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C.E. Macaulay



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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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      MO15.10 - ELF3 is a novel oncogene frequently activated by genetic and epigenetic mechanisms in lung adenocarcinoma (ID 1024)

      17:10 - 17:15  |  Author(s): C.E. Macaulay

      • Abstract
      • Presentation
      • Slides

      Background
      Lung cancer remains the cause of the most cancer-related deaths each year, with a 5 year survival rate of less than 15%. The predominant type of lung cancer is non-small cell lung cancer, and the majority of these cases consist of the adenocarcinoma (AC) histology. Oncogenes such as EGFR and KRAS are well defined drivers of AC, but in approximately 50% of cases the driver alterations are unknown. Furthermore, not all defined drivers are drugable. Additional oncogenes are clearly involved in driving this subtype, and must be elucidated to better understand AC biology and improve treatment. ELF3 is an member of the E-Twenty Six (ETS) transcription factor family, which includes several well known oncogenes such as ETS1. Expression of ELF3 is uniquely epithelial-specific, with high expression in fetal but not adult lung tissue. ELF3 overexpression has been reported in a handful of clinical AC cases and cell lines, however a comprehensive analysis of the extent and impact of this overexpression is lacking. Therefore we conducted a multi-'omic, functional analysis of ELF3, and hypothesize ELF3 represents a novel oncogene in lung AC.

      Methods
      ELF3 was interrogated in a multidimensional integrative manner by assessing copy number (SNP 6.0), methylation (Illumina HM27), and expression (Illumina) data from a panel of 83 AC tumors and matched adjacent non-malignant tissues. ELF3 expression was also assessed in The Cancer Genome Atlas (TCGA) public database. Stable ELF3 mRNA knock-down models were established in AC cell lines with high ELF3 expression, and these models were used to assess the role of ELF3 in cell viability and proliferation via MTT and BrdU incorporation assay, respectively. Knock-down models were also used to assess the impact of ELF3 overexpression on tumor growth in vitro and in vivo by soft agar colony formation assay and flank injections of NOD-SCID mice. Subcellular localization of ELF3 was determined by western blot and confirmed with immunofluorescence. In addition, an ELF3 overexpression model was established in immortalized Human Bronchial Epithelial Cells (HBECs) to assess proliferation and soft agar colony formation in a non-malignant model system.

      Results
      ELF3 was found to be frequently overexpressed in our cohort (72%) and the TCGA cohort (80%). This upregulation correlated significantly with high frequencies of sequence gain (49%) and hypomethylation (71%), often seen within the same tumor. In fact, 82% of tumors with ELF3 overexpression had concurrent gain and/or hypomethylation of the ELF3 locus. Knock-down of ELF3 in cell models led to significantly reduced cell viability and proliferation. Western blot and IF revealed ELF3 to be predominantly located in the nucleus, indicating ELF3 likely behaves through its transcription factor activity. A similar hyperproliferative phenotype was seen in the HBEC ELF3 overexpression models.

      Conclusion
      The high frequency of ELF3 overexpression (>70%) observed in lung AC is accompanied by frequent DNA-level selection events. The affect of ELF3 on cell proliferation suggests that ELF3 is a novel oncogene in lung AC. Further studies are warranted to determine the mechanism by which ELF3 drives hyperproliferation and potentially other oncogenic functions to define novel drugable targets for this disease.

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    O18 - Cancer Control and Epidemiology II (ID 133)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Prevention & Epidemiology
    • Presentations: 1
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      O18.01 - Multi-'omic analysis of an arsenic-associated lung squamous cell carcinoma reveals specific DNA level signatures (ID 283)

      10:30 - 10:40  |  Author(s): C.E. Macaulay

      • Abstract
      • Presentation
      • Slides

      Background
      Chronic low-level exposure to arsenic is an emerging cancer risk factor in many parts of the world, including North America. The lung is one anatomical site prominently affected by the carcinogenic effects of arsenic, evident by the striking incidence of lung cancer in never smokers with chronic exposure. Histologically, arsenic related lung tumors are indistinguishable from those induced by other lung carcinogens, and molecularly, arsenic specific DNA copy-number, methylation and expression changes have been identified. Arsenic mediated carcinogenesis occurs through a combination of molecular mechanisms; however, high resolution, multi-'omic analyses of arsenic related tumors have been difficult due to the lack of fresh frozen samples required to obtain high quality DNA and RNA. In this study, we sought to characterize global changes in DNA sequence and methylation levels and their impacts on gene expression in a lung tumor from a patient with chronic arsenic exposure (As-LUSC).

      Methods
      Tumor and non-malignant lung tissues were obtained from a never smoker with lung squamous cell carcinoma (LUSC) who had no family history of lung cancer and 50 years of chronic exposure to high levels of arsenic-contaminated drinking water. Whole genome sequencing was performed and the tumor's mutational signature was compared to those observed in 194 previously characterized NSCLC tumors from the cancer genome atlas (TCGA). DNA methylation was measured using high density methylation arrays and gene expression by RNA sequencing.

