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Greg L. Stewart
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MA 15 - Lung Cancer Biology II (ID 670)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Biology/Pathology
- Presentations: 1
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MA 15.14 - Long Non-Coding RNA Disruption in Lung Adenocarcinoma Reveals Novel Mechanisms of Metastasis (ID 8659)
17:10 - 17:15 | Author(s): Greg L. Stewart
- Abstract
- Presentation
Background:
Identifying the drivers of metastasis will yield new molecular targets for prognostics and therapeutics. Long non-coding RNAs (lncRNAs) are known to regulate gene transcription through their influence on the expression of nearby (cis) and distant (trans) genes. Emerging evidence suggests that lncRNAs are involved in key cellular processes, presenting an opportunity for large-scale identification of lncRNA genes critical to lung cancer progression. Here we investigate the contribution of this class of non-coding RNA to lung adenocarcinoma (LUAD) metastasis.
Method:
Stage T1 and T2 tumours with (N≥1 and/or M≥1) and without (N=0 and M=0) metastasis were examined for expression comparisons. Sequencing data from 265 non-metastatic and 130 metastatic tumours obtained from The Cancer Genome Atlas were used as our discovery cohort. Results were validated in 20 non-metastatic and 10 metastatic tumour samples microdissected to 90% purity and sequenced using the Illumina Hi-Seq platform. Normalized sequence read count comparisons were performed (Mann Whitney U-Test, FDR-BH p<0.05) to identify lncRNAs significantly deregulated in metastatic samples. LncRNAs over- and under-expressed in metastatic LUAD were compared to nearby protein-coding-target genes to identify putative mechanisms of regulation in cis.
Result:
We discovered 150 lncRNAs to be significantly differentially expressed between metastatic and non-metastatic tumours, including lncRNAs with previously described oncogenic roles in lung cancer, such as Lung Cancer Associated Transcript 1 and H19. As individual lncRNAs can positively or negatively regulate target-gene expression, it is noteworthy that we identified potential protein-coding-target genes that display both concordant and discordant expression patterns with specific lncRNAs. For example, we discovered the upregulation of linc00942 in metastatic LUAD (FDR-BH p=0.001) and the concordant overexpression of its corresponding protein-coding-target gene, ELKS/RAB6-Interacting/CAST Family Member 1 (ERC1) (FDR-BH p=0.02). Further, metastatic LUAD samples stratified by linc00942 expression also display corresponding elevation of ERC1 (p=0.0002), which holds true in the validation cohort. ERC1 (an upstream member of the NF-κB signaling pathway) is implicated in cell migration and focal adhesion, and displays deregulated expression in a number of cancer types. Thus, overexpression of linc00942 may act as a novel positive cis-regulator of ERC1, promoting metastasis.
Conclusion:
This work has led to the discovery of a large number of lncRNA genes deregulated in metastatic LUAD, suggesting that altered lncRNA expression contributes functionally to malignant progression. Understanding cis- or trans-mediated mechanisms of gene deregulation enacted by metastasis-associated lncRNAs will present novel opportunities for diagnosis and treatment.
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P2.02 - Biology/Pathology (ID 616)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P2.02-017 - Aberrant Expression of Long Non-Coding RNAs from Pseudogene Loci Highlights Alternative Mechanisms of Cancer Gene Regulation (ID 10231)
09:30 - 09:30 | Presenting Author(s): Greg L. Stewart
- Abstract
Background:
Less than half of lung adenocarcinoma (LUAD) patients harbour clinically actionable driver genes, emphasizing the need to explore alternative mechanisms of cancer gene deregulation. Long non-coding RNAs (lncRNAs) have emerged as important players in cell biology, and can be exploited by tumours to drive the hallmarks of cancer. Pseudogenes are DNA sequences that are defunct relatives of their functional protein-coding parent genes but retain high sequence homology. Interestingly, 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. We hypothesize that this phenomenon occurs more broadly than previously realized, and that these events provide an alternative mechanism of cancer gene deregulation in LUAD tumourigenesis that has clinical implications.
