Inducible and repressable oncogene-addicted hepatocellular carcinoma in Tet-on xmrk transgenic zebrafish

Background & Aims

Liver cancer, mainly hepatocellular carcinoma, is a major malignancy and currently there are no effective treatment protocols due to insufficient understanding of hepatocarcinogenesis. As a potentially high-throughput and cost-effective experimental model, the zebrafish is increasingly recognized for disease studies. Here, we aim at using the zebrafish to generate a convenient hepatocellular carcinoma model.

Methods

Using the Tet-on system for liver-specific expression of fish oncogene xmrk, a hyperactive version of epidermal growth factor receptor homolog, we have generated transgenic zebrafish with inducible development of liver cancer.

Results

Liver tumors were rapidly induced with 100% penetrance in both juvenile and adult xmrk transgenic fish. Histological examination indicated that they all showed features of hepatocellular carcinoma. The induced liver tumors regressed rapidly upon inducer withdrawal. During the tumor induction stage, we detected increased cell proliferation and activation of Xmrk downstream targets Erk and Stat5, which were important for liver tumorigenesis as proved by inhibition experiments. When tumors regressed, there were decreased phosphorylated Erk and Stat5 accompanied with an increase in apoptosis.

Conclusions

Our zebrafish model demonstrates the potential of a hyperactivated epidermal growth factor receptor pathway in initiating heptocarcinogenesis. It provides clear evidence for the requirement of only a single oncogene for HCC initiation and maintenance and is thus a convenient model for further investigation of oncogene addiction and future anti-cancer drug screening.

Introduction

Hepatocellular carcinoma (HCC) is one of the deadliest malignancies in the world. Currently, treatment of HCCs is limited by the lack of early diagnostic tools, high recurrence after resection and paucity of curative therapies at advanced stages [1]. Furthermore, HCC is usually heterogeneous and its development is a complicated multi-step process, making it difficult to identify driver factors and classify subgroups. Therefore, it is imperative to understand not only the differences among HCC subgroups, but also the dynamic molecular mechanisms underlying HCC progression. Rodent models have been the gold standard for human diseases, but their high cost for high-throughput studies greatly limits its potential for detailed analyses. In the past decade, the zebrafish has become an increasingly popular model for human diseases because of its economy in animal husbandry and potential of high-throughput studies [2], [3]. By comparative transcriptomic analyses, we previously showed a high similarity between human and carcinogen-induced zebrafish liver cancers and thus validated the zebrafish model for assisting human cancer studies [4], [5]. So far, several cancer models have been established in zebrafish by the transgenic approach, such as leukemia, melanoma, embryonal rhabdomyosarcoma, and others [6], [7], [8]. However, there is still no such defined liver cancer model in zebrafish. Although the liver is a major target organ for carcinogens [9], it is difficult to rely on carcinogen-induced tumors for detailed analyses and therapeutic development as these fish are difficult to maintain and the underlying genetic and epigenetic changes may differ between individual tumors. Thus, a transgenic model for zebrafish HCC is desirable to enable long-term investigation of hepatocarcinogenesis and development of therapeutic tools.

In humans, two factors play major roles in HCC pathogenesis, i.e. chronic inflammation with cirrhosis accompanied by hepatic regeneration and mutations in oncogenes/tumor suppressor genes. Both factors have been linked to changes in several signaling pathways such as growth factors (VEGF, EGF, IGF, and HGF)/MAPKs, PI3K/AKT/mTOR, TGFb/STAT, and Wnt/β-catenin [10]. Among these, epidermal growth factor receptor (EGFR) and its ligand EGF have become attractive targets for liver cancer therapy in humans. Activation of EGFR has been correlated to the poor prognosis of HCC [11] and recognized as a link between inflammation signals and HCC formation [12]. Currently, several small chemical inhibitors and one monoclonal antibody targeting the EGFR pathway are undergoing clinical trials for HCC treatment [13].

Xmrk is a naturally occurring mutated form of the EGFR isoform EGFRb in fish of the genus Xiphophorus (platyfish and swordtails), and two mutations in its extracellular domain result in the constitutive autophosphorylation and activation of downstream signals [14]. If overexpressed in pigment cells, Xmrk can induce melanoma formation in both Xiphophorus and transgenic medaka [15], [16]. Thus, if specifically expressed in hepatocytes, it may also cause liver cancer.

So far, most tumor producing transgenic zebrafish have been generated by constitutively expressed oncogenes. However, these transgenic lines are often difficult to maintain for in-depth long-term studies. Thus, a conditional transgenic system is desirable for developing cancer models. Here we used the Tet-on system to generate transgenic zebrafish to express xmrk under a liver specific fabp10 promoter [17]. We demonstrate rapidly inducible HCC from both juvenile and aged adult fish. Moreover, withdrawal of the inducer led to rapid regression of HCC, making it an attractive model to investigate initiation, progression and regression of HCC.