A transposon-based insertional mutagenesis screen for hepatocellular carcinoma (HCC)-induced metastasis in mice

Abstract

Metastasis, spread of the primary tumors to distant sites, is the main lethal attribute of cancer. Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths in the world. Distant metastasis is frequently diagnosed in HCC patients, reducing one's life expectancy and the efficacy of targeted therapies. Meanwhile, treatments available for advanced and metastatic HCC patients are very limited. To unravel the underlying genetic mechanism(s) of HCC-related metastasis and establish novel therapeutic targets, a Sleeping Beauty (SB) insertional mutagenesis screen, which recapitulated the human liver cancer disease with manifesting lung metastases, enabled us to identify metastasis-associated candidate genes. Truncated epidermal growth factor receptor (Egfr) arising from identical premature termination at Egfr intron 24 by SB transposition was detected in some of the primary tumors and most of the metastatic derivatives, suggesting the clonal origin of the metastases. In addition, a truncated version of actinin alpha 2 (Actn2) resulted from SB transposition upstream of exon 15 was found exclusively in metastases, indicating Actn2 evolution may be important in modulating the metastatic progression in HCC. Clinically, alpha actinin 2 (ACTN2) was highly dysregulated and associated in HCC patients with signs of vascular invasion or distant metastasis in The Cancer Genome Atlas (TCGA) online resource, providing an insight of the potential function of Actn2 during HCC-related metastasis. Furthermore, validation of the function of candidate genes in SB-based reverse genetic screen on both alpha actinin 2 (ACTN2) and ankyrin repeat domain 17 (Ankd17) identified in a previous study for HCC-related metastasis, was performed. Both both in vivo and in vitro overexpression experiments proved their promoting effect in HCC-associated metastasis. Work on exploring their independent metastatic mechanisms were still in process. Our results demonstrated the significance of SB forward and reverse genetics in modeling human liver malignancy and its progression. The random integration of SB mutagenic transposon allows for unbiased in vivo forward genetic screens for novel metastatic gene discovery whereas SB gene delivery allows rapid assessment on the pathogenic properties of candidate genes in our reverse genetic studies.