• The BH3-only protein BID impairs the p38-mediated stress response and promotes hepatocarcinogenesis during chronic liver injury in mice.

      Orlik, Johanna; Schüngel, Sven; Buitrago-Molina, Laura Elisa; Marhenke, Silke; Geffers, Robert; Endig, Jessica; Lobschat, Katharina; Rössler, Stephanie; Goeppert, Benjamin; Manns, Michael P; et al. (2015-09)
      Apoptosis is critical for maintaining tissue homeostasis, and apoptosis evasion is considered as a hallmark of cancer. However, increasing evidence also suggests that proapoptotic molecules can contribute to the development of cancer, including liver cancer. The aim of this study was to further clarify the role of the proapoptotic B-cell lymphoma 2 homology domain 3 (BH3)-only protein BH3 interacting-domain death agonist (BID) for chronic liver injury (CLI) and hepatocarcinogenesis (HCG). Loss of BID significantly delayed tumor development in two mouse models of Fah-mediated and HBsTg-driven HCG, suggesting a tumor-promoting effect of BID. Liver injury as well as basal and mitogen-stimulated hepatocyte proliferation were not modulated by BID. Moreover, there was no in vivo or in vitro evidence that BID was involved in DNA damage response in hepatocytes and hepatoma cells. Our data revealed that CLI was associated with strong activation of oxidative stress (OS) response and that BID impaired full activation of p38 after OS.
    • The degree of liver injury determines the role of p21 in liver regeneration and hepatocarcinogenesis in mice.

      Buitrago-Molina, Laura Elisa; Marhenke, Silke; Longerich, Thomas; Sharma, Amar Deep; Boukouris, Aristeidis E; Geffers, Robert; Guigas, Bruno; Manns, Michael P; Vogel, Arndt; Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany. (2013-09)
      Hepatocellular carcinoma (HCC) frequently arises in the context of chronic injury that promotes DNA damage and chromosomal aberrations. The cyclin-dependent kinase inhibitor p21 is an important transcriptional target of several tumor suppressors, which promotes cell cycle arrest in response to many stimuli. The aim of this study was to further delineate the role of p21 in the liver during moderate and severe injury and to specify its role in the initiation and progression of HCC. Deletion of p21 led to continuous hepatocyte proliferation in mice with severe injury allowing animal survival but also facilitated rapid tumor development, suggesting that control of compensatory proliferation by high levels of p21 is critical to the prevention of tumor development. Unexpectedly, however, liver regeneration and hepatocarcinogenesis was impaired in p21-deficient mice with moderate injury. Mechanistically, loss of p21 was compensated by activation of Sestrin2, which impaired mitogenic mammalian target of rapamycin (mTOR) signaling and activated cytoprotective Nrf2 signaling. Conclusion: The degree of liver injury and the strength of p21 activation determine its effects on liver regeneration and tumor development in the liver. Moreover, our data uncover a molecular link in the complex mTOR, Nrf2, and p53/p21-signaling network through activation of Sestrin2, which regulates hepatocyte proliferation and tumor development in mice with liver injury. (Hepatology 2013;53:1143-1152).
    • Dysregulated serum response factor triggers formation of hepatocellular carcinoma.

      Ohrnberger, Stefan; Thavamani, Abhishek; Braeuning, Albert; Lipka, Daniel B; Kirilov, Milen; Geffers, Robert; Authenrieth, Stella E; Römer, Michael; Zell, Andreas; Bonin, Michael; et al. (2015-03)
      The ubiquitously expressed transcriptional regulator serum response factor (SRF) is controlled by both Ras/MAPK (mitogen-activated protein kinase) and Rho/actin signaling pathways, which are frequently activated in hepatocellular carcinoma (HCC). We generated SRF-VP16(iHep) mice, which conditionally express constitutively active SRF-VP16 in hepatocytes, thereby controlling subsets of both Ras/MAPK- and Rho/actin-stimulated target genes. All SRF-VP16(iHep) mice develop hyperproliferative liver nodules that progresses to lethal HCC. Some murine (m)HCCs acquire Ctnnb1 mutations equivalent to those in human (h)HCC. The resulting transcript signatures mirror those of a distinct subgroup of hHCCs, with shared activation of oncofetal genes including Igf2, correlating with CpG hypomethylation at the imprinted Igf2/H19 locus.
    • Global micro RNA expression profiling in the liver biopsies of Hepatitis B Virus infected patients suggests specific miRNA signatures for viral persistence and hepatocellular injury.

      Singh, Avishek Kumar; Rooge, Sheetalnath Babasaheb; Varshney, Aditi; Vasudevan, Madavan; Bhardwaj, Ankit; Venugopal, Senthil Kumar; Trehanpati, Nirupama; Kumar, Manoj; Geffers, Robert; Kumar, Vijay; et al. (2017-11-30)
      Hepatitis B virus (HBV) can manipulate the miRNA regulatory networks in infected cells to create a permissive environment for viral replication, cellular injury, disease onset and its progression. The aim of the present study was to understand the miRNA networks and their target genes in the liver of hepatitis B patients involved in HBV replication, liver injury and liver fibrosis. We investigated differentially expressed miRNAs by microarray in the liver biopsy samples from different stages of HBV infection and liver disease [immune tolerant (IT; n= 8); acute viral hepatitis (AVH; n=8); no fibrosis (n=16); early (F1+F2) (n=19) or late fibrosis (F3+F4) (n=14) and healthy controls (n=7)]. The miRNA expression levels were analyzed by the unsupervised principal component analysis (PCA) and hierarchical clustering. Analysis of miRNA-mRNA regulatory networks identified 17 miRNAs and 18 target gene interactions with four distinct nodes each representing a stage-specific gene regulation during disease progression. The IT group showed elevated miR-199a-5p, miR-221-3p and Let-7a-3p levels which could target genes involved in innate immune response and viral replication. In AVH group, miR-125b-5p and miR-3613-3p were up whereas miR-940 was down which might affect cell proliferation via STAT3 pathway. In early fibrosis, miR-34b-3p, miR-1224-3p and miR-1227-3p were up while miR-499a-5p was down which together, possibly mediate chronic inflammation. In advanced fibrosis, miR-1, miR-10b-5p, miR-96-5p, miR-133b and miR-671-5p were up while miR-20b-5p and miR-455-3p were down, possibly allowing chronic disease progression. Interestingly, only 8 of 17 liver-specific miRNAs exhibited a similar expression pattern in patient sera.
    • 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; et al. (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.
    • Posttranscriptional destabilization of the liver-specific long noncoding RNA HULC by the IGF2 mRNA-binding protein 1 (IGF2BP1).

      Hämmerle, Monika; Gutschner, Tony; Uckelmann, Hannah; Ozgur, Sevim; Fiskin, Evgenij; Gross, Matthias; Skawran, Britta; Geffers, Robert; Longerich, Thomas; Breuhahn, Kai; et al. (2013-05-31)
      Selected long noncoding RNAs (lncRNAs) have been shown to play important roles in carcinogenesis. Although the cellular functions of these transcripts can be diverse, many lncRNAs regulate gene expression. In contrast, factors that control the expression of lncRNAs remain largely unknown. Here we investigated the impact of RNA binding proteins on the expression of the liver cancer-associated lncRNA HULC (highly up-regulated in liver cancer). First, we validated the strong up-regulation of HULC in human hepatocellular carcinoma. To elucidate posttranscriptional regulatory mechanisms governing HULC expression, we applied an RNA affinity purification approach to identify specific protein interaction partners and potential regulators. This method identified the family of IGF2BPs (IGF2 mRNA-binding proteins) as specific binding partners of HULC. Depletion of IGF2BP1, also known as IMP1, but not of IGF2BP2 or IGF2BP3, led to an increased HULC half-life and higher steady-state expression levels, indicating a posttranscriptional regulatory mechanism. Importantly, HULC represents the first IGF2BP substrate that is destabilized. To elucidate the mechanism by which IGF2BP1 destabilizes HULC, the CNOT1 protein was identified as a novel interaction partner of IGF2BP1. CNOT1 is the scaffold of the human CCR4-NOT deadenylase complex, a major component of the cytoplasmic RNA decay machinery. Indeed, depletion of CNOT1 increased HULC half-life and expression. Thus, IGF2BP1 acts as an adaptor protein that recruits the CCR4-NOT complex and thereby initiates the degradation of the lncRNA HULC. Conclusion: Our findings provide important insights into the regulation of lncRNA expression and identify a novel function for IGF2BP1 in RNA metabolism. (Hepatology 2013).