| Abstract |
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.
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