The role of the EphA1 receptor tyrosine kinase during embryogenesis and cancer

The University of Queensland2005

Abstract

The Eph Receptor Tyrosine Kinases form the largest subfamily of RTKs, with 14 receptors currently identified in mammals. Interaction of Ephs with their membrane bound ephrin ligands initiates bidirectional signalling pathways that regulate cell movements involved in axon guidance, vascular networking and the formation of embryonic tissue boundaries. While the complete range of physiological functions for Eph proteins is still emerging, elevated expression in discrete regions of the developing embryo and low levels in mature adult tissues are consistent with a critical role in embryogenesis. In addition to their role as guidance and adhesion molecules during normal development, Ephs and ephrins have been implicated in tumourigenesis, where they may perform similar functions during tumour invasion and metastasis. Hence, the overall aims of this thesis were to examine the role of the Eph-ephrin system during the early embryonic development of mice, and to investigate the effects of these genes on colon carcinogenesis. In order to define the functional role of Eph and ephrin proteins during early vertebrate development it was first necessary to elucidate the gene expression patterns. Expression of the EphA1 receptor and its high-affinity ligands, ephrin AI and ephrin A3, were characterised from 4.5 to 12.5 dpc using RNA in situ hybridisation and histological techniques. These results showed complex, dynamic patterns of expression for EphA1, ephrin A1 and ephrin A3 that imply novel functions for these genes during the earliest phases of development. The identification of receptors and ligands with overlapping or complementary expression was of particular interest, as it suggests the presence of authentic receptor-ligand pairs of in vivo functional significance. Although Ephs and ephrins are frequently overexpressed in a wide range of human malignancies, the molecular mechanisms underlying activation of expression in tumours have not yet been identified. Colorectal carcinoma is the most common cancer affecting humans and the Eph-ephrin system features extensively in the normal physiology and pathology of this organ. In order to investigate the relationship between the expression characteristics and the tumorigenic capacity of colon carcinomas, Quantitative PCR was used to develop an Eph-ephrin expression profile of cells, followed by Microarray Analysis to determine the broader expression characteristics. To complement these experiments, the generation of a transgenic mouse model of colon cancer that conditionally over-expresses the EphA1 receptor was also undertaken. This has enabled the identification of transcriptome variations commonly associated with changes in Eph-ephrin expression, which may contribute to the pathogenesis of cancer. The consensus class of gene expression changes identified in these experiments revealed biologically important features of tumourigenesis and metastasis, with particular emphasis on the Rho family of small GTPases. As colonic carcinomas demonstrate unpredictable metastatic behaviour associated with poor prognosis in humans, the identification of molecular derangements contributing to this behaviour may have profound clinical significance. Collectively, these results provide further evidence that the Eph-ephrin system functions as a key regulator of the cell-cell interactions that are critical to early vertebrate embryogenesis and make an important contribution to the developing theories that aim to understand the role of Eph and ephrin genes in the neoplastic process.

Abstract

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