its potential as a prognostic marker.

      Conclusion

      These results suggest that UHRF1 is a potential therapeutic target for KRAS mutated NSCLC and that it is a prognostic marker for NSCLC.