Oncogenic Determination of a Broad Spectrum of Phenotypes of Hepatocyte-Derived Mouse Liver Tumors

Activation of the phosphoinositide 3-kinase–AKT, Yes-associated protein (YAP), and MYC pathways is involved in human liver cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). However, the nature of the interactions among these pathways has remained poorly understood. Herein, we demonstrate the coordination of these pathways during the formation of mouse liver tumors induced by hepatocyte-specific somatic integration of myristoylated AKT, mutant YAP, Myc, or their combinations. Although the introduction of YAP or Myc alone was inefficient in inducing tumors, these proteins accelerated tumorigenesis induced by AKT. The generated tumors demonstrated various histological features: low-grade HCC by AKT/Myc, CC by AKT/YAP, and high-grade HCC by AKT/Myc/YAP. CC induced by AKT/YAP was associated with activation of the Notch pathway. Interestingly, the combination of Myc and YAP generated tumors composed of hepatoblast/stem-like cells expressing mRNA for Afp, Dlk1, Nanog, and Sox2 and occasionally forming immature ducts. Finally, immunohistochemical analysis revealed that human HCC and CC were predominantly associated with phosphorylation of S6 and glycogen synthase kinase-3β, respectively, and >60% of CC cases were positive for both phosphorylated glycogen synthase kinase--3β and YAP. Our study suggests that hepatocyte-derived tumors demonstrate a wide spectrum of tumor phenotypes, including HCC, CC, and hepatoblastoma-like, through the combinatory effects of the oncogenic pathways and that the state of the phosphoinositide 3-kinase–AKT pathway is a key determinant of differentiation. Activation of the phosphoinositide 3-kinase–AKT, Yes-associated protein (YAP), and MYC pathways is involved in human liver cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). However, the nature of the interactions among these pathways has remained poorly understood. Herein, we demonstrate the coordination of these pathways during the formation of mouse liver tumors induced by hepatocyte-specific somatic integration of myristoylated AKT, mutant YAP, Myc, or their combinations. Although the introduction of YAP or Myc alone was inefficient in inducing tumors, these proteins accelerated tumorigenesis induced by AKT. The generated tumors demonstrated various histological features: low-grade HCC by AKT/Myc, CC by AKT/YAP, and high-grade HCC by AKT/Myc/YAP. CC induced by AKT/YAP was associated with activation of the Notch pathway. Interestingly, the combination of Myc and YAP generated tumors composed of hepatoblast/stem-like cells expressing mRNA for Afp, Dlk1, Nanog, and Sox2 and occasionally forming immature ducts. Finally, immunohistochemical analysis revealed that human HCC and CC were predominantly associated with phosphorylation of S6 and glycogen synthase kinase-3β, respectively, and >60% of CC cases were positive for both phosphorylated glycogen synthase kinase--3β and YAP. Our study suggests that hepatocyte-derived tumors demonstrate a wide spectrum of tumor phenotypes, including HCC, CC, and hepatoblastoma-like, through the combinatory effects of the oncogenic pathways and that the state of the phosphoinositide 3-kinase–AKT pathway is a key determinant of differentiation. As the third leading cause of cancer death in the world, primary liver cancer is refractory to various treatment modalities. This finding may be partly explained by its wide genetic variations, as reflected by diverse phenotypes and histological types.1Zucman-Rossi J. Villanueva A. Nault J.C. Llovet J.M. Genetic landscape and biomarkers of hepatocellular carcinoma.Gastroenterology. 