Method:
Illumina HiSeq reads were processed and aligned to the ENSEMBL annotation file in order to derive the most complete set of both protein-coding and non-coding genes. Two datasets were selected due to their paired nature, complete with both LUAD and non-malignant lung profiles (TCGA n=108, BCCA n=72). LncRNAs were filtered based on positional overlap within pseudogene loci, and a Wilcoxon sign-rank test was run to identify lncRNAs with significantly altered expression between paired tumour and normal tissues (FDR p<0.05). To identify lncRNAs that likely regulate protein-coding parent gene expression in trans, tumours were ranked by lncRNA expression, and protein-coding parent gene expression of top and bottom ranked tertiles was compared by Mann Whitney U-test (p<0.05). Survival analysis was performed using a Cox proportional hazard model.
Result:
Our analysis has identified 129 lncRNAs expressed from pseudogene loci that were significantly deregulated in LUAD in both datasets. Remarkably, many of these deregulated lncRNAs (i) were expressed from the loci of pseudogenes related to known cancer genes, (ii) had expression that significantly correlated with protein-coding parent gene expression, and (iii) protein-coding parent gene expression was significantly associated with survival. For example, RP11-182J1.1 is a lncRNA expressed from a pseudogene to EGLN1, a previously described cancer gene involved in regulation of tumour hypoxia. RP11-182J1.1 was underexpressed in LUAD and significantly positively correlated with EGLN1 expression. In addition, EGLN1 was significantly associated with patient survival (p=1.2e-08) emphasizing the clinical potential of these lncRNAs.
Conclusion:
This work uncovers evidence to suggest the lncRNA-pseudogene-protein-coding gene axis is a prominent mechanism of cancer gene regulation. Further characterization of this understudied gene regulatory mechanism could lead to novel therapies that silence oncogenes or reactivate tumour suppressor genes.
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P2.02-022 - Alternative Regulation of Cancer-Associated Genes through Modulation of Long Non-Coding RNAs (ID 8658)
09:30 - 09:30 | Author(s): Greg L. Stewart
- Abstract
Background:
Uncovering novel mechanisms of cancer-gene regulation may reveal new actionable targets to direct the treatment of patients who do not harbour targetable molecular drivers of lung cancer. Long non-coding RNAs (lncRNAs), are a class of transcripts that hold an emerging role in cell biology, particularly in gene regulation. These genes have since been implicated in cancer-associated phenotypes, and may represent attractive therapeutic intervention points; however, prediction of downstream regulatory targets of lncRNAs has been impeded due to their complex tertiary structure. Recently, a subset of lncRNAs has been shown to regulate the expression of neighbouring protein-coding genes in cis. Here we take a novel approach to identify lncRNAs deregulated in lung adenocarcinoma (LUAD) and examine their roles in the expression modulation of their cancer-associated protein-coding cis-partner genes.
Method:
RNA-sequencing was performed on 36 LUAD tumour samples with matched adjacent non-malignant tissue obtained via microdissection to 90% purity. Significantly deregulated lncRNAs and neighbouring protein-coding genes were identified by comparison of matched tumour and non-malignant normalized read counts (Wilcoxon Signed-Rank Test, FDR-BH<0.05). Fifty LUAD tumours with paired normal tissue from The Cancer Genome Atlas (TCGA) were used to validate these findings. Cox-Proportional Hazard analysis was performed on both datasets to assess survival associations of significantly deregulated lncRNAs.
Result:
Our approach revealed greater than 500 lncRNAs that were significantly deregulated between LUAD and matched normal tissues. Many of these lncRNAs have neighbouring protein-coding genes that also display deregulated expression patterns. Of particular interest are the protein-coding-target genes that have been previously implicated in cancer, including OIP5, which is involved in chromatin segregation, as well as HMGA1, which contributes to cell transformation and metastasis. In both of these cases, the neighbouring lncRNA is significantly underexpressed while the protein-coding gene is significantly overexpressed, suggesting a negative regulatory function of the lncRNA. Moreover, survival analyses revealed that patients with high expression of either OIP5 or HMGA1 had significantly shorter overall survival. Strikingly, patients with low expression of the lncRNA near OIP5 also displayed poorer overall survival, illustrating the clinical opportunity that these genes present.
Conclusion:
Our results highlight the landscape of lncRNA deregulation in LUAD and uncover a role of these non-coding transcripts in the cis-regulation of neighbouring protein-coding genes, many of which have been described in cancer and predict patient survival. Further characterization of this alternative lncRNA-mediated cancer-gene regulatory mechanism may reveal novel therapeutic targets that may improve treatment for LUAD patients without well defined molecular drivers.
