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M.J. Pajares



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    MINI 34 - RNA and miRNA (ID 162)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI34.11 - Identification and Functional Characterization of Non-Small Cell Lung Cancer-Associated Splice Variants and Splicing Factors (ID 2101)

      18:30 - 20:00  |  Author(s): M.J. Pajares

      • Abstract
      • Presentation
      • Slides

      Background:
      Deregulation of alternative splicing has become a hallmark of cancer. In non-small cell lung cancer (NSCLC), the biological importance of splicing is evidenced by the identification of aberrant RNA transcripts associated with somatic mutations in genes encoding splicing factors. We have previously developed ExonPointer, an algorithm optimized to detect differential splicing cassette events from data obtained in microarrays containing probes in exons and junctions. Our present objective was to apply this technology for the identification and characterization of cancer-associated splice variants and splicing factors in NSCLC.

      Methods:
      We applied ExonPointer for the identification of differential splice forms in lung cancer tissues (8 adenocarcinomas, 13 squamous cell carcinomas and 1 large cell carcinoma) and matched normal lung. We validated the events by RT-PCR and used bioinformatics tools, such as DAVID and Ingenuity Pathway Analysis, for cluster enrichment analyses. siRNA knockdown of specific splice isoforms was used for functional analyses. Prognostic studies were performed by immunohistochemistry in 127 primary tissues from patients with NSCLC.

      Results:
      The validation rate for the top 20 differentially expressed splice events identified by ExonPointer was 70%. Gene cluster analyses using the first 250 events showed a significant enrichment of cancer-related clusters such as Cellular Growth and Proliferation, or Cell Death and Survival. Among the validated genes, we identified Extended synaptotagmin-2 (ESYT2). ESYT2 is a membrane protein that mediates fibroblast growth factor receptor-1 (FGFR1) endocytosis and actin dynamics. A significantly different pattern of ESYT2 alternative splicing was found in primary lung tumors as compared to normal lung tissue (p<0.001). In particular, an isoform containing an extra exon was overexpressed in cancer tissues, while the expression of the canonical isoform was decreased. We found a significant correlation between the splicing pattern of ESYT2 and the expression of FGFR1 in a panel of 43 lung cancer cell lines (r=-0.724, p<0.001). Using siRNA downregulation, we analyzed the implication of the ESYT2 isoforms in tumor biology and demonstrated a distinct role of the splice isoforms in actin and tubulin cytoskeleton organization. We also searched for splicing factors responsible for the splicing of ESYT2. We found that the ratio of ESYT2 isoforms correlated with the expression of the splicing factor QKI in lung cancer cell lines (r=-0.793, p<0.001). Moreover, in vitro downregulation of QKI markedly affected ESYT2 splicing. Interestingly, we found a significant enrichment of QKI targets in the list of differentially spliced genes identified by ExonPointer (p<0.001), suggesting that this factor is a critical regulator of splicing in lung cancer. Finally, we observed a significant downregulation of QKI expression in primary NSCLC compared to adjacent normal lung cancer cells (p<0.001), and an association between the nuclear expression of this factor and disease-free survival (HR=0.61; 95%CI=0.35-1.05) or overall survival (HR=0.44; 95%CI=0.21-0.94).

      Conclusion:
      Using a novel analytical tool we have identified new splicing variants with functional relevance in lung cancer. Moreover, changes in splicing events in lung primary tumors were found to be largely regulated by the splicing factor QKI, a potential tumor suppressor gene downregulated in NSCLC and associated with the prognosis of the disease.

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