A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer

Kathryn A. O’Donnella,b,1,2, Vincent W. Kengc,d,e, Brian Yorkf, Erin L. Reinekef, Daekwan Seog, Danhua Fanc,h, Kevin A. T. Silversteinc,h, Christina T. Schruma,b, Wei Rose Xiea,b,3, Loris Mularonii,j, Sarah J. Wheelani,j, Michael S. Torbensonk, Bert W. O’Malleyf, David A. Largaespadac,d,e, and Jef D. Boekea,b,i,2 Departments of aMolecular Biology and Genetics, iOncology, jDivision of Biostatistics and Bioinformatics, and kPathology, and bThe High Throughput Biology Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; cMasonic Cancer Center, dDepartment of Genetics, Cell Biology, and Development, eCenter for Genome Engineering, and hBiostatistics and Bioinformatics Core, University of Minnesota, Minneapolis, MN 55455; fDepartment of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; and gLaboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 AUTHOR SUMMARY

Emerging data from cancer ge- functions are altered. This ap- fi nome-sequencing studies have Sleeping Beauty (SB) transposase proach identi ed at least 16 demonstrated that human genes/loci that contribute to liver tumors exhibit tremendous Transposon array with gene trap (GT) tumor development. complexity and heterogeneity in We next validated that the the number and nature of iden- genes identified in the SB screen tified mutations (1). Based on contribute to tumor initiation these findings, there is an in- SB mobilization and/or progression using in vitro creasing need for in vivo vali- and in vivo cancer model sys- dation of genes whose altered tems. First, we used liver pro- function contributes to cancer Transposon insertion Gene truncation and/or genitor cells that form tumors pathogenesis. Transposons are activation only after sustaining additional DNA sequences that can insert genetic alterations. Candidate themselves into new locations gene function was inhibited us- within the genome and thereby ing shRNAs, and cells were serve as powerful mutagens. transplanted into recipient mice Sleeping Beauty (SB), a member to assess tumor-forming poten- of the Tc1/mariner superfamily tial. These experiments vali- of DNA transposons, is highly Sequence common transposon dated the tumor-suppressor active in mammalian cells (2). A insertions to identify activity of several genes, in- growing body of evidence has tumor-promoting genes cluding Nuclear receptor coac- revealed that the SB system is an tivator 2 (Ncoa2/Src-2), Zinc efficient tool for cancer gene Fig. P1. The Sleeping Beauty (SB) DNA transposon system is based on finger transcription factor (Zfx), discovery (3, 4). We have per- the use of two transgenic mouse lines, one harboring multiple copies and Beta-Dystrobrevin (Dtnb). formed a forward genetic screen of the mutagenic transposon containing sequences that can disrupt Next, we compared our SB hit using the SB system to identify gene function and the other carrying a transposase, which is an enzyme list with an expression-profiling that binds to the transposon ends and catalyzes its mobilization to genes that accelerate liver tu- dataset from a large cohort fi new sites. To identify mutations that cooperate with Myc in liver morigenesis in mice. Our nd- tumorigenesis, we performed a forward genetic screen with a mouse of human liver tumors to de- ings reveal genes and pathways model of Myc-induced liver cancer. We sequenced insertions in early- termine the relevance of the that participate in tumorigenesis developing liver tumors and identified at least 16 genes/loci defined CISs to human liver cancer and therefore may provide tar- as CISs that contribute to accelerated tumor development. Functional pathogenesis. We found that gets for liver cancer therapy. studies were then performed to validate CISs, revealing a tumor- several of the genes identified in In this study, we sought to suppressor role for several of these genes in liver cancer. identify genes that, when mu- tated, contribute to accelerated Author contributions: K.A.O., D.A.L., and J.D.B. liver tumor development (Fig. P1). Specifically, a screening ap- designed research; K.A.O., V.W.K., B.Y., E.L.R., C.T.S., W.R.X., and B.W.O. performed re- proach was designed to recover mutations that cooperate with search; M.S.T. and D.A.L. contributed new reagents/analytic tools; K.A.O., B.Y., E.L.R., D.S., MYC, one of the most commonly dysregulated genes in human D.F., K.A.S., L.M., and S.J.W. analyzed data; and K.A.O. and J.D.B. wrote the paper. fl malignancy. We bred mice containing an active SB transposon to The authors declare no con ict of interest. mice that develop MYC-induced liver cancer. A cohort of mice This article is a PNAS Direct Submission. containing the SB transposon, a transposase, and the MYC on- Data deposition: The sequences from this study have been submitted to the National fi Center for Biotechnology Information (NCBI) Short Read Archive, http://www.ncbi.nlm. cogene expressed speci cally in liver cells was generated. Control nih.gov/sra (accession no. SRA051795.1). mice expressed MYC but lacked an active SB transposon. A sig- 1 Present address: Department of Molecular Biology, University of Texas Southwestern nificantly higher percentage of animals harboring the active SB Medical Center, Dallas, TX 75390. element developed liver tumors compared with controls. We used 2To whom correspondence may be addressed. E-mail: Kathryn.ODonnell@UTSouthwestern. high-throughput sequencing to characterize transposon insertion edu or [email protected]. sites in tumors and to identify regions of the genome that har- 3Present address: Department of Molecular and Human Genetics, Graduate Program in bored insertions at a statistically significantly greater frequency Cell and Molecular Biology, Baylor College of Medicine, Houston, TX 77030. than expected by chance. These common insertion sites (CISs) See full research article on page E1377 of www.pnas.org. mark genes that are likely to accelerate tumorigenesis when their Cite this Author Summary as: PNAS 10.1073/pnas.1115433109.