      Results
      The As-LUSC exhibited alterations typical of LUSC, such as copy number gains at 3q26 (SOX2 locus) and expression of squamous markers including up-regulation of KRT6B, DSG3, MMP12, KRT5, and down-regulation of PDK4, which are consistent with LUSC histology. However, the As-LUSC harbored a low number of point mutations (only 49 non-synonymous mutations affecting coding DNA sequences) and had a remarkably high fraction of T>G/A>C and low fraction of C>A/G>T transversions, which are features uncharacteristic of LUSCs that suggest arsenic is associated with a distinct mutational spectrum. Furthermore, at the gene level, we identified a G>C mutation in TP53 which is rare in lung tumors (<0.2%) but has been observed in other arsenic-related malignancies. Clustering analysis using ~450,000 methylation probes revealed that the As-LUSC methylation profile was completely distinct from never smoker LUSCs from the TCGA. Of interest, the As-LUSC exhibited lower levels of methylation at CpG islands sores that are not associated with genes, although have been described to exhibit cell type specific methylation patterns.

      Conclusion
      By applying whole genome sequencing, methylation and expression profiling of a LUSC from a never-smoker patient chronically exposed to arsenic, we identified a distinct mutational spectrum and methylation pattern in the As-LUSC. Our results support the concept that arsenic induces lung cancers through mechanisms different from tobacco smoke and other carcinogens. Further study of the mutational profiles of additional arsenic-related cancers is warranted and may yield valuable insight into arsenic associated tumourigenesis, leading to the development of novel diagnostic and therapeutic targets for environmental monitoring and treatment.

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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.02-004 - Differential pathway disruption in lung adenocarcinomas from current and never smokers - A multi-omics data integration analysis (ID 1072)

      09:30 - 09:30  |  Author(s): C.E. Macaulay

      • Abstract

      Background
      Lung cancers in smokers and never smokers (NS) are distinct clinical diseases. Specific molecular differences identified in these two groups include: EGFR and KRAS mutation, DNA methylation levels at specific loci, and most recently, global mutation spectra. However, much remains to be understood about the biology driving lung tumourigenesis in smokers and NS in order to improve treatment outcome. To date, no multi-dimensional integrative genomics (i.e. multi-omics) analysis designed to specifically compare current (CS) and NS lung tumours has been performed. We hypothesize that a multi-omics analysis which considers each tumour as its own unique perturbed system (as opposed to a grouped approach) will reveal molecular mechanisms of lung adenocarcinoma (AC) biology that are common or different in CS and NS.

      Methods
      Copy number, DNA methylation, and gene expression profiles were generated for lung AC and matched non-malignant lung tissues from 34 CS and 30 NS. PCR was performed to determine EGFR and KRAS mutation status. Copy number, methylation and expression alterations were integrated for 14,000 genes on an individual tumour basis. Disrupted genes were ranked according to the magnitude of alterations they exhibited using a novel algorithm we developed denoted MITRA. Of the genes scored by MITRA, those ranking in the 99th and 1st (top) percentiles for up- and downregulation, respectively, were subjected to Ingenuity Pathway Analysis (IPA). IPA was performed separately on all 64 lung tumours and pathway results for CS and NS were compared.

      Results
      We identified 361 genes that ranked in the top percentiles for up- or downregulation in at least 20% of the lung ACs we assessed. Identification of recurrent RASSF1A downregulation, and EGFR upregulation predominantly in NS demonstrates the ability of our ranking algorithm to prioritize genes known to be involved in lung tumour biology using multi-dimensional genomics data. To determine cellular pathways and functions likely deregulated as a consequence of gene disruption, we performed IPA on each tumour and determined the frequency of individual pathway disruption across tumours. This analysis revealed 88 annotated pathways with a minimum disruption frequency of 15% in either or both CS and NS. Commonly affected pathways involved: adhesion and extravasation implicating tumour invasion and migration; various catabolic and anabolic processes implicating cell metabolism; and several specific signaling pathways including atherosclerosis and Wnt/β-catenin signaling implicating inflammation and cell proliferation. Comparison of the pathways identified in CS and NS revealed 13 differentially disrupted pathways (Fisher's Exact test p < 0.05 and disruption frequency difference > 15%). Eleven pathways were preferentially disrupted in CS and affected metabolic, immune response, and inflammatory pathways. Anandamine degradation and ephrin receptor signaling were preferential to NS.

      Conclusion
      Our novel, multi-omics tumour system based approach revealed genes prominently disrupted in CS and NS lung AC which were associated with several cellular pathways commonly or differentially disrupted in these two groups. Pathways affected by genes disrupted at both the DNA and RNA level may contribute to the distinct clinical characteristics associated with CS and NS lung cancer and may serve as targets for intervention.