Phosphoproteomic profiling and targeting of the PI3K/Akt/mTOR and MAPK pathways in ovarian cancer
Tashkandi, Ghassan Yousuf
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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.