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V.D. Martinez



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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)

      15:14 - 15:20  |  Author(s): V.D. Martinez

      • 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)

      15:20 - 15:26  |  Author(s): V.D. Martinez

      • 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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    P1.02 - Poster Session with Presenters Present (ID 454)

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 1
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      P1.02-073 - Characterizing the Genomes of Lung Adenocarcinomas from Never Smokers Reveals SHPRH as a Novel Candidate Tumour Suppressor Gene (ID 4772)

      14:30 - 14:30  |  Author(s): V.D. Martinez

      • Abstract

      Background:
      Approximately 15 to 25% of lung adenocarcinomas (LAC) arise in never smokers. They develop through mechanisms distinct from those that affect smokers and are associated with unique histological and molecular characteristics. A significant fraction of LACs in never smokers do not have mutations in known oncogenic driver genes such as EGFR/ALK/KRAS. Furthermore, mutations in oncogenic driver genes appear to be insufficient for tumorigenesis, suggesting that additional alterations are required.

      Methods:
      To address these issues, we used whole-exome sequencing to comprehensively study 15 LACs from never smokers - seven “triple negative” tumors (with normal EGFR/ALK/KRAS) and eight EGFR-mutant tumors - with the goal of identifying novel mutant genes in these subsets. To identify mutated genes that confer a selective advantage a multistep approach was used to filter variants based on gene expression level, background mutation rate and gene size. Targeted sequencing of 180 genes in the original 15 and an extended panel of 85 tumor/normal pairs validated these alterations and indicated their prevalence in LAC. Sequence data was integrated with copy number and gene expression levels to determine mechanisms, and consequences, of gene disruption. Animal and cell models were used to functionally validate identified genes of interest and explore their role in LAC biology.

      Results:
      32 unique genes demonstrated significant evidence of conferring a selective advantage including known oncogenes (EGFR/ERBB2/MET) and tumor suppressor genes (p53/RB1/ATM). In addition, RNA-seq revealed fusions involving RET or ROS1 in one tumour each. The variations in MET consisted of truncating and splice-site mutations that we are currently investigating in transgenic mouse models. Pathway analysis indicated frequent mutation in genes implicated in PI3-kinase signaling, RNA splicing and histone modification. Importantly, we identified the hemizygous and homozygous loss of multiple genes from chromosome arm 6q - a genetic locus associated with familial lung cancer susceptibility - including a novel candidate tumor suppressor gene, SHPRH, based on its high frequency of biallelic disruption. SHPRH is an evolutionarily conserved E3-ligase that mediates crucial processes related to DNA repair. We found that SHPRH silencing increased transformation of normal lung cells, increased DNA damage and induced cell cycle changes while SHPRH inhibition sensitized LAC cells to topoisomerase II and PARP inhibitors.

      Conclusion:
      SHPRH inactivation may induce genetic alterations that cooperate with mutations in driver oncogenes to promote LAC development. Together, this work will expand our understanding of LAC initiation and progression in never smokers and may offer new biomarkers for response to therapy.

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

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 2
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      P2.01-022 - A PIWI-Interacting RNAs Co-Expression Networks as a Prognostic Factor in Lung Cancer (ID 5812)

      14:30 - 14:30  |  Author(s): V.D. Martinez

      • Abstract

      Background:
      PIWI-interacting RNAs (piRNAs) are small (24-32 nucleotides) non-coding RNAs. Their functions, widely conserved across species, are associated to epigenetic control of gene expression and maintenance of genomic stability by the repression of mobile elements. In humans, >23,000 piRNAs are known, showing tissue-specific expression patterns. While the aberrant expression of individual piRNAs has been identified in some cancer types, the role of piRNA co-expression networks in the development of lung tumors and their utility as molecular markers remains unexplored. By analyzing over 7000 piRNA transcriptomes from human tumors and non-malignant tissues, we have identified lung cancer (LC) specific expression networks associated with clinically-relevant tumor features and patient prognosis.

