Tumour invasion is the key element in the high rate of mortality and morbidity in glioma patients. Increased expression, in neoplastic cells, of receptor tyrosine kinases (RTKs) of the TAM family (comprising Tyro3, Axl and MerTK), has been reported in several cancers including gliomas, in which they play a key role in tumour invasion. Axl RTK, with molecular weight of 120-140 kDa, mediates glioma cell adhesion and invasion through a variety of ways. Within the scope of this thesis, Western blotting, quantitative polymerase chain reaction (qRT-PCR) and glioblastoma multiforme (GBM) tissue microarray revealed an upregulation of Axl and Tyro3 in brain tumours and two adult GBM cell lines, SNB-19 and UP007. Both cell lines were treated with the TAM ligand Gas6 and/or the specific Axl small molecule inhibitor BGB324, and analysed in assays for survival, 3D colony growth, motility, migration and invasion. Western blotting was used to detect protein expression and signal protein phosphorylation. In both cell lines, BGB324 inhibited specifically phosphorylation of Axl as well as Akt kinase further downstream. BGB324 also inhibited survival and proliferation of both cell lines in a concentration-dependent manner, as well as completely suppressing migration and invasion. Axl inhibition by BGB324 also sensitised GBM cells to golden standard chemotherapeutic agent temozolomide. Furthermore, novel, unconventional activation mechanisms for the TAMs in human GBM cells were investigated. With the use of Western blotting, co-immunoprecipitation and in vitro kinase assays, Axl was shown to both interact with and be activated by the RTK EGFR. With the aid of qRT-PCR screens, EGFR was shown to promote GBM cell invasion through the Axl/TIMP1/MMP9 signalling axis. Additionally, heterodimerisation of Axl with its sister RTK, Tyro3, in GBM cells was confirmed using co-immunoprecipitation assays; the functional significance of this complex was determined to be promotion of GBM cell survival. In conclusion, this thesis demonstrates the importance of TAM signalling in GBM, identifies novel molecular pathways employed by GBM cells for their survival, growth and spread, and thereby further strengthens the case for targeting TAM receptors as a novel therapeutic approach to combat both primary tumours as well as secondary tumours arising from drug resistance.
|Date of Award||May 2016|
|Supervisor||Sassan Hafizi (Supervisor), Qian An (Supervisor) & Geoff Pilkington (Supervisor)|