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  current news   Press   selected story    
     
  13 December 2013  
  Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model
 
 



Authors
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 Benoukraf8, 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.

1  Institute Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR),    Biopolis, Singapore.
2  Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
3  Clinical Imaging Research Centre, National University of Singapore, Centre for Translation Medicine,    Singapore, Singapore Bioimaging Consortium.
4  Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore.
5  Department of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The    Netherlands.
6  Singapore Immunology Network (SIgN), A*STAR, Biopolis, Singapore.
7  National Cancer Centre, Singapore.
8  Cancer Science Institute of Singapore, National University of Singapore.
9  Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City,    Iowa, USA.
10 Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center,    Houston, Texas, USA.
11 Present address: The Methodist Hospital Research Institute, Houston, Texas, USA.
12 These authors contributed equally to this work.

 Correspondence should be addressed to N.G.C. (ncopeland@tmhs.org).

Published in Nature Genetics on 8 December 2013.

Abstract

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.

Figure legend:

Identification of driver genes for HCC in the Liver-SB/HBV screen
a, Kaplan-Meier survival curves for male and female mice of all four combinations of genotypes. Liver-SB/HBsAg (SB/HBV), Liver-SB (SB), HBsAg transgene (HBV) and littermate control mice carrying an inactive transposon (no transposase) and no HBsAg transgene (control). Female median survival was 79.1 weeks for Liver-SB/HBsAg and 103.7 weeks for Liver-SB (Logrank test p<10-4). Male median survival was 70.4 weeks for Liver-SB/HBsAg, 94.9 weeks for Liver-SB, and 88.4 for HBsAg mice (Logrank test p<10-4). b, Hepatocellular carcinoma in a male Liver-SB/HBsAg mouse at 63.6 weeks of age. Macroscopic picture showing multiple large liver tumors. c, Common insertion sites (CIS) were called from the indicated numbers of randomly chosen Liver-SB/HBV tumors. The numbers of tumor samples used were plotted against the genomic bases covered. For each combination of tumors the median values and 25th and 75th percentiles were plotted. Near saturation of the screen is seen with a theoretical asymptote (in orange) at 248,023,943 bases with an increasing number of samples.  The percentage saturation obtained with 100 samples is 75.4%. d, Many Liver-SB/HBV CIS genes are deregulated or mutated in human cancer. Repartition of the 42.5% Liver-SB/HBV CIS genes found to be either mutated or misexpressed in human HCC. A total of 1224 CISs from the Liver-SB/HBV screen were associated with a gene mutated or misexpressed in human HCC. e, Mapping of genomic and metabonomic data to metabolic pathways disrupted in HCC. To understand the metabolic pathways disrupted in HCC at the genetic, mRNA, and metabolite levels, we used data from the Liver-SB/HBV screen (Supplementary Table 1c), human expression data, and metabolomic results (Supplementary Table 7). Metabolites are written with larger font size, and genes with smaller font size. Red or blue fonts mean the gene expression or metabolite amount is increased or decreased, respectively, in HCC tissue versus adjacent non-tumor tissue. CIS genes listed in Supplementary Table 1c are labeled with *. This map focuses on glycolysis, TCA cycle, glutaminolysis, and the pentose phosphate (PP) pathway. Despite the increased glucose uptake (Glut1/2) and consumption, the tumor cells predominantly expressed the pyruvate kinase M2 isoform (Pkm2), which converts phosphoenolpyruvate (PEP) to pyruvate less efficiently than Pkm1. This promotes the accumulation and shuttling of glycolytic intermediates such as glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and fructose-1,6-biphosphate 2 (FBP) to the pentose phosphate (PP) pathway and macromolecular synthesis. Thus, this termination of the glycolysis metabolic pathway together with dysregulation of Pcx and Pdk1/2, would prevent pyruvate from entering the TCA cycle. This would also lead to a truncated TCA cycle and insufficient glucose-dependent citrate production. However, most of the TCA cycle genes (Idh1/2, Sdha, Fh, Odgh) and corresponding intermediates are up-regulated in tumor cells, except for the down-regulation of the aconitase gene family (Aco1/2), suggesting the TCA cycle is at least partially activated. Our results imply an activation of glutaminolysis (Cat2, Gls, Glud1/2) converting glutamine to α-ketoglutarate to replenish TCA cycle intermediates. Effective maintenance of citrate synthesis is possible through reductive carboxylation of glutamine-derived α-ketoglutarate by Idh1/2. These adaptations around glycolysis and the TCA cycle could allow rapid generation of both ATP for bioenergetics and important metabolites for biosynthesis. In addition, the breakdown of citrate by Acly could constitute a primary source of acetyl-CoA for fatty acid and lipid synthesis. Moreover, up-regulation of Ldha, could convert glutamine-derived pyruvate to lactate that is excreted by Mct4 transporter outside the cells.