CARACTERÍSTICAS BIOMÉDICAS, PSICOLÓGICAS, CONDUCTUALES Y SOCIALES

which are all important processes in metastasis, it was important to investigate what signalling cascades resulted in this phenotype. The involvement of PI3K pathway via AKT/PKB in modulating tumour migration and invasion is known (Kim et al, 2001; Xu et al, 2013; Li et al, 2015). Furthermore, reports show that a chemokine receptor-ligand CXCR4-CXCL12 signalling axis promotes tumour invasion and EMT by inducing AKT phosphorylation (Lv et al, 2015), however very little is known about SGK3. As mentioned before, the CXCR4-CXCL12 signalling axis is well known in promoting invasion and migration (Guo et al, 2015) and its overexpression correlates with metastatic potential of tumour cells (Balkwill, 2004). Consequently, overexpression of CXCR4 is associated with poor outcome in a number of cancers (Otsuka et al, 2011; Zhao et al, 2015). An E3 ubiquitin ligase, AIP4, has been suggested to be involved in controlling the degradation of membrane-bound receptors, including CXCR4 by targeting it for lysosomal degradation and in fact, its degradation was dependent on AIP4 activity (Marchese et al, 2003a, 2003b). The characteristic PX domain of SGK3 results in its endosomal location, where it is activated and it has been reported that SGK3 co-localises and interacts with AIP4 and consequently

attenuates its function as an E3 ubiquitin ligase (Slagsvold et al, 2006). It was therefore hypothesized that SGK3 was mediating its migration and invasion effect through CXCR4. These studies were initiated by first showing that AIP4/CXCR4 and SGK3 interact, using immunoprecipitation studies. The studies focused primarily on the IGROV1 cells as opposed to SKOV3, which were primarily utilized for all other studies because they had high endogenous SGK3 levels, so higher levels were immunoprecipitated in IGROV1 to assess protein interactions, with the results showing that SGK3 did interact with CXCR4.

To investigate this further, AIP4 was knocked down in the first instance. The rationale behind this was that AIP4 knockdown cells would migrate at a rate similar to SGK3wt overexpression as knockdown of AIP4 would result in more CXCR4 expression as a result of decreased targeting of CXCR4 for lysosomal degradation. From the migration assays, it was observed that AIP4 knockdown resulted in a faster migration rate compared to non-targeting control as did the SGK3wt overexpression compared to the empty vector (figure 41A), which had similar migration rates to the non-targeting so it was a fair comparison between the two transfections. The difference in migration rates between the AIP4 knockdown and SGK3wt overexpression is not surprising because there will be other mechanisms at play, which regulate CXCR4 expression such as HER2 overexpression, which will be discussed in detail further on.

It was then hypothesized that knocking down AIP4 in combination with SGK3wt overexpression should increase migration faster than either transfection alone because both scenarios result in increased CXCR4 expression, and as expected, combining the two transfections did increase the the rate of migration compared to the two individually. The graph in figure 41B shows the empty vector and the non-targeting controls; a combination of both the controls were also included in the experiment and they behaved similar to the two controls alone so for simplicity this was not added to the graph due to the number of conditions already on the graph. Although these experiments were not statistically significant, this was due to scale variation between experiments, even though similar trends were observed in each experiment.

As the experiments with AIP4 appeared to follow our hypothesis, CXCR4 was then knocked down using siRNA. The resulting lack of CXCR4 should slow down migration if these phenotypes are being

implemented through the CXCR4-CXCL12 signalling axis. The results indicate that knockdown of CXCR4 does slow down migration when compared to the SGK3wt overexpression. However, there appears to be a noticeable difference between the behavior of the two siRNAs used in this study (figure 42A). This could be due to transfection efficiency differences between the two oligonucleotides, however, the siRNA (#8) that is slowing down the migration rate most effectively has less knockdown compared to the other siRNA (#7) when compared on a western blot (figure 42B). For more clinical relevance, a specific CXCR4 inhibitor, AMD3100 was utilized for these studies. Interestingly, AMD3100 treatment slowed down migratory capacity of SKOV3 cells compared to the SGK3wt overexpressed cells, returning the migration rate to levels comparable with the empty vector control. Additionally, it was also observed that treating the empty vector control with AMD3100 increased migration compared to the untreated empty vector control, though not to the same level as SGK3wt overexpression. This could be due to ineffective CXCR4 inhibition in the empty vector condition as is visible in the western blot in figure 43B. Collectively, the data indicates that SGK3 is modulating increased migration and possibly invasion at least in part through the CXCR4-CXCL12 signalling axis because inhibiting this pathway does adversely affect the rate of migration, however there are likely other pathways through which SGK3 is modulating these phenotypes as inhibiting the CXCR4 pathway did not result in complete impairment of the migratory capacity of the cells.

Although all reports propose a role for AIP4 in regulating proteosomal degradation of CXCR4 protein, a role for SGK3 was also thought to be entirely due to its positive regulation of CXCR4 protein by preventing its degradation. However interestingly, it has been observed that both SGK3 and AIP4 appear to regulate CXCR4 at transcriptional level too. It is observed that SGK3 overexpression increases CXCR4 mRNA level in addition to protein level (figure 44C) and knockdown of AIP4 also appears to decrease CXCR4 levels however this effect is not significant (Supplementary figure 7).