Ras Transformation In Epithelial Cells Research Articles

Loss of Transgelin in Breast and Colon Tumors and in RIE-1 Cells by Ras Deregulation of Gene Expression through Raf-independent Pathways

Activated Ras but not Raf can transform RIE-1 and other epithelial cells, indicating the critical importance of Raf-independent effector function in Ras transformation of epithelial cells. To elucidate the nature of these Raf-independent activities, we utilized representational difference analysis to identify genes aberrantly expressed by Ras through Raf-independent mechanisms in RIE-1 cells. We identified a total of 22 genes, both known and novel, whose expression was either activated or abolished by Ras but not Raf. The genes up-regulated encode proteins involved in protein or DNA synthesis, regulation of protease activity, or ligand binding, whereas those genes down-regulated encode actin cytoskeletal-, extracellular matrix-, and gap junction-associated proteins, and transmembrane receptor- or cytokine-like proteins. These results suggest that a key function of Raf-independent signaling involves deregulation of gene expression. We further characterized transgelin as a gene whose expression was abolished by Ras. Transgelin was identified previously as a protein whose expression was lost in virally transformed cell lines. We show that this loss is regulated at the level of gene expression and that both Raf-dependent and Raf-independent pathways are required to cause Ras down-regulation of transgelin in RIE-1 cells, whereas Raf alone is sufficient to cause its loss in NIH 3T3 fibroblasts. We also found that Ras-dependent and Ras-independent mechanisms can cause the down-regulation of transgelin in human breast and colon carcinoma cells lines and patient-derived tumor samples. We conclude that loss of transgelin gene expression may be an important early event in tumor progression and a diagnostic marker for breast and colon cancer development.

Dominant negative mutants of mitogen-activated protein kinase pathway

One of the gene families most commonly found mutated in human tumors is the ras gene family. Ras has been shown to regulate several pathways that contribute to cellular transformation, including the Rac and Rho pathways and the Raf-MEK-MAPK pathway (MEK, MAPK/ERK kinase; MAPK, mitogen activated protein kinase; ERK, extracellular signal-regulated kinase).Raf-MEK-MAPK pathway is necessary for Ras transformation in epithelial cells. The immediate biochemical effect of expressing dominant negative MEK in the tumor cells is a reduction in endogenous Raf-1 and MEK-1 activity through the formation of nonproductive complexes between endogenous Raf-1 and dominant negative MEK. An unexpected finding is that there is no direct correlation between dominant negative MEK expression levels and the percentage reduction of MEK activity in the cells. The most striking aspect of the in vivo studies is the finding that the inhibitory effect of dominant negative MEK on the measured end points was dose dependent. It was consistently observed that clones expressing the highest levels of dominant negative MEK formed the smallest tumors and the fewest metastases and exhibited significant increases in survival. However, it could be argued that high levels of dominant negative MEK might nonspecifically interfere with pathways other than the Raf- MEK-MAPK pathway and perhaps affect in vivo tumor growth. This possibility is negated by the observation that clones expressing low levels of dominant negative MEK were able to elicit all the biological responses measured, suggesting that altered MEK activity alone can have a profound effect on biological outcome.

Activation of the Raf-1/MAP kinase cascade is not sufficient for Ras transformation of RIE-1 epithelial cells.

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.