Transposon Mutagenesis Identifies Genes Driving Hepatocellular Carcinoma in a Chronic Hepatitis B Mouse Model
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ARTICLES Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model Emilie A Bard-Chapeau1, Anh-Tuan Nguyen1, Alistair G Rust2, Ahmed Sayadi1, Philip Lee3, Belinda Q Chua1, Lee-Sun New4, Johann de Jong5, Jerrold M Ward1, Christopher K Y Chin1, Valerie Chew6, Han Chong Toh7, Jean-Pierre Abastado6, Touati Benoukraf 8, Richie Soong8, Frederic A Bard1, Adam J Dupuy9, Randy L Johnson10, George K Radda3, Eric Chun Yong Chan4, Lodewyk F A Wessels5, David J Adams2, Nancy A Jenkins1,11,12 & Neal G Copeland1,11,12 The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC, we performed a near-saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers and a very large number (2,860) of candidate later stage drivers that were enriched for genes that are mutated, deregulated or functioning in signaling pathways important for human HCC, with a striking 1,199 genes being linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC. Nearly 500,000 people are diagnosed with HCC each year, and their genome-wide association studies map to these distal enhancers9, rais- overall 5-year survival rate is below 12%. The highest incidence of ing the possibility that noncoding mutations in these distal elements HCC is in regions in which infection with HBV is endemic, and men might also substantially contribute to cancer and potentially explain- Nature America, Inc. All rights reserved. America, Inc. Nature 10,11 4 are two to four times more likely to develop HCC than women. HCC ing why some tumors have few or no mutated cancer genes , even related to infection with HBV has also become the fastest-rising cause after extensive genome characterization. Tumors with a paucity of © 201 of cancer-related death in the United States during the past two dec- mutated genes might also have mutations in very infrequently mutated ades. Although the use of emerging sequencing and genomics tech- genes that we do not yet have the statistical power to detect. nologies has identified many mutated and/or differentially expressed One method for identifying these missing cancer genes, as well as genes in HCC, these techniques have also uncovered a surprising to validate the hundreds of candidate cancer genes already described, npg amount of intratumor and intertumor heterogeneity1–5. For these rea- is through comparative genomics involving transposon-based inser- sons, and because important DNA mutations can be concealed among tional mutagenesis12. It has recently become possible to mobilize the the large number of passenger mutations present in these tumors, Sleeping Beauty (SB) transposon in essentially any mouse tissue at it has been difficult to identify the complete complement of driver high enough frequencies to induce virtually any kind of cancer13–15. genes for HCC. Epigenetic silencing of tumor suppressor genes also Mutagenic SB transposons carry a strong promoter for activating frequently occurs in tumors6,7. This fact, combined with recent stud- proto-oncogenes and transcriptional stop cassettes for inactivating ies showing that there may be thousands of haploinsufficient tumor tumor suppressor genes, and the transposons therefore tag cancer genes suppressor genes8, makes the identification of all driver genes for in tumor cells. Human tumor genomes are complex, with multiple HCC even more difficult. Published reports from the Encyclopedia operative mutagenic processes. By contrast, transposons tag cancer of DNA Elements (ENCODE) project have also identified millions of genes directly, thus facilitating their identification. functional elements, many of which are transcription factor binding Here we sought to obtain a comprehensive list of genes that are sites that regulate the expression of genes often located hundreds of functionally necessary to trigger HCC by performing a large-scale kilobases away9. Nearly 70% of disease-associated SNPs identified in SB transposon mutagenesis screen13,14. Because the major etiology of 1Institute Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Biopolis, Singapore. 2Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. 3Clinical Imaging Research Centre, National University of Singapore, Centre for Translation Medicine, Singapore Bioimaging Consortium, Singapore. 4Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore. 5Department of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands. 6Singapore Immunology Network (SIgN), A*STAR, Biopolis, Singapore. 