Liver-Specific Expressions of HBx and src in the p53 Mutant Trigger Hepatocarcinogenesis in Zebrafish

Abstract

Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.

Up‐regulation of golgi α‐mannosidase IA and down‐regulation of golgi α‐mannosidase IC activates unfolded protein response during hepatocarcinogenesis

“…Although many HBx transgenic mouse models developed HCC, there was no cirrhosis or fibrosis before HCC formation. HBx transgenic zebrafish in p53 mutant, src transgenic fish or edn1 transgenic fish developed steatosis to fibrosis, hyperplasia, and dysplasia prior to developing HCC 24, 26. Therefore, to study the effects of MAN1A1 and MAN1C1 on liver tumorigenesis in vivo , transgenic zebrafish expressing MAN1A1 or MAN1C1 under the control of the liver tissue‐specific promoter fabp10a were generated and examined for the progression of hepatocarcinogenesis.…”

“…Oil Red O and Sirius Red staining were performed as described 26. Detailed protocols and reagents are included in the Supporting Materials and Methods.…”

Zebrafish: An Important Tool for Liver Disease Research

“…105 In a modified approach that used an inducible transgenic system, agents that targeted MEK, PI3K, and mTOR were tested for their efficacy in reducing growth of ras-induced tumors. 105 Other oncogenes, including myc 111 and src , 112 and hepatitis B and C viral proteins 113 have been overexpressed in zebrafish hepatocytes. Coexpression of HBX and HCV core protein cause cancer in zebrafish, primarily fibrosis-associated intrahepatic cholangiocarcinoma, 113 whereas mice typically develop HCC.…”

Genetic alterations in hepatocellular carcinoma: An update

“…HBx has also been shown to bind to p53 and to block p53 -sequence-specific DNA-binding and p53 -dependent transcription, ultimately blocking p53 -mediated apoptosis[126]. HBx and TP53 mutations have been suggested to synergistically contribute to the formation of HCC in animal models[127]. These findings suggest that HBx is involved in the etiology of TP53 mutations during the molecular pathogenesis of HCC.…”