Emergence of the Dedifferentiated Phenotype in Hepatocyte‐Derived Tumors in Mice: Roles of Oncogene‐Induced Epigenetic Alterations

Hepatocellular carcinoma often reactivates the genes that are transiently expressed in fetal or neonatal livers. However, the mechanism of their activation has not been elucidated. To explore how oncogenic signaling pathways could be involved in the process, we examined the expression of fetal/neonatal genes in liver tumors induced by the introduction of myristoylated v‐akt murine thymoma viral oncogene (AKT), HRas proto‐oncogene, guanosine triphosphatase (HRAS(V12)), and MYC proto‐oncogene, bHLH transcription factor (Myc), in various combinations, into mouse hepatocytes in vivo. Distinct sets of fetal/neonatal genes were activated in HRAS‐ and HRAS/Myc‐induced tumors: aldo‐keto reductase family 1, member C18 (Akr1c18), glypican 3 (Gpc3), carboxypeptidase E (Cpe), adenosine triphosphate‐binding cassette, subfamily D, member 2 (Abcd2), and trefoil factor 3 (Tff3) in the former; insulin‐like growth factor 2 messenger RNA binding protein 3 (Igf2bp3), alpha fetoprotein (Afp), Igf2, and H19, imprinted maternally expressed transcript (H19) in the latter. Interestingly, HRAS/Myc‐induced tumors comprised small cells with a high nuclear/cytoplasmic ratio and messenger RNA (mRNA) expression of delta‐like noncanonical Notch ligand 1 (Dlk1), Nanog homeobox (Nanog), and sex determining region Y‐box 2 (Sox2). Both HRAS‐ and HRAS/Myc‐induced tumors showed decreased DNA methylation levels of Line1 and Igf2 differentially methylated region 1 and increased nuclear accumulation of 5‐hydroxymethylcytosine, suggesting a state of global DNA hypomethylation. HRAS/Myc‐induced tumors were characterized by an increase in the mRNA expression of enzymes involved in DNA methylation (DNA methyltransferase [Dnmt1, Dnmt3]) and demethylation (ten‐eleven‐translocation methylcytosine dioxygenase 1 [Tet1]), sharing similarities with the fetal liver. Although mouse hepatocytes could be transformed by the introduction of HRAS/Myc in vitro, they did not express fetal/neonatal genes and sustained global DNA methylation, suggesting that the epigenetic alterations were influenced by the in vivo microenvironment. Immunohistochemical analyses demonstrated that human hepatocellular carcinoma cases with nuclear MYC expression were more frequently positive for AFP, IGF2, and DLK1 compared with MYC‐negative tumors. Conclusion: The HRAS signaling pathway and its interactions with the Myc pathway appear to reactivate fetal/neonatal gene expression in hepatocytic tumors partly through epigenetic alterations, which are dependent on the tumor microenvironment.