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Title: Correlating genetic change with changes in cell behaviour in squamous cell carcinoma of the head and neck : focus on inactivating NOTCH1 mutations
Author: Pritchard, Ashley Jane
ISNI:       0000 0004 7224 0177
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2018
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Squamous Cell Carcinoma of the Head and Neck (SCCHN) is a heterogeneous cancer at the genetic and cellular levels. Whole exome-sequencing studies have identified inactivating mutations in NOTCH1 in 11-15% of SCCHN. It was the aim of this research to correlate genetic changes in NOTCH1 expression with changes in cell behaviour. A human oral SCC line harbouring two truncating NOTCH1 mutations was rescued via lentiviral transduction of the NOTCH intracellular domain (NICD). In vitro assays provided evidence to suggest that expression of NICD was able to inhibit proliferation, clonogenicity and cell migration into a scratch wound. At high expression levels, NICD also promoted differentiation. Microarray data supported the in vitro findings and further associated NOTCH1 expression with increased cell death and decreased angiogenesis. SERPINE 1 was highlighted as a potential regulator of NICD-mediated changes in cell behaviour. A high number of histone related genes were also found to be down-regulated in response to increased NICD expression. These findings were not explored further in this thesis but are of interest for future work. Microarray analysis also identified SPANXA1/2 as the most up-regulated gene in response to NICD expression. The fold change increase of SPANXA1/2 was 3x that of any other gene. In order to investigate the functional consequence of SPANXA1/2 expression, a shRNA system was used to knockdown SPANXA1/2 in the NICD-rescued cell line. Primary findings provide evidence to associate the expression of SPANXA1/2 with a potential increase in cell proliferation, increased cell migration into a scratch wound and a change in cell morphology. Current literature suggests that changes in cell morphology may be associated with the inhibition of epithelial to mesenchymal transition (EMT) and the induction of mesenchymal to epithelial transition (MET). However, data is preliminary and further studies are required to confirm this association. A murine xenograft model was established to assess tumour formation in vivo. The expression of NICD resulted in significantly larger tumours. However, no difference in proliferation was recorded over the two hour period prior to sacrifice. Histological observations also suggest that the expression of NICD may be associated with reduced keratin pearl formation and increased areas of what pathologically presents as comedo-type necrosis. Finally, the expression of NICD was not found to give rise to any significant difference in angiogenesis. Together, these findings suggest a tumour suppressor role for NOTCH1 in SCCHN.
Supervisor: Maher, John ; Watt, Fiona Mary Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available