Dual inhibition of phosphoinositide 3-kinase and mammalian target of rapamycin as a novel therapeutic approach in human ovarian carcinoma

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Mammalian target of rapamycin (mTOR) plays a central role in regulating cell growth and cell cycle progression, and is considered a promising therapeutic target for cancers with phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway activation, including ovarian cancer. However, recent studies have demonstrated that mTOR inhibition can increase levels of phosphorylated Akt (pAkt) due to the stabilization of insulin receptor substrate. This effect can limit the efficacy of mTOR inhibitors due to the growth promoting and anti-apoptotic effects of pAkt. Thus, we hypothesized that concomitant inhibition of PI3K and mTOR would enhance the effect of mTOR inhibition. We tested the effect of the mTOR inhibitor RAD001 (everolimus) and the dual pan-PI3K/mTOR inhibitor NVP-BEZ235 as single agents and in combination on three human ovarian cancer cell lines, SKOV3 and IGR-OV1 with constitutively active PI3K/Akt/mTOR signaling, and OVCAR5 cells with a low level of pathway activation. In SKOV3 cells, treatment with 10 nM RAD001 or 50 nM NVP-BEZ235 as single agents induced only a moderate reduction of cell viability by XTT assay (31% and 38%, respectively). However, combination treatment with RAD001 and NVP-BEZ235 reduced cell viability by 68%. Similar results were found in IGR-OV1 and OVCAR5 cells. The enhanced effects of dual inhibition were confirmed using the inhibitors rapamycin (mTOR) and LY294002 (PI3K). Two-way ANOVA statistical analyses showed that at low concentrations the interaction between RAD001 and NVP-BEZ235 was synergistic. Immunoblotting confirmed that 10 nM RAD001 induced a modest increase in pAkt levels in SKOV3, and up to 3-fold in OVCAR5, but inhibited pAkt in IGR-OV1 cells. NVP-BEZ235 at 50 nM decreased pAkt by up to 75% in SKOV3 and IGROV1 cells. These findings suggest that the cell viability response to dual inhibition of mTOR and PI3K is independent of the activation status of the PI3K/Akt/mTOR pathway and the effect of RAD001 on Akt phosphorylation. In cell cycle distribution assays, treatment of cells with RAD001 and NVP-BEZ235 in combination caused a significantly greater increase in the proportion of cells in G₀/G₁ compared to single agent treatment. Furthermore, in the LSL-K-Ras G12D/+/PTEN loxP/loxP murine ovarian cancer model that is based on conditional deletion of the tumor suppressor gene PTEN and concomitant expression of constitutively active K-Ras, oral administration of either RAD001 (10 mg/kg) or NVP-BEZ235 (40 mg/kg) resulted in significantly reduced S6 ribosomal protein phosphorylation for RAD001, and decreased Akt phosphorylation for NVP-BEZ235 in tumor tissue of treated mice. In conclusion, our results indicate that the efficacy of mTOR inhibition can be significantly enhanced by concomitant inhibition of Akt phosphorylation. Dual inhibition of mTOR and PI3K could be utilized as a promising new therapeutic strategy in ovarian cancer.