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B.C. Minatel



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    MA02 - RNA in Lung Cancer (ID 377)

    • Event: WCLC 2016
    • Type: Mini Oral Session
    • Track: Biology/Pathology
    • Presentations: 1
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      MA02.03 - Expression of Oncofetal miRNAs Inactivates NFIB, a Developmental Transcription Factor Linked to Tumour Aggressiveness in Lung Adenocarcinoma (ID 5224)

      14:32 - 14:38  |  Author(s): B.C. Minatel

      • Abstract
      • Presentation
      • Slides

      Background:
      Fetal and tumour development share striking similarities, such as intense cell proliferation, angiogenesis, increased cell motility, and immune evasion. Molecular regulators, including microRNAs (miRNAs), play important roles in both fetal lung development and in the malignant transformation of adult lung cells. Consequently, investigation of lung tumour biology in the context of lung development may reveal key regulatory mechanisms that tumours hijack from normal development, which potentially play critical roles in the pathology of lung cancer.

      Methods:
      131 pairs of non-small cell lung cancer (NSCLC) tumour and non-malignant lung tissues and 15 human fetal lung tissue samples were profiled by miRNA-sequencing. Genes controlled by the oncofetal miRNAs identified were first investigated by miRNA-Data-Integration-Portal (mirDIP) prediction, followed by luciferase-reporter assays. Associations between patient survival and mRNA expression of oncofetal miRNA-gene targets were evaluated in independent samples (>1,400 cases) across multiple NSCLC cohorts. Immunohistochemical analysis of oncofetal miRNA targets was performed on 96 lung adenocarcinoma (LUAD) specimens.

      Results:
      We describe for the first time a comprehensive characterization of miRNA expression in human fetal lung tissue, and identified numerous miRNAs that recapitulate their fetal expression patterns in NSCLC. Nuclear Factor I/B (NFIB), a transcription factor essential for lung development, was identified as being frequently targeted by these oncofetal miRNAs. Overexpression of the oncofetal miRNA miR-92b-3p, significantly reduced NFIB levels in vitro. Concordantly, analysis of NFIB expression in multiple NSCLC cohorts revealed its frequent underexpression in tumours (~40-70%). This is in contrast with its recurrent oncogenic overexpression recently reported in SCLC. Low expression of NFIB was significantly associated with poorer survival in LUAD patients but not in squamous cell carcinoma patients, consistent with the functional role of NFIB in distal lung cell differentiation (i.e., precursor cells of LUAD). Furthermore, an NFIB-related gene signature was identified in LUAD tumours, comprising several well-known lung differentiation markers (e.g., TTF-1, SFTPB, ABCA3). The underexpression of NFIB protein was ultimately validated in LUAD specimens, which also revealed that tumours presenting lower levels of this transcription factor are associated with higher grade, biologically more aggressive LUAD (invasive mucinous, micropapillary and solid subtypes).

      Conclusion:
      This work has revealed a prominent mechanism for the downregulation of NFIB, a transcription factor essential for lung differentiation, which we found to be associated with aggressive phenotypes of LUAD and consequently, poor patient survival. Restoration of NFIB expression, specifically in LUAD, has the potential to induce lung cell differentiation and thereby reduce tumour aggressiveness.

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

    • Event: WCLC 2016
    • Type: Poster Presenters Present
    • Track: Biology/Pathology
    • Presentations: 3
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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): B.C. Minatel

      • 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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      P2.01-024 - Expression of miR-106 Paralogs Improves Prognostic Value of Mesenchymal Signatures but Only miR-106b Promotes Invasiveness (ID 6250)

      14:30 - 14:30  |  Author(s): B.C. Minatel

      • Abstract

      Background:
      Improved understanding of the molecular mechanisms driving lung cancer progression can lead to novel therapeutic strategies to improve the currently poor patient treatment outcome. Deregulation of microRNA (miRNA) expression in malignant cells activates molecular pathways that drive tumor progression such as epithelial-mesenchymal transition (EMT). We identify miRNA paralogs, miR-106a and miR-106b, to be elevated in metastatic lung adenocarcinoma (LUAD). We assess whether these two highly similar miRNAs share the same functions in vitro, and measure how their elevated expression increases invasiveness or induces EMT in LUAD tumor.

      Methods:
      MiRNA expression was obtained from small RNA sequencing data derived from clinical primary LUAD specimens and paired non-malignant tissues (60 localized, 27 with lymph node invasion). Non-invasive, epithelial LUAD cell lines with low endogenous miR-106a/b levels were transfected and co-transfected with overexpression vectors for miR-106a and miR-106b. Invasiveness of experimentally-modulated tumor cells was assessed in vitro by Boyden chamber assay and in vivo using a zebrafish model, and expression of EMT markers was determined by Western Blot. Predicted miRNA targets were identified using mirDIP portal. To identify putative genetic mechanisms of mir-106a/b overexpression, DNA copy number, methylation, and Gene Set Enrichment Analysis (GSEA) were performed. Clinical associations were computed in an independent cohort of TCGA LUAD samples.

      Results:
      Both miR-106 paralogs were significantly overexpressed in LUAD samples with lymph node invasion. However, increased expression of miR-106b alone or together with miR-106a, but not miR-106a alone, enhanced metastatic phenotypes, and correlated with increased mesenchymal and decreased epithelial marker expression. Predicted targets include EP300, a transcriptional activator of E-cadherin, and members of the TGFβ signaling pathway. Copy number and methylation status did not correlate with miRNA expression; however, GSEA analysis revealed enrichment of E2F transcription factor targets in LUAD with high expression of either miR-106 paralogs. Furthermore, expression of miR-106 paralogs was significantly positively correlated with E2F1 and E2F2, suggesting that upstream regulation by E2F is a potential mechanism. Interestingly, miR-106a and miR-106b expression was associated with poor survival and advanced stage when stratified by mesenchymal marker vimentin.

      Conclusion:
      Although both miR-106a and miR-106b are overexpressed in metastatic LUAD, the strongest prognostic association was found in LUAD with a mesenchymal expression signature and high expression of both miRNAs. Our cell models suggest that miR-106b may play a direct role in EMT, with miR-106a influencing tumor progression via alternative mechanisms. Inhibition of one or both of these miRNAs may provide a strategy for treating advanced stage disease.

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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): B.C. Minatel

      • 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.