The Role Of ECT2 In Astroctyoma Invasion and Stress Granule Formation

In this thesis, the oncogenic role of the cytokinetic RhoA guanine nucleotide exchange factor, ECT2 in astrocytoma is studied. ECT2 is overexpressed in astrocytoma cells, and correlates with poor patient prognosis and increasing astrocytoma grade. ECT2 is a nuclear protein in non- cancerous cells. Here, it is demonstrated that ECT2 is aberrantly located in the cytoplasm of astrocytoma cells at the leading edge of the cell where it activates Rac1 and Cdc42 but not RhoA. Interestingly, ECT2 overexpression results in mesenchymal-amoeboid transition with ECT2 localizing to the retraction bleb in amoeboid cells. Using ECT2 immunoprecipitation followed by mass spectrometry, I identified the novel ECT2 interactor, RASAL2. RASAL2 is a RasGap know to be an inhibitor of breast cancer growth and metastasis. SiRNA-mediated loss of RASAL2 leads to highly invasive astroctyoma cells with an amoeboid phenotype and increased RhoA activity. This suggests that RASAL2 may modulate ECT2 specificity toward critical Rho GTPases. I also demonstrate that ECT2 has a novel role in stress granule formation. Stress granule formation results in translational reorganization that allows cells to survive acute stress such as hypoxia and radiation. ECT2 colocalizes with stress granules and co-immunoprecipites TIAR under stress. SiRNA-mediated silencing of ECT2 results in increased sensitivity to cellular apoptosis and stress granules in a less mature peri-nuclear location, however stress granule number and size are unchanged. The upstream activator of the ECT2 pathway, AurkB, was then examined. I demonstrate for the first time that AurkB and its downstream target phospho- Histone 3 are phosphorylated with cellular stress. Chemical and siRNA mediated interference with AurkB phosphorylation leads to the formation of fewer stress granule of a smaller size and with less TIAR aggregation. Using RNA immunoprecipitation of stress granules, I show that iii mRNA from multiple cellular pathways are contained in astrocytoma stress granules. These pathways include those subserving cellular migration, cellular metabolism, cell death and post translational and transcriptional modifiers. Utilizing a doxycycline-inducible expressing siRNA system, I show that mice injected with ECT2 siRNA live longer, with fewer tumours and decreased invasion than mice injected with NS siRNA controls. Taken together, these data suggest that ECT2 is a putative astrocytoma oncogene that warrants further investigation as a therapeutic target in astrocytoma.Ph.D