To identify new tumor-suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome-wide methylation profiling and vertical integration with arraybased comparative genomic hybridization (aCGH), as well as expression data from a cohort of well-characterized human hepatocellular carcinomas (HCCs). Bisulfite-converted DNAs from 63 HCCs and 10 healthy control livers were analyzed for the methylation status of more than 14,000 genes. After defining the differentially methylated genes in HCCs, we integrated their DNA copy-number alterations as determined by aCGH data and correlated them with gene expression to identify genes potentially silenced by promoter hypermethylation. Aberrant methylation of candidates was further confirmed by pyrosequencing, and methylation dependency of silencing was determined by 5-aza-2 0 -deoxycytidine (5-aza-dC) treatment. Methylation profiling revealed 2,226 CpG sites that showed methylation differences between healthy control livers and HCCs. Of these, 537 CpG sites were hypermethylated in the tumor DNA, whereas 1,689 sites showed promoter hypomethylation. The hypermethylated set was enriched for genes known to be inactivated by the polycomb repressive complex 2, whereas the group of hypomethylated genes was enriched for imprinted genes. We identified three genes matching all of our selection criteria for a tumor-suppressor gene (period homolog 3 [PER3], insulin-like growth-factor-binding protein, acid labile subunit [IGFALS], and protein Z). PER3 was down-regulated in human HCCs, compared to peritumorous and healthy liver tissues. 5-aza-dC treatment restored PER3 expression in HCC cell lines, indicating that promoter hypermethylation was indeed responsible for gene silencing. Additionally, functional analysis supported a tumor-suppressive function for PER3 and IGFALS in vitro. Conclusion: The present study illustrates that vertical integration of methylation data with high-resolution genomic and transcriptomic data facilitates the identification of new tumor-suppressor gene candidates in human HCC. (HEPATOLOGY 2012;56:1817-1827 H epatocellular carcinoma (HCC) is the fifthmost frequent cancer worldwide and has a poor prognosis. 1 Various etiologies have been linked to HCC development, most of which cause chronic liver damage and finally lead to liver cirrhosis.The most prevalent etiological factors are chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, chronic alcohol consumption, and, in certain geographical areas, aflatoxin B1 food contamination. 2 Approximately 10% of HCC patients lack viral

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.

Polo-like kinase (PLK) proteins play critical roles in the control of cell cycle progression, either favoring or inhibiting cell proliferation, and in DNA damage response. Although either overexpression or down-regulation of PLK proteins occurs frequently in various cancer types, no comprehensive analysis on their function in human hepatocellular carcinoma (HCC) has been performed to date. In the present study, we define roles for PLK1, PLK2, PLK3, and PLK4 during hepatocarcinogenesis. Levels of PLK1, as assessed by means of real-time reverse-transcription PCR and western blot analysis, were progressively increased from nonneoplastic surrounding liver tissues to HCC, reaching the highest expression in tumors with poorer outcome (as defined by the length of patients' survival) compared with normal livers. In sharp contrast, PLK2, PLK3, and PLK4 messenger RNA and protein expression gradually declined from nontumorous liver to HCC, with the lowest levels being detected in HCC with shorter survival. In liver tumors, PLK2-4 down-regulation was paralleled by promoter hypermethylation and/or loss of heterozygosity at the PLK2-4 loci. Subsequent functional studies revealed that PLK1 inhibition led to suppression of cell growth in vitro, whereas opposite effects followed PLK2-4 silencing in HCC cell lines. P olo-like kinase (PLKs) proteins play pivotal roles in cell cycle progression and response to DNA damage. 1 Four members of this family of serine/threonine kinases were identified: PLK1, PLK2 (also known as SNK), PLK3 (also known as FNK or PRK), and PLK4 (or SAK). 1 PLKs are characterized by a highly conserved Nterminal serine/threonine kinase domain and one or two polo boxes in the C-terminal region, which are crucial for subcellular localization and binding of specific phosphopeptides. 2 Expression of PLKs is tightly regulated during the cell cycle. 1 PLK1 is inhibited by numerous checkpoint genes, whereas PLK2-4 genes are activated by spindle checkpoints and DNA damage. 1,3 Despite the high sequence homology among the four members of the PLK family, their functions seem to diverge. PLK1 is involved mainly in the control of the G2/M phase, by promoting CDC25C phosphatase activity with subsequent activation of CyclinB1/CdK1 complex, and the degradation of early mitotic inhibitor-1 (EMI1), which inhibits the activated Anaphase-Promoting Complex/Cyclosome. 1 PLK2 and PLK3 were identified as serum-inducible growth responsive genes and are implicated in the stress-response. 4 Analysis of PLK2 knockout mice indicated that PLK2 is implicated in embryonic development and cell cycle regulation, as confirmed by recent findings showing an involvement of PLK2 in promotion of S-

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...

Mouse Double Minute homolog 4 (MDM4) gene upregulation often occurs in human hepatocellular carcinoma (HCC), but the molecular mechanisms responsible for its induction remain poorly understood. Here, we investigated the role of the phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog/mammalian target of Rapamycin (PI3K/AKT/mTOR) axis in the regulation of MDM4 levels in HCC. The activity of MDM4 and the PI3K/AKT/mTOR pathway was modulated in human HCC cell lines via silencing and overexpression experiments. Expression of main pathway components was analyzed in an AKT mouse model and human HCCs. MDM4 inhibition resulted in growth restraint of HCC cell lines both in vitro and in vivo. Inhibition of the PI3K-AKT and/or mTOR pathways lowered MDM4 protein levels in HCC cells and reactivated p53-dependent transcription. De-ubiquitination by ubiquitin-specific protease 2a and AKT-mediated phosphorylation protected MDM4 from proteasomal degradation and increased AKT protein stability. The eukaryotic elongation factor 1A2 (EEF1A2) was identified as an upstream inducer of PI3K supporting MDM4 stabilization. Also, we detected MDM4 protein upregulation in an AKT mouse model and a strong correlation between the expression of EEF1A2, activated/phosphorylated AKT, and MDM4 in human HCC (each rho>.8, P<.001). Noticeably, a strong activation of this cascade was associated with shorter patients' survival. Conclusions The EEF1A2/PI3K/AKT/mTOR axis promotes the protumorigenic stabilization of the MDM4 protooncogene in human HCC via a post-transcriptional mechanism. The activation level of the EEF1A2/PI3K/AKT/mTOR/MDM4 axis significantly influences the survival probability of HCC patients in vivo and may thus represent a promising molecular target.

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.

Fast growth and deregulation of G1 and S phases characterize preneoplastic and neoplastic liver lesions of genetically susceptible F344 rats, whereas a G1-S block in lesions of resistant BN rats explains their low progression capacity. However, signal transduction pathways responsible for the different propensity of lesions from the 2 rat strains to evolve to malignancy remain unknown. Here, we comparatively investigated the role of Ras/Erk pathway inhibitors, involved in growth restraint and cell death, in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis. Moderate activation of Ras, Raf-1 and Mek proteins was paralleled in both rat models by strong induction of Dab2 and Rkip inhibitors. Levels of Dusp1, a specific ERK inhibitor, increased only in BN rat lesions, leading to modest ERK activation, whereas a progressive Dusp1 decline occurred in corresponding lesions from F344 rats and was accompanied by elevated ERK activation. Furthermore, a gradual increase of Rassf1A/Nore1A/ Mst1-driven apoptosis was detected in both rat strains, with highest levels in BN hepatocellular carcinoma (HCC), whereas loss of Dab2IP, a protein implicated in ASK1-dependent cell death, occurred only in F344 rat HCC, resulting in significantly higher apoptosis in BN than F344 HCC. Taken together, our results indicate a control of the Ras/Erk pathway and the pro-apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways by HCC susceptibility genes. Dusp1 possesses a prominent role in the acquisition of the phenotype resistant to HCC by BN rats, whereas late activation of RassF1A/Nore1A and Dab2IP/Ask1 axes is implicated in the highest apoptosis characteristic of BN HCC. ' 2008 Wiley-Liss, Inc.Key words: hepatocellular carcinoma; Dusp1; Erk; RassF1A/ Nore1A; signal transduction; genetic predisposition Hepatocellular carcinoma (HCC) is the fifth commonest malignant tumor worldwide, and the third cause of cancer-caused death. 1,2Highest HCC prevalence occurs in Africa and Asia, but HCC incidence is rising also in Western countries, due to the increasing rates of alcoholic liver disease and hepatitis C.1,2 Surgery, including transplantation, is still the only potentially curative treatment of HCC, but recurrence rate is high and long-term survival is rare. The prediction of the risk of HCC recurrence and prognosis could be useful to guide surgery and chemotherapy. Moreover, the discovery of genetic and molecular events during hepatocarcinogenesis could lead to the identification of molecular markers of the disease, which could eventually be used as therapeutic targets.Hepatocarcinogenesis is characterized by the deregulation of the balance between proliferation and cell death by apoptosis, with decrease in some proapoptotic signals (i.e. Fas, p53, Bax or Bid), and up-regulation of antiapoptotic signals.3 Recent work on the genetic susceptibility to liver cancer led to mapping different hepatocarcinogenesis susceptibility and resistance genes in mice and rats, controlling the growth, progression and redifferentiation (r...

Mounting evidence underlines the role of inducible nitric oxide synthase (iNOS) in hepatocellular carcinoma (HCC) development, but its functional interactions with pathways involved in HCC progression remain uninvestigated. Here, we analyzed in preneoplastic and neoplastic livers from Fisher 344 and Brown Norway rats, possessing different genetic predisposition to HCC, in transforming growth factor-alpha (TGF-alpha) and c-Myc-TGF-alpha transgenic mice, characterized by different susceptibility to HCC, and in human HCC: (i) iNOS function and interactions with nuclear factor-kB (NF-kB) and Ha-RAS/extracellular signal-regulated kinase (ERK) during hepatocarcinogenesis; (ii) influence of genetic predisposition to liver cancer on these pathways and role of these cascades in determining a susceptible or resistant phenotype and (iii) iNOS prognostic value in human HCC. We found progressive iNos induction in rat and mouse liver lesions, always at higher levels in the most aggressive models represented by HCC of rats genetically susceptible to hepatocarcinogenesis and c-Myc-TGF-alpha transgenic mice. iNOS, inhibitor of kB kinase/NF-kB and RAS/ERK upregulation was significantly higher in HCC with poorer prognosis (as defined by patients' survival length) and positively correlated with tumor proliferation, genomic instability and microvascularization and negatively with apoptosis. Suppression of iNOS signaling by aminoguanidine led to decreased HCC growth and NF-kB and RAS/ERK expression and increased apoptosis both in vivo and in vitro. Conversely, block of NF-kB signaling by sulfasalazine or short interfering RNA (siRNA) or ERK signaling by UO126 caused iNOS downregulation in HCC cell lines. These findings indicate that iNOS cross talk with NF-kB and Ha-RAS/ERK cascades influences HCC growth and prognosis, suggesting that key component of iNOS signaling could represent important therapeutic targets for human HCC.