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Common fusion transcripts identified in colorectal cancer cell lines by high-throughput RNA sequencing.

ABSTRACT: Colorectal cancer (CRC) is the third most common cancer disease in the Western world, and about 40% of the patients die from this disease. The cancer cells are commonly genetically unstable, but only a few low-frequency recurrent fusion genes have so far been reported for this disease. In this study, we present a thorough search for novel fusion transcripts in CRC using high-throughput RNA sequencing. From altogether 220 million paired-end sequence reads from seven CRC cell lines, we identified 3391 candidate fused transcripts. By stringent requirements, we nominated 11 candidate fusion transcripts for further experimental validation, of which 10 were positive by reverse transcription-polymerase chain reaction and Sanger sequencing. Six were intrachromosomal fusion transcripts, and interestingly, three of these, AKAP13-PDE8A, COMMD10-AP3S1, and CTB-35F21.1-PSD2, were present in, respectively, 18, 18, and 20 of 21 analyzed cell lines and in, respectively, 18, 61, and 48 (17%-58%) of 106 primary cancer tissues. These three fusion transcripts were also detected in 2 to 4 of 14 normal colonic mucosa samples (14%-28%). Whole-genome sequencing identified a specific genomic breakpoint in COMMD10-AP3S1 and further indicates that both the COMMD10-AP3S1 and AKAP13-PDE8A fusion transcripts are due to genomic duplications in specific cell lines. In conclusion, we have identified AKAP13-PDE8A, COMMD10-AP3S1, and CTB-35F21.1-PSD2 as novel intrachromosomal fusion transcripts and the most highly recurring chimeric transcripts described for CRC to date. The functional and clinical relevance of these chimeric RNA molecules remains to be elucidated.

SUBMITTER: Nome T

PROVIDER: S-EPMC3799197 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Common fusion transcripts identified in colorectal cancer cell lines by high-throughput RNA sequencing.

Nome Torfinn T Thomassen Gard Os GO Bruun Jarle J Ahlquist Terje T Bakken Anne C AC Hoff Andreas M AM Rognum Torleiv T Nesbakken Arild A Lorenz Susanne S Sun Jinchang J Barros-Silva João Diogo JD Lind Guro E GE Myklebost Ola O Teixeira Manuel R MR Meza-Zepeda Leonardo A LA Lothe Ragnhild A RA Skotheim Rolf I RI

Translational oncology 20131001 5

Colorectal cancer (CRC) is the third most common cancer disease in the Western world, and about 40% of the patients die from this disease. The cancer cells are commonly genetically unstable, but only a few low-frequency recurrent fusion genes have so far been reported for this disease. In this study, we present a thorough search for novel fusion transcripts in CRC using high-throughput RNA sequencing. From altogether 220 million paired-end sequence reads from seven CRC cell lines, we identified ...[more]

PMID: 24151535

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Project description:BACKGROUND:Gene fusions arising from chromosomal translocations have been implicated in cancer. However, the role of gene fusions in BRCA1-related breast cancers is not well understood. Mutations in BRCA1 are associated with an increased risk for breast cancer (up to 80% lifetime risk) and ovarian cancer (up to 50%). We sought to identify putative gene fusions in the transcriptomes of these cancers using high-throughput RNA sequencing (RNA-Seq). METHODS:We used Illumina sequencing technology to sequence the transcriptomes of five BRCA1-mutated breast cancer cell lines, three BRCA1-mutated primary tumors, two secretory breast cancer primary tumors and one non-tumorigenic breast epithelial cell line. Using a bioinformatics approach, our initial attempt at discovering putative gene fusions relied on analyzing single-end reads and identifying reads that aligned across exons of two different genes. Subsequently, latter samples were sequenced with paired-end reads and at longer cycles (producing longer reads). We then refined our approach by identifying misaligned paired reads, which may flank a putative gene fusion junction. RESULTS:As a proof of concept, we were able to identify two previously characterized gene fusions in our samples using both single-end and paired-end approaches. In addition, we identified three novel in-frame fusions, but none were recurrent. Two of the candidates, WWC1-ADRBK2 in HCC3153 cell line and ADNP-C20orf132 in a primary tumor, were confirmed by Sanger sequencing and RT-PCR. RNA-Seq expression profiling of these two fusions showed a distinct overexpression of the 3' partner genes, suggesting that its expression may be under the control of the 5' partner gene's regulatory elements. CONCLUSIONS:In this study, we used both single-end and paired-end sequencing strategies to discover gene fusions in breast cancer transcriptomes with BRCA1 mutations. We found that the use of paired-end reads is an effective tool for transcriptome profiling of gene fusions. Our findings suggest that while gene fusions are present in some BRCA1-mutated breast cancers, they are infrequent and not recurrent. However, private fusions may still be valuable as potential patient-specific biomarkers for diagnosis and treatment.

Project description:UnlabelledIn an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPCs), we employed our high-throughput RNA sequencing (RNA-seq) analysis pipeline, RNA CoMPASS, to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC and Epstein-Barr virus (EBV) researchers. Sequencing data sets from both CNE1 and HONE1 were found to contain reads for human papillomavirus 18 (HPV-18). Subsequent real-time reverse transcription-PCR (RT-PCR) analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed patterns identical to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, as well as HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR-based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell-specific L1 retrotransposon insertion, suggesting that these three HPV-18(+) NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq-based gene level SNV analysis. Taking all of these findings together, we conclude that a widespread HeLa contamination may exist in many NPC cell lines, and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq-based approach for cell authentication.ImportanceNasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of Epstein-Barr virus (EBV). Using an RNA-seq-based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and the lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq-based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.

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Common fusion transcripts identified in colorectal cancer cell lines by high-throughput RNA sequencing.

Publications

Common fusion transcripts identified in colorectal cancer cell lines by high-throughput RNA sequencing.

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