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-