      Methods:
      We developed a custom small-RNA sequence analysis pipeline to generate >7,000 human piRNA transcriptomes. piRNA expression baseline was deduced from 6,378 piRNA transcriptomes (non-malignant/tumors) from 11 organ sites. In lungs, we analyzed 1,082 tumors and 209 non-malignant samples from two cohorts: BC Cancer Agency (BCCA) and The Cancer Genome Atlas (TCGA). Network analysis was performed using the weighted gene co-expression network analysis (WCGNA). We evaluated tumour aggressiveness by considering correlation to several clinical parameters, including stage, number of mutations, nodal/distant metastasis, and overall/disease-free survival. piRNA survival signatures were identified using a Cox Proportional Hazard model.

      Results:
      A subset of piRNA showed robust expression in somatic tissues. Expressed piRNAs display organ-specific patterns and mainly map to coding transcripts, suggesting a role in regulation of gene expression. In lungs, 204 piRNAs were consistently expressed in both LC cohorts. Tumor piRNA expression profiles are markedly different from their non-malignant counterparts (133 piRNAs were differentially expressed). The patterns differ between the adenocarcinoma and squamous cell carcinoma, and were influenced by smoking status. Network-based analysis identified piRNA expression changes in two modules of piRNAs are associated with aggressiveness tumor features, such as increased number of mutations, tumor size and nodal metastasis. Finally, combined expression of piRNAs define signatures associated with patient overall and recurrence free survival.

      Conclusion:
      We provide evidence of somatic, tissue-specific human piRNA expression. In lungs, aberrant expression patterns are associated with well-established etiological factors of cancer and seem to contribute to lung cancer subtype-specific biology. We discover that specific piRNA-based expression patterns characterize aggressive lung tumors and also exhibit prognostic value. The unique expression patterns of piRNAs offer an opportunity to better understand lung cancer-specific biology as well as develop novel prognostic markers for clinical application.

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      P2.01-037 - Molecular Biology Underlying COPD and Lung Cancer Converge on FOXM1 Network (ID 5773)

      14:30 - 14:30  |  Author(s): V.D. Martinez

      • Abstract

      Background:
      Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease associated with an up to 10-fold increased risk of lung cancer (LC). COPD and LC share common etiologies including genetic susceptibilities and risk factors, such as smoking. This study systematically characterizes the molecular overlap between COPD and LC.

      Methods:
      Small airway gene expression data was obtained from subjects with spirometry measures (n=267) (GSE37147). Genome-wide, multi-omics data for lung adenocarcinoma (LUAD) tumor and non-malignant lung tissues from two cohorts (TCGA, n=515; BCCA, n=90) was analyzed. Weighted correlation network analysis (WGCNA) was applied to identify clusters (modules) of highly correlated genes across airway expression profiles. Combined module expression (eigengene scores) were used to: 1) identify modules negatively associated with FEV~1~ and 2) calculate module preservation in lung tumors. Signaling network, pathway and gene ontology analyses were performed using IID, pathDIP, ClueGo and PARADIGM. Known and predicted protein-protein physical interactions (PPIs) were obtained from IID. Network analysis and visualization was performed in NAViGaTOR.

      Results:
      A module of 31 genes significantly co-expressed across small airways was negatively associated with FEV~1~ and preserved in LUAD tumors. Genes in this module were enriched in functions associated with cell cycle progression, and known and/or predicted to physically interact in the protein complex critical to mediating G2/M progression. The forkhead transcription factor FOXM1 network was the most highly perturbed entity across 515 LUAD tumors. FOXM1 is an essential mitotic protein, known to regulate expression of genes involved in cell cycle progression, as well as stress response to ROS and DNA damage, angiogenesis and metastasis. COPD-related airway mRNA changes and genes highly altered at the DNA and mRNA level in LUAD tumors directly converge on the FOXM1 regulated mitotic complex proteins and/or FOXM1 transcription factor network.

      Conclusion:
      FOXM1 is overexpressed in multiple cancer types where it is correlated with poor prognosis and oncogenic transformation of epithelia through induction of genomic instability. The convergence of COPD and LUAD changes on this network may underlie increased LC risk in COPD patients, warranting further exploration as a target for COPD treatment and/or LC prevention or treatment.