Heterogeneous beta-catenin activation is sufficient to cause hepatocellular carcinoma in zebrafish

Abstract

Up to 41% of hepatocellular carcinomas (HCCs) result from activating mutations in the CTNNB1 gene encoding β-catenin. HCC-associated CTNNB1 mutations stabilize the β-catenin protein, leading to nuclear and/or cytoplasmic localization of β-catenin and downstream activation of Wnt target genes. In patient HCC samples, β-catenin nuclear and cytoplasmic localization are typically patchy, even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in β-catenin activation are not well understood. To define mechanisms of β-catenin-driven HCC initiation, we generated a Cre-lox system that enabled switching on activated β-catenin in (1) a small number of hepatocytes in early development; or (2) the majority of hepatocytes in later development or adulthood. We discovered that switching on activated β-catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/β-catenin signaling heterogeneity later in hepatocarcinogenesis, we performed RNA-seq analysis of zebrafish β-catenin-driven HCC. At the single-cell level, 2.9% to 15.2% of hepatocytes from zebrafish β-catenin-driven HCC expressed two or more of the Wnt target genes axin2, mtor, glula, myca and wif1, indicating focal activation of Wnt signaling in established tumors. Thus, heterogeneous β-catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis.

MicroRNAs in Animal Models of HCC

“…Up to 40% of human HCCs result from activating mutations in the gene encoding for β-catenin (CTNNB1). The zebrafish model was used to prove that activated β-catenin expression in a small subset of hepatocytes is sufficient to drive HCC initiation [209]. Zebrafish are effective as disease models because of their high fecundity, low housing cost, and transparency, which allows live imaging and is useful to explore mechanisms by which HBV and HCV induce HCC.…”

Genetic Engineering of Zebrafish in Cancer Research

“…Additionally, the large number of CreER T2 expressing lines [ 224 ] offers a strong potential in terms of generation of zebrafish cancer models. However, only few applications of the conditional CreER T2 technology have been reported so far in the field of cancer modelling [ 225 , 226 ]. Kalasekar et al [ 225 ] generated a model of hepatocellular carcinoma based on the CreER T2 -mediated conditional expression of an activated version of β-catenin (Xla.Ctnnb1 S33A, S37A, T41A, S45A , and hereafter-named Ctnnb1 ACT ).…”

“…However, only few applications of the conditional CreER T2 technology have been reported so far in the field of cancer modelling [ 225 , 226 ]. Kalasekar et al [ 225 ] generated a model of hepatocellular carcinoma based on the CreER T2 -mediated conditional expression of an activated version of β-catenin (Xla.Ctnnb1 S33A, S37A, T41A, S45A , and hereafter-named Ctnnb1 ACT ). In this system, a Tg(fapb10a:CreER T2 ) line expressing Cre recombinase fused to the modified LBD of the estrogen receptor downstream of the hepatocyte-specific fabp10a promoter is used as a driver line, whereas a Tg(fapb10a:loxP-BFP-loxP-Ctnnb1 ACT ) transgenic line serves as a switch target line.…”

“…Furthermore, hepatocellular carcinoma penetrance was significantly lower in the CreER T2 -based model (about 30%) than in the constitutive Tg(fabp10a:Ctnnb1 ACT ) model in which activated β-catenin is directly under the control of the fabp10a promoter (about 85%) [ 147 ]. One possible explanation for the difference in penetrance is that activated β-catenin is expressed slightly earlier in the constitutive Tg(fabp10a:Ctnnb1 ACT ) model than in the CreER T2 -based model due to the time required to transcribe and translate CreER T2 and to excise the BFP/STOP cassette preventing the expression of the activated β-catenin [ 225 ]. Then, the potential leakiness and the possible reduced efficacy of the CreER T2 system imply the requirement for further improvements before its wider application to zebrafish cancer modelling.…”

Cooperation between liver-specific mutations of pten and tp53 genetically induces hepatocarcinogenesis in zebrafish

“…In addition, liver-specific expression of oncogenic kras V12 can drive robust liver tumorigenesis in transgenic zebrafish [ 33 ]. A recent study also indicated that heterogeneous β-catenin activation is sufficient to cause HCC in zebrafish [ 34 ]. Another study reported that gankyrin transgenic zebrafish spontaneously developed persistent hepatocyte damage, steatosis, cholestasis, cholangitis, fibrosis, and hepatic tumours at 7–12 months of age [ 35 ].…”