Summary DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.

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.

Amplification and/or activation of the c-Myc protooncogene is one of the leading genetic events along hepatocarcinogenesis. The oncogenic potential of c-Myc has been proven experimentally by the finding that its overexpression in the mouse liver triggers tumor formation. However, the molecular mechanism whereby c-Myc exerts its oncogenic activity in the liver remains poorly understood. Here, we demonstrate that the mammalian target of rapamycin complex 1 (mTORC1) cascade is activated and necessary for c-Myc dependent hepatocarcinogenesis. Specifically, we found that ablation of Raptor, the unique member of the mTORC1 complex, strongly inhibits c-Myc liver tumor formation. Also, p70S6K/ribosomal protein S6 (RPS6) and eukaryotic translation initiation factor 4E-binding protein 1/eukaryotic translation initiation factor 4E (4EBP1/eIF4E) signaling cascades downstream of mTORC1 are required for c-Myc-driven tumorigenesis. Intriguingly, microarray expression analysis revealed the upregulation of multiple amino acid transporters, including SLC1A5 and SLC7A6, leading to robust uptake of amino acids, including glutamine, into c-Myc tumor cells. Subsequent functional studies showed that amino acids are critical for activation of mTORC1, as their inhibition suppressed mTORC1 in c-Myc tumor cells. In human HCC specimens, levels of c-Myc directly correlate with those of mTORC1 activation as well as of SLC1A5 and SLC7A6. Conclusion Our current study indicates that an intact mTORC1 axis is required for c-Myc-driven hepatocarcinogenesis. Thus, targeting mTOR pathway or amino acid transporters may be an effective and novel therapeutic option for the treatment of HCC with activated c-Myc signaling.

Activation of the AKT/mTOR cascade and overexpression of c-Met have been implicated in the development of human hepatocellular carcinoma (HCC). To elucidate the functional crosstalk between the two pathways, we generated a model characterized by the combined expression of activated AKT and c-Met in the mouse liver. Co-expression of AKT and c-Met triggered rapid liver tumor development and mice required to be euthanized within 8 weeks after hydrodynamic injection. At the molecular level, liver tumors induced by AKT/c-Met display activation of AKT/mTOR and Ras/MAPK cascades as well as increased lipogenesis and glycolysis. Since a remarkable lipogenic phenotype characterizes liver lesions from AKT/c-Met mice, we determined the requirement of lipogenesis in AKT/c-Met driven hepatocarcinogenesis using conditional Fatty Acid Synthase (FASN) knockout mice. Of note, hepatocarcinogenesis induced by AKT/c-Met was fully inhibited by FASN ablation. In human HCC samples, coordinated expression of FASN, activated AKT, and c-Met proteins was detected in a subgroup of biologically aggressive tumors. Altogether, our study demonstrates that co-activation of AKT and c-Met induces HCC development that depends on the mTORC1/FASN pathway. Suppression of mTORC1 and/or FASN might be highly detrimental for the growth of human HCC subsets characterized by concomitant induction of the AKT and c-Met cascades.

Background & Aims Cumulating evidence underlines the crucial role of aberrant lipogenesis in human hepatocellular carcinoma (HCC). Here, we investigated the oncogenic potential of fatty acid synthase (FASN), the master regulator of de novo lipogenesis, in the mouse liver. Methods FASN was overexpressed in the mouse liver, either alone or in combination with activated N-Ras, c-Met, or SCD1, via hydrodynamic injection. Activated AKT was overexpressed via hydrodynamic injection in livers of conditional FASN or Rictor knockout mice. FASN was suppressed in human hepatoma cell lines via specific small interfering RNA. Results Overexpression of FASN, either alone or in combination with other genes associated with hepatocarcinogenesis, did not induce histological liver alterations. In contrast, genetic ablation of FASN resulted in the complete inhibition of hepatocarcinogenesis in AKT-overexpressing mice. In human HCC cell lines, FASN inactivation led to a decline in cell proliferation and a rise in apoptosis, which were paralleled by a decrease in the levels of phosphorylated/activated AKT, an event controlled by the mammalian target of rapamycin complex 2 (mTORC2). Downregulation of AKT phosphorylation/activation following FASN inactivation was associated with strong inhibition of rapamycin-insensitive companion of mTOR (Rictor), the major component of mTORC2, at post-transcriptional level. Finally, genetic ablation of Rictor impaired AKT-driven hepatocarcinogenesis in mice. Conclusions FASN is not oncogenic per se in the mouse liver, but is necessary for AKT-driven hepatocarcinogenesis. Pharmacological blockade of FASN might be highly useful in the treatment of human HCC characterized by activation of the AKT pathway.