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Adam Patrick Sage
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MA 15 - Lung Cancer Biology II (ID 670)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Biology/Pathology
- Presentations: 2
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MA 15.12 - Expanding the Lung Small RNA Transcriptome: Discovery of Unannotated microRNAs with Roles in Development and Tumourigenesis (ID 9100)
17:00 - 17:05 | Author(s): Adam Patrick Sage
- Abstract
- Presentation
Background:
MicroRNAs (miRNAs) are key regulators of gene expression. They participate in many biological and pathological processes, from organ development to malignant transformation. Their functions are widely conserved, involving post-transcriptional silencing of gene expression. Over 2500 mature miRNA sequences have been identified in humans; however, recent studies have showed that the number of annotated miRNAs represent only a fraction of the total pool of existing miRNAs, suggesting that there are still many potentially undiscovered biologically relevant miRNAs encoded by the human genome. Here, we perform a comprehensive study to identify novel miRNA sequences expressed in non-malignant lung tissues, as well as samples from developmental stages and pathological conditions.
Method:
A total of 422 samples were included in this analysis. First, 209 non-malignant samples from two cohorts (BCCA, n=118 and TCGA, n=91) were analyzed using our customized small RNA sequence analysis pipeline. Sequence reads were aligned to the hg38 build of the human genome (STAR algorithm) and novel miRNAs were predicted using mirDeep2. The results were compared to miRNA databases and further filtered by abundance and for miRNA-compatible structure. The same procedure was applied to matched tumours (n=209) and samples derived from fetal lungs (n=4). The biological relevance of the novel sequences was investigated by assessing their expression in tumours and fetal samples, together with gene target prediction and tissue-specific protein-protein interaction (PPI) network analyses using IID.
Result:
Our study discovered the expression of 294 novel miRNA sequences in lung tissue, significantly expanding the current human lung miRNA transcriptome. These novel miRNAs showed similar nucleotide composition and genomic distribution compared to known miRNAs, providing additional evidence of their miRNA-compatible nature. Interestingly, a subset of these miRNAs were also found to be expressed in tumour and fetal samples, indicating that they might play important roles in organ development and tumourigenesis. Likewise, target prediction analysis revealed that these novel miRNAs are involved in key cellular processes including cell proliferation, migration and survival, as well as pathways known to be deregulated in cancer, as comprehensively analyzed using pathDIP.
Conclusion:
Our study has significantly expanded the lung small RNA transcriptome, and provided evidence that the novel miRNAs are involved in molecular networks relevant to lung biology and pathology. These results also highlight their specific roles in developmental regulation and malignant transformation, suggesting their role as biological regulators and implicating their potential as therapeutic targets.
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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 | Presenting Author(s): Adam Patrick Sage
- 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.
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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P1.02 - Biology/Pathology (ID 614)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.02-006 - Arsenic Promotes Persistent Alterations in the Lung PiRNA Transcriptome to Target Epigenetic Pathways (ID 9567)
09:30 - 09:30 | Author(s): Adam Patrick Sage
- Abstract
Background:
Chronic exposure to arsenic leads to the onset of different diseases, including lung cancer. Arsenic-induced lung tumors have been associated with a high-frequency of lung squamous-cell carcinomas among never smokers (a rare epidemiological pattern), suggesting a unique underlying biology. Epigenetic alterations are known to play a role in this process; however, detailed mechanisms are not yet fully elucidated. Piwi-interacting RNAs (piRNAs), a novel class of small non-coding RNAs (sncRNAs), play a key role in epigenetic regulation and maintenance of genome integrity. Here, we examine the impact of different arsenic species in the human piRNA transcriptome, using lung cell models mirroring chronic, low dose exposure. We also investigate the interaction network of deregulated piRNAs and identified biological pathways potentially affected.
Method:
One normal lung (HBEC) and two lung cancer cell lines: A459 (adenocarcinoma) and H520 (squamous-cell carcinoma) were grown in 10 ppm of sodium arsenite (AsIII) or arsenate (AsV) for six passages. Total RNA was extracted at different time points and sequenced. piRNA expression was deduced using our custom sncRNA analysis pipeline, which interrogates >23K piRNA-encoding human loci. piRNA/DNA binding prediction was performed using two different algorithms (miRanda/ThermoBLAST). Network analysis was performed using Partek Pathways.
