Bussell, Katrin Natalie; (2001) Regulation and functional analysis of EphA2 in normal and Ras-transformed mammary epithelium. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Members of the Erythropoietin-producing hepatoma (Eph) family of receptor tyrosine kinases are dynamically expressed during embryogenesis, with roles in topographic mapping, hindbrain segmentation and neural crest cell migration thought to be mediated by repulsive/anti-adhesive cues induced by interaction with their membrane-bound ligands, known as ephrins. Formerly known as epithelial cell kinase (eck), EphA2 is expressed primarily in cells of epithelial origin in the adult, including the mammary gland. Results presented in this thesis demonstrate that elevated steady-state levels of catalytically active EphA2 protein were characteristic of mammary tumours arising in WAPras transgenic mice. These mice express v-Ha-Ras under the control of the mammary-specific whey acidic protein (WAP) promoter. Furthermore, a mouse mammary epithelial cell line expressing v-Ha- Ras also displayed elevated levels of EphA2 mRNA and protein. This implies a potential role for EphA2 in tumourigenesis, and provided a highly relevant in vitro cell culture model in which further investigation revealed downregulation of EphA2 in response to mitogen-activated protein kinase (MAPK) pathway inhibition. Analysis of mice bearing an insertional mutation at the EphA2 locus crossed with WAPras transgenic mice revealed continued upregulation of EphA2 protein in mammary tumours, in contrast to the significant deficiency in EphA2 protein in other adult tissues of these double mutant mice. This suggests a potentially complex mechanism for the transcriptional regulation of EphA2. Overexpression of recombinant EphA2 in a non-transformed mammary epithelial cell line resulted in a 'rounded' cellular morphology, implicating EphA2 in the regulation of cell-extracellular matrix interactions. Furthermore, recombinant ephrinA1, cloned and expressed as a source of cognate ligand for EphA2, also influenced the cytoskeleton. Finally, receptor activation by ephrinA1 induced transient inhibition of MAPK activity, suggesting the presence of an EphA2-MAPK regulatory feedback loop, which may be overcome by oncogenic Ras to upregulate EphA2 and thus confer anti-adhesive properties on tumourigenic cells.

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