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Activation of an endogenous retrovirus-associated long non-coding RNA in human adenocarcinoma.

Gibb EA, Warren RL, Wilson GW, Brown SD, Robertson GA, Morin GB, Holt RA - Genome Med (2015)

Bottom Line: Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs).ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency.Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR.

View Article: PubMed Central - PubMed

Affiliation: Genome Sciences Centre, British Columbia Cancer Agency, 675 West 10th Ave, Vancouver, British Columbia V5Z 1L3 Canada ; Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V6T 1Z4 Canada.

ABSTRACT

Background: Long non-coding RNAs (lncRNAs) are emerging as molecules that significantly impact many cellular processes and have been associated with almost every human cancer. Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs). ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency. The aberrant regulation of ERVs associated with lncRNAs (ERV-lncRNAs), coupled with the diverse range of lncRNA functions, creates significant potential for ERV-lncRNAs to impact cancer biology.

Methods: We used RNA-seq analysis to identify and profile the expression of a novel lncRNA in six large cohorts, including over 7,500 samples from The Cancer Genome Atlas (TCGA).

Results: We identified the tumor-specific expression of a novel lncRNA that we have named Endogenous retroViral-associated ADenocarcinoma RNA or 'EVADR', by analyzing RNA-seq data derived from colorectal tumors and matched normal control tissues. Subsequent analysis of TCGA RNA-seq data revealed the striking association of EVADR with adenocarcinomas, which are tumors of glandular origin. Moderate to high levels of EVADR were detected in 25 to 53% of colon, rectal, lung, pancreas and stomach adenocarcinomas (mean = 30 to 144 FPKM), and EVADR expression correlated with decreased patient survival (Cox regression; hazard ratio = 1.47, 95% confidence interval = 1.06 to 2.04, P = 0.02). In tumor sites of non-glandular origin, EVADR expression was detectable at only very low levels and in less than 10% of patients. For EVADR, a MER48 ERV element provides an active promoter to drive its transcription. Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR. The sequence and structure of the EVADR locus is highly conserved among Old World monkeys and apes but not New World monkeys or prosimians, where the MER48 insertion is absent. Conservation of the EVADR locus suggests a functional role for this novel lncRNA in humans and our closest primate relatives.

Conclusions: Our results describe the specific activation of a highly conserved ERV-lncRNA in numerous cancers of glandular origin, a finding with diagnostic, prognostic and therapeutic implications.

No MeSH data available.


Related in: MedlinePlus

A long non-coding RNA is highly activated in colorectal tumors. (a) Expression of EVADR in tumor and adjacent normal tissue from 65 patients with colorectal carcinoma. For each subject (x-axis) tumor is shown in red and normal tissue in grey. The dashed line indicates an arbitrary minimum expression threshold of 10 FPKM. A total of 21 patients demonstrated robust (>10 FPKM) levels of EVADR expression. Normal colon tissue was generally not found to express EVADR or it was expressed at low levels (<10 FPKM). (b) Schematic representation of the chromosomal location of the EVADR gene locus. Arrowheads indicate the orientation of transcription. Bar plots indicate mean expression levels for respective genes across all 65 tumor (COL91A, 0.25 ± 1.11 FPKM; EVADR, 14.4 ± 24.5 FPKM; FAM135A, 7.2 ± 3 FPKM (mean ± SD)) and normal samples (COL91A, 0.19 ± 0.53 FPKM; EVADR, 0.50 ± 1.52 FPKM; FAM135A, 5 ± 2.6 FPKM (mean ± SD)). *P < 0.00001; paired t-test. (c) Exon structure and primer locations for the lncRNA EVADR. (d) Representative gel image showing EVADR transcript levels in colorectal tumor (T32) and matched normal (N32) tissue samples for patient 32, measured by RT-PCR. The letter L indicates the molecular ladder.
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Fig1: A long non-coding RNA is highly activated in colorectal tumors. (a) Expression of EVADR in tumor and adjacent normal tissue from 65 patients with colorectal carcinoma. For each subject (x-axis) tumor is shown in red and normal tissue in grey. The dashed line indicates an arbitrary minimum expression threshold of 10 FPKM. A total of 21 patients demonstrated robust (>10 FPKM) levels of EVADR expression. Normal colon tissue was generally not found to express EVADR or it was expressed at low levels (<10 FPKM). (b) Schematic representation of the chromosomal location of the EVADR gene locus. Arrowheads indicate the orientation of transcription. Bar plots indicate mean expression levels for respective genes across all 65 tumor (COL91A, 0.25 ± 1.11 FPKM; EVADR, 14.4 ± 24.5 FPKM; FAM135A, 7.2 ± 3 FPKM (mean ± SD)) and normal samples (COL91A, 0.19 ± 0.53 FPKM; EVADR, 0.50 ± 1.52 FPKM; FAM135A, 5 ± 2.6 FPKM (mean ± SD)). *P < 0.00001; paired t-test. (c) Exon structure and primer locations for the lncRNA EVADR. (d) Representative gel image showing EVADR transcript levels in colorectal tumor (T32) and matched normal (N32) tissue samples for patient 32, measured by RT-PCR. The letter L indicates the molecular ladder.

