Role of hepatocyte growth factor and its receptor MET in the malignant transformation of human fibroblasts

It is now generally recognized that cancer is the result of a multistep process, which involves the activation of oncogenes and the inactivation of tumor suppressor genes. In cancer cells, activation of oncogenes occurs mainly by mutations or over-expression. Conventionally, only mutated genes have been considered as candidate cancer-related genes. Recent advances in various techniques have identified many more genes that are altered in expression in cancer cells than are mutated. Non-mutated genes with stably altered expression patterns may be a key component of the cancer puzzle and may ultimately be traced back to aberrations in upstream genes, such as those encoding transcription factors.; The goal of my research has been to investigate the role of hepatocyte growth factor gene (hgf) and its receptor, the met gene, in the malignant transformation of human fibroblasts. I utilized human fibroblastic cells transformed to malignancy in culture in the Carcinogenesis Laboratory as well as human fibrosarcoma cell lines derived from patients' tumors. My first observation was that the MET protein was expressed at three to nine fold higher levels in ten out of 11 human fibrosarcoma cell lines tested, compared to several foreskin-derived normal human fibroblast cell lines. To determine whether these higher levels of expression play a causal role in the malignant transformation of human fibroblasts, I chose two human fibrosarcoma cell lines and down-regulated their expression of MET and/or HGF using chimeric transgenes consisting of U1 small nuclear RNA and hammerhead ribozymes targeting met and/or hgf. When injected into athymic mice, cell strains with reduced MET and/or HGF expression exhibited reduced frequency and increased latency in tumor formation and growth. These results directly demonstrate a strong dependence on endogenous high levels of MET and/or HGF expression for both tumor formation and growth of human fibrosarcomas.; I also examined the mechanism(s) responsible for MET over-expression in human fibrosarcoma cell lines. Gene amplification was not found to be important for such over-expression. Instead, a majority of the fibrosarcoma cell lines with high levels of MET showed coordinately high levels of transcription factor Sp1. Deletion analysis and site-directed mutagenesis of the met promoter revealed that the tandem Sp1 sites in the proximal promoter region are important for the transcription of the met gene. Two human fibrosarcoma cell lines with high levels of MET and Sp1 exhibited much higher met promoter-luciferase activity than did two normal human fibroblast cell lines with low levels of MET and Sp1. Furthermore, transfection of Sp1 cDNA into a normal human fibroblast cell line resulted in a dose-dependent increase in the met promoter activity, whereas transfection of a human fibrobsarcoma cell line with an Sp1 decoy to interfere with and inhibit Sp1 binding to DNA led to a dramatic reduction in MET expression. These data demonstrate that transcriptional up-regulation by Sp1 is a major mechanism for MET over-expression in human fibrosarcomas. The results suggest that when highly expressed, Sp1 functions as an oncoprotein.