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A novel mode of TGF-beta signalling through Smad1/Smad5 phosphorylation: mechanism and functional role in cancer

Final Report Summary - SMAD1/5 AND CANCER (A novel mode of TGF-beta signalling through Smad1/Smad5 phosphorylation: mechanism and functional role in cancer)

TGF-beta is a cytokine that regulates diverse biological functions, mainly by regulating the Smad transcription factors. Historically, TGF-beta has been reported to signal specifically through Smad2 and Smad3 and the co-Smad Smad4, but in our lab we have observed that TGF-beta is also able to phosphorylate Smad1, Smad5 and Smad8 (Daly et al. 2008, Mol Cell Biol, 28, 6889-6902), which are normally thought to be activated only downstream of BMPs and GDFs (ligands from the same superfamily). The aim of this project was to study the mechanism and functional relevance of this poorly understood mode of TGF-beta signalling.

In the first part of the project the aim was to understand the molecular mechanisms of TGF-beta signalling through Smad1/5/8. The TGF-beta superfamily uses two types of receptor: type II, which recognise the ligands and then recruit type I receptors, which are responsible for phosphorylating the Smads. Specific receptors have been linked to TGF-beta or BMPs. I have performed knock down and overexpression studies and in vitro kinase assays to determine which receptors are required for TGF-beta to signal through Smad1/5/8.

The simultaneous phosphorylation of Smad1/5/8 and Smad2/3 in response to TGF-beta results in the production of novel mixed R-Smad complexes. Using Biomolecular Fluorescence Complementation assays, I have been able for the first time to observe these mixed R-Smad complexes.

The second part of the project was directed at answering key questions regarding the function of this novel pathway, mainly in cancer. Using shRNA, I have generated a stable EpRas cell line with very low levels of Smad1. We are studying the capability of these cells to induce tumours in xenograft assays in mice and to colonize lungs in nude mice.

The differences observed in cells with low expression levels of Smad1 in vivo compared with wild type cells prompted me to study which specific set of genes are regulated by phosphorylated Smad1/5/8-comprising complexes in response to TGF-beta. I am performing Binding Site Selection assays to determine the sequence specificity of the mixed R-Smad complexes. I am also performing RNAseq using EpRas cells in which Smad1/5 are downregulated. In response to TGF-beta, a number of genes are differentially expressed when these two Smads are knocked down.

Smad1/5/8 can be phosphorylated and activated in response to TGF-beta. I have shown that canonical TGF-beta receptors are not sufficient for this, but type I BMP receptors are additionally required. I have shown that this leads to the formation of new mixed R-Smad complexes comprising Smad2 and Smad1, as well as Smad4 and Smad1 in response to TGF-beta.

Functionally, I have observed that when Smad1 expression is reduced, malignant EpRas cells partially lose their capacity to form tumours in nude mice, and preliminary data suggest that they form less aggressive tumours in lungs. Thus, it is likely that these Smad1/5/8-containing complexes formed in response to TGF-beta regulate a specific set of genes. I have proved this using RNAseq, although validation of the target genes is still required.

Once I determine the specificity and capacity of Smad1/5/8 to control gene expression upon TGF-beta stimulation, I will have a complete picture of this novel branch of signalling from receptors to transcription. The tumourigenesis studies will complement the mechanistic view of the pathway. The evidence suggests that this novel branch of TGF-beta signalling is associated specifically with the pro-tumourogenic effects of TGF-beta in cancer. Therefore, my findings could be extremely important to understand the dual role that TGF-beta plays in cancer.