Liang Hongyan, O'Reilly Sandra, Liu Youhua, Abounader Roger, Laterra John, Maher Veronica M, McCormick J Justin
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824-1302, USA.
Int J Oncol. 2004 May;24(5):1057-67.
Changes in expression of hepatocyte growth factor (HGF) and its receptor, MET, are associated with formation and malignant progression of human tumors. In the present study, 10 of 11 human fibrosarcoma cell lines tested expressed significantly higher levels of MET than were found in a series of normal human fibroblast lines. Still more significant, MET was constitutively phosphorylated in all 11 fibrosarcoma lines, whereas the normal fibroblasts exhibited very low levels of the phosphorylated form. All the cell lines expressed HGF mRNA. To determine the role of MET and/or HGF in tumorigenesis, a fibrosarcoma line expressing high levels of MET protein and low levels of HGF/NK2 mRNA was stably transfected with a hammerhead ribozyme targeting MET. In addition, a fibrosarcoma line expressing high levels of both MET protein and HGF/NK2 mRNA was transfected with a ribozyme targeting MET, or with a ribozyme targeting MET and another targeting HGF. The transfectant cell lines no longer formed tumors, or did so at a greatly reduced frequency and/or longer latency. Because Sp1 is a transcription factor for MET, we assayed the cell lines for their level of Sp1 protein. Sp1 was markedly overexpressed in 7 of the 11 fibrosarcoma lines compared to normal fibroblast lines. Deletion analysis and site-directed mutagenesis of the MET promoter revealed that tandem Sp1 sites in the proximal promoter are critical for transcription of MET. Increased expression of Sp1 in a normal human fibroblast line containing a MET promoter-luciferase construct resulted in a dose-dependent increase in luciferase. Conversely, inhibition of Sp1 binding to DNA in a fibrosarcoma cell line, using an Sp1 decoy, dramatically reduced MET expression. Taken together, these results indicate that in human fibrosarcoma cells, high levels of the phosphorylated form of MET are required for tumor formation and that Sp1 can function to control the level of MET.
肝细胞生长因子（HGF）及其受体MET的表达变化与人类肿瘤的形成和恶性进展相关。在本研究中，所检测的11个人类纤维肉瘤细胞系中有10个表达的MET水平明显高于一系列正常人成纤维细胞系。更显著的是，在所有11个纤维肉瘤细胞系中MET持续磷酸化，而正常成纤维细胞中磷酸化形式的水平非常低。所有细胞系均表达HGF mRNA。为了确定MET和/或HGF在肿瘤发生中的作用，用靶向MET的锤头状核酶稳定转染了一个表达高水平MET蛋白和低水平HGF/NK2 mRNA的纤维肉瘤细胞系。此外，用靶向MET的核酶或靶向MET和另一个靶向HGF的核酶转染了一个同时表达高水平MET蛋白和HGF/NK2 mRNA的纤维肉瘤细胞系。转染后的细胞系不再形成肿瘤，或者形成肿瘤的频率大大降低和/或潜伏期延长。因为Sp1是MET的转录因子，我们检测了细胞系中Sp1蛋白的水平。与正常成纤维细胞系相比，11个纤维肉瘤细胞系中有7个明显过表达Sp1。对MET启动子的缺失分析和定点诱变表明，近端启动子中的串联Sp1位点对MET的转录至关重要。在含有MET启动子 - 荧光素酶构建体的正常人成纤维细胞系中Sp1表达增加导致荧光素酶呈剂量依赖性增加。相反，在纤维肉瘤细胞系中使用Sp1诱饵抑制Sp1与DNA的结合，显著降低了MET的表达。综上所述，这些结果表明在人类纤维肉瘤细胞中，肿瘤形成需要高水平的磷酸化MET，并且Sp1可以发挥作用控制MET的水平。