7966–7967 | PNAS | May 22, 2012 | vol. 109 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1115433109 Downloaded by guest on September 29, 2021 our screen exhibit dysregulated expression in human hepatocel- functional effect as increasing G6Pase activity, is antitumori- PNAS PLUS lular carcinoma (HCC). genic. Based on these findings, loss of Ncoa2 may promote liver Among the genes identified in our screen, Ncoa2 was of par- tumorigenesis at least in part through a subsequent reduction ticular interest. Ncoa2 is a member of the p160 family of tran- in G6Pase activity, which would be expected to increase glyco- scriptional coactivators that activates expression of Glucose 6 lytic flux by increasing levels of glucose-6-phosphate. Thus, re- phosphatase (G6Pase) and other important genes by acting in duction of Ncoa2 and G6Pase may contribute to the metabolic concert with nuclear receptors such as RORα. G6Pase is a rate- reprogramming of cancer cells and fuel tumor growth. However, limiting enzyme that dephosphorylates glucose-6-phosphate, the Ncoa2 loss of function also leads to broader dysregulation of initial substrate for glycolysis. Analysis of gene-expression data gene expression in liver (5), including the altered expression of from human HCC revealed that low expression of NCOA2 and genes involved in signal transduction and cell death. Therefore, its target G6PC are associated with poor patient survival. Fur- Ncoa2 likely functions as a tumor suppressor by controlling thermore, we documented that genetic ablation of Ncoa2/Src-2 the activity of multiple pathways and targets relevant to hep- in mice promotes liver tumorigenesis in a carcinogen-induced atocarcinogenesis. Future studies will focus on the detailed fi hepatocarcinogenesis model. These ndings establish a tumor- mechanistic dissection of NCOA2-mediated tumor suppression. suppressor role for Ncoa2 in HCC and demonstrate that trans- poson-mediated mutagenesis screens in mice can identify clini- 1. Jones S, et al. (2008) Core signaling pathways in human pancreatic cancers revealed by cally relevant genes that participate in the pathogenesis of global genomic analyses. Science 321:1801–1806. human cancer. 2. Ivics Z, Hackett PB, Plasterk RH, Izsvák Z (1997) Molecular reconstruction of Sleeping The role of Ncoa2 in regulating glucose metabolism may play Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91: 501–510. a role in its antitumorigenic activity. Mice lacking Ncoa2 develop 3. Collier LS, Carlson CM, Ravimohan S, Dupuy AJ, Largaespada DA (2005) Cancer gene glycogen storage disease type 1 (Von Gierke’s disease) and ex- discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. hibit decreased activity of G6pase. Similarly, a genetic deficiency Nature 436:272–276. of G6PC leads to Von Gierke’s disease in humans and in mice. 4. Dupuy AJ, Akagi K, Largaespada DA, Copeland NG, Jenkins NA (2005) Mammalian − − Patients who have Von Gierke’s disease and G6Pase / mice mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436:221–226. develop liver adenomas and are at high risk of progression to 5. Jeong JW, et al. (2006) The genomic analysis of the impact of steroid receptor HCC. Moreover, inhibition of hexokinase, which has the same coactivators ablation on hepatic metabolism. Mol Endocrinol 20:1138–1152. MEDICAL SCIENCES

O’Donnell et al. PNAS | May 22, 2012 | vol. 109 | no. 21 | 7967 Downloaded by guest on September 29, 2021