Result:
Overall, 691 piRNAs were expressed. Persistent changes in piRNA expression over time were identified, with specific patterns associated with the different arsenic species. In HBECs (non-malignant lung tissue), 14 piRNAs were persistently upregulated and 16 downregulated in response to AsIII. Similarly, 6 were up- and 11 downregulated when the same cells were exposed to AsV. Only 1 piRNA, DQ598008, was commonly upregulated in response to both arsenic species, while 4 piRNAs were commonly downregulated. Lung cancer cell lines follow the same arsenic species-specific trends, with a high subtype-specificity indicating these species maintain a role during lung tumor development. Remarkably, we found an enrichment of genes associated with methyltransferase activities predicted to be targeted by piRNAs altered by AsIII (a biologically-relevant form of arsenic), evidencing their role in arsenic-related carcinogenic mechanisms.
Conclusion:
Arsenic induces persistent alterations in the lung sncRNA transcriptome, particularly piRNAs, impacting pathways linked to epigenetic regulation. Together, these results provide insights into sncRNA-related mechanisms in arsenic-induced lung carcinogenesis. Moreover, different arsenic species induce distinct alteration patterns, highlighting the relevance of the source of exposure. piRNAs, as with other sncRNAs, are stable in biofluids, circulating tumour cells, and archival clinical materials. Therefore, piRNAs hold great promise as potential exposure and monitoring biomarkers for arsenic-related health effects.
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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 | Author(s): Adam Patrick Sage
- 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 | Presenting Author(s): Adam Patrick Sage
- 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.
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.02-094 - Identification of Oncofetal piRNAs in Lung (ID 9107)
09:30 - 09:30 | Author(s): Adam Patrick Sage
- Abstract
Background:
PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs (distinct from microRNAs) that recognize complementary DNA sequences in the nucleus. Their primary functions involve epigenetic control of gene transcription and maintenance of genomic stability through repression of mobile elements. Recent observations of cancer type specific aberrant expression have raised the possibility of a role for piRNAs in lung cancer. Here we characterize piRNA transcriptomes of fetal, adult and tumour lung tissue to identify fetal piRNA genes that are silenced in normal adult lung and re-activated in cancer. Our goal is to identify oncofetal piRNAs, which might represent ideal cancer therapeutic targets, as they are absent in normal adult lung tissue.
Method:
We determined piRNA expression profiles from small-RNA sequencing libraries using an in-house pipeline. All sequence reads were aligned to the hg38 build of the human genome. The expression levels of fetal lung samples (n=25) and two tumour/non-malignant paired cohorts (BCCA, n=118 and TCGA, n=91; derived from non-small cell lung cancer cases) were compared. piRNAs not expressed in non-malignant samples but with comparable expression in both fetal and tumour tissues were classified as oncofetal piRNAs. In order to identify the biological functions of the identified oncofetal piRNAs, we performed piRNA/DNA binding prediction using the miRanda algorithm adjusted for piRNA-specific features.
Result:
Our results provide a comprehensive characterization of piRNA expression in both normal and tumour lung tissues, as well as an unique piRNA expression profile of fetal lung tissues. A subset of the piRNA pool expressed in lung tissues are similarly expressed between fetal lung and lung tumours, but are absent in non-malignant tissue, implying that tumour initiation might involve the reactivation of developmental pathways. More importantly, target prediction analysis revealed that the identified oncofetal piRNAs are involved in key cellular processes, such as cell proliferation, migration and survival.
Conclusion:
Our study provides an unique and comprehensive characterization of the piRNA pool of lung tissues, as well as the identification of specific similarities in piRNA expression during both organ and tumour development. These similarities between fetal and tumour tissues might represent a promising avenue for the identification of strong biomarkers or optimal therapeutic targets with little toxicity for the treatment of lung cancer. Therefore, our study provides new and promising insights for lung tumour biology and may aid in the development of novel therapeutic approaches.
