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Greg L. Stewart
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MA24 - Genomic Evolution, KEAP 3 and More Non-Coding RNA (ID 928)
- Event: WCLC 2018
- Type: Mini Oral Abstract Session
- Track: Biology
- Presentations: 2
- Moderators:
- Coordinates: 9/26/2018, 10:30 - 12:00, Room 205 BD
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MA24.06 - Long Non-Coding Rna Expression Patterns Delineate Infiltrating Immune Cells in the Lung Tumour Microenvironment (ID 13978)
11:05 - 11:10 | Author(s): Greg L. Stewart
- Abstract
- Presentation
Background
The tumour microenvironment is characterized by complex interactions between different cell types, including immune cells that may exhibit pro- or anti-tumour effects. Sequencing and deconvolution techniques present opportunities to identify immune cell composition of bulk tumour data; similarly, these have renewed an interest in the non-coding transcriptome and its regulation of immune- and tumour-biology. Numerous long non-coding RNAs (lncRNAs; >200nt) have emerged as regulators of tumour initiation, progression, and metastasis. Additionally, several immune-related lncRNAs mediate fine-level regulation to balance pro- and anti-inflammatory phenotypes; yet, the landscape of lncRNA expression in human immune cells remains uncharacterized. Thus, delineating these multifaceted regulatory networks is critical to cancer immunology, particularly in immunogenic malignancies such as lung cancer.
a9ded1e5ce5d75814730bb4caaf49419 Method
RNA-sequencing data of purified immune-cell subsets (CD8+ T, CD4+ T, B, Monocytes, Neutrophils, and Natural Killer) obtained from flow-sorted healthy peripheral blood samples were probed for lncRNA expression. Sequencing reads were aligned to the hg38 reference genome and quantified to Ensembl v89, yielding 4919 expressed lncRNAs. These immune-associated lncRNAs were correlated with immune cell infiltrate in tumour and paired non-malignant lung adenocarcinoma samples (n=54, The Cancer Genome Atlas) as estimated by the proportion of consistent immune-associated methylation profiles, denoted by leukocytes unmethylation for purity (LUMP) scores.
4c3880bb027f159e801041b1021e88e8 Result
We observed that lncRNA expression patterns display a greater degree of cell-type specificity than protein-coding genes in immune cells. In fact, 676 lncRNAs had detectable expression in exclusively one cell type. We uncovered previously-uncharacterized lncRNAs that have expression patterns suggestive of immune-regulatory roles. Compared with lung tumour samples, 19 immune-associated lncRNAs were significantly negatively correlated with LUMP scores (r<-0.400, BH-p<0.0100), 17 of which were also strongly positively correlated with CD45 gene expression (r>0.400, BH-p<0.0100) suggesting expression from immune rather than tumour cells. For instance, the lncRNA USP30-AS1 is significantly downregulated in tumours (average fold-change=2.96, BH-p=6.88*10-13), suggesting its relevance to tumour biology; however, higher transcript expression is correlated with decreased LUMP score (r=-0.685, BH-p=1.02*10-4), illustrating its specificity to immune cells.
8eea62084ca7e541d918e823422bd82e Conclusion
Here, we present an atlas of cell-type specific lncRNAs in human immune cells. Our data suggest a functional relevance of lncRNAs to the biology of the tumour microenvironment, and the necessary consideration of tumour purity when examining non-coding RNA expression in order to avoid conclusions confounded by immune cells in bulk tumour data. Thus, we provide a resource for further elucidation of genomic links between immune and malignant cells, which may aid the development of future prognostic and therapeutic strategies.
6f8b794f3246b0c1e1780bb4d4d5dc53Only 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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MA24.07 - A Novel cis-Acting lncRNA Controls HMGA1 Expression in Lung Adenocarcinoma (ID 13979)
11:10 - 11:15 | Author(s): Greg L. Stewart
- Abstract
- Presentation
Background
High mobility group A1 (HMGA1) chromatin remodeling protein is enriched in several aggressive cancer types, including NSCLC, where mRNA and protein expression are markedly increased. Additionally, high HMGA1 expression has been associated with poor overall survival and chemotherapy resistance. While HMGA1 is deregulated in lung cancer, the mechanisms that mediate its expression are only beginning to emerge. Long non-coding RNAs (lncRNAs), are a class of transcripts have been implicated in the onset of cancer-associated phenotypes in tumourigenesis and metastasis. Recently, an emerging class of lncRNAs - cis-acting - has been shown to regulate the expression of neighbouring protein-coding genes, including oncogenes and tumour suppressor genes. Thus, lncRNAs may represent novel actionable therapeutic intervention points in known cancer driving pathways. Here we investigate the role of a cis-acting lncRNA, RP11.513I15.6, its deregulation in NSCLC, and its relationship with HMGA1.
a9ded1e5ce5d75814730bb4caaf49419 Method
LncRNA transcriptomes were deduced from RNA-sequences of 36 microdissected tumour and matched non-malignant tissues. Normalized sequence read counts were used to identify transcripts with significantly deregulated expression (Wilcoxon Signed-Rank Test, BH-p<0.05). Sequencing data obtained from The Cancer Genome Atlas were analyzed to validate these results. SiRNA-mediated knockdown of lncRNA candidates identified in these analyses were performed in a non-malignant lung epithelial cell line (BEAS-2B). Quantitative real-time PCR quantified the effects of lncRNA knockdown on the expression of neighbouring cancer-associated protein-coding genes.
