The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SBmediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/ Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamineinduced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy. T ransposable elements (TEs) are powerful genetic tools that are widely used in insertional mutagenesis screens because of the facile identification of transposon-induced mutations. The application of transposon-based approaches to cancer gene identification provides opportunities to study the consequences of specific mutations in the context of tumor cell initiation, progression, and maintenance in well-defined, genetically engineered mouse models. Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of DNA transposons, is highly active in mammalian cells (1). A growing body of evidence has demonstrated that SB is an efficient tool for cancer gene discovery when used in forward genetic screens in mice (2, 3).The SB system is based on the use of two transgenic mouse lines, one harboring a transposase and the other with a concatemerized mutagenic transposon containing sequences that can disrupt gene function through either gain-of-function or loss-offunction mechanisms. The transposase binds to the transposon ends and catalyzes its mobilization to new sites. The effect of ubiquitous transposition in mice is the development of T-cell leukemia and brain tumors (3, 4). In contrast, restricted expression of SB transposase accelerates fibrosarcoma development in p19 Arf −/− mice (2).Recently, this approach has been modified through the conditional activation of the SB transposase in specific tissues using Cre/LoxP technology (5, 6). In one of these studies, an SB screen identified 19 genes that accelerate liver cancer induced by expression of a dominant-negative Trp53 transgene. However, the SB system has not been used previously to identify genes that cooperate with oncogenes that initiate liver tumorigenesis.Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide and is the third leading cause of death from cancer (7,8). In the majority of cases, it occurs in a setting of chronic inflammation or cirrhosis. Although numerous genomic alterations have been documented in liver cancer, the key genetic alterations that drive hepatocellular transforma...