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 (HRASV12), 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.