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L. Pikor



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    MO15 - Novel Genes and Pathways (ID 89)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO15.09 - Amplification of YEATS4, a novel oncogene in NSCLC, inhibits the p53 pathway and increases resistance to cisplatin (ID 1073)

      16:15 - 17:45  |  Author(s): L. Pikor

      • Abstract
      • Presentation
      • Slides

      Background
      Characterization of lung cancer genomes has revealed a number of genes critical to tumorigenesis (e.g. EGFR, KRAS, EML4-ALK), resulting in significant changes to the treatment of lung cancer and an increase in survival for a subset of patients. These successes have prompted the search for additional driver alterations, leading to the discovery of a number of recurrently mutated or amplified genes and gene fusions with promising clinical utility. Distinguishing the key mechanisms and causal events driving tumorigenesis will lead not only to a better understanding of lung cancer phenotypes and biology, but also to new molecular markers and therapeutic targets. Using an integrative analysis of gene expression and copy number data to identify novel candidate oncogenes, we identified the chromosomal region at 12q13-15, and more specifically, the putative transcription factor YEATS4 (YEATS domain containing 4) as frequently amplified and overexpressed in NSCLC. Amplification of YEATS4 has been reported in dedifferentiated liposarcomas and in the earliest stages of glioma and astrocytoma.

      Methods
      Copy number profiles were generated for 261 NSCLC tumors (169 adenocarcinomas (AC) and 92 squamous cell carcinomas (SqCC)) and expression profiles for a subset of tumors with matched non-malignant tissue. Recurrent DNA amplifications were identified using the GISTIC algorithm. Copy number data were integrated with gene expression data to identify genes frequently amplified and overexpressed (defined as a 2-fold difference in expression between tumor and matched non-malignant tissue). The functional significance of YEATS4 was assessed by lentiviral knockdown in lung cancer cell lines with and without YEATS4 amplification and ectopic expression in human bronchial epithelial cells (HBECs). In vitro and in vivo assays measuring proliferation, anchorage independent growth, senescence, apoptosis, drug sensitivity and tumor growth were used to assess the phenotypic effect of YEATS4 gene expression manipulation.

      Results
      YEATS4 is gained or amplified and concomitantly overexpressed in over 20% of NSCLC tumors, with similar frequencies of amplification in both AC and SqCC. Although frequently co-amplified with MDM2, amplification of YEATS4 was observed to occur in the absence of MDM2 amplification, suggesting it is not merely a passenger event. Overexpression of YEATS4 in HBECs abrogated senescence, whereas knockdown reduced cell proliferation, impaired colony formation and induced cellular senescence in cell lines with YEATS4 amplification. Western blotting revealed increased p21, cleaved PARP and p53 in knockdown lines compared to empty vector controls, implicating YEATS4 as a negative regulator of the p21-p53 pathway. Moreover, YEAST4 expression was found to correlate with cisplatin sensitivity, as overexpression increased resistance and knockdown conferred sensitivity. Consistent with our in vitro findings, tumor size and growth were significantly reduced in mice injected with YEATS4 knockdown cells relative to control mice. Furthermore, survival analysis revealed that patients expressing high levels of YEATS display poorer outcomes.

      Conclusion
      Our findings reveal YEATS4 as a novel candidate oncogene frequently amplified and overexpressed in NSCLC. Gene expression manipulation resulted in distinct phenotypic changes consistent with oncogenic function, and suggesting YEATS4 amplification is a novel mechanism contributing to NSCLC tumorigenesis.

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    P3.02 - Poster Session 3 - Novel Cancer Genes and Pathways (ID 149)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.02-006 - Downregulation of the candidate tumor suppressor gene SIRPA induces senescence mediated by Rb and p27 and is associated with mutation of EGFR (ID 1074)

      09:30 - 16:30  |  Author(s): L. Pikor

      • Abstract

      Background
      The epidermal growth factor receptor (EGFR) signaling pathway is involved in numerous biological processes including proliferation and apoptosis, migration/invasion, and angiogenesis, and has emerged as one of the most important and frequently deregulated pathways in NSCLC. The discovery of oncogenic, activating mutations in the tyrosine kinase domain of EGFR and DNA amplification of EGFR have led to the development of multiple targeted therapeutics against this pathway. While effective at prolonging survival, these targeted therapies are only applicable to a subset of patients (~15-20%) that harbour these alterations and resistance to treatment ultimately develops. As multiple genomic and epigenomic mechanisms can disrupt genes, a comprehensive understanding of the genetic alterations affecting genes within this pathway is required. An integrative, multi-dimensional genomics approach can detect genes disrupted by multiple mechanisms which may otherwise be overlooked if only a single genomic dimension were assessed, improving the ability to identify causal genetic events and decipher downstream consequences.

      Methods
      A multi-dimensional integrative analysis of copy number, DNA methylation and gene expression profiles on 77 adenocarcinomas and matched non-malignant tissue, was performed to investigate the complement of genetic alterations affecting the EGFR pathway. Novel candidate genes were validated in external datasets and immunohistochemical analysis of a tissue microarray was used to verify disruption at the protein level and to correlate expression with clinical features. The tumor suppressive effects of SIRPA were assessed by stable knockdown and in vitro assays on a panel of lung cancer cell lines. The effect of SIRPA downregulation on TKI sensitivity was assessed by dose response assays.

      Results
      Of the 35 genes examined, 11 were aberrantly expressed in over 50% of tumors, with 6 (RRAS, SIRPA, PIK3R1, TGFA, ERBB2 and EGFR) ranking in the 95th percentile of altered genes. Of these genes, all but SIRPA are known to be frequently disrupted in NSCLC and play a role in tumorigenesis. SIRPA is a transmembrane protein that negatively regulates receptor tyrosine kinsase activity and is frequently downregulated at both the mRNA and protein level in NSCLC tumors and cell lines. Underexpression of SIRPA is associated with EGFR mutations and is more prominent in adenocarcinoma than squamous cell carcinoma. Downregulation of SIRPA enhanced colony formation and wound healing but impaired viability and suppressed proliferation. Interestingly, SIRPA knockdown induced a senescent phenotype through the accumulation of p27 and Rb in its unphosphorylated state thereby blocking progression of the cell cycle. These results suggest senescence induced by SIRPA downregulation is a tumor suppressive mechanism that must be overcome to develop tumors.

      Conclusion
      Our integrative analysis of the EGFR pathway revealed SIRPA as one of the most frequently deregulated genes within the pathway. SIRPA functions as a tumor suppressor gene, controlling a number of biological functions through the inhibition of singaling pathways downstream of EGFR. To our knowledge, this is the first study to report a role for SIRPA in NSCLC tumorigenesis.