Calipel Armelle, Mouriaux Frédéric, Glotin Anne-Lise, Malecaze François, Faussat Anne-Marie, Mascarelli Frédéric
INSERM U598 and IFR58, Institut Biomédical des Cordeliers, Paris 75006, France.
J Biol Chem. 2006 Apr 7;281(14):9238-50. doi: 10.1074/jbc.M600228200. Epub 2006 Feb 1.
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.
约66%的皮肤黑色素瘤中存在突变型B-Raf介导的ERK1/2组成性激活。相比之下，B-RAF的激活突变在眼黑色素瘤中很少见。本研究旨在确定野生型B-Raf（(WT)B-Raf）在葡萄膜黑色素瘤细胞生长中的作用。我们使用源自葡萄膜黑色素瘤原发性肿瘤的细胞系来评估(WT)B-Raf在细胞增殖中的作用，并对其上游调节因子和下游效应器进行表征。表达(WT)B-Raf和(WT)Ras的黑色素瘤细胞系以相似的增殖速率生长，显示ERK1/2的组成性激活，并且与表达激活型V600E突变（(V600E)B-Raf）的黑色素瘤细胞系具有相似水平的B-Raf表达和B-Raf激酶活性。它们对MEK1/2的药理学抑制在细胞增殖和转化方面与(V600E)B-Raf细胞同样敏感。siRNA介导的Raf-1缺失不影响ERK1/2激活，而siRNA介导的B-Raf缺失通过抑制ERK1/2激活使细胞增殖降低多达65%，无论B-Raf的突变状态如何。cAMP依赖性蛋白激酶（PKA）的药理学抑制和siRNA介导的PKA缺失极大地降低了(WT)B-Raf细胞中的B-Raf活性、ERK1/2激活和细胞增殖，而对(V600E)B-Raf细胞没有影响，这表明PKA在介导(WT)B-Raf/ERK信号以促进葡萄膜黑色素瘤细胞生长中起关键作用。此外，PKA的失活或缺失不影响Rap-1活性，Rap-1缺失也不影响B-Raf活性或ERK1/2激活。这排除了Rap1在(WT)B-Raf细胞中PKA介导的B-Raf/ERK激活中的作用。最后，我们证明了细胞周期蛋白D1在介导PKA/(WT)B-Raf信号以促进细胞增殖中的重要性。总之，我们的结果表明PKA/B-Raf途径是针对表达(WT)B-Raf的葡萄膜黑色素瘤治疗策略的潜在靶点。
