title: Extracellular signal-regulated kinase-dependent proliferation is mediated through the protein kinase A/B-Raf pathway in human uveal melanoma cells.
abstract: Mutated B-Raf-mediated constitutive activation of ERK1/2 is involved in about 66% of cutaneous melanoma. By contrast, activating mutations in B-RAF are rare in ocular melanoma. This study aimed to determine the role of wild-type B-Raf ((WT)B-Raf) in uveal melanoma cell growth. We used cell lines derived from primary tumors of uveal melanoma to assess the role of (WT)B-Raf in cell proliferation and to characterize its upstream regulators and downstream effectors. Melanoma cell lines expressing (WT)B-Raf and (WT)Ras grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as melanoma cell lines expressing the activating V600E mutation ((V600E)B-Raf). They were equally as sensitive to pharmacological inhibition of MEK1/2 for cell proliferation and transformation as (V600E)B-Raf cells. siRNA-mediated depletion of Raf-1 did not affect either ERK1/2 activation, whereas siRNA-mediated depletion of B-Raf reduced cell proliferation by up to 65% through the inhibition of ERK1/2 activation, irrespective of the mutational status of B-Raf. Pharmacological inhibition of cAMP-dependent protein kinase (PKA) and siRNA-mediated depletion of PKA greatly reduced B-Raf activity, ERK1/2 activation, and cell proliferation in (WT)B-Raf cells, whereas it did not affect (V600E)B-Raf cells, demonstrating a key role of PKA in mediating (WT)B-Raf/ERK signaling for uveal melanoma cell growth. Moreover, inactivation or depletion of PKA did not affect Rap-1 activity, and Rap-1 depletion did not affect either B-Raf activity or ERK1/2 activation. This ruled out a role for Rap1 in the PKA-mediated B-Raf/ERK activation in (WT)B-Raf cells. Finally, we demonstrated the importance of cyclin D1 in mediating PKA/(WT)B-Raf signaling for cell proliferation. Altogether, our results suggest that the PKA/B-Raf pathway is a potential target for therapeutic strategies against (WT)B-Raf-expressing uveal melanoma.
authors.affiliation: INSERM U598 and IFR58, Institut Biomédical des Cordeliers, Paris 75006, France.
authors.forename: Armelle
authors.initials: A
authors.lastname: Calipel
authors.forename: Frédéric
authors.initials: F
authors.lastname: Mouriaux
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Glotin
authors.forename: François
authors.initials: F
authors.lastname: Malecaze
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Faussat
authors.forename: Frédéric
authors.initials: F
authors.lastname: Mascarelli
mesh_terms: D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019938:Cyclin D1 / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases; D006801:Humans; D007527:Isoenzymes; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D014604:Uveal Neoplasms / Q000235:genetics / Q000473:pathology*
chemical_list: D007527:Isoenzymes; D019938:Cyclin D1; D000242:Cyclic AMP; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3