Kadife, Elif (2018) The Functional and Biological Implications of EphB4 Receptor Overexpression and Knockout in Colorectal Cancer. PhD thesis, Victoria University.

Abstract

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in Australia and globally. Early detection and intervention is vital for the longevity of patients with any cancer, however, this appears to be most challenging with CRC, as it is largely asymptomatic. For this reason, most cases are not diagnosed until the cancer has metastasised, primarily to the liver. At this late stage of diagnosis, 5- year patient’s survival is predicted to be less than 10%. However, even when CRC is diagnosed and treated in the initial stages of neoplastic growth, high recurrence rates in patients still present as a serious issue. The problems associated with treatment and recurrence raise the need to identify molecular targets, so that specific and aggressive therapeutic interventions may be designed and developed. One such potential target is the erythropoietin-producing hepatocellular B4 (EphB4) receptor. The Ephs constitute the largest family of tyrosine kinase receptors. The activation of Eph receptors is achieved through association with their corresponding cell- bound ‘Eph receptor interacting’ (Ephrin) ligands. The signalling by the Eph receptors and their membrane-bound ligands, the Ephrins, is unique among the tyrosine kinases as both the receptor and ligand are found on the cell surface. Bidirectional interaction results in the phenomena of ‘forward’ signalling via the Eph receptor carrying cells and ‘reverse’ signalling in those cells expressing the Ephrin ligands. Several members of the Eph receptor receptor family, including EphB4, have been implicated with progression of many different types of cancer. However, EphB4 receptor’s contribution towards CRC yields the most contradictory findings. Some studies suggest that EphB4 is upregulated in late and metastatic stages of CRC, while others argue that EphB4 expression is often silenced in the progressive state of the disease. Due to the promising results achieved in other types of cancers, it is important to elucidate the role of EphB4 receptors in CRC in order to develop more specific and aggressive cancer therapies. The overall aim of this study is to elucidate the influence of EphB4 receptor expression on the development and progression of CRC. To achieve this, we used modified derivatives of multiple human and a mouse CRC cell line in in vitro and in vivo experiments. In vitro experiments were utilised to study effects of EphB4 overexpression and knockout on proliferative aptitude, migratory and invasive abilities of human and mouse CRC cells. In vivo subcutaneous models of CRC were used to evaluate the ability of high, low and knockdown of EphB4 receptor expression to influence morphological changes, rate of growth, vascularization and tumour-stromal interactions. The time course and rate of metastasis of CRC cells to the liver were studied in in vivo orthotopic and intra-splenic metastasis models. The level of EPHB4 and EPHRINB2 expression was investigated using databases to determine their correlation with survival and disease-free outcomes of CRC patients. The results of this study provide evidence that high EphB4 receptor expression significantly increases the rate of proliferation, migration and invasion of CRC cells in vitro, and enhances tumour growth in vivo due to enhanced vascularisation. Knockout of EphB4 expression reduces these effects. EphrinB2 appears to inhibit proliferation in cells overexpressing EphB4 and its expression correlates with poor patient outcome.

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