Effective in vivo targeting of the mammalian target of rapamycin pathway in malignant peripheral nerve sheath tumors

Abstract

Malignant peripheral nerve sheath tumors (MPNST) are chemoresistant sarcomas with poor 5-year survival that arise in patients with neurofibromatosis type 1 (NF1) or sporadically. We tested three drugs for single and combinatorial effects on collected MPNST cell lines and in MPNST xenografts. The mammalian target of rapamycin complex 1 inhibitor RAD001 (Everolimus) decreased growth 19% to 60% after 4 days of treatment in NF1 and sporadic-derived MPNST cell lines. Treatment of subcutaneous sporadic MPNST cell xenografts with RAD001 significantly, but transiently, delayed tumor growth, and decreased vessel permeability within xenografts. RAD001 combined with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib caused additional inhibitory effects on growth and apoptosis in vitro, and a small but significant additional inhibitory effect on MPNST growth in vivo that were larger than the effects of RAD001 with doxorubicin. RAD001 plus erlotinib, in vitro and in vivo, reduced phosphorylation of AKT and total AKT levels, possibly accounting for their additive effect. The results support the consideration of RAD001 therapy in NF1 patient and sporadic MPNST. The preclinical tests described allow rapid screening strata for drugs that block MPNST growth, prior to tests in more complex models, and should be useful to identify drugs that synergize with RAD001.

Figure 1

RAD001 and erlotinib response in MPNST cell lines. A, Western blot of lysates from human MPNST-designated cell lines probed with anti-T389 S6K1 (pS6K), anti-total S6K (S6K), and β-actin as a loading control. NHSC, normal human Schwann cells. The effect of RAD001 (B) and erlotinib (C) at designated doses on the MPNST cell lines ST8814, STS26T, S462, ST88-3, and T265p21 after 4 d of treatment evaluated by MTS assay. Results are plotted as percentage growth compared with vector control.

Figure 2

MPNST cell line growth is decreased by exposure to doxorubicin or to the combination of RAD001 and erlotinib. The proliferation of five MPNST cell lines, ST8814 (black columns), ST88-3 (hatched columns), T265 (gray columns), STS26T (horizontal lines), and S462 (white columns), was measured using a MTS assay. In each chart, the value shown on the Y axis designates percentage of growth compared with no treatment. A, the cell lines were grown in the presence of designated concentrations of doxorubicin (Dox) for 48 or 96 h. B, cells were pretreated with either RAD001 ( + ) or 0.05%ethanol (–) for 24 h before adