• Methylome analysis and integrative profiling of human HCCs identify novel protumorigenic factors.

      Neumann, Olaf; Kesselmeier, Miriam; Geffers, Robert; Pellegrino, Rossella; Radlwimmer, Bernhard; Hoffmann, Katrin; Ehemann, Volker; Schemmer, Peter; Schirmacher, Peter; Lorenzo Bermejo, Justo; Longerich, Thomas; Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany. (2012-11)
      To identify new tumor-suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome-wide methylation profiling and vertical integration with array-based 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'-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.
    • Protumorigenic role of Timeless in hepatocellular carcinoma.

      Elgohary, Nahla; Pellegrino, Rossella; Neumann, Olaf; Elzawahry, Heba M; Saber, Magdy M; Zeeneldin, Ahmed A; Geffers, Robert; Ehemann, Volker; Schemmer, Peter; Schirmacher, Peter; Longerich, Thomas; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-02)
      The mammalian timeless (TIM) protein interacts with proteins of the endogenous clock and essentially contributes to the circadian rhythm. In addition, TIM is involved in maintenance of chromosome integrity, growth control and development. Thus, we hypothesized that TIM may exert a potential protumorigenic function in human hepatocarcinogenesis. TIM was overexpressed in a subset of human HCCs both at the mRNA and the protein level. siRNA-mediated knockdown of TIM reduced cell viability due to the induction of apoptosis and G2 arrest. The latter was mediated via CHEK2 phosphorylation. In addition, siRNA-treated cells showed a significantly reduced migratory capacity and reduced expression levels of various proteins. Mechanistically, TIM directly interacts with the eukaryotic elongation factor 1A2 (EEF1A2), which binds to actin filaments to promote tumor cell migration. siRNA-mediated knockdown of TIM reduced EEF1A2 protein levels thereby affecting ribosomal protein biosynthesis. Thus, overexpression of TIM exerts oncogenic function in human HCCs, which is mediated via CHEK2 and EEF1A2.