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F. Mascarelli, A. Calipel, A.–L. Glotin, F. Cohen–Behar, F. Mouriaux; Differential Roles of Raf–1 and B–Raf in Uveal Melanoma Tumorigenesis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4615.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Few or no mutations have been found in the genes encoding proteins of the major intracellular signaling pathways controlling cell proliferation in uveal melanomas, including those affected in cutaneous melanomas (B–RafV599E mutation). We recently demonstrated the role of the SCF/c–Kit/ERK signaling pathway in the uveal melanoma cell tumorigenesis (J. Biol. Chem., 2004, 279: 31769–79), suggesting that uveal melanoma tumorigenesis occurs preferentially through growth factor autocrine loop and is not the result of function gain of mutated signaling proteins. Therefore, we investigated the role of wild–type B–Raf and wild type Raf–1 in the control of the activation of ERK1/2 signaling for uveal melanoma cell proliferation. Methods: The levels of Raf–1, B–Raf , and ERK1/2 were analyzed by Western blotting in uveal melanocytes and four melanoma cells (two expressing wild type B–Raf and two expressing B–RafV599E). The roles of Raf–1 and the two forms of B–Raf on ERK1/2 activation and cell proliferation were investigated by downregulating their respective expression using siRNA and ODN antisens (AS) strategies, and assessing ERK activation and cell proliferation using Western blot analysis and the MTT colorimetric method, respectively. Results:Strong and sustained ERK1/2 activation of ERK1/2 was observed in both melanoma cells expressing B–RafV599E and wild–type B–Raf. Si RNA–mediated inhibition of B–RafV599E and wild–type B–Raf protein expression abolished both ERK1/2 activation and cell proliferation. In contrast, si RNA–or ODN AS–mediated inhibition of Raf–1 protein expression did not affect ERK1/2 activation, but greatly reduced cell viability of uveal melanoma cells, irrespective of the B–Raf mutation status. Conclusions: Our data strongly suggest that growth factor–induced cell proliferation is mediated by wild–type B–Raf through the activation of ERK1/2, whereas cell survival is under the control of Raf–1 in uveal melanoma cells. Targeting both B–Raf and Raf–1 may be therefore of interest in the design of therapeutic strategies for the control of uveal melanoma growth.
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