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G. Stewart



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    MA02 - RNA in Lung Cancer (ID 377)

    • Event: WCLC 2016
    • Type: Mini Oral Session
    • Track: Biology/Pathology
    • Presentations: 2
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      MA02.08 - Deregulation of Cis-Acting Long Non-Coding RNAs in Non-Small Cell Lung Cancer (ID 6303)

      14:20 - 15:50  |  Author(s): G. Stewart

      • 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 17%. Targeted therapeutics have been developed against drivers of the lung adenocarcinoma (AC) subtype, but are relevant only to the proportion of patients harbouring these genetic aberrations, emphasizing the need to explore alternative mechanisms of AC development. Natural antisense transcripts (NATs) are long non-coding RNA (lncRNA) products expressed from the opposite strand of coding mRNAs. NATs can function in cis or trans to regulate the transcriptional activity of their cognate gene partner in either a positive or negative fashion. Here we take a novel approach to identify cis- NATs deregulated in lung AC, and explore the function of these genes with regards to their protein coding partner genes.

      Methods:
      We performed RNA-sequencing on a set of 36 lung AC and matched non-malignant lung tissues. A sign-rank test was used to identify NATs and partner genes with significantly altered expression between tumor and matched normal tissues. These findings were validated in an external dataset of 50 lung AC tumors with matched non-malignant tissue obtained from The Cancer Genome Atlas (TCGA). Survival analysis was performed using a Cox Proportional hazard model, as well as the log-rank method.

      Results:
      Analysis of Illumina Hi-seq data from TCGA revealed the majority (79%) of deregulated sense-antisense partnerships observed in AC displayed concordant regulation. However, several discordant cis-NAT pairs were identified including an antisense to OPA INTERACTING PROTEIN 5 (OIP5), a gene required for chromatin segregation, as well as an antisense to HIGH MOBILITY GROUP A1 (HMGA1) a gene involved in the metastatic progression of many cancer types. Both the antisense to OIP5 (OIP5-AS1) as well as the antisense to HMGA1, (HMGA1-AS1) were significantly underexpressed in AC, while we find the overlapping protein coding partner genes to be significantly overexpressed, suggesting that these genes may negatively regulate their sense counterparts. In addition both OIP5 and HMGA1 are significantly associated with 5-year survival. Patients with higher expression levels of either of these genes had a significantly shorter overall survival time than patients with low expression levels, highlighting the potential clinical importance of these genes.

      Conclusion:
      This study characterizes the landscape of antisense expression in AC and highlights novel mechanisms of oncogene regulation through natural antisense transcripts. Characterizing these oncogene regulatory mechanisms could uncover therapeutic intervention points and further our understanding of AC biology.

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      MA02.09 - Long Non-Coding RNA Expression from Pseudogene Loci as a Novel Mechanism of Cancer Gene Regulation (ID 6287)

      14:20 - 15:50  |  Author(s): G. Stewart

      • Abstract
      • Presentation
      • Slides

      Background:
      The advent of next generation sequencing has lead to the discovery of the functional importance of non-coding RNAs (ncRNAs) in a wide variety of cellular processes, and these genes can be exploited by tumours to drive the hallmarks of cancer. Pseudogenes are DNA sequences that are defunct relatives of their functional parent genes but retain high sequence homology. Long non-coding RNAs (lncRNAs) have been shown to regulate protein-coding genes; however, complex folding patterns make lncRNA function difficult to predict. Several lncRNAs expressed from pseudogene loci have been shown to regulate the protein-coding parent genes of these pseudogenes in trans due to sequence complementarity. The biological impact of this mechanism has not been investigated in lung adenocarcinoma (LUAD). We hypothesize that expression changes in lncRNAs expressed from pseudogene loci can affect the expression of corresponding protein-coding parent genes in trans, and that these events provide an alternative mechanism of cancer gene deregulation in LUAD tumourigenesis.

      Methods:
      We analysed RNA-seq data from 50 LUAD with matched non-malignant tissue obtained from the TCGA for both protein-coding and non-coding gene expression. Significantly differentially expressed lncRNAs located within pseudogene loci were identified by sign-rank test (p<0.001). Mann Whitney U-tests were used to identify lncRNA-parent gene pairs which significantly correlated expression, and survival analysis was performed using a Cox proportional hazard model.

      Results:
      Our analysis has identified 172 lncRNAs expressed from pseudogene loci that were significantly deregulated in LUAD. Remarkably, many of these lncRNAs were expressed from the loci of pseudogenes related to known cancer genes. One of these lncRNAs, CTD-2583A14.8, was expressed from a pseudogene to ubiquitin-conjugating enzyme E2C (UBE2C), which regulates tumor growth, apoptosis, and angiogenesis through phospho-ERK1/2. We find CTD-2583A14.8 as well as the UBE2C parent gene to be significantly upregulated in LUAD tumours compared to matched normal tissue. Furthermore, tumours with higher levels of CTD-2583A14.8 have significantly higher levels of UBE2C expression than tumours with low levels of CTD-2583A14.8, indicating that CTD-2583A14.8 may positively regulate UBE2C in trans.

      Conclusion:
      Here we show expression of lncRNAs within pseudogene loci is deregulated in LUAD, and can correlate with the expression of their protein-coding counterparts. Many of these genes associated with this putative lncRNA-pseudogene-protein-coding axis have previously been implicated in cancer. Therefore, this represents an alternative mechanism of cancer gene deregulation, and may represent novel therapeutic intervention points for the treatment of LUAD.

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    P2.01 - Poster Session with Presenters Present (ID 461)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P2.01-023 - Deregulation of Small Non-Coding RNAs at the DLK1-DIO3 Imprinted Locus Predicts Lung Adenocarcinoma Patient Outcome (ID 6142)

      14:30 - 15:45  |  Author(s): G. Stewart

      • Abstract
      • Slides

      Background:
      Deregulation of small RNAs at the imprinted DLK1-DIO3 locus has been linked to lung adenocarcinoma (LUAD) patient outcome. While the contribution of microRNAs (miRNAs) is established, the role of Piwi-interacting RNAs (piRNAs), small RNAs involved in epigenetic regulation of gene transcription, is unexplored. We quantified expression of piRNAs and miRNAs mapping to this locus in two independent cohorts of LUAD and assessed the ability of a combined miRNA/piRNA signature to improve patient outcome stratification.

      Methods:
      Expression levels (RPKM) for miRNA/piRNA were determined from small RNA sequencing experiments from two cohorts (TCGA, n=154, 5-year follow up; BCCA, n=77, 8-year follow up). Associations with patient overall survival (OS) and recurrence free survival (RFS) were calculated by inputting miRNA and piRNA expression combinations into a Cox proportional hazard model. Risk scores were calculated by multiplying the expression value for each gene by its hazard coefficient, and summed per sample. Risk scores were ranked and divided into tertiles for log-rank survival analysis. DNA-level piRNA targets were predicted using MiRanda based on sequence complementarity in the region 3.5kb upstream of the transcription-start site of all human transcripts from ENSEMBL. Transcript-level miRNA targets were predicted using the miRDIP algorithm, which integrates 13 miRNA target prediction algorithms and six miRNA prediction databases.

      Results:
      Only 7 out of 138 piRNAs mapping to the locus were expressed. A combined miRNA/piRNA signature improved both OS and RFS predictions compared to signatures of miRNAs or piRNAs alone. In TCGA, log-rank analysis of risk groups indicated only the miRNA/piRNA signature significantly stratified patients (OS p=0.0038, RFS p=0.0229) into low, intermediate, and high risk groups compared to separated miRNA or piRNA signatures. Similarly, in the BCCA dataset, only the combined miRNA/piRNA signature significantly stratified high, intermediate, and low risk groups (p=0.0019). Target prediction of piRNAs and miRNAs from the signature indicated that 34 genes may be regulated at both the DNA (piRNA) and mRNA (miRNA) level.

      Conclusion:
      We find the combination of miRNA and piRNA expression derived from the DLK1-DIO3 locus produces a superior stratification of patient outcome than either metric alone. While the contribution of miRNAs to patient risk stratification is established, the improved model performance derived from the addition of piRNAs adds another layer of gene regulation at the DNA-level. Model performance is optimal when these two small RNA species are considered simultaneously; suggesting their coordinated biological effects as a result of deregulation at this locus in LUAD.

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