Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition

Abstract

Mammalian target of rapamycin (mTOR) regulates cellular processes important for progression of human cancer. RAD001 (everolimus), an mTORC1 (mTOR/raptor) inhibitor, has broad antitumor activity in preclinical models and cancer patients. Although most tumor lines are RAD001 sensitive, some are not. Selective mTORC1 inhibition can elicit increased AKT S473 phosphorylation, involving insulin receptor substrate 1, which is suggested to potentially attenuate effects on tumor cell proliferation and viability. Rictor may also play a role because rictor kinase complexes (including mTOR/rictor) regulate AKT S473 phosphorylation. The role of raptor and rictor in the in vitro response of human cancer cells to RAD001 was investigated. Using a large panel of cell lines representing different tumor histotypes, the basal phosphorylation of AKT S473 and some AKT substrates was found to correlate with the antiproliferative response to RAD001. In contrast, increased AKT S473 phosphorylation induced by RAD001 did not correlate. Similar increases in AKT phosphorylation occurred following raptor depletion using siRNA. Strikingly, rictor down-regulation attenuated AKT S473 phosphorylation induced by mTORC1 inhibition. Further analyses showed no relationship between modulation of AKT phosphorylation on S473 and T308 and AKT substrate phosphorylation patterns. Using a dual pan-class I phosphatidylinositol 3-kinase/mTOR catalytic inhibitor (NVP-BEZ235), currently in phase I trials, concomitant targeting of these kinases inhibited AKT S473 phosphorylation, eliciting more profound cellular responses than mTORC1 inhibition alone. However, reduced cell viability could not be predicted from biochemical or cellular responses to mTORC1 inhibitors. These data could have implications for the clinical application of phosphatidylinositol 3-kinase/mTOR inhibitors.

Figure 1

Antiproliferative response to RAD001 correlates with basal activation of the AKT pathway but not with AKT phosphorylation response following RAD001 treatment. A, left, basal expression levels of phospho-AKT S473, phospho-GSK3β S9, and phospho-TSC2 T1462 in a mixed panel of human cancer cell lines was measured by immunoblot analysis. Cell lines are listed according to their sensitivity to RAD001 treatment (antiproliferative IC₅₀: listed in Table 1). Right, Spearman rank order correlation analysis of AKT and AKT substrate phosphorylation status was done based on the IC₅₀ values for RAD001. R values ± 0.5 and P < 0.05 are considered statistically relevant. B, cells were treated for the indicated times with vehicle control (DMSO) or 20 nmol/L RAD001. Cell lines are listed according to ascending IC₅₀ values for RAD001 (antiproliferative IC₅₀: listed in Table 1). Total protein lysates were subjected to immunoblot analysis. RAD001-induced effects are shown for AKT S473 and AKT T308 phosphorylation. C, AKT levels are used as a loading control.

Figure 2

mTORC1 inhibition after treatment with RAD001 or raptor siRNA has differential effects on the phosphorylation of AKT substrates. Cells were either treated with 20 nmol/L RAD001 for 48 h (A) or transfected with raptor (B) or rictor (C) siRNA for 72 h. Total protein extracts were analyzed by immunoblot for phosphorylation of the AKT substrates—GSK3β, FoxO1/3a, and TSC2.

Figure 3

RAD001-induced AKT S473 phosphorylation is dependent on rictor. Cell lines were transfected with 20 nmol/L control siRNA or siRNA against rictor or raptor for 72 h. Treatment within these 72 h, with vehicle control (DMSO) or 20 nmol/L RAD001, was according to the indicated time points. A, siRNA transfection efficiently down-regulates raptor and rictor protein expression in all lines tested. B, rictor down-regulation attenuates RAD001-induced AKT S473 phosphorylation in all lines. Raptor down-regulation induces basal AKT S473 phosphorylation and enhances the effects of RAD001. AKT T308 phosphorylation is independently regulated, showing attenuation of RAD001-induced effects by rictor down-regulation only in some of the cell lines tested. C, dephosphorylation of S6 on S235/236 as a control for RAD001-induced inhibition of mTORC1 signaling. AKT protein levels serve as loading controls.

Figure 4

Targeting mTORC1/2 and PI3K efficiently inhibits AKT S473 phosphorylation. A, MCF7 and BT474 cells were treated for 4 and 24 h with vehicle control (DMSO), 20 nmol/L RAD001, and/or 50 nmol/L NVP-BEZ235. NVP-BEZ235 inhibits mTORC1 signaling (S6 and 4E-BP1 phosphorylation). Combination treatment inhibits basal and RAD001-induced AKT S473 phosphorylation. B, MCF7 and BT474 cells transfected with siRNA (20 nmol/L) for 48 h were treated for another 24 h with DMSO, 20 nmol/L RAD001, and/or 50 nmol/L NVP-BEZ235. Reduced rictor expression or NVP-BEZ235 treatment attenuates AKT S473 phosphorylation induced after treatment with RAD001 or raptor siRNA. C, TSC2^+/+ and TSC2^−/− MEFs were treated with DMSO, 20 nmol/ L RAD001, and/or 50 nmol/L NVP-BEZ235 for 4 h. NVP-BEZ235 inhibits basal and RAD001-induced AKT S473 phosphorylation.

Figure 5

Effects of RAD001 and NVP-BEZ235 on cell proliferation and viability in MEF and tumor cell lines. The effect of RAD001 and NVP-BEZ235 treatment (alone and in combination) on relative cell proliferation and viability was evaluated using the YO-PRO assay in TSC2^+/+ (A) and TSC2^−/− (B) MEFs and MCF7 (C) and BT474 (D) breast tumor cells. Cells were treated for 72 h with the indicated concentrations of either RAD001 and/or NVP-BEZ235. Effects on proliferation and cell viability were statistically evaluated using two-way ANOVA (with Tukey test). In all cases, a representative experiment is shown.