Yayon A, Ma Y S, Safran M, Klagsbrun M, Halaban R
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
Oncogene. 1997 Jun 26;14(25):2999-3009. doi: 10.1038/sj.onc.1201159.
Basic Fibroblast Growth Factor (bFGF/FGF2) is thought to play a decisive role in malignant progression. Aberrant expression of bFGF causes constitutive autocrine activation of its cognate receptor and autonomous growth of human melanoma cells or bFGF transformed fibroblasts in culture. It remains to be determined, however, whether the endogenous bFGF confers growth advantage to tumors and what are the downstream targets of the activated FGF receptor critical for its transforming capacity. We therefore transfected metastatic melanoma cells and bFGF transformed mouse fibroblasts with a dominant-negative mutant of the murine FGF receptor 1 (fgfr1/flg), comprising the extracellular and transmembrane domains but lacking the intracellular kinase domain (dnflg). Reverse transcriptase-PCR, 125I-bFGF binding and affinity labeling analyses show that the truncated receptor is targeted to the membrane and is expressed at much higher levels than the endogenous receptor in all of the selected clones. Expression of the dnflg dramatically reduces the basal as well as bFGF induced growth of these cells in vitro and also suppresses their tumorigenic potential in nude mice. The expression of the dnflg does not significantly alter the general level of tyrosyl-phosphorylated proteins in the trunsduced melanoma cells. Rather, a major downstream affected target is a Src-family kinase, whose activity, determined by an in vitro immune kinase assay, is stimulated in normal melanocytes by exogenous bFGF, and is markedly reduced in the dnflg-expressing melanoma cells. The present study demonstrates that direct interference with the activity of FGF receptors has a deleterious effect on cell proliferation and survival in vitro and in vivo leading to the suppression of melanoma tumor progression possibly through the inactivation of a Src-family kinase.
碱性成纤维细胞生长因子（bFGF/FGF2）被认为在恶性进展中起决定性作用。bFGF的异常表达导致其同源受体的组成型自分泌激活以及人黑色素瘤细胞或培养中bFGF转化的成纤维细胞的自主生长。然而，内源性bFGF是否赋予肿瘤生长优势以及激活的FGF受体对其转化能力至关重要的下游靶点是什么，仍有待确定。因此，我们用鼠源FGF受体1（fgfr1/flg）的显性负性突变体转染转移性黑色素瘤细胞和bFGF转化的小鼠成纤维细胞，该突变体包含细胞外和跨膜结构域但缺乏细胞内激酶结构域（dnflg）。逆转录聚合酶链反应、125I-bFGF结合和亲和标记分析表明，截短的受体靶向细胞膜，并且在所有选定的克隆中表达水平比内源性受体高得多。dnflg的表达显著降低了这些细胞在体外的基础生长以及bFGF诱导的生长，并且还抑制了它们在裸鼠中的致瘤潜力。dnflg的表达并未显著改变转导的黑色素瘤细胞中酪氨酸磷酸化蛋白的总体水平。相反，一个主要的下游受影响靶点是Src家族激酶，通过体外免疫激酶测定确定其活性，在外源性bFGF刺激下在正常黑素细胞中被激活，而在表达dnflg的黑色素瘤细胞中明显降低。本研究表明，直接干扰FGF受体的活性对体外和体内细胞增殖和存活具有有害影响，可能通过Src家族激酶的失活导致黑色素瘤肿瘤进展的抑制。
