Sleeping Beauty Insertional Mutagenesis in Mice Identifies Drivers of Steatosis-Associated Hepatic Tumors

Abstract

Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis, and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis-associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance.

Molecular profiling of nonalcoholic fatty liver disease-associated hepatocellular carcinoma using SB transposon mutagenesis

“…Tschida et al ( 46 ) reported an SB mutagenesis screen performed in the context of the alcohol-induced steatotic liver. In these studies, Tschida et al identified 203 candidate steatosis-associated CCGs and a number of cancer driver pathways, including Wnt/β-catenin and PKA/cAMP.…”

Transposon Insertion Mutagenesis in Mice for Modeling Human Cancers: Critical Insights Gained and New Opportunities

“…Tol2 (isolated from medaka fish) and insect-derived PB and Minos have also been used in mutagenesis in vertebrates such as the mouse and zebrafish [16][17][18]. Sleeping Beauty (SB) is derived from elements cloned from salmonid fish and has been widely used in insertional mutagenesis screens in mice [19][20][21][22][23][24][25][26][27][28][29] and shown to be active in other vertebrates including cultured cell lines, rats, zebrafish, and Xenopus [19,[30][31][32].…”

“…Transposon mutagenesis offers an opportunity to reflect these changes in how we model cancer in the mouse. For example, Tschida et al used SB insertion mutagenesis to model hepatocellular carcinoma in the context of steatosis or accumulation of fat in the liver [27]. By comparing steatosis-associated drivers to drivers found in another screen with normal diet [65], the authors were able to identify steatosis-specific drivers.…”

A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

“…Mutagenic SB transposons were developed to modify expression of endogenous genes in specific ways depending on their relative position and orientation, as previously described (Dupuy 2010). This feature, combined with our experience analyzing insertion site data from 15 SB-induced models of cancer Mann et al 2012;Wu et al 2012;Keng et al 2013;Quintana et al 2013;Rogers et al 2013;Zanesi et al 2013;Bard-Chapeau et al 2014;Mann et al 2015;Takeda et al 2015;Montero-Conde et al 2017;Morris et al 2017;Suarez-Cabrera et al 2017;Tschida et al 2017;Riordan et al 2018), led us to develop an algorithm to predict the functional impact that recurrent transposon insertion has on a given gene. This approach evaluates clustered transposon insertions within each candidate locus to determine if there is a bias for insertion in the same orientation as the gene, indicative of an over-expression mechanism, or if the transposons are randomly orientated, suggesting a gene disruption mechanism (see Supplemental Methods).…”