4c3880bb027f159e801041b1021e88e8 Result
Our analyses identified RP11.513I15.6, an undescribed lncRNA neighbouring HMGA1, to be significantly downregulated in adenocarcinoma (>2-fold downregulation in 81.5% of cases). This observation was confirmed in our validation cohort. HMGA1 expression was found to be anticorrelated with RP11.513I15.6, as tumours with downregulated RP11.513I15.6 displayed significant overexpression of HMGA1. This suggested that this lncRNA may be a key negative regulator of HMGA1. In vitro experiments demonstrated siRNA-mediated inhibition of RP11.513I15.6 in immortalized lung epithelial cells resulted in a significant increase in the expression of HMGA1 mRNA and protein. Taken together, our results suggest that RP11.513I15.6 is a novel cis-acting lncRNA that negatively regulates HMGA1, and may contribute mechanistically to the maintenance of cancer phenotypes.
8eea62084ca7e541d918e823422bd82e Conclusion
We have discovered a novel, 18-fold downregulated transcript that is anti-correlated with expression of HMGA1, a well established oncogene. In vitro studies support the hypothesis that this transcript, RP11.513I15.6, is a cis-acting lncRNA as siRNA-mediated inhibition led to upregulation of neighbouring HMGA1. Characterizing this oncogene regulatory mechanism will not only further our understanding of cancer biology, but could uncover a novel therapeutic intervention point.
6f8b794f3246b0c1e1780bb4d4d5dc53Only 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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P2.03 - Biology (Not CME Accredited Session) (ID 952)
- Event: WCLC 2018
- Type: Poster Viewing in the Exhibit Hall
- Track:
- Presentations: 1
- Moderators:
- Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall
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P2.03-18 - X-Inactivation Specific Transcript (XIST)-Mediated miRNA Sequestration In NSCLC (ID 12307)
16:45 - 18:00 | Author(s): Greg L. Stewart
- Abstract
Background
Long non-coding RNA (lncRNA; >200nt) transcripts have been recently recognized as crucial regulators of gene expression. XIST is a prototypical lncRNA involved in cis-silencing of an X chromosome in females; however, there are conflicting reports of lncRNA-mediated regulation in cancer biology and therapeutics through “sponging” of single miRNAs that cause upregulation of canonical miRNA-target genes. XIST-associated sex-specific differences afford the opportunity to study lncRNA sponging systems within a cancer type. Existing studies considering one miRNA in singular cancer types do not account for important biological considerations, including sex and localization of miRNA and XIST transcripts. Here, we detail an in-depth and unbiased pipeline for the discovery of candidate miRNA and gene targets in lncRNA sponging, and validate the miRNA that may be mediating these interactions in XIST.
a9ded1e5ce5d75814730bb4caaf49419 Method
We performed a comprehensive analysis of the role of XIST in the positive regulation of protein-coding genes using male and female lung adenocarcinoma (LUAD) samples as a model. To find genes regulated by XIST-mediated miRNA sponging, we correlated all Ensembl-annotated genes with XIST expression in female LUAD (n=307; rho>0.4, p<0.05). Using a specialized algorithm based on binding energies and sequence homology, we assessed the potential binding of all miRNAs against target genes and XIST. We then determine the best candidates for sponging by XIST using XIST-high (female and male) and XIST-low (male) systems, and validate the presence of these candidate miRNAs in the nucleus.
4c3880bb027f159e801041b1021e88e8 Result
Our analysis yielded 543 genes that may be defended from miRNAs by XIST (DMX), with a predicted 804 miRNAs targeting both DMX genes and XIST. We compared the changes in miRNA-DMX relationships in XIST-high and XIST-low systems and discovera high-confidence set of 13 miRNA-DMX gene pairs. Interestingly, 5 of these miRNA-DMX gene pairs involve miRNA with validated nuclear localization.
8eea62084ca7e541d918e823422bd82e Conclusion
A massive number of lncRNA sponging studies exist, but most only consider one miRNA in a singular type of cancer, are biased in their selection of this target, and limited in biological context. By analyzing the transcriptome of female and male LUAD, we show that the XIST-miRNA-DMX sponging axis is affected by expression of sex-specific genes and number of shared miRNA binding sites on DMX genes. Importantly, we identify that the miRNAs that mediate the XIST-DMX gene axis are enriched in the nucleus, co-localizing with XIST. In summary, our analysis provides both a comprehensive methodology for studying cancer-related miRNA sponge lncRNAs, and suggests the relevance of XIST in lung cancer.
6f8b794f3246b0c1e1780bb4d4d5dc53
