Analysis of transformation of mammary epithelial cells by activated M-Ras

The apparent absence of activating mutations within the p21 Ras genes in human breast cancer raised the possibility of involvement of other Ras family genes in this tumor type. It had been proposed that activating mutations in R-Ras2 (TC21) may have a role in breast tumors and work within the laboratory had identified elevated levels of R-Ras3 (M-Ras) in breast cancer cell-lines. To investigate this idea I expressed M-Ras in a functional murine mammary epithelial cell-line, scp2. Expression of activated mutants of M-Ras, but not wild type M-Ras, in scp2 cells resulted in epithelial to mesenchymal transition (EMT) and oncogenic transformation. Cells expressing constitutively active M-Ras exhibited a loss of epithelial markers and a gain of mesenchymal markers. These cells continued to grow in the absence of serum and gained the capacity for anchorage-independent growth in methylcellulose. Furthermore, unlike the parental cells, they failed to form differentiated mammospheres on Matrigel and instead formed branched networks of cells.; When injected into mice scp2 cells expressing activated M-Ras rapidly formed tumors. Expression of activated p21 Ras also resulted in EMT and tumorigenesis, although there was evidence that high levels of expression were toxic to this cell type. Tumors derived from scp2 cells expressing activated M-Ras or p21 Ras exhibited activation of both PKB and ERK. When expressed at similar levels Q71L M-Ras and G12V H-Ras resulted in comparable rates of tumor formation, although higher levels of expression of the weaker G22V M-Ras mutant were required. These data indicate that expression of activated M-Ras was sufficient for oncogenic transformation of a murine mammary epithelial cell-line.; I further analyzed the mechanisms by which activated M-Ras leads to transformation in this cell type using mutations within the effector loop of Q71L M-Ras. Mutations at the equivalent positions within the effector loop of activated p21 Ras have been shown to bind restricted subsets of effectors. Expression of the E47G/Q71L M-Ras construct in epithelial cells resulted in transformation and colony formation. It was shown that this mutant interacted strongly with the Ras effectors Nore-1 and RPM. Furthermore, cells expressing E47G/Q71L exhibited activation of ERK and PKB at levels similar to Q71L M-Ras alone. However, cells expressing the T45S or Y50C mutants of Q71L M-Ras remained epithelial.; I found that expression of activated PKB or Raf alone did not effect the epithelial morphology of cells. However, pharmacological inhibition of PI3K or MEK1 resulted in reduced growth and altered morphology respectively of epithelial cells expressing either activated M-Ras or p21 Ras. I addressed the role of Ral and Rap in epithelial cell transformation and found no phenotypic effect of activation of these GTPases alone, suggesting that it may be combinations of these pathways which are important. Lastly, I looked at inducible expression of activated M-Ras to determine the requirement for continued M-Ras expression to maintain the transformed phenotype. I found that repression of activated M-Ras expression in scp2 cells resulted in a partial reversion of phenotype, suggesting that elements of the observed phenotype do require continued activation of M-Ras.