Novel Fusion Transcripts in Human Gastric Cancer Revealed by Transcriptome Analysis
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Oncogene (2014) 33, 5434–5441 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Novel fusion transcripts in human gastric cancer revealed by transcriptome analysis H-P Kim1,10, G-A Cho1,10, S-W Han1,2, J-Y Shin3,4, E-G Jeong1, S-H Song1, W-C Lee3,5, K-H Lee1,2, D Bang6, J-S Seo3,4,5,7,8, J-Il Kim3,4,5,7 and T-Y Kim1,2,9 Gene fusion is involved in the development of various types of malignancies. Recent advances in sequencing technology have facilitated identification of gene fusions and have stimulated the research of this field in cancer. In the present study, we performed next-generation transcriptome sequencing in order to discover novel gene fusions in gastric cancer. A total of 282 fusion transcript candidates were detected from 12 gastric cancer cell lines by bioinformatic filtering. Among the candidates, we have validated 19 fusion transcripts, which are 7 inter-chromosomal and 12 intra-chromosomal fusions. A novel DUS4L–BCAP29 fusion transcript was found in 2 out of 12 cell lines and 10 out of 13 gastric cancer tissues. Knockdown of DUS4L–BCAP29 transcript using siRNA inhibited cell proliferation. Soft agar assay further confirmed that this novel fusion transcript has tumorigenic potential. We also identified that microRNA-coding gene PVT1, which is amplified in double minute chromosomes in SNU-16 cells, is recurrently involved in gene fusion. PVT1 produced six different fusion transcripts involving four different genes as fusion partners. Our findings provide better insight into transcriptional and genetic alterations of gastric cancer: namely, the tumorigenic effects of transcriptional read-through and a candidate region for genetic instability. Oncogene (2014) 33, 5434–5441; doi:10.1038/onc.2013.490; published online 18 November 2013 Keywords: gene fusion; fusion transcript; gastric cancer; transcriptome sequencing; next-generation sequencing INTRODUCTION In the present study, we used paired end transcriptome Gene fusion is one of the common oncogenic activation sequencing, which is an efficient tool for detecting fusion genes. mechanisms involved in the development of various types of This approach allowed us to detect 23 fusion transcripts, including malignancies including leukaemia, lymphoma, sarcoma and chimeric RNAs and fusion genes, all of which, to our knowledge, are prostate cancer.1 More importantly, gene fusions can produce novel. Fusion partner genes included hepatocyte growth factor important druggable targets, which make detection of gene receptor (MET), cyclin-dependent kinase12 (CDK12) and fibroblast fusion as an essential part of diagnosis and treatment strategy for growth factor receptor 2 (FGFR2) that are potential druggable some malignancies. Representative example is imatinib targeting targets. We also confirmed that transcriptional read-through of BCR-ABL tyrosine kinase, which has entirely changed treatment dihydrouridine synthase 4-like (DUS4L)–B-cell receptor-associated paradigm of chronic myelogenous leukaemia.2 Recent success of protein 29 (BCAP29) can be tumorigenic. Transcriptional read- crizotinib in nonsmall cell lung cancer harbouring EML4-ALK fusion through has previously been neglected because of the lack of has further underscored the importance of identifying novel gene genetic changes that occur and has never been functionally studied. fusions and effective targeted agents.3,4 In addition, we found the plasmacytoma variant translocation 1 (Pvt- Although other methods for detecting genetic alterations have 1) oncogene (PVT1), amplified and located on double minute (DM) been used, it was the advent of next-generation sequencing that chromosome, was involved in six different fusions. This suggests stimulated the fusion gene research in epithelial cancers. that PVT1 is associated with genetic instability resulting in gene Transcriptome sequencing has been the main contributor to fusions with various partners. Taken together, transcriptome fusion gene studies, for example, on breast,5,6 prostate7,8 and sequencing provides various novel fusion transcript candidates in gastric cancers.9 gastric cancer, and functional studies of these candidates contribute Gastric cancer is one of the most common and deadly to enhanced understanding of gastric carcinogenesis. cancers.10,11 Several gastric cancer-related fusion genes have been reported;9,12 however, the impact of gene fusion in gastric cancer has not been thoroughly studied. As with the discoveries of RESULTS fusion genes in other tumours, the identification of additional Detection and filtration of gene fusion candidates fusion genes in gastric cancer might provide new perspectives for We sequenced the transcriptomes of 12 gastric cancer cell lines its categorisation and understanding. (Supplementary Table S1) and detected 282 gene fusion 1Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea; 2Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea; 3Genomic Medicine Institute, Medical Research Center, Seoul National University, Seoul, Korea; 4Psoma Therapeutic Inc, Seoul, Korea; 5Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Korea; 6Department of Chemistry, College of Science, Yonsei University, Seoul, Korea; 7Department of Biochemistry, Seoul National University College of Medicine, Seoul, Korea; 8Macrogen Inc., Seoul, Korea and 9Department of Molecular Medicine & Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University College of Medicine, Seoul, Korea. Correspondence: Dr T-Y Kim, Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Korea. E-mail: [email protected] 10These authors contributed equally to the work. Received 4 April 2013; revised 1 October 2013; accepted 4 October 2013; published online 18 November 2013 Novel fusion transcripts in gastric cancer H-P Kim et al 5435 candidates (Figure 1). From the initial list of 282 gene fusion splicing or trans-splicing, were filtered out at the beginning step candidates, we discarded candidates with fewer than 10 junction because the main focus of these studies was on genetic changes. reads because fewer junction reads are likely indicators of false However, recent studies have suggested that RNA chimera positives. In addition, we observed false positive results in about TMEM79-SMG5 is generated by splicing in prostate cancer by 50 candidate fusions, with less than 10 reads. After this step, only deep sequencing.8 Similarly, SLC45A3–ELK4 fusion transcript can 22 candidates remained; however, we added back PVT1–ATE1, arise from cis-splicing of adjacent genes without corresponding PVT1–PPAPDC1A and CD44–FGFR2 because, in terms of their loci, DNA changes, suggesting that chimeric transcript is a novel class these three candidates were closely related to the already selected of gene fusion.13 On the basis of these, we included fusion PVT1–PDHX and PVT1–APIP fusion genes. In contrast, two transcript in our study. candidates were excluded as they are known to encode In order to define genetic rearrangement status of the 19 fusion hypothetical proteins. Thus, the final list contained 23 gene fusion candidates, we performed long-range PCR in the genomic DNA of candidates (Figure 2). Apart from SNU-216, the other 11 cell lines cancer cell lines. We confirmed that two fusion transcripts (MED1– exhibited one or more fusion transcripts. CDK12 and CAPZA2–MET) were results of gene fusion (Supplementary Text S2). In contrast, long-range PCR revealed that five fusion transcripts are produced by read-through Validation of gene fusion candidates mechanism (Table 1). Although we were unable to validate RT–PCR and Sanger sequencing across fusion junctions were genetic rearrangement in remaining 12 fusion transcripts owing to conducted to confirm 23 candidates identified by transcriptome the technical limitations of long-range PCR, the 7 inter-chromo- sequencing; 19 of the 23 candidates were validated with 7 inter- somal fusion transcripts are most likely resulting from genetic chromosomal and 12 intra-chromosomal fusions (Table 1 and rearrangements. The genetic status of the five intra-chromosomal Supplementary Text S1). Five read-through fusion transcripts were fusion transcripts needs to be investigated using alternative among the 12 intra-chromosomal fusions. In most gene fusion technique in future studies. In the genome, MED1 and CDK12 are studies, chimeric RNAs, which are generated by read-through/ neighbours, but are located on different strands with opposite Figure 1. Circos plot representing the fusion events in 12 gastric cancer cell lines. Transcriptome analysis was conducted on 12 gastric cancer cell lines: SNU-1, SNU-5, SNU-16, SNU-216, SNU-484, SNU-601, SNU-620, SNU-638, SNU-668, SNU-719, MKN45 and NCI-N87. Each connection with a line in a circle indicates a gene fusion in the cell line. & 2014 Macmillan Publishers Limited Oncogene (2014) 5434 – 5441 Novel fusion transcripts in gastric cancer H-P Kim et al 5436 orientations; therefore, MED1–CDK12 fusion can be classified as cis Next, to investigate their relationships with tumours, we intra-chromosomal fusion gene.14 CAPZA2 and MET are examined the expression of the fusion transcripts in 13 primary neighbours on the same strand. Unlike most transcriptional gastric cancer tissues and their matched adjacent normal mucosa read-throughs, however,