TGF-BETA AND CANCER · Regulating TGF-beta signalling at the transcriptional level and in cancer

Regulating TGF-beta signalling at the transcriptional level and in cancer

In my proposal, there were two main objectives: a) Identifying components involved in Smad2/3-mediated transcription, which in the midterm was redefined as elucidating the mechanisms by which TGFb mediates inhibition of BMP induced transcription. b) Identifying how Smad7 confers an increased risk for colon cancer. A description of the work performed I setup, screened and validated the potential hits from an siRNA library of chromatin modifying proteins. The hits were further validated by confocal microscopy, promoter-reporter assays, western blotting and QPCR. I also validated candidates from an siRNA screen for ubiquitinating/deubiquitinating enzymes. However none of the potential hits from either of the screens made it through all the validation steps. The second part of the proposal turned out to be technically impossible due to a lack of reagents sensitive enough to detect endogenous Smad7 in the system used. During my studies I realised that TGFb has an immediate inhibitory effect on BMP-induced transcription. In order to elucidate the mechanism by which TGFb mediates this effect, I generated two stable luciferase promoter-reporter MDA-MB-231 breast cancer cell lines responsive to either TGFb or BMP. These cell lines were used in promoter-reporter studies coupled with siRNA knockdowns to identify components required for the TGFb-mediated inhibition of BMP signalling. A microarray study was performed to identify BMP/TGFb regulated genes in vivo. I have also used Electro Mobility Shift Assays (EMSA) to study if TGFb influences the interaction between BMP activated Smads and DNA. Co-immunoprecipitation studies have been used to elucidate the effect of TGFb on BMP-mediated protein-protein interactions. A description of the main results achieved so far The finding that TGFb signalling inhibits BMP-induced transcription is both novel and unexpected. This inhibition was seen in all cell lines tested and using either cells with a stably integrated luciferase promoter-reporter construct or in transient assays. In the same type of assays, BMP signalling had no effect on TGFb-induced transcription. A microarray experiment identified a specific subset of genes as being both BMP-induced and negatively regulated by TGFb. These results were confirmed by QPCR and I could show that TGFb-mediated inhibition is independent of new protein synthesis. Inhibiting TGFb type I receptor kinase activity totally abolishes the effect of TGFb on BMP-induced transcription, indicating that receptor activation and downstream signalling is required. By using siRNA-mediated knockdown and the cell lines described above, I could show that the effect of TGFb is independent of Smad7, a well known inhibitor of both the BMP and TGFb pathway. BMP signalling induces the phosphorylation and nuclear accumulation of Smad1/5/8 whereas TGFb induces the activation and nuclear accumulation of Smad2/3. In the nucleus, both the BMP- and the TGFb-activated Smads form complexes with Smad4. It has been suggested that Smad4 is a limiting component in the TGFb/BMP signalling pathway. In the presence of overexpressed Smad4, TGFb was still able to inhibit BMP signalling arguing against this hypothesis. Interestingly siRNA knockdown experiments indicate that TGFb inhibits BMP signalling in a Smad3- and not Smad2-dependent way. The exact mechanism how this Smad3 dependent inhibition occurs is under investigation. Expected final results TGFbs and BMPs are critically important for embryonic development and still play important roles in tissue homeostasis in the adult. Disruptions in these pathways have been implicated in various human disorders ranging from fibrosis to cancer. My results will contribute to further the understanding how tissue homeostasis is maintained and also how BMP signalling contributes to cancer when TGFb signalling is disrupted. I expect my final results to be published in a high impact scientific journal.

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