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K.S.S. Enfield
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MA02 - RNA in Lung Cancer (ID 377)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Biology/Pathology
- Presentations: 2
- Moderators:E. Brambilla, M. Noguchi
- Coordinates: 12/05/2016, 14:20 - 15:50, Stolz 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): K.S.S. Enfield
- Abstract
- Presentation
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): K.S.S. Enfield
- Abstract
- Presentation
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.
Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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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
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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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): K.S.S. Enfield
- 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): K.S.S. Enfield
- 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.
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P2.01-065 - Quantification of Tumour-Immune Cell Spatial Relationships in the Lung Tumour Microenvironment Using Single Cell Profiling (ID 6104)
14:30 - 14:30 | Author(s): K.S.S. Enfield
- Abstract
Background:
How clinical-genomic features of the lung tumour microenvironment (TME) influence immune-checkpoint-blockade therapy is not well understood. Immunohistochemistry (IHC) is necessary to decipher cell-cell relationships that cannot be observed by bulk tumour profiling. In this pilot study, we assess whether immune cell phenotypes and spatial relationships differ between lung adenocarcinoma (LUAD) from smokers/non-smokers, KRAS/EGFR mutation, or with stage and tumour size using a novel multicolour IHC quantitative pathology method that enables in situ single cell profiling within the TME.
Methods:
Two consecutive sections from 21 cases of LUAD were stained with multicolour IHC panels to assess immune cell composition (CD8, CD3, CD79a) and T-cell exhaustion (CD8, PD1, PDL1). Hyperspectral images were captured as directed by a pathologist and analyzed using software developed in-house. The software segments individual cell boundaries based on haematoxylin stain. IHC stain positivity thresholds were applied based on intensity. Tumour and immune cells were classified into groups based on IHC staining. Interactions between specific groups were quantified by assessing the frequency and variance of the spatial relationship of each group vs. all other groups. Voronoi tessellation, based on cell centres, was used to define “next to”. Group counts and relationships were then compared with clinical features using a Student’s t-test or Kruskal-Wallis test.
Results:
A greater number of cells expressed PDL1 in KRAS+ LUAD. While the total number of CD8+PD1+ T-cells did not differ between KRAS+ and EGFR+ LUAD, there was an observed increased proximity between PDL1+ cells and CD8+PD1+ T-cells in KRAS+ LUAD. In EGFR+ LUAD, CD8+ T-cells that did not express PD1 were primarily localized in PDL1 negative regions. Both EGFR+ LUAD and never smokers harbored a higher proportion of CD8- T-cells and CD3-CD8+ immune cells. Both immune cell types were frequently localized in clusters with CD8+ T-cells. KRAS+ LUAD and smokers had increased B-cell counts. No significant associations of PD1 and PDL1 expression were found with stage; however, there was a statistically significant increase in proximity between varied immune cell types as stage and tumour size increased.
Conclusion:
Our method enabled identification of specific cell-cell spatial relationships within LUAD that are associated with smoking history and KRAS/EGFR mutation. Despite limited sample size, we observed an increased proximity between PDL1+ cells and CD8+PD1+ T-cells in KRAS+ LUAD. TME single cell profiling and cell sociology is a promising method to improve stratification of patients for immune-checkpoint-blockade therapies and opens new avenues to explore the complex cell-cell interactions within the TME.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-049 - ELF3 Overexpression Leads to Oncogenic Reprogramming of Protein Interactions Exposing Therapeutically Actionable Targets (ID 5807)
14:30 - 14:30 | Author(s): K.S.S. Enfield
- Abstract
Background:
Emerging evidence has implicated ELF3 involvement in cancer signaling pathways. To determine the biological basis to pursue ELF3 as a novel therapeutic target, we investigated the role of ELF3 in lung adenocarcinoma (LUAD). Using a multi-omics approach in two independent cohorts of LUAD we (a) discover genetic mechanisms driving aberrant expression of this oncogene, (b) identify the protein-protein-interaction (PPI) partners of ELF3, and (c) determine the specific functions of ELF3 in LUAD using model systems.
Methods:
Comprehensive, multi-omic data was collected from the BC Cancer Research Centre (BCCRC), The Cancer Genome Atlas (TCGA), and several mouse models of LUAD tumourigenesis. ELF3 cellular localization was visualized by immunofluorescence. ELF3 knock-down and overexpression was achieved by lentiviral vector delivery for in vitro and in vivo assays. Physical protein-protein interaction (PPI) networks obtained from IID were overlaid onto cancer and non-malignant gene expression data from TCGA and 11 restructured datasets from Gene Expression Omnibus. PPIs were interrogated to investigate malignancy-associated ELF3 interactions. Pathway analysis was performed using pathDIP. Survival analysis was performed using the log-rank method.
Results:
ELF3 was significantly overexpressed in both cohorts, remarkably in >70% of cases (p=1.64E-21). However, mutation of known upstream regulators was not sufficient to explain the frequency of ELF3 overexpression. Instead, the ELF3 locus underwent frequent (>80%) genetic alteration including focal amplification and promoter hypomethylation, which corresponded with increased expression. ELF3 was predominantly localized to the nucleus, consistent with its transcription factor function. Analysis of PPI networks indicated highly LUAD-specific ELF3 interactions whereby loss and gain of interactions lead to reprogramming of LUAD transcriptional networks, including loss of TNFα pathway, and gain of TGFβ pathway, PI3K pathway, and translesion (DNA repair) pathway interactions. Furthermore, EGFR, KRAS, and MYC transgenic models of LUAD tumourigenesis all displayed a marked increase (6 to 8-fold) in ELF3 expression signifying its importance to LUAD of varied genetic backgrounds. In culture, ELF3 regulated proliferation, viability and anchorage-independent growth. In animal models, ELF3 knock-down cells underwent negative clonal selection, suggesting ELF3 expression is beneficial to tumour growth. Clinically, high expression of ELF3 was associated with poor survival regardless of tumour stage.
Conclusion:
Overexpression of ELF3 reprograms protein-protein-interactions in LUAD leading to the activation of cancer-specific pathways, and producing oncogenic phenotypes. Depletion of ELF3 with shRNAs reverses tumour cell growth, suggesting ELF3 is a promising therapeutic target. In addition to ELF3, interruption of cancer-specific PPIs also represents a therapeutically actionable strategy.
