Multiple aspects of the phenotype of mammary epithelial cells transformed by expression of activated M-Ras depend on an autocrine mechanism mediated by hepatocyte growth factor/scatter factor.

Multiple aspects of the transformed phenotype induced in a murine mammary epithelial cell line scp-2 by expression of activated G22V M-Ras, including maintainance of cell number at low density, anchorage-independent growth, invasion of Matrigel, and secretion of matrix metalloproteinases (MMP) 2 and 9, were dependent on an autocrine mechanism. Conditioned medium from dense cultures of scp-2 cells expressing G22V M-Ras, but not from parental cells, induced activation of Erk and Akt in cells expressing G22V M-Ras, maintained the cell number and promoted anchorage-independent growth of cells expressing G22V M-Ras (although not the parental cells), and induced scattering of MDCK cells. The latter activities were blocked by neutralizing antibodies to hepatocyte growth factor/scatter factor (HGF/SF) and could be mimicked by HGF/SF. Anti-HGF/SF antibodies also inhibited invasion of Matrigel, and the production of MMP-2 and MMP-9, together with urokinase-type plasminogen activator, was secreted by G22V M-Ras scp-2 cells but not by parental cells. Invasion of Matrigel was blocked by an inhibitor of MMPs, BB94, and by the mitogen-activated protein kinase kinase 1/2 kinase inhibitor PD98059 but was only marginally affected by the phosphatidylinositol 3-kinase inhibitor LY294002. Autocrine HGF/SF was thus critical for expression of key features of the phenotype of mammary epithelial cells transformed by expression of activated M-Ras.

Expression of activated M-Ras in a murine mammary epithelial cell line induces epithelial-mesenchymal transition and tumorigenesis.

The expression of activated mutants of M-Ras (G22V or Q71L), but not wild-type M-Ras, in a murine mammary epithelial cell line, scp2, resulted in epithelial-mesenchymal transition (EMT) and oncogenic transformation. Cells expressing constitutively active M-Ras continued to grow in the absence of serum and exhibited a loss of the epithelial markers cytokeratin, E-cadherin and beta-catenin, together with a gain of the mesenchymal marker vimentin, a loss of contact inhibition in monolayer growth and a gain of the capacity for anchorage-independent growth. Moreover, unlike the parental cells, they failed to form differentiated mammospheres on Matrigel and instead formed branched networks of cells that grew and invaded the Matrigel. The expression of activated p21 Ras (G12V H-Ras or Q61K N-Ras) also resulted in EMT and tumorigenesis, although there was evidence that expression of higher levels was toxic. Tumors derived from scp2 cells expressing activated M-Ras exhibited activation of Akt and of ERK. The levels of expression of Q71L M-Ras and G12V H-Ras required for tumorigenesis were comparable, although higher levels of the weaker G22V M-Ras mutant were selected for in vivo. These data indicate that the expression of activated mutants of M-Ras was sufficient for oncogenic transformation of a murine mammary epithelial cell line.