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A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma

Abstract

We describe a system that permits conditional mobilization of a Sleeping Beauty (SB) transposase allele by Cre recombinase to induce cancer specifically in a tissue of interest. To demonstrate its potential for developing tissue-specific models of cancer in mice, we limit SB transposition to the liver by placing Cre expression under the control of an albumin enhancer/promoter sequence and screen for hepatocellular carcinoma (HCC)–associated genes. From 8,060 nonredundant insertions cloned from 68 tumor nodules and comparative analysis with data from human HCC samples, we identify 19 loci strongly implicated in causing HCC. These encode genes, such as EGFR and MET, previously associated with HCC and others, such as UBE2H, that are potential new targets for treating this neoplasm. Our system, which could be modified to drive transposon-based insertional mutagenesis wherever tissue-specific Cre expression is possible, promises to enhance understanding of cancer genomes and identify new targets for therapeutic development.

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Figure 1: Accelerated tumorigenesis in p53-deficient livers compared with nonpredisposed livers of male mice.
Figure 2: Immunohistochemical analyses of liver adenomas.
Figure 3: Frequent mutagenic transposon insertions into epidermal growth factor receptor (Egfr) and EGFR interacting genes associated with HCC.
Figure 4: SB-induced tumorigenesis reveals clonal relationships between primary and metastatic derivatives.
Figure 5: The Fah-deficient mouse model validates the oncogenic potential of truncated EGFR.

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Acknowledgements

The authors wish to thank Christine E. Nelson, Stefanie S. Breitbarth, Michelle K. Gleason and Geoff Hart for their excellent technical support; Jason B. Bell for performing the hydrodynamic injections; and Heidi Gruelich, Dana Farber Cancer Institute, for her kind gift pBabe-Puro-LTR-EGFR. We are also grateful to the Minnesota Supercomputing Institute for providing extensive computational resources (hardware and systems administration support) used to carry out the sequence analysis. A.V. is supported by a Sheila Sherlock fellowship from the European Association for the Study of the Liver. L.S.C. is supported by a 1K01CA122183-01 grant from the National Cancer Institute. N.G.C., N.A.J. and L.T. are supported by the Department of Health and Human Services, National Institutes of Health and the National Cancer Institute. J.M.L. is supported by the US National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK076986-01), Spanish National Institute of Health (SAF-2007-61898) and Samuel Waxman Cancer Research Foundation. D.A.L. is supported by U01 CA84221 and R01 CA113636 grants from the National Cancer Institute.

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Correspondence to David A Largaespada.

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Competing interests

Sleeping Beauty (SB) technology was exclusively licensed to Discovery Genomics (DGI, Minneapolis), cofounded by D.A.L., who has an equity interest in DGI and is an unpaid scientific advisor to DGI. DGI, which is pursuing the use of SB for human gene therapy, contributed neither money nor personnel to the present work. The work reported in this paper is not related to DGI's future plans. The University of Minnesota has filed a patent application on the process of using transposons, such as SB, to find cancer genes in laboratory animals using a forward genetic approach like the one described here. D.A.L., L.S.C., A.J.D., N.A.J. and N.G.C. are named inventors on the patent.

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Figures 1–6, Tables 1–3, Methods (PDF 2700 kb)

Supplementary Data

8,060 non-redundant insertion sites (Excel file) (XLS 6327 kb)

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Keng, V., Villanueva, A., Chiang, D. et al. A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma. Nat Biotechnol 27, 264–274 (2009). https://doi.org/10.1038/nbt.1526

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