Liver-specific androgen receptor knockout attenuates early liver tumor development in zebrafish

Hankun Li,Yan Li,Jeng-Wei Lu,Zhiyuan Gong,Xiaojing Huo

doi:10.1038/s41598-019-46378-3

Abstract

Hepatocellular carcinoma (HCC) is one of the most severe cancer types and many genetic and environmental factors contribute to the development of HCC. Androgen receptor (AR) signaling is increasingly recognized as one of the important factors associated with HCC. Previously, we have developed an inducible HCC model in kras transgenic zebrafish. In the present study, to investigate the role of AR in liver tumor development, we specifically knocked out ar gene in the liver of zebrafish via the CRISPR/Cas9 system and the knockout zebrafish was named L-ARKO for liver-specific ar knockout. We observed that liver-specific knockout of ar attenuated liver tumor development in kras transgenic zebrafish at the early stage (one week of tumor induction). However, at the late stage (two weeks of tumor induction), essentially all kras transgenic fish continue to develop HCC irrespective of the absence or presence of ar gene, indicating an overwhelming role of the driver oncogene kras over ar knockout. Consistently, cell proliferation was reduced at the early stage, but not the late stage, of liver tumor induction in the kras/L-ARKO fish, indicating that the attenuant effect of ar knockout was at least in part via cell proliferation. Furthermore, androgen treatment showed acceleration of HCC progression in kras fish but not in kras/L-ARKO fish, further indicating the abolishment of ar signalling. Therefore, we have established a tissue-specific ar knockout zebrafish and it should be a valuable tool to investigate AR signalling in the liver in future.

Highlights

Hepatocellular carcinoma (HCC) is the most common type of malignant liver cancer and causes millions of deaths annually
In our previously published hepatocyte transcriptomic data derived from WT and kras transgenic zebrafish[22], we found that ar transcripts were presented at a moderately abundant levels (1–7 transcripts per million transcript) in all samples including 6 dpf larvae and male and female adult fish (Supplementary Fig. S1A)
Since the fabp10a www.nature.com/scientificreports promoter is only active in hepatocytes, the primary liver cells constituting about 70% of total number of liver cells[35]

Summary

Result

Generation and characterization of liver-specific ar knockout (L-ARKO) transgenic zebrafish. At 2 wpi, essentially all the kras and kras/L-ARKO fish had HCC phenotype while the WT and L-ARKO control fish remained normal liver morphology and histology (Fig. 4C,E) These observations suggested that the liver-specific ar knockout deterred liver tumor progression only at the early stage of tumorigenesis and the ar knockout effect was eventually overcome by the prevailing expression of oncogenic kras. Cell proliferation in kras zebrafish increased significantly after KT11 treatment compared with that in the kras fish following Dox treatment (Fig. 6A,C), suggesting that KT11 promoted the cell proliferation during liver tumor progression. In kras/L-ARKO zebrafish, cell proliferation was lower than those in kras fish with or without KT11 treatment (Fig. 6A,C) and, after KT11 treatment, cell proliferation was not significantly increased (Fig. 6A,C) These observations suggested that the effect of KT11 was attenuated by liver-specific ar knockout in male kras zebrafish, further confirming the effect of ar knockout

Discussion

Findings

Methods