Mounting evidence underlines the role of genomic hypomethylation in the generation of genomic instability (GI) and tumorigenesis, but whether DNA hypomethylation is required for hepatocellular carcinoma (HCC) development and progression remains unclear. We investigated the correlation between GI and DNA methylation, and influence of methionine metabolism deregulation on these parameters and hepatocarcinogenesis in c-Myc and cMyc/Tgf-a transgenic mice and human HCCs. S-adenosyl-L-methionine/S-adenosylhomocysteine ratio and liver-specific methionine adenosyltransferase (MatI/III) progressively decreased in dysplastic and neoplastic liver lesions developed in c-Myc transgenic mice and in human HCC with better (HCCB) and poorer (HCCP) prognosis (based on patient's survival length). Deregulation of these parameters resulted in a rise of global DNA hypomethylation both in c-Myc and human liver lesions, positively correlated with GI levels in mice and humans, and inversely correlated with the length of survival of HCC patients. No changes in MATI/ III and DNA methylation occurred in c-Myc/Tgf-a lesions and in a small human HCC subgroup with intermediate prognosis, where a proliferative activity similar to that of c-Myc HCC and HCCB was associated with low apoptosis. Upregulation of genes involved in polyamine synthesis, methionine salvage and downregulation of polyamine negative regulator OAZ1, was highest in c-Myc/Tgf-a HCCs and HCCP. Our results indicate that alterations in the activity of MAT/I/III, and extent of DNA hypomethylation and GI are prognostic markers for human HCC. However, a small human HCC subgroup, as c-Myc/Tgf-a tumors, may develop in the absence of alterations in DNA methylation. ' 2007 Wiley-Liss, Inc.Key words: DNA hypomethylation; hepatocellular carcinoma; genomic instability; prognosis; transgenic mice Hepatocellular carcinoma (HCC), is the fifth most frequent human cancer, with the highest frequency in sub-Saharan Africa and far eastern Asia, where hepatitis B virus and hepatitis C virus infections are endemic and food is contaminated by Aflatoxin B1. 1 HCC incidence is rising in Europe and United States, presumably due to increased incidence of hepatitis C virus infection, cirrhosis related to Type II diabetes, and alcoholic hepatitis.2 HCC is rapidly fatal, since most patients at risk are not diagnosed in time and amenable to potentially curative treatments, i.e., partial liver resection or transplantation. 1,2 Hepatocarcinogenesis is characterized by the accumulation of various alterations in oncogenes and oncosuppressor genes.3 This event is presumably due to the increased tendency of initiated cells to acquire mutations following dysregulation of the mechanisms preserving genome integrity, a condition known as genomic instability 4 (GI). Indeed, a number of studies have demonstrated the progressive appearance of GI, such as point mutations, loss of heterozygosity and chromosomal alterations from preneoplastic liver to HCC.5 In addition, microsatellite instability occurs in HCC, although l...

Sprouty2 (Spry2), a negative feedback regulator of the Ras/mitogen-activated protein kinase (MAPK) pathway, is frequently down-regulated in human hepatocellular carcinoma (HCC). We tested the hypothesis that loss of Spry2 cooperates with unconstrained activation of the c-Met protooncogene to induce hepatocarcinogenesis via in vitro and in vivo approaches. We found coordinated down-regulation of Spry2 protein expression and activation of c-Met as well as its downstream effectors extracellular signal-regulated kinase (ERK) and v-akt murine thymoma viral oncogene homolog (AKT) in a subset of human HCC samples with poor outcome. Mechanistic studies revealed that Spry2 function is disrupted in human HCC via multiple mechanisms at both transcriptional and post-transcriptional level, including promoter hypermethylation, loss of heterozygosity, and proteosomal degradation by neural precursor cell expressed, developmentally down-regulated 4 (NEDD4). In HCC cell lines, Spry2 overexpression inhibits c-Met-induced cell proliferation as well as ERK and AKT activation, whereas loss of Spry2 potentiates c-Met signaling. Most importantly, we show that blocking Spry2 activity via a dominant negative form of Spry2 cooperates with c-Met to promote hepatocarcinogenesis in the mouse liver by sustaining proliferation and angiogenesis. The tumors exhibited high levels of activated ERK and AKT, recapitulating the subgroup of human HCC with a clinically aggressive phenotype. Conclusion: The occurrence of frequent genetic, epigenetic, and biochemical events leading to Spry2 inactivation provides solid evidence that Spry2 functions as a tumor suppressor gene in liver cancer. Coordinated deregulation of Spry2 and c-Met signaling may be a pivotal oncogenic mechanism responsible for unrestrained activation of ERK and AKT pathways in human hepatocarcinogenesis. (HEPATOLOGY 2010;52:506-517) H uman hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, with limited treatment options and high mortality rate.

