Assessment of NORE1A as a putative tumor suppressor in human neuroblastoma.
The putative tumor suppressor NORE1A (RASSF5) is a member of the Ras association domain family and is commonly inactivated in human cancer. The closely related gene family member and functional collaborator RASSF1A is a bona fide tumor suppressor and is frequently involved in neuroblastoma. In the present study, we sought to investigate the role of NORE1A in human neuroblastoma. A panel of tumors (36 neuroblastomas and 4 ganglioneuromas) and neuroblastoma cell lines was assessed for NORE1A gene expression by Taqman quantitative RT-PCR. Promoter methylation was quantitatively determined by methylation sensitive pyrosequencing. The antitumourigenic role was functionally investigated in Nore1a transfected SK-N-BE (2) cells by fluorescent inhibition of caspase activity and BrdU incorporation assays. Neuroblastoma cells showed very low or absent NORE1A mRNA expression, which could not be reversed by trichostatin A or 5-aza-cytidine treatments. Neuroblastoma tumors showed suppressed NORE1A gene expression that was particularly pronounced in cases without MYCN amplification or 1p loss. Methylation of the NORE1A promoter was not observed in primary tumors and only one out of seven neuroblastoma cell lines displayed weak partial methylation. Transient expression of Nore1a resulted in enhanced apoptosis and delayed cell cycle progression. In conclusion NORE1A appears to be strongly suppressed in neuroblastic tumors and reconstitution of its expression diminishes the tumorigenic phenotype. Promotor methylation is not a common mechanism responsible for NORE1A transcriptional suppression in this tumor type.
Geli
Janos
J
Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.
Kogner
Per
P
Department of Woman and Child Health, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.
Lanner
Fredrik
F
Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Natalishvili
Natalia
N
Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.
Juhlin
Christofer
C
Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.|Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.
Kiss
Nimrod
N
Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.
Clark
Geoffrey J
GJ
JG Brown Cancer Center, University of Louisville, Louisville, KY 40202.
Ekström
Tomas J
TJ
Department of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.
Farnebo
Filip
F
Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Larsson
Catharina
C
Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital-Solna, SE-171 76 Stockholm, Sweden.
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000378:metabolism; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D005729:Ganglioneuroma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016147:Genes, Tumor Suppressor / Q000187:drug effects; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D006877:Hydroxamic Acids; C454042:RASSF5 protein, human; D012333:RNA, Messenger; C012589:trichostatin A; D020559:Monomeric GTP-Binding Proteins; D001973:Bromodeoxyuridine; D001374:Azacitidine