The Ras effectors NORE1A and RASSF1A are frequently inactivated in pheochromocytoma and abdominal paraganglioma.
NORE1A (RASSF5) and RASSF1A are newly described Ras effectors with tumour suppressor functions. Both molecules are frequently inactivated in various cancers. In this study, we aimed to explore the potential involvement of NORE1A and RASSF1A in pheochromocytoma and abdominal paraganglioma tumorigenesis. A panel of 54 primary tumours was analysed for NORE1A and RASSF1A mRNA expression by TaqMan quantitative RT-PCR. Furthermore, NORE1A and RASSF1A promoter methylation was assessed by combined bisulphite restriction endonuclease assay and methylation-sensitive Pyrosequencing respectively. The anti-tumorigenic role of NORE1A was functionally investigated in Nore1A-transfected PC12 rat pheochromocytoma cells by fluorescent inhibition of caspase activity and soft agar assays. Significantly suppressed NORE1A and RASSF1A mRNA levels were detected in primary tumours compared with normal adrenal medulla (P<0.001). Methylation of the NORE1A promoter was not observed in primary tumours. On the other hand, 9% (5/54) of the primary tumours examined showed RASSF1A promoter methylation greater than 20% as detected by Pyrosequencing. Methylation of the RASSF1A promoter was significantly associated with malignant behaviour (P<0.05). Transient expression of Nore1a resulted in enhanced apoptosis and impaired colony formation in soft agar. Our study provides evidence that NORE1A and RASSF1A are frequently suppressed in pheochromocytoma and abdominal paraganglioma. Silencing of NORE1A contributes to the transformed phenotype in these tumours.
Geli
Janos
J
Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital Solna, SE-171 76 Stockholm, Sweden. janos.geli@ki.se
Kiss
Nimrod
N
Lanner
Fredrik
F
Foukakis
Theodoros
T
Natalishvili
Natalia
N
Larsson
Olle
O
Kogner
Per
P
Höög
Anders
A
Clark
Geoffrey J
GJ
Ekström
Tomas J
TJ
Bäckdahl
Martin
M
Farnebo
Filip
F
Larsson
Catharina
C
D000008:Abdominal Neoplasms / Q000235:genetics*; D048868:Adaptor Proteins, Signal Transducing; D000368:Aged; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008875:Middle Aged; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics*; D016716:PC12 Cells; D010235:Paraganglioma / Q000235:genetics*; D010673:Pheochromocytoma / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013447:Sulfites / Q000494:pharmacology; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C411762:RASSF1 protein, human; C454042:RASSF5 protein, human; D012333:RNA, Messenger; D013447:Sulfites; D025521:Tumor Suppressor Proteins; D020559:Monomeric GTP-Binding Proteins; C042345:hydrogen sulfite