Mounting epidemiological evidence supports a role for insulin-signaling deregulation and diabetes mellitus in human hepatocarcinogenesis. However, the underlying molecular mechanisms remain unknown. To study the oncogenic effect of chronically elevated insulin on hepatocytes in the presence of mild hyperglycemia, we developed a model of pancreatic islet transplantation into the liver. In this model, islets of a donor rat are transplanted into the liver of a recipient diabetic rat, with resulting local hyperinsulinism that leads to the development of preneoplastic lesions and hepatocellular carcinoma (HCC). Here, we investigated the metabolic and growth properties of the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (AKT/mTOR) pathway, a major downstream effector of insulin signaling, in this model of insulin-induced hepatocarcinogenesis. We found that activation of insulin signaling triggers a strong induction of the AKT/ mTOR cascade that is paralleled by increased synthesis of fatty acids, cholesterol, and triglycerides, induction of glycolysis, and decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic and neoplastic liver lesions, when compared with the healthy liver. AKT/mTOR metabolic effects on hepatocytes, after insulin stimulation, were found to be mTORC1 dependent and independent in human HCC cell lines. In these cells, suppression of lipogenesis, glycolysis, and the pentose phosphate pathway triggered a strong growth restraint, despite insulin administration. Noticeably, metabolic abnormalities and proliferation driven by insulin were effectively reverted using the dual PI3K/mTOR inhibitor, NVP-BEZ235, both in vitro and in vivo. Conclusions: The present results indicate that activation of the AKT/mTOR cascade by unconstrained insulin signaling induces a defined module of metabolic alterations in hepatocytes contributing to aberrant cell growth. Thus, inhibition of AKT/mTOR and related metabolic changes might represent a novel preventive and therapeutic approach to effectively inhibit insulin-induced hepatocarcinogenesis. (HEPATOLOGY 2012;55:1473-1484 H uman hepatocellular carcinoma (HCC) incidence is rapidly rising in Western Europe and the United States.1 However, the wellestablished causal relationship between chronic hepatitis B and C infection and hepatocarcinogenesis does not fully explain this escalation in HCC occurrence, with one quarter of the cases remaining idiopathic. Recently, nonalcoholic steatohepatitis (NASH) has received attention for its potential causal role in hepatocarcinogenesis.2-5 Indeed, several case-control studies indicate that HCC patients with cryptogenic cirrhosis show clinical and demographic features suggestive of NASH, as compared with age-and sex-matched HCC patients of viral or alcoholic etiology.

Alterations in global DNA methylation are implicated in several pathobiological processes. The tissues stored as paraffin blocks represent an important source of DNA for retrospective genetic and epigenetic analysis on a large scale. Therefore, we developed the first capillary electrophoresis method able to measure global methylation in formalin-fixed, paraffin-embedded (FFPE) DNA extracts. A field-amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of cytosine and 5-methylcytosine released following DNA hydrolysis by means of formic acid was employed. Analytes were baseline-separated within 8 min by using 300 mM tris(hydroxymethyl)aminomethane phosphate pH 3.75 as the running buffer. With use of electrokinetic injection the limit of detection (LOD) in real sample was 0.1 nM, thus improving by about 400-fold the LOD of the previously described methods based on capillary electrophoresis. Sample extraction and purification were optimized so that evaluation of the DNA methylation degree was possible starting from 0.5-1 μg of DNA with intra- and interassay relative standard deviations for the 5-methylcytosine to total cytosine ratio of 2.0 and 3.2%, respectively. Because of its high accuracy and throughput, our method will be useful for large-scale applications to determine the implications of genomic DNA methylation levels in tumorigenesis.

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