The Sleeping Beauty Transposon System For Forward And Reverse Genetic Studies Of Liver Cancer

Abstract

Hepatocellular carcinoma (HCC) is the second leading cause of death from cancer globally and increasing in prevalence. HCC usually occurs in the context of chronic liver damage. The specific genetic alterations promoting HCC have been difficult to identify due to the genetic heterogeneity of HCC and the prevalence of large chromosome aberrations. To study the pathogenesis of HCC, we used Sleeping Beauty (SB) transposons for both a forward genetic screen and reverse genetic studies of genes identified in HCC mouse models, altered in human HCC, or components of the Hepatitis B virus, a major HCC risk factor. Hepatic steatosis is a common chronic liver disease linked to HCC development. We used conditional SB transposon insertional mutagenesis in mice with diet and ethanol induced hepatic steatosis to model steatosis-associated HCC and perform a forward genetic screen for molecular drivers. We compared the results from this screen to human HCC data from multiple sources. We found an increase in HCC in females in both mice and humans with hepatic steatosis, reducing the typical male sex bias of HCC. We identified over 200 genes candidate steatosis-associated HCC genes in mice, many of which are altered in human steatosis and alcohol associated HCC. We identified an association between protein kinase A/cyclic AMP signaling pathway alterations and steatosis-associated HCC, and NAT10 overexpression and HCC, both of which could potentially be targeted therapeutically. We used SB transposon-based gene delivery for reverse genetic studies testing candidate oncogenes identified in human HCC, complementary mouse models including the steatosis-associated HCC screen, and Hepatitis B viral genes for their roles in promoting liver tumorigenesis. These studies revealed new oncogenic roles for genes tested in normal livers, in mice of both sexes, in steatotic livers, and in fibrotic livers. We generated several new mouse models of HCC that could be used for mechanistic studies or therapeutic testing.