Mentions: To identify lncRNAs associated with colorectal cancer, we performed whole transcriptome sequence analysis on 65 tumor and matched normal colorectal poly(A)-selected RNA-seq libraries generated in a previously described study [33]. From these data we identified two Ensembl-curated yet uncharacterized lncRNAs, ENSG00000222041 and ENSG00000237643, which were strongly upregulated in colorectal tumors compared with matched normal control tissues. We did not characterize ENSG00000222041 further because its differential expression was less substantial than that of ENSG00000237643, and because it had a complex alternative splicing pattern that complicated further analysis. Further scrutiny of ENSG00000237643 (which we named EVADR for Endogenous retroViral-associated ADenocarcinoma RNA) revealed significantly increased expression of this lncRNA in tumors (14.4 ± 24.5 FPKM (mean ± standard deviation (SD))) compared with matched normal control tissues, where it was typically not expressed (0.50 ± 1.52 FPKM (mean ± SD)) (Figure 1a; P = 2.8e-05; t-test). Genomic analysis revealed a predicted 397-nucleotide lncRNA with three exons and a single transcript isoform located on chromosome 6, 91.8 kb downstream of Col9A1 and 13.5 kb upstream of FAM135A (Figure 1b). Despite high expression of EVADR in tumor samples, we did not observe differential expression of genes flanking EVADR (Figure 1b). We designed primers to amplify a 200 bp region of the mature EVADR transcript and validated the expression of this lncRNA in the colorectal tumor and normal tissues using RT-PCR (Figure 1c,d; Additional file 2). To corroborate our observation that EVADR was not expressed in non-malignant tissue, we measured EVADR expression in 16 normal adult human tissues finding only weak expression in lung and prostate tissues (Figure S3A in Additional file 1). Expanding these analyses to 15 cell lines, we found that EVADR was highly expressed (122 FPKM) in the chronic myeloid leukemia K562 cell line, but its expression was extremely low (<1 FPKM) in the others, including in the H1-HESC embryonic stem cell line (Figure S3B in Additional file 1), where it was 0.04 FPKM. These data demonstrate that EVADR is selectively expressed.Figure 1


Activation of an endogenous retrovirus-associated long non-coding RNA in human adenocarcinoma.

Gibb EA, Warren RL, Wilson GW, Brown SD, Robertson GA, Morin GB, Holt RA - Genome Med (2015)

A long non-coding RNA is highly activated in colorectal tumors. (a) Expression of EVADR in tumor and adjacent normal tissue from 65 patients with colorectal carcinoma. For each subject (x-axis) tumor is shown in red and normal tissue in grey. The dashed line indicates an arbitrary minimum expression threshold of 10 FPKM. A total of 21 patients demonstrated robust (>10 FPKM) levels of EVADR expression. Normal colon tissue was generally not found to express EVADR or it was expressed at low levels (<10 FPKM). (b) Schematic representation of the chromosomal location of the EVADR gene locus. Arrowheads indicate the orientation of transcription. Bar plots indicate mean expression levels for respective genes across all 65 tumor (COL91A, 0.25 ± 1.11 FPKM; EVADR, 14.4 ± 24.5 FPKM; FAM135A, 7.2 ± 3 FPKM (mean ± SD)) and normal samples (COL91A, 0.19 ± 0.53 FPKM; EVADR, 0.50 ± 1.52 FPKM; FAM135A, 5 ± 2.6 FPKM (mean ± SD)). *P < 0.00001; paired t-test. (c) Exon structure and primer locations for the lncRNA EVADR. (d) Representative gel image showing EVADR transcript levels in colorectal tumor (T32) and matched normal (N32) tissue samples for patient 32, measured by RT-PCR. The letter L indicates the molecular ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: A long non-coding RNA is highly activated in colorectal tumors. (a) Expression of EVADR in tumor and adjacent normal tissue from 65 patients with colorectal carcinoma. For each subject (x-axis) tumor is shown in red and normal tissue in grey. The dashed line indicates an arbitrary minimum expression threshold of 10 FPKM. A total of 21 patients demonstrated robust (>10 FPKM) levels of EVADR expression. Normal colon tissue was generally not found to express EVADR or it was expressed at low levels (<10 FPKM). (b) Schematic representation of the chromosomal location of the EVADR gene locus. Arrowheads indicate the orientation of transcription. Bar plots indicate mean expression levels for respective genes across all 65 tumor (COL91A, 0.25 ± 1.11 FPKM; EVADR, 14.4 ± 24.5 FPKM; FAM135A, 7.2 ± 3 FPKM (mean ± SD)) and normal samples (COL91A, 0.19 ± 0.53 FPKM; EVADR, 0.50 ± 1.52 FPKM; FAM135A, 5 ± 2.6 FPKM (mean ± SD)). *P < 0.00001; paired t-test. (c) Exon structure and primer locations for the lncRNA EVADR. (d) Representative gel image showing EVADR transcript levels in colorectal tumor (T32) and matched normal (N32) tissue samples for patient 32, measured by RT-PCR. The letter L indicates the molecular ladder.
Mentions: To identify lncRNAs associated with colorectal cancer, we performed whole transcriptome sequence analysis on 65 tumor and matched normal colorectal poly(A)-selected RNA-seq libraries generated in a previously described study [33]. From these data we identified two Ensembl-curated yet uncharacterized lncRNAs, ENSG00000222041 and ENSG00000237643, which were strongly upregulated in colorectal tumors compared with matched normal control tissues. We did not characterize ENSG00000222041 further because its differential expression was less substantial than that of ENSG00000237643, and because it had a complex alternative splicing pattern that complicated further analysis. Further scrutiny of ENSG00000237643 (which we named EVADR for Endogenous retroViral-associated ADenocarcinoma RNA) revealed significantly increased expression of this lncRNA in tumors (14.4 ± 24.5 FPKM (mean ± standard deviation (SD))) compared with matched normal control tissues, where it was typically not expressed (0.50 ± 1.52 FPKM (mean ± SD)) (Figure 1a; P = 2.8e-05; t-test). Genomic analysis revealed a predicted 397-nucleotide lncRNA with three exons and a single transcript isoform located on chromosome 6, 91.8 kb downstream of Col9A1 and 13.5 kb upstream of FAM135A (Figure 1b). Despite high expression of EVADR in tumor samples, we did not observe differential expression of genes flanking EVADR (Figure 1b). We designed primers to amplify a 200 bp region of the mature EVADR transcript and validated the expression of this lncRNA in the colorectal tumor and normal tissues using RT-PCR (Figure 1c,d; Additional file 2). To corroborate our observation that EVADR was not expressed in non-malignant tissue, we measured EVADR expression in 16 normal adult human tissues finding only weak expression in lung and prostate tissues (Figure S3A in Additional file 1). Expanding these analyses to 15 cell lines, we found that EVADR was highly expressed (122 FPKM) in the chronic myeloid leukemia K562 cell line, but its expression was extremely low (<1 FPKM) in the others, including in the H1-HESC embryonic stem cell line (Figure S3B in Additional file 1), where it was 0.04 FPKM. These data demonstrate that EVADR is selectively expressed.Figure 1

