Current evidence indicates that neoplastic nodules induced in liver of Brown Norway (BN) rats genetically resistant to hepatocarcinogenesis are not prone to evolve into hepatocellular carcinoma. We show that BN rats subjected to diethylnitrosamine/2-acetylaminofluorene/partial hepatectomy treatment with a "resistant hepatocyte" protocol displayed higher number of glutathione-S-transferase 7-7(؉) hepatocytes when compared with susceptible Fisher 344 (F344) rats, both during and at the end of 2-acetylaminofluorene treatment. However, DNA synthesis declined in BN but not F344 rats after completion of reparative growth. Upregulation of p16 INK4A , Hsp90, and Cdc37 genes; an increase in Cdc37-Cdk4 complexes; and a decrease in p16 INK4A -Cdk4 complexes occurred in preneoplastic liver, nodules, and hepatocellular carcinoma of F344 rats. These parameters did not change significantly in BN rats. E2f4 was equally expressed in the lesions of both strains, but Crm1 expression and levels of E2f4-Crm1 complex were higher in F344 rats. H uman and rodent hepatocarcinogenesis is characterized by the progressive development of foci of altered hepatocytes (FAH), neoplastic nodules, and hepatocellular carcinoma (HCC). 1,2 Epidemiological evidence suggests a polygenic predisposition for human HCC. 2 Studies on rodents allowed mapping the loci responsible for the progression of preneoplastic hepatocytes to malignancy. [3][4][5][6][7] However, the nature and temporal occurrence of the events underlying resistance to HCC remain uncertain. The evaluation of these mechanisms could help in understanding the molecular pathways affected by susceptibility genes to evaluate cancer risk and identify potentially reversible phases of carcinogenesis.Previous research has shown cell cycle deregulation in neoplastic liver lesions induced by the "resistant hepatocyte" protocol in susceptible Fisher 344 (F344) rats. [8][9][10] Lower or no changes were found in resistant Brown Norway (BN) rats in which overexpression of p16 INK4A , a well-known inhibitor of the cyclin-dependent kinase (Cdk) 4/6, occurs. Forma- PH, partial hepatectomy; BrdU, siRNA, small interfering RNA. From the

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Sustained activation of extracellular signal-regulated kinase (ERK) has been detected previously in numerous tumors in the absence of RAS-activating mutations. However, the molecular mechanisms responsible for ERK-unrestrained activity independent of RAS mutations remain unknown. Here, we evaluated the effects of the functional interactions of ERK proteins with dual-specificity phosphatase 1 (DUSP1), a specific inhibitor of ERK, and S-phase kinase-associated protein 2 (SKP2)/ CDC28 protein kinase 1b (CKS1) ubiquitin ligase complex in human hepatocellular carcinoma (HCC). Levels of DUSP1, as assessed by real-time reverse transcription-PCR and Western blot analysis, were significantly higher in tumors with better prognosis (as defined by the length of patients' survival) when compared with both normal and nontumorous surrounding livers, whereas DUSP1 protein expression sharply declined in all HCC with poorer prognosis. In the latter HCC subtype, DUSP1 inactivation was due to either ERK/SKP2/CKS1-dependent ubiquitination or promoter hypermethylation associated with loss of heterozygosity at the DUSP1 locus. Noticeably, expression levels of DUSP1 inversely correlated with those of activated ERK, as well as with proliferation index and microvessel density, and directly with apoptosis and survival rate. Subsequent functional studies revealed that DUSP1 reactivation led to suppression of ERK, CKS1, and SKP2 activity, inhibition of proliferation and induction of apoptosis in human hepatoma cell lines. Taken together, the present data indicate that ERK achieves unrestrained activity during HCC progression by triggering ubiquitin-mediated proteolysis of its specific inhibitor DUSP1. Thus, DUSP1 may represent a valuable prognostic marker and ERK, CKS1, or SKP2 potential therapeutic targets for human HCC.

P revious studies on genetic predisposition to hepatocellular carcinoma (HCC) of rats led to identification of 4 hepatocarcinogenesis susceptibility (Hcs1-4) loci, and 7 resistance (Hcr1-7) loci 1,2 (De Miglio et al., unpublished data). Resistance alleles, dominantly transmitted to the progeny, 3,4 apparently modify the activity of susceptibility loci. Recent evidence suggests the presence of at least 3 oncosuppressor genes at Hcr1 locus. 4 Molecular mechanisms underlying these effects are unknown. Available evidence suggests the existence of a relatively stable genome in neoplastic lesions of resistant rats, as shown by the absence of c-myc amplification in the lesions of a resistant Wistar rat strain, which instead is present in susceptible Fisher 344 (F344) rats. 5,6 Moreover, allelic imbalance occurs, at several chromosomes, in HCCs of susceptible (F344 x Wistar Furth)F1 rats, 7 but not in those of resistant BFF1 rats. 8 HCCs induced in LFF1 rats, generated by crossing the susceptible F344 and Long-Evans strains, show allelic imbalance at Hcs1, Hcr1, and Hcr6. 4 c-myc is located at Hcs1 in a segment syntenic to human chromosomal regions in which frequent allelic gain occurs. 9 These observations suggest that mechanisms controlling cell growth are differently affected in neoplastic liver lesions of susceptible and resistant rats.Overexpression of c-myc in c-myc-and c-myc/Tgf-␣-transgenic mice is associated with deregulation of the pRb-E2F pathway. 10 Interaction of c-Myc with various cell cycle components occurs in in vitro growing cells. 11,12 Enzymes controlling cell cycle include cyclin-dependent kinases (CDKs), activated by binding to the cyclins. 13 Complexes of CDK4 and CDK6 with D-type cyclins are required for the G1 phase progression. Further progression through G1 requires cyclin E, and passage through

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...

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Forkhead box M1B is a determinant of rat susceptibility to hepatocarcinogenesis and sustains ERK activity in human HCC ABSTRACT Background and aim: Previous studies indicate unrestrained cell cycle progression in liver lesions from hepatocarcinogenesis-susceptible Fisher 344 (F344) rats and a block of G 1 -S transition in corresponding lesions from resistant Brown Norway (BN) rats. Here, the role of the Forkhead box M1B (FOXM1) gene during hepatocarcinogenesis in both rat models and human hepatocellular carcinoma (HCC) was assessed. Methods and results: Levels of FOXM1 and its targets were determined by immunoprecipitation and real-time PCR analyses in rat and human samples. FOXM1 function was investigated by either FOXM1 silencing or overexpression in human HCC cell lines. Activation of FOXM1 and its targets (Aurora Kinose A, Cdc2, cyclin B1, Nek2) occurred earlier and was most pronounced in liver lesions from F344 than BN rats, leading to the highest number of Cdc2-cyclin B1 complexes (implying the highest G 2 -M transition) in F344 rats. In human HCC, the level of FOXM1 progressively increased from surrounding nontumorous livers to HCC, reaching the highest levels in tumours with poorer prognosis (as defined by patients' length of survival). Furthermore, expression levels of FOXM1 directly correlated with the proliferation index, genomic instability rate and microvessel density, and inversely with apoptosis. FOXM1 upregulation was due to extracellular signal-regulated kinase (ERK) and glioblastoma-associated oncogene 1 (GLI1) combined activity, and its overexpression resulted in increased proliferation and angiogenesis and reduced apoptosis in human HCC cell lines. Conversely, FOXM1 suppression led to decreased ERK activity, reduced proliferation and angiogenesis, and massive apoptosis of human HCC cell lines. Conclusions: FOXM1 upregulation is associated with the acquisition of a susceptible phenotype in rats and influences human HCC development and prognosis.