Lehmann, Kerstin Elisabeth; (2001) Regulation of epithelial cell transformation and survival by Raf activation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Oncogenic Ras genes are activated by point mutation in about 30% of human tumours, being one of the most frequent mutations in human cancers. Activation of the Raf/MAPK pathway by Ras plays a major role in the establishment of cell transformation allowing cells to evade apoptosis and negative cell cycle control. In many advanced human tumours of epithelial origin, Ras was found to co-operate with TGFβ in promoting tumour progression and cells undergo an epithelial to mesenchymal transition, termed EMT, in which the epithelial phenotype, characterised by strong cell-cell adhesion and cell polarity, is lost. The acquisition of a mesenchymal phenotype is characterised by reduced cell-cell contacts and induction of invasive growth. In order to analyse the contribution of the Raf/MAPK pathway on changes in the epithelial cell-morphology during cell transformation, an inducible Raf-1 fusion protein was stably expressed in MDCK cells, which allowed to monitor the progression of cellular events. Activation of Raf was sufficient to cause disruption of cell-cell junctions. Furthermore, sustained activation of Raf was sufficient to cause EMT, thereby inducing down-regulation of E-cadherin expression. Raf activation promoted invasive growth in collagen gels, which was dependent on the establishment of autocrine TGFβ signalling, whose secretion was induced by Raf. During EMT, cells were rendered insensitive to anti-proliferative TGFβ effects. Furthermore, expression of the TGFβ effector Smad3 was down-regulated in MDCK cells which have undergone EMT, and re-expression of Smad3 rendered these cells sensitive to growth inhibition by TGFβ. Apoptosis induced by TGFβ in MDCK cells was prevented by short-term Raf activation at a time point at which cells were still susceptible for TGFβ-induced growth arrest. The mechanism by which short-term Raf activation prevents TGFβ-induced apoptosis in MDCK cells differs from the one observed in MDCK cells which had undergone EMT due to sustained Raf activation. While short-term activation of Raf did not prevent release of cytochrome c from mitochondria, it was abolished by sustained Raf activation, presumably due to expression of Bcl-XL. The anti-apoptotic potential of Raf was not restricted to TGFβ-induced apoptosis, but protected epithelial cells from a variety of pro-apoptotic stimuli, suggesting a more general survival mechanism mediated by Raf.

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