Dual inhibition of the PI3K/AKT/mTOR pathway suppresses the growth of leiomyosarcomas but leads to ERK activation through mTORC2: biological and clinical implications

Abstract

The PI3K/AKT/mTOR pathway plays a crucial role in the development of leiomyosarcomas (LMSs). In this study, we tested the efficacy of dual PI3K/mTOR (BEZ235), PI3K (BKM120) and mTOR (everolimus) inhibitors in three human LMS cell lines. In vitro and in vivo studies using LMS cell lines showed that BEZ235 has a significantly higher anti-tumor effect than either BKM120 or everolimus, resulting in a greater reduction in tumor growth and more pronounced inhibitory effects on mitotic activity and PI3K/AKT/mTOR signaling. Strikingly, BEZ235 but neither BKM120 nor everolimus markedly enhanced the ERK pathway. This effect was reproduced by the combination of BKM120 and everolimus, suggesting the involvement of mTORC2 via a PI3K-independent mechanism. Silencing of RICTOR in LMS cells confirmed the role of mTORC2 in the regulation of ERK activity. Combined treatment with BEZ235 and GSK1120212, a potent MEK inhibitor, resulted in synergistic growth inhibition and apoptosis induction in vitro and in vivo. These findings document for the first time that dual PI3K/mTOR inhibition in leiomyosarcomas suppress a negative feedback loop mediated by mTORC2, leading to enhanced ERK pathway activity. Thus, combining a dual PI3K/mTOR inhibitor with MEK inhibitors may be a relevant approach to increase anti-tumor activity and prevent drug resistance in patients with LMS.

Keywords: BEZ235; BKM120; PI3K/AKT/mTOR pathway; everolimus; leiomyosarcomas.

Figure 1. Immunohistochemical (IHC) staining against p-S6RPser240/244 and PTEN in leiomyosarcoma (LMS) disease tissues and cell lines

Immunohistochemical staining pictures of LMS tissue samples with anti-p-S6RP^ser240/244 and anti-PTEN antibodies and of cell line pellets with anti-p-S6RP^ser240/244. Endothelial cells (positive control) are indicated by black arrows.

Figure 2. Antiproliferative and apoptotic activities of BEZ235, BKM120 and everolimus (EVE) in LMS cell lines

Growth curves indicating growth inhibition of the 3 LMS cell lines (IB112, 134 and 136) after treatment for 72 hours (A). Representative pictures of the clone formation assay for cell lines treated for 72 hours by PI3K/mTOR pathway inhibitors at their IC₅₀ value (B). The average percentage of monoclonal cell number for each cell line (C).

Figure 3. Effect of BEZ235, BKM120 and everolimus (EVE) on LMS cell apoptosis

Representative dot-plot diagrams of flow cytometry with annexin V/PI for LMS cells treated for 72 hours with BEZ235, BKM120 and EVE at 10-fold IC₅₀ values (A). Percentage of apoptotic cells after treatment for 72 hours with 3 drug concentrations (1/10 IC₅₀, IC₅₀ value, 10-fold IC₅₀) (B). Data are presented as the mean ± SEM of three independent experiments. *, drug p < 0.05 vs. control; ***, drug p < 0.001 vs. control (two-way ANOVA).

Figure 4. Downstream kinase inhibition by BEZ235, BKM120 and everolimus (EVE) in LMS cell lines

Representative western blotting of active kinase and total kinase levels of the PI3K/mTOR and MAPK pathway with GAPDH as a loading control (A). Cells were treated for 72 hours at the IC₅₀ value of each drug. Representative signal intensities for p-ERK1/2^{thr202/tyr204} were normalized to those for total ERK in each cell line (B). Data are presented as the mean ± SEM of three independent experiments.

Figure 5. Anti-tumor effect of BEZ235, BKM120 and everolimus (EVE) on human IB136 cell xenografts in Ragγ2C−/− mice

Curves of tumor volume progression during 3 weeks of treatment (A) Mice were randomly assigned to receive 40 mg/kg BEZ235, 50 mg/kg GDC-0941, 5 mg/kg everolimus or vehicle. The data points represent an average from 8 mice (bars, SEM). *p < 0.05; ***p < 0.001, two-way ANOVA. Immunohistochemical staining images of tumor samples treated with anti-p-S6RP^ser240/244 and anti-Ki-67 antibodies (B).

Figure 6. Effect of RICTOR silencing on the PI3K and MAPK downstream signaling pathways in the IB134 cell line

Representative western blotting of active kinase and total kinase levels of the PI3K/mTOR and MAPK pathway with GAPDH as a loading control (A). LMS cell lines were incubated with BEZ235 and RNAi against RICTOR for 72 hours. Representation of the signal intensities for RICTOR and p-ERK1/2^{thr202/tyr204} were normalized to those for GAPDH and ERK, respectively (B). Data are presented as the mean ± SEM of two independent experiments.

Figure 7. Synergistic activity of the BEZ235 and GSK1120212 combination in IB112, 134 and 136 LMS cell lines

Isobologram analysis of the combination of BEZ235 and GSK1120212 in LMS cells (A). Combination index (CI) values for each cell line were calculated using the method developed by Chou and Talalay and are represented by a red point. Representative dot-plot diagrams of the flow cytometry results with annexin V/PI for LMS cells treated with BEZ235 and GSK1120212 either alone or in combination (B). Percentage of apoptotic cells after respective treatments (C). Immunoblotting analysis of active kinase and total kinase levels of the PI3K/mTOR and MAPK pathway with GAPDH as a loading control (D). Cells were treated for 72 hours at the IC₅₀ value of each drug. Data are presented as the mean ± SEM of three independent experiments. **p < 0.01; ***p < 0.001, two-way ANOVA.

Figure 8. Synergistic anti-tumor effect of the BEZ235 and GSK1120212 combination in human IB136 cell xenografts in Ragγ2C−/− mice

Curves of tumor volume progression during 3 weeks of treatment (A). Mice were randomly treated with vehicle, 10 mg/kg BEZ235, 0.5 mg/kg GSK1120212 or a combination of both drugs. The data points represent an average from 10 mice (bars, SEM). ***p < 0.001, two-way ANOVA. Kaplan-Meier curves for tumor doubling times (B). Immunohistochemical staining images of tumor samples treated with the anti-Ki-67 antibody (C).