Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference
Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference
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DOI：
10.1038/sj.onc.1207812
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发表时间：
2004-08-12
期刊：
影响因子：
8
通讯作者：
Kawakami, Y
中科院分区：
文献类型：
--
作者：
Sumimoto, H;Miyagishi, M;Kawakami, Y
参与有丝分裂原活化蛋白激酶（MAPK）途径的分子的致癌突变提供了介导包括黑色素瘤在内的各种癌症的肿瘤生长和侵袭的信号。在66%的黑色素瘤中发现的BRAF体细胞突变具有NIH3T3转化能力，体外激酶活性升高。我们尝试在10个黑色素瘤细胞系中用HIV慢病毒载体介导RNA干扰（RNAi），所述HIV慢病毒载体特异于野生型或BRAF的最常见突变形式（V599E），并且发现RNAi在体外和体内抑制大多数黑色素瘤细胞系的生长，这伴随着BRAF蛋白和ERK磷酸化的降低。有趣的是，突变的BRAF（V599E）特异性siRNA仅抑制具有该突变的黑素瘤细胞系的生长和MAPK活性。此外，BRAF RNAi抑制黑色素瘤细胞的基质胶侵袭，伴随着基质金属蛋白酶活性和β（1）整合素表达的降低。这些结果阐明了突变的BRAF（V599E）通过MAPK活化基本上参与黑色素瘤细胞的恶性表型，并且是黑色素瘤治疗的有吸引力的分子靶标。慢病毒介导的针对癌基因突变的RNAi技术有望成为肿瘤基因治疗的有力手段。
Oncogenic mutations of molecules involved in the mitogen-activated protein kinase (MAPK) pathways provide signals mediating both tumor growth and invasion in various cancers including melanomas. BRAF somatic mutations, found in 66% of melanomas, have NIH3T3 transforming ability with the elevated kinase activity in vitro. We attempted to mediate RNA interference (RNAi) with HIV lentiviral vectors specific for either wild type or the most frequently mutated form of BRAF (V599E) in 10 melanoma cell lines, and found that RNAi inhibited the growth of most melanoma cell lines in vitro as well as in vivo, which was accompanied by decrease of both BRAF protein and ERK phosphorylation. Interestingly, the mutated BRAF (V599E)-specific siRNA inhibited the growth and MAPK activity of only melanoma cell lines with this mutation. Furthermore, BRAF RNAi inhibited matrigel invasion of melanoma cells accompanied with a decrease of matrix metalloproteinase activity and beta(1) integrin expression. These results clarify that the mutated BRAF (V599E) is essentially involved in malignant phenotype of melanoma cells through the MAPK activation and is an attractive molecular target for melanoma treatment. The lentivirus-mediated RNAi specific for oncogenic mutations may be a powerful technique for gene therapy of cancer.