Mitogen-activated protein (MAP) kinase kinase (MAPKK) activates MAP kinase in a signal transduction pathway that mediates cellular responses to growth and differentiation factors. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells by prolonging the activated state of MAPKK and of components downstream in the signaling pathway. To test this hypothesis, constitutively active MAPKK mutants were designed that had basal activities up to 400 times greater than that of the unphosphorylated wild-type kinase. Expression of these mutants in mammalian cells activated AP-1-regulated transcription. The cells formed transformed foci, grew efficiently in soft agar, and were highly tumorigenic in nude mice. These findings indicate that constitutive activation of MAPKK is sufficient to promote cell transformation.
The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). NIH 3T3 cells express HGF/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH cells are invasive in vitro and display enhanced protease activity necessary for the invasive phenotype. In experimental and spontaneous metastasis assays, Metmu or HMH cells metastasize to the lung, but lower numbers of subcutaneously injected Metmu and HMH cells produced invasive tumors in the heart, diaphragm, salivary gland, and retroperitoneum. It has been reported elsewhere that Met expression increased with tumor passage in athymic nude mice, and these tumor explants show enhanced activity in the metastasis assays. Autocrine-mediated Met-HGF/SF signal transduction in NIH 3T3 mesenchymal cells may provide an important system for understanding the biological process of metastasis.
Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector of cells expressing the Met tyrosine kinase receptor. Although HGF/SF is synthesized by mesenchymal cells and acts predominantly on epithelial cells, we have recently demonstrated that human sarcoma cell lines often inappropriately express high levels of Met and respond mitogenically to HGF/SF. In the present report we show that HGF/SF-Met signalling in the human leiomyosarcoma cell line SK-LMS-1 enhances its in vivo tumorigenicity, an effect for which the mitogenicity of this signalling pathway is likely to play a role. In addition, we found that HGF/SF-Met signalling dramatically induces the in vitro invasiveness and in vivo metastatic potential of these cells. We have studied the molecular basis by which HGF/SF-Met signalling mediates the invasive phenotype. A strong correlation has previously been demonstrated between the activation of the urokinase plasminogen activator (uPA) proteolysis network and the acquisition of the invasive-metastatic phenotype, and we show here that HGF/SF-Met signalling significantly increases the protein levels of both uPA and its cellular receptor in SK-LMS-1 cells. This results in elevated levels of cell-associated uPA and enhanced plasmin-generating ability by these cells. These studies couple HGF/SF-Met signalling to the activation of proteases that mediate dissolution of the extracellular matrix-basement membrane, an important property for cellular invasionmetastasis.
The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). It was previously shown that, like the oncogenic tpr-met, the mouse met proto-oncogene transforms NIH 3T3 cells. We have established NIH 3T3 cells stably expressing both human (Methu) and mouse (Metmu) met proto-oncogene products. The protein products are properly processed and appear on the cell surface. NIH 3T3 cells express endogenous mouse HGF/SF mRNA, suggesting an autocrine activation mechanism for transformation by Metmu. However, the tumor-forming activity of Methu in NIH 3T3 cells is very low compared with that of Metmu, but efficient tumorigenesis occurs when Methu and HGF/SFhu are coexpressed. These results are consistent with an autocrine transformation mechanism and suggest further that the endogenous murine factor inefficiently activates the tumorigenic potential of Methu. The tumorigenicity observed with reciprocal chimeric human and mouse receptors that exchange external ligand-binding domains supports this conclusion. We also show that HGF/SFhu expressed in NIH 3T3 cells produces tumors in nude mice.
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