Ege, NM; (2016) The Regulation and Function of YAP1. Doctoral thesis , UCL (University College London).

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Abstract

Yes-associated protein 1 (YAP1) is a transcriptional regulator that was first described as part of the Hippo signalling cascade. In the last five years, several others regulators have been identified, placing YAP1 at the centre of a much more complex network. One common feature of these different regulatory mechanisms is that YAP1 localises either in the nucleus bound to transcription factors promoting the expression of target genes or in the cytoplasm sequestered into diverse complexes. In vivo studies reveal that YAP1 can promote tumour initiation, progression and metastasis by controlling proliferation and survival of cancer cells. In addition to cancer cells, tumours contain many other cell types such as cancer-associated fibroblasts (CAFs). These cells show a high ability to promote invasion of cancer cells by remodelling the extra-cellular matrix. One possible origin for CAFs is through the local conversion of resident NFs, however the mechanism of this conversion is not fully understood. This thesis aims to understand the function and regulation of YAP1 in fibroblasts during normal development and tumour progression. Analyses of gene expression showed that YAP1 transcriptional activity was upregulated in CAFs compared to NFs. Treatment with actomyosin inhibitors revealed that this upregulation was dependent on the actomyosin cytoskeleton network suggesting a role for mechanotransduction in YAP1 regulation. This high transcriptional activity was associated with a nuclear translocation of YAP1 in CAFs compared to NFs. In order to determine the dynamics of the protein sub-cellular localisation, YAP1 was fused to EYFP fluorescent protein. Photobleaching experiments revealed a high mobility of YAP1 in the nucleus and the cytoplasm in both NFs and CAFs. These experiments also highlighted a constant and fast nucleocytoplasmic shuttling of the protein. Mathematical modelling analyses suggested a difference in the export rates from the nucleus to the cytoplasm, NFs showing a higher export rate than CAFs. This approach was extended to study the localisation, dynamics, and function of five YAP1 mutants. Analyses of the mutants showed differences in nuclear mobility and in export rates compared to the WT protein. Overall these data suggest the major roles of nuclear binding partners as well as export machinery in the regulation of YAP1 dynamics in NF1 and CAF1. In parallel, the role of YAP1 in fibroblasts during mouse development was assessed. Deletion of YAP1 in PDGFRα+ cells led to a lethal phenotype appearing at E11.5 due to haemorrhage in the head suggesting a crucial role of YAP1 protein in mesenchymal cells during normal vasculature development.

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