Bottom Line: Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs).ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency.Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR.

View Article: PubMed Central - PubMed

Affiliation: Genome Sciences Centre, British Columbia Cancer Agency, 675 West 10th Ave, Vancouver, British Columbia V5Z 1L3 Canada ; Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V6T 1Z4 Canada.

ABSTRACT

Background: Long non-coding RNAs (lncRNAs) are emerging as molecules that significantly impact many cellular processes and have been associated with almost every human cancer. Compared to protein-coding genes, lncRNA genes are often associated with transposable elements, particularly with endogenous retroviral elements (ERVs). ERVs can have potentially deleterious effects on genome structure and function, so these elements are typically silenced in normal somatic tissues, albeit with varying efficiency. The aberrant regulation of ERVs associated with lncRNAs (ERV-lncRNAs), coupled with the diverse range of lncRNA functions, creates significant potential for ERV-lncRNAs to impact cancer biology.

Methods: We used RNA-seq analysis to identify and profile the expression of a novel lncRNA in six large cohorts, including over 7,500 samples from The Cancer Genome Atlas (TCGA).

Results: We identified the tumor-specific expression of a novel lncRNA that we have named Endogenous retroViral-associated ADenocarcinoma RNA or 'EVADR', by analyzing RNA-seq data derived from colorectal tumors and matched normal control tissues. Subsequent analysis of TCGA RNA-seq data revealed the striking association of EVADR with adenocarcinomas, which are tumors of glandular origin. Moderate to high levels of EVADR were detected in 25 to 53% of colon, rectal, lung, pancreas and stomach adenocarcinomas (mean = 30 to 144 FPKM), and EVADR expression correlated with decreased patient survival (Cox regression; hazard ratio = 1.47, 95% confidence interval = 1.06 to 2.04, P = 0.02). In tumor sites of non-glandular origin, EVADR expression was detectable at only very low levels and in less than 10% of patients. For EVADR, a MER48 ERV element provides an active promoter to drive its transcription. Genome-wide, MER48 insertions are associated with nine lncRNAs, but none of the MER48-associated lncRNAs other than EVADR were consistently expressed in adenocarcinomas, demonstrating the specific activation of EVADR. The sequence and structure of the EVADR locus is highly conserved among Old World monkeys and apes but not New World monkeys or prosimians, where the MER48 insertion is absent. Conservation of the EVADR locus suggests a functional role for this novel lncRNA in humans and our closest primate relatives.

Conclusions: Our results describe the specific activation of a highly conserved ERV-lncRNA in numerous cancers of glandular origin, a finding with diagnostic, prognostic and therapeutic implications.

No MeSH data available.


Related in: MedlinePlus