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D.A. Rowbotham
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MA02 - RNA in Lung Cancer (ID 377)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Biology/Pathology
- Presentations: 1
- 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)
14:20 - 15:50 | Author(s): D.A. Rowbotham
- 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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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- 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 - 15:45 | Author(s): D.A. Rowbotham
- 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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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 - 15:45 | Author(s): D.A. Rowbotham
- 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.
