Mutation of B-Raf in human choroidal melanoma cells mediates cell proliferation and transformation through the MEK/ERK pathway.
The BRAF gene, encoding a mitogen-activated protein kinase kinase kinase, is mutated in several human cancers, with the highest incidence occurring in cutaneous melanoma. The activating V599E mutation accounted for 80% of all mutations detected in cutaneous melanoma cell lines. Reconstitution experiments have shown that this mutation increases ectopically expressed B-Raf kinase activity and induces NIH3T3 cell transformation. Here we used tumor-derived cell lines to characterize the activity of endogenous mutated B-Raf protein and assess its specific role in transformation. We show that three cell lines (OCM-1, MKT-BR, and SP-6.5) derived from human choroidal melanoma, the most frequent primary ocular neoplasm in humans, express B-Raf containing the V599E mutation. These melanoma cells showed a 10-fold increase in endogenous B-RafV599E kinase activity and a constitutive activation of the MEK/ERK pathway that is independent of Ras. This, as well as melanoma cell proliferation, was strongly diminished by siRNA-mediated depletion of the mutant B-Raf protein. Moreover, blocking B-RafV599E-induced ERK activation by different experimental approaches significantly reduced cell proliferation and anchorage-independent growth of melanoma cells. Finally, quantitative immunoblot analysis allowed us to identify signaling and cell cycle proteins that are differentially expressed between normal melanocytes and melanoma cells. Although the expression of signaling molecules was not sensitive to U0126 in melanoma cells, the expression of a cluster of cell cycle proteins remained regulated by the B-RafV599E/MEK/ERK pathway. Our results pinpoint this pathway as an important component in choroidal melanoma cell lines.
Calipel
Armelle
A
Institut Biomédical des Cordeliers, INSERM U450, 15 rue de l'Ecole de Médecine, 75006 Paris, France.
Lefevre
Gaelle
G
Pouponnot
Celio
C
Mouriaux
Frédéric
F
Eychène
Alain
A
Mascarelli
Frédéric
F
D018797:Cell Cycle Proteins / Q000032:analysis; D002455:Cell Division; D002830:Choroid Neoplasms / Q000473:pathology*; D020869:Gene Expression Profiling; D006801:Humans; D048369:MAP Kinase Kinase 1; D008545:Melanoma / Q000473:pathology*; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D015513:Oncogene Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D048493:Proto-Oncogene Proteins B-raf; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000235:genetics; D014407:Tumor Cells, Cultured; D018631:ras Proteins
D018797:Cell Cycle Proteins; D015513:Oncogene Proteins; D034741:RNA, Small Interfering; C482119:BRAF protein, human; D017346:Protein Serine-Threonine Kinases; D048493:Proto-Oncogene Proteins B-raf; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins