Evidence for a role of Met-HGF/SF during Ras-mediated tumorigenesis/metastasis CP Webb, GA Taylor, M Jeers, M Fiscella, M Oskarsson, JH Resau and GF Vande Woude ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, PO Box B, Frederick, Maryland, 21702, USA Aberrations in Met-hepatocyte growth factor/scatter factor (HGF/SF) signaling have been implicated in the acquisition of tumorigenic and metastatic phenotypes. Here we show that murine NIH3T3 and C127 cells transformed by the Ras oncogene overexpress the Met receptor, resulting in enhanced HGF/SF-mediated responses in vitro including invasion through basement membrane. Accompanying the increase in Met in ras- transformed NIH3T3 cells, there is a decrease in endogenous HGF/SF expression as previously observed in cells exogenously overexpressing Met. However, subcutaneously grown tumors and experimental lung metastases derived from these cells express signi®cantly higher levels of endogenous HGF/SF together with high levels of Met. These results suggest Met-HGF/SF signaling enhances tumor growth and metastasis of Ras-transformed NIH3T3 cells. Keywords: Ras; Met; HGF/SF; tumorigenesis; metas- tasis Introduction The product of the met proto-oncogene is the transmembrane tyrosine kinase p190 Met (Park et al., 1986), which has been identi®ed as the receptor for hepatocyte growth factor/scatter factor (HGF/SF) (Bottaro et al., 1991). HGF/SF is a polypeptide growth factor produced predominantly by cells of mesenchymal origin, which elicits a variety of eects in vitro on target cells expressing Met including the induction of cell proliferation, dierentiation, invasion and motility (reviewed by Rubin et al., 1993; Vande Woude et al., 1997). While HGF/SF-Met signaling has been shown to be important in a number of normal physiological processes, we and others have shown that aberrant HGF/SF-Met signaling leads to the develop- ment of tumors and metastases (reviewed by Jeers et al., 1996b). In addition, Met and/or HGF/SF are frequently found to be overexpressed in a variety of tumor tissues, providing further evidence for their involvement in tumor formation/metastasis (reviewed by Jeers et al., 1996b). Members of the Ras family of small GTPases (H-, K- and N-Ras) are frequently mutated (activated) in a number of human tumors (Bos, 1989), and in addition, oncogenic mutants of Ras have been shown to confer both tumorigenic and metastatic phenotypes to a variety of cell types in culture (Bondy et al., 1985; Greig et al., 1985; Muschel et al., 1985; Thorgeirsson et al., 1985). Although a number of downstream genetic events have been characterized in Ras-transformed cells, precisely which of these are important for the acquisition of the tumorigenic and metastatic pheno- types are poorly understood (Chambers and Tuck, 1993). In this paper we demonstrate that two Ras- transformed murine cell lines, NIH3T3 and C127 cells, express higher levels of Met when compared with their non-transformed counterparts. As a result, Ras- transformed cells are signi®cantly more responsive to HGF/SF in vitro. In addition we show that Ras- transformed NIH3T3 cells expressing high levels of Met express signi®cantly lower levels of endogenous HGF/SF. However, both tumors and experimental lung metastases derived from injecting these cells into athymic nude mice express high levels of both Met and HGF/SF. These results suggest that autocrine Met- HGF/SF signaling enhances Ras-mediated tumorigeni- city and metastasis of NIH3T3 cells and may be important for understanding the genetic events leading to tumorigenesis and metastasis. Results Met is overexpressed in Ras-transformed cells Stable cell lines of NIH3T3 and C127 cells expressing the human HA-tagged V12-Harvey ras oncogene were generated as described in Materials and methods. Expression of the Ras oncoprotein was con®rmed by Western blotting using an antibody against the HA epitope (Figure 1a). To determine the level of Met expression in Ras-transformed cells, Met proteins were immunoprecipitated and analysed by Western blotting using an antibody against the C terminus of murine Met. As shown in Figure 1b, Ras transformed NIH3T3 and C127 cells both expressed higher levels of Met compared to the non-transformed counterparts. In NIH3T3 cells, this increase was most notable in the p140 Met mature b- chain (containing the tyrosine kinase domain) and less so in the p170 Met precursor (Iyer et al., 1990). In Ras transformed C127 cells, both p140 Met and p170 Met were increased above parental levels. As a consequence of overexpressing Met, the level of p140 Met tyrosine phosphorylation was substantially higher in the Met immunoprecipitates from Ras-transformed cells follow- ing HGF/SF stimulation, when compared with non- transformed parental cells (Figure 1b). In addition, we noted reduced basal Met phosphorylation in Ras- transformed NIH3T3 cells when compared with Correspondence: GF Vande Woude Received 9 February 1998; revised 18 May 1998; accepted 18 May 1998 Oncogene (1998) 17, 2019 ± 2025 ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc