Phosphoproteomic profiling and targeting of the PI3K/Akt/mTOR and MAPK pathways in ovarian cancer
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Date
08/07/2017Item status
Restricted AccessRestricted Access
Embargo end date
31/12/2100Author
Tashkandi, Ghassan Yousuf
Metadata
Abstract
The PI3K/Akt/mTOR and MAPK pathways are frequently altered in
ovarian cancer cells, making them potential candidates for targeted therapy.
A more complete understanding of the complex interactions between the
different proteins within the two pathways would assist in developing more
effective treatment strategies to help overcome therapy resistance. The
purpose of this project was to understand the phosphoproteomic changes in
response to PI3K/mTOR inhibition in ovarian cancer cells and to identify
potential mechanisms that may lead to targeted therapy resistance.
To investigate the effect of inhibiting PI3K/mTOR at the cellular level in
ovarian cancer, PI3K (LY294002), mTOR (rapamycin) and dual PI3K/mTOR
(BEZ235) inhibitors were used to treat a panel of ovarian cancer cell lines. All
tested cells, irrespective of PI3K/Akt/mTOR and MAPK pathways
mutational status, responded to the three inhibitors. BEZ235 treatment
produced greater cell inhibition than the monotargeted agents, while PTENmutated
cell lines were more responsive to mTOR blockade than inhibition
of PI3K alone. The phosphoproteomic changes in the cell lines were
evaluated over a time course after treatment with the inhibitors, stimulated
by heregulin, and studied using reverse phase protein array analysis. The
results revealed that the decreased expression of pAkt (Thr308) appears to be
a biomarker of sensitivity for LY294002 and BEZ235 in both PEO4 and A2780
cells, while upregulation of pAkt (Ser473) is an indicator for effective
rapamycin treatment within the same cell lines. Increased pAkt (Ser473)
expression after rapamycin treatment in PEO4 cells is believed to be due to
the S6K1-mTORC2-Akt feedback loop. It was observed that pERK was
upregulated upon BEZ235 treatment, which suggested the presence of cross
talk between the PI3K/Akt/mTOR and MAPK pathways. A combination of
BEZ235 and PD-0325901 (MEK inhibitor) treatments inhibited both pAkt
(Ser473) and pERK, which also produced significant inhibition in cell
proliferation compared to monotherapy treatment. The data also revealed a
novel finding in ovarian cancer that prolonged (24h) treatment with
rapamycin sensitises mTORC2 in PEO4 cells under heregulin stimulation.
Moreover, network correlation and clustering analysis using the
phosphoproteomic data identified significant correlations between the
expression of pmTOR (Ser2481), and both p-cRaf (Ser259 and Ser338). Sin1
knockdown was performed in PEO4 cells and showed significant
downregulation in the expression of pAkt (Ser473) and upregulation in
pERK expression, indicating the role of Sin1 to regulate both the
PI3K/Akt/mTOR and MAPK pathways potentially via mTORC2 and Ras.
Phosphoproteomic profiling was performed on 469 ovarian cancer tissue
samples using TMA and IHC analysis. Several significant associations were
discovered between the phosphoproteomic data and the different
clinicopathological parameters. High expression of pmTOR (Ser2448) was
correlated with poorer overall survival in patients with ovarian
endometrioid carcinoma compared to patients with low expression
(p<0.024). This implies that pmTOR (Ser2448) expression may potentially be
a prognostic marker for patients with ovarian endometrioid carcinoma.
In conclusion, I present dynamic phosphoproteomic profiling of the
PI3K/Akt/mTOR and MAPK pathways in ovarian cancer, suggesting novel
feedback loops and cross talk that could play a role in resistance mechanisms
to these therapies. Combination treatment showed an additive effect on cell
growth offering an approach to overcome drug resistance.