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K.S. Enfield



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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)

      16:15 - 17:45  |  Author(s): K.S. Enfield

      • 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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    O12 - Lung Cancer Biology II (ID 87)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      O12.03 - Pseudogenes as miRNA sponges in non-small cell lung cancer (ID 3455)

      10:30 - 12:00  |  Author(s): K.S. Enfield

      • Abstract
      • Presentation
      • Slides

      Background
      Lung cancer is the most common cause of cancer death worldwide, with a five-year survival of less than 15%. This poor therapeutic outcome is largely due to complex molecular backgrounds as well as typically late stage at diagnosis, with most patients presenting with unresectable local tumours or metastatic disease. While mutations of driver genes is a well known mechanism of tumorigenesis, approximately half of all non-small cell lung cancer (NSCLC) tumours harbour no known actionable oncogenic drivers, emphasizing the need to explore alternative mechanisms. New sequencing technologies have allowed investigation of previously unexplored areas of the genome and revealed that several classes of non coding RNAs (ncRNAs), those with no protein product, are involved in tumourigenesis, emphasizing the need for further exploration and study. MicroRNAs (miRNAs) have emerged as major players in lung carcinogenesis, displaying both oncogenic and tumor suppressive functions through translational inhibition of genes containing miRNA target sequences. Pseudogenes are non-coding relatives of protein-coding genes that contain a high degree of sequence similarity with their parent genes, thus sharing many of the same miRNA target sequences. As a result, when overexpressed, a pseudogene can function as a miRNA "decoy" protecting its parent gene from miRNA-mediated translational inhibition. DNA copy number (CN) alterations (gain of oncogenes/loss of tumour suppressors), is a major molecular mechanism driving cancer. Like protein coding genes, CN alterations can influence ncRNA expression levels, and several pseudogenes have been reported to be deregulated at the CN level in other cancer types. We hypothesize that pseudogenes of lung cancer-related genes are deregulated at the CN level in NSCLC.

      Methods
      Global CN profiles for 83 lung adenocarcinomas, and 12 squamous cell carcinomas, as well as paired adjacent non-malignant tissues were generated on the Affymetrix SNP 6.0 array. Frequencies of DNA CN alterations were assessed at candidate pseudogene loci (gain>2.3 copies, loss<1.7 copies). Candidate pseudogenes (1) have a parent gene that has been previously reported to play a role in cancer biology, (2) are expressed in human tissue, and (3) share at least one conserved miRNA binding site with its parent gene.

      Results
      Several pseudogenes for OCT4 (octamer-binding transcription factor 4), an early embryonic transcription factor, were found to be frequently gained (46.9-34.9%), and could protect OCT4 from miRNA-mediated translational inhibition. Additionally, pseudogenes for E2F3 (E2F Transcription Factor 3), a potent cell cycle regulator, as well as those for the well known lung cancer oncogene BRAF, were found to have high frequencies of CN alteration (36.1%, and 19.2%, respectively). These high frequencies of alteration suggest that these pseudogenes play an important role in NSCLC.

      Conclusion
      These results suggest that pseudogenes are clonally selected for at the DNA level, and pseudogene-mediated protection of oncogenic transcripts from miRNA-mediated translational inhibition may represent a novel mechanism of oncogenicity in NSCLC. Analyses of pseudogene expression and corresponding parent gene protein level in cell models will yield insight into how this class of ncRNA affects tumourigenesis, potentially leading to improvements in early detection, diagnosis, and treatment.

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    P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.02-020 - MicroRNA deregulation associated with node positive lung adenocarcinoma (ID 3426)

      09:30 - 16:30  |  Author(s): K.S. Enfield

      • Abstract

      Background
      Lung cancer is the leading cause of cancer-related deaths worldwide, with poor survival largely attributed to late stage of disease at diagnosis and frequent metastasis. Typically, tumor cells colonize the regional lymph nodes before spreading to distant sites, significantly dampening prognosis. The presence and number of affected nodes can be detected by routine imaging procedures, but occasionally, additional affected nodes are not detected until time of surgery, which impacts staging. The identification of biomarkers that stratify node positive (NP) and node negative (NN) patients could improve detection of aggressive disease, while a deeper understanding of the underlying biology of NP lung cancer could lead to the design of novel therapeutic interventions. MicroRNAs (miRNAs) are major regulators of gene expression that control a wide range of cellular processes, and have been shown to be excellent biomarker candidates due to their stability in biofluids. The role of miRNA deregulation in NP lung cancer is poorly understood; therefore, we sought to compare miRNA expression levels of lung adenocarcinoma (AC) cases with and without nodal involvement in order to identify miRNAs associated with tumor aggressiveness. We hypothesize that a subset of miRNAs are specifically deregulated in NP AC, representing potential biomarkers, and that identification of miRNA-mRNA interaction networks will shed light on the biological mechanisms of tumor spread that may be therapeutically exploited.

      Methods
      A panel of 45 NN and 28 NP primary AC tumors were collected along with paired adjacent non-malignant tissues. All samples underwent expression profiling of miRNA (Illumina GAIIx small RNA sequencing) and mRNA (Illumina microarray). NN and NP groups were analyzed separately as follows: matched tumor and normal miRNA normalized read count comparisons were performed (Wilcoxon Signed-Rank test, Bejamini-Hochberg corrected p<0.05), and miRNAs that were significantly deregulated in tumors were further investigated. miRNA fold changes (FC) were calculated on a case by case basis, and FC values were then compared between groups (Mann Whitney U Test p<0.05). miRNAs that (i) displayed a minimum 2-fold frequency of disruption >50% of NP cases, (ii) had a median FC>2 in the NP group, (iii) had a Signed-Rank corrected p<0.05, and (iv) had a MWU p<0.05 were considered to be NP-specific. Target prediction analysis of these miRNAs was then performed, and mRNA expression data was utilized to ensure predicted target expression was anti-correlated with miRNA expression. Candidate targets were input into Ingenuity Pathway Analysis to determine pathways implicated in NP AC.

      Results
      Fourteen miRNAs were NP-specific according to our criteria. These miRNAs included those previously associated with biology and metastasis of epithelial cancers, as well as miRNAs novel to lung AC. Target analysis implicated several pathways and functions previously associated with a metastatic phenotype such as the TGFβ pathway.

      Conclusion
      These results indicate that miRNA expression differs between NP and NN lung AC, further demonstrating the involvement of miRNAs in the regulation of the metastatic process. The role of these miRNAs as prognostic markers and serum biomarkers, as well as the mechanism of action of these miRNAs in AC biology must be further explored.