ABSTRACT
Recently, we found epigenetic silencing of the Ras effector genes NORE1B and/or RASSF1A in 97% of the hepatocellular carcinoma (HCC) investigated. This is strong evidence that the two genes are of major significance in hepatocarcinogenesis. Although RASSF1A serves as a tumor suppressor gene, the functions of NORE1B are largely unknown. Here, we studied the role of NORE1B for growth and transformation of cells. To understand the molecular mechanisms of action of the gene, we used the wild-type form and deletion mutants without the NH(2) terminus and CENTRAL domain, the Ras association (RA) domain, or the COOH-terminal SARAH-domain. Intact RA and SARAH-domains were found to be necessary for NORE1B (a) to increase the G(0)-G(1) fraction in hepatoma cells, (b) to suppress c-Myc/Ha-Ras-induced cell transformation, and (c) to interact closely with RASSF1A, as determined with fluorescence resonance energy transfer. In further studies, cell cycle delay by NORE1B was equally effective in hepatocyte cell lines with wild-type or mutant Ras suggesting that NORE1B does not interact with either Ras. In conclusion, NORE1B suppresses replication and transformation of cells as effectively as RASSF1A and thus is a putative tumor suppressor gene. NORE1B interacts physically with RASSF1A and functional loss of one of the interacting partners may lead to uncontrolled growth and transformation of hepatocytes. This may explain the frequent epigenetic silencing of NORE1B and/or RASSF1A in HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , Cell Transformation, Neoplastic/genetics , Liver Neoplasms/genetics , Monomeric GTP-Binding Proteins/genetics , Tumor Suppressor Proteins/genetics , Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle/genetics , Cell Growth Processes/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic/metabolism , Cytoplasm/metabolism , Gene Deletion , Gene Silencing , Genes, Tumor Suppressor , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Monomeric GTP-Binding Proteins/metabolism , RNA, Small Interfering/genetics , Transfection , Tumor Suppressor Proteins/metabolism , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
BACKGROUND/AIMS: Activins A and E negatively regulate hepatic cell number by inhibiting cell replication and inducing apoptosis. Follistatin and follistatin-like 3 bind activins and antagonise their biological activities. Aim of our study was to investigate, whether activins and follistatins may play a role in hepatocarcinogenesis. METHODS: Expression levels of follistatin, follistatin-like 3, and activin subunits beta(A) as well as beta(E) were investigated in chemically induced rat and human liver tumours by real-time PCR and immunohistochemistry. In addition, the effects of follistatin and activin A on DNA synthesis of normal as well as preneoplastic hepatocytes and hepatoma cells were analysed. RESULTS: Follistatin was overexpressed while both activin subunits were downregulated in the majority of rat and human liver tumours. Follistatin-like 3 expression was low in normal but enhanced in malignant rat liver. In human normal liver, in contrast, it was abundantly expressed but downregulated in liver cancer. Administration of follistatin to normal and preneoplastic hepatocytes stimulated DNA synthesis preferentially in preneoplastic rat hepatocytes, whereas activin A repressed it. CONCLUSIONS: The balanced expression of follistatins and activins becomes deregulated during hepatocarcinogenesis. The sensitivity of preneoplastic hepatocytes to activin signals suggests the activin/follistatin system as promising target for therapeutic intervention.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Follistatin-Related Proteins/metabolism , Follistatin/metabolism , Inhibin-beta Subunits/metabolism , Liver Neoplasms/metabolism , Animals , Carcinoma, Hepatocellular/physiopathology , DNA/biosynthesis , Down-Regulation/physiology , Hepatocytes/physiology , Humans , Immunohistochemistry , Liver Neoplasms/physiopathology , Male , Models, Animal , Polymerase Chain Reaction/methods , Rats , Up-Regulation/physiologyABSTRACT
BACKGROUND/AIMS: In human hepatocellular carcinoma (HCC) the ras-proto-oncogene is rarely mutated. We therefore studied the possible inactivation of the putative tumor-suppressors and ras-associating proteins, NORE1A, NORE1B, and RASSF1A in HCCs by mutation or epigenetic gene silencing through promoter-CpG hypermethylation. METHODS: SSCP-analyses, sequencing, and methylation-specific PCR were performed in 28 fibrotic/cirrhotic livers and 40 HCCs. RESULTS: The sequence of NORE1A/B exhibited no deviations and that of the RASSF1A gene a non-silent polymorphism ( approximately 10% of cases) and a missense mutation (one HCC). Both alterations may affect the growth-inhibiting capability of RASSF1A. Epigenetic inactivation of NORE1B was found in 62% of the HCCs and in hepatocarcinoma-cell lines due to considerable promoter-methylation of the gene. Methylation was detected also for RASSF1A in HCCs and hepatocarcinoma cell-lines. As a result, 97% of the HCCs revealed epigenetic silencing of NORE1B, RASSF1A, or both. In contrast every third fibrotic/cirrhotic liver only exhibited silencing of one or both genes. CONCLUSIONS: The candidate tumor suppressor genes NORE1B and RASSF1A are epigenetically down-regulated alone in at least 62%, or in combination in 97% of the HCCs studied. This indicates a frequent and critical event in hepatocarcinogenesis, which may allow HCCs to subverse growth-control in the presence of an unaltered Ras.
