Activation of β-Catenin and Yap1 in Human Hepatoblastoma and Induction of Hepatocarcinogenesis in Mice

Abstract

Background & Aims Aberrant activation of βcatenin and Yes-associated protein 1 (Yap1) signaling pathways have been associated with development of multiple tumor types. Yap functions as a transcriptional co-activator by interacting with TEAD DNA binding proteins. We investigated the interactions among these pathways during hepatic tumorigenesis. Methods We used immunohistochemical analysis to determine expression of β-catenin and Yap1 in liver cancer specimens collected from patients in Europe and the US, consisting of 104 hepatocellular carcinoma (HCC), 62 intrahepatic cholangiocarcinoma (ICC), and 94 hepatoblastoma samples. We assessed βcatenin and Yap1 signaling and interactions in hepatoblastoma cell lines ((HuH6, HepG2, HepT1, HC-AFW1, HepG2, and HC-AFW1); proteins were knocked down with small interfering (si)RNAs and effects on proliferation and cell death were measured. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β catenin ( N90-βcatenin) and Yap1 (YapS127A) in livers of mice; tissues were collected and histologic and immunohistochemical analyses were performed. Results We observed nuclear localization of βcatenin and Yap1 in 79% of hepatoblastoma samples, but not in most HCC or ICC tissues. Yap1 and β catenin co-precipitated in hepatoblastoma but not HCC cells. siRNA-mediated knockdown of Yap1 or β catenin in hepatoblastoma cells reduced proliferation in an additive manner. Knockdown of Yap1 reduced its ability to co-activate transcription with βcatenin; βcatenin inhibitors inactivated Yap1. Overexpression of constitutively active forms of Yap1 and βcatenin in mouse liver led to rapid tumorigenesis, with 100% mortality by 11 weeks. Tumors cells expressed both proteins, and human hepatoblastoma cells expressed common targets of their 2 signaling pathways. Yap1 binding of TEAD factors was required for tumorigenesis in mice. Conclusions β catenin and the transcriptional regulator Yap1 interact physically and are activated in most human hepatoblastoma tissues; overexpression of activated forms of these proteins in livers of mice leads to rapid tumor development. Further analysis of these mice will allow further studies of these pathways in hepatoblastoma pathogenesis and could lead to the identification of new therapeutic targets.

Prolactin prevents hepatocellular carcinoma by restricting innate immune activation of c-Myc in mice

“…However, the association between serum PRL and HCC in patients was correlative only and did not take into account circulating E2. Additionally, the in vitro experiments relied on HepG2 cells, a cell line derived from hepatoblastoma and not HCC (48), and which preferentially express PRLR-L over PRLR-S (Fig. S1B).…”

Coordinated Activities of Multiple Myc-dependent and Myc-independent Biosynthetic Pathways in Hepatoblastoma

“…In contrast to the germ line APC mutations associated with familial HB and CRC, ϳ85% of sporadic HBs harbor acquired ␤-catenin mutations, mostly in exon 3-encoded amino acids, that also relieve ␤-catenin of its APC-dependent regulation and allow for its constitutive nuclear localization (9,10).…”

“…Most sporadic HBs also demonstrate nuclear localization of Yes-associated protein (YAP), a component of the Hippo tumor suppressor pathway that communicates with the Wnt/␤-catenin pathway (10,11). Hippo regulates the size, proliferation, and survival of normal organs and tissues and affects tumor growth (12).…”

Hepatoblastoma modeling in mice places Nrf2 within a cancer field established by mutant β-catenin

“…Moreover, as tumors frequently show hyperstabilization of Myc, mRNA profiling of HBs in the absence of follow up analysis of Myc at the protein level runs the risk of underestimating and diminishing the significance of this oncogenic transcription factor in HB (63,64). However, in light of the fact that Myc emerged as the dominant driver of HBs in this study and that Myc is overexpressed in chemically induced mouse HBs (65) as well as in HBs and HCCs that develop in mouse models following expression of oncogenes other than Myc (33,36), we propose that Myc serves as a central signaling hub for an array of other oncogenic HB drivers, making it a potentially important therapeutic target in HB that could benefit from the array of promising new drugs that target Myc and its network (66,67). Therefore, the feasibility of adopting Myc inhibitory strategies for the treatment of pediatric liver cancers and other embryonal tumors with evidence of Myc amplification/ overexpression should be explored.…”

“…In this setting, perinatal activation of the Albumin-Myc transgene likely counteracts the developmentally programmed decline in endogenous Myc to sustain the proliferation and survival of immature mutant β-catenin-expressing hepatic cells that would otherwise arrest or die (31). However, this is unlikely to be the case in Tet-O-Myc mice or other mouse models that develop HBs following oncogene activation in mature hepatocytes (32,33), raising the question of whether HB transcriptomes and immunoprofiles reflect a hepatoblast cell of origin or the end result of dedifferentiation or reprogramming of mature hepatocytes (34). While an abundance of experimental evidence indicates that aberrant activation of Wnt/β-catenin alone is not sufficient for HB development, this has recently been challenged by the creation of a genetically engineered mouse strain that develops HBs and HCCs when mutant β-catenin is targeted to rare hepatic progenitor cells that express the CBP/p300 transcriptional coactivator protein and Myc target, Cited1 (35,36).…”