2015; 149: 1226-1239.e4Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar Although hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CC) are prototypical epithelial liver cancers, there are many intermediate variants [combined hepatocellular cholangiocarcinoma (cHCC-CC)] and tumors reminiscent of immature (fetal) liver [hepatoblastoma (HB)]. On morphological and immunohistochemical grounds, it has been hypothesized that HCC and CC are derived from hepatocytes and bile ducts/ductules (cholangiocytes), respectively. Some cHCC-CC subtypes and a fraction of HCCs have been regarded as tumors of immature bipotential cells or liver stem/progenitor cells with hepatoblastic features, which have been postulated to present in the adult liver.2Komuta M. Spee B. Vander Borght S. De Vos R. Verslype C. Aerts R. Yano H. Suzuki T. Matsuda M. Fujii H. Desmet V.J. Kojiro M. Roskams T. 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Demartis M.I. Ribback S. Utpatel K. Dombrowski F. Evert M. Calvisi D.F. Chen X. Co-activation of PIK3CA and Yap promotes development of hepatocellular and cholangiocellular tumors in mouse and human liver.Oncotarget. 2015; 6: 10102-10115Crossref PubMed Scopus (46) Google Scholar Thus, liver tumors derived from mature hepatocytes demonstrate a wide spectrum of phenotypes from HCC to CC, possibly encompassing cHCC-CC and HB. In this study, we explored the interactions of the PI3K-AKT, YAP, and Myc pathways in hepatocytic tumorigenesis and phenotypic determination, using a combination of Sleeping Beauty (SB) transposon-mediated oncogene integration and hydrodynamic tail vein injection (HTVi) in mice. Herein, we demonstrate that the activation of the PI3K-AKT pathway alone in hepatocytes induces HCC with bile ductular transdifferentiation and that the simultaneous activation of the Myc or YAP pathway with the PI3K-AKT pathway promotes hepatocarcinogenesis with unidirectional differentiation, thus resulting in HCC and CC, respectively. Furthermore, unless the PI3K-AKT pathway is active, coactivation of the Myc and YAP pathways generates dedifferentiated liver tumors reminiscent of HB composed of hepatoblast-like cells with a high nuclear/cytoplasmic ratio. C57BL/6J mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). The mice were euthanized under deep anesthesia, and the livers were removed for further examination. The protocols used for animal experimentation were approved by the Animal Research Committee, Asahikawa Medical University (Asahikawa, Japan). All animal experiments adhered to the criteria outlined in the Guide for the Care and Use of Laboratory Animals.25Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals.Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar An SB13 transposase-expressing vector [pT2/C-Luc/PGKSB13; Addgene plasmid #20207 deposited by Dr. John Ohlfest (University of Minnesota)], a myristoylated AKT-hemagglutinin (HA)-expressing transposon cassette vector [pT3-EF1α-myrAKT-HA; Addgene plasmid #31789 deposited by Dr. Xin Chen (University of California, San Francisco)], and a mutant YAP-expressing transposon cassette vector (pT3-EF1α-FLAG-YAPS127A; Addgene plasmid #46049 deposited by Dr. Xin Chen) were obtained from Addgene (Cambridge, MA). cDNA fragments of Notch1 intracellular domain (N1ICD), Notch2 intracellular domain (N2ICD), and enhanced green fluorescent protein were amplified from p3xFlag-N1ICD [Addgene plasmid #20183 deposited by Dr. Raphael Kopan (University of Cincinnati), Addgene], p3xFlag-N2ICD [Addgene plasmid #20184 deposited by Dr. Raphael Kopan (University of Cincinnati); Addgene], and pCMV–enhanced green fluorescent protein (Takara Bio, Kusatsu, Japan), respectively. A full-length Myc fragment was amplified from the cDNA of diethylnitrosamine-induced mouse liver tumors. These cDNA fragments were cloned into the pT3-EF1α plasmid with the Gateway system (ThermoFisher Scientific, Waltham, MA). LoxP sites were removed from pT3-EF1α-myrAKT-HA and pT3-EF1α-FLAG-YAPS127A using the GENEART Site-Directed Mutagenesis System (ThermoFisher Scientific) (pT3-myrAkt-ΔΔLoxP and pT3-YAPS127A-ΔΔLoxP, respectively). All plasmids were amplified and purified using the EndoFree Plasmid Maxi kit (Qiagen, Hilden, Germany).