Mechanisms of Smad regulation that determine the specificity of TGF-responses during tumorigenesis.

Transforming growth factor beta (TGF-beta) regulates many diverse biological processes including proliferation, motility, differentiation and survival. Aberrant TGF-beta signalling is also involved in tumour development. TGF-beta signals predominantly through a receptor complex, comprising of ALro and TpRII, to activate the R-Smads. Smad2 and Smad3. These activated R-Smads form complexes with Smad4 to regulate the transcription of TGF-beta inducible genes, which results in a huge array of biological responses. This has raised the question of the mechanism by which signalling specificity and diversity is generated. To address this question. I have used a model tumour cell system developed in Hartmut Beug's laboratory. The parental cell line. FpH4. is a non-tumorigenic. mouse mammary epithelial cell line that undergoes growth inhibition and apoptosis in response to TGF-beta. However. FpH4 cells that have been transformed by stable expression of oncogenic Ras (EpRas cells) undergo an Epithelial-to-Mesenchymal Transition (HMD in response to TGF-beta and form rapidly growing tumours in mice. Initially. I established the TAP-tagging purification system to identity new Smad-interacting partners in HpH4 and EpRas cells. Unfortunately, despite great efforts, no novel Smad partners were found using this method. I then went on to characterise the components of the TGF-beta pathway in this model tumour system. This work revealed that Smad3 is downregulated in EpRas cells compared with EpH4 cells and also that Smad3 expression is cell cycle regulated. Both transcriptional regulation and stability play a role in determining Smad3 expression. Low Smad3 levels in EpRas cells may account for their inability to growth arrest in response to TGF-beta. I have also observed C-terminal phosphorylation of Smad 1/5 as a result of autocrine Bone Morphogenetic Protein (BMP) signalling and also upon TGF-beta stimulation. Again, this is regulated at the cell cycle level. The Smad complexs formed as a result of Smad1 phosphorylation by TGF-beta are distinct from those formed by Smad1 activated by BMP. Molecular analysis of these signalling pathways has led to a model whereby TGF-beta induced Smad1 acts in concert with Smad2 and Smad3 to induce transcription of a unique set of genes, which adds a level of complexity and specificity to the TGF-beta signalling pathway.