Miller, DSJ; (2016) The mechanism and function of TGF-β superfamily signalling dynamics. Doctoral thesis , UCL (University College London).

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Abstract

The signalling dynamics of different transforming growth factor-β (TGF-β) superfamily members vary considerably. I have fully characterised these and shown them to be driven by the dynamics of receptor activation, internalisation, trafficking and degradation. For canonical TGF-β, receptors are completely depleted from the cell surface in response to just five minutes of signalling, leaving cells in a refractory state where they cannot respond to further acute stimulation until ligand has been depleted from the micro-environment and receptors allowed to recover. This refractory state was mechanistically dissected and then mathematically modelled by a collaborator. This model made testable predictions about the dynamics of signalling in complex environments. Several whole genome siRNA screens were performed to identify novel regulators of TGF-β signalling. I identified trafficking through the ESCRT complex as the route of degradation for TGF-β, Activin and BMP receptors. Knockdown of ESCRT components leads to a persistence of SMAD phosphorylation in response to TGF-β signalling and an increase in the downstream biological outputs of the pathway. In environments such as the tumour niche, cells are continuously exposed to TGF-β family ligands, but SMAD phosphorylation levels remain high. Because in cell culture models long-term exposure to TGF-β leads to an attenuated phospho-SMAD2 response, I reasoned that the pathway must be wired differently in these tumour cells or alternative TGF-β superfamily ligands must be present. This led me to the discovery that in cancer-associated fibroblasts (CAFs) derived from the MMTV-PyMT mouse mode of breast cancer, the only TGF-β ligands produced are the Activins, which I have shown to have a functional role in CAF activity. In addition, the primary tumour and cells derived from it also express high levels of Activin. By characterising long-term TGF-β superfamily signalling dynamics, it has been possible to identify novel regulatory mechanisms and functions of the TGF-β pathway, which may lead to therapeutic outcomes.

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