7National Cancer Centre, Singapore. 8Cancer Science Institute of Singapore, National University of Singapore, Singapore. 9Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA. 10Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA. 11Present address: The Methodist Hospital Research Institute, Houston, Texas, USA. 12These authors contributed equally to this work. Correspondence should be addressed to N.G.C. ([email protected]). Received 21 May; accepted 8 November; published online 8 December 2013; doi:10.1038/ng.2847 24 VOLUME 46 | NUMBER 1 | JANUARY 2014 NATURE GENETICS ARTICLES human HCC is chronic infection with HBV16, we mobilized SB in the identified because of local transposon hopping, and we therefore livers (liver-SB)17 of transgenic mice predisposed to develop HCC as a excluded CISs on this chromosome). A gene-centric CIS-calling result of expression of the toxic HBV surface antigen (HBsAg) in their method (gCIS) that looks for a higher density of transposon inser- livers (called liver-SB/HBsAg mice)18. By using this mouse model, tions within the coding regions of all RefSeq genes than predicted we aimed to identify genes that could cooperate with HBV-induced by chance20 identified 2,525 gCIS genes (Supplementary Table 1a), liver inflammation in the induction of HCC, which is similar to that whereas a non–gene centric Gaussian kernel convolution (GKC) occurring in most human HCC. method21 that looks for a higher density of insertions within fixed kernel widths of 15–240 kb (ref. 22) identified 2,041 CISs containing RESULTS 2,103 genes (Supplementary Table 1b). There was an 83% overlap HBV-associated HCC mouse model for mutagenesis screening between the CISs identified by these two methods (P < 2 × 10−16, χ2 Liver-SB/HBsAg transgenic mice develop chronic liver inflamma- with Yates correction), and together they identified 2,871 CIS loci tion with associated reactive hyperplasia (Fig. 1a and Supplementary containing 2,881 genes (Supplementary Table 1c and Supplementary Fig. 1), hepatocytomegaly and ground glass hepatocytes (Fig. 1b), Fig. 5a). In vitro transposition cell culture assays have suggested that similarly to human chronic hepatitis B and the previously charac- SB is a random insertional mutagen and that the only requirement for terized HBsAg mice18. Liver-SB/HBsAg modifications lead to the insertion is a TA dinucleotide12. Although the GKC method scans the appearance of preneoplastic foci, which are visible from 19.7 weeks entire cancer genome for CISs, most CISs were located within or in of age, followed by hepatocellular adenoma and trabecular HCC close proximity to genes, providing additional evidence that SB tar- (Supplementary Fig. 2). SB induced a cooperative tumorigenic gets the coding regions of genes that confer a selective advantage for effect with HBsAg, as liver-SB/HBsAg mice displayed reduced sur- tumor growth. gCISs not identified by GKC often contained very large vival (Fig. 1c). We also noted a tendency toward larger numbers of (>300 kb) or small (<10 kb) genes and were probably missed in part tumors (Supplementary Fig. 2) and more advanced-stage disease because of the use of fixed kernel widths (Supplementary Fig. 5b,c). compared to HBsAg mice alone (Fig. 1d). To estimate the genetic coverage of this screen, we randomly To identify the genes mutated by SB that cooperate with HBsAg- selected subgroups of 10–220 tumors from the 228 total tumors and associated inflammation in tumor induction, we PCR amplified and then used GKC to identify the CISs for each subgroup. We then plot- sequenced the transposon insertion sites from 250 tumors harvested ted the number of tumors in each subgroup against the genomic base from 34 mice19, which yielded 328,687 sequence reads and identified pairs covered by all the CISs identified for each subgroup. The curve an average of 1,315 unique transposon insertion sites per tumor. By plateaued before reaching 100 tumors, indicating that the screen comparing the location of the transposon insertions in all tumors, was approaching saturation with as few as a 100 tumors (Fig. 2a,b). we found a few tumors that were genetically related. We removed Adding more tumors merely identified additional CISs of lower these tumors, leaving 228 genetically unrelated tumors, which frequency (Supplementary Figs. 6 and 7). Consistent with this notion we subsequently used for downstream analysis (Supplementary of saturation, this screen identified most of the CIS genes found pre- Figs. 3 and 4). viously in much smaller-scale HCC transposon screens performed in p53 mutant17 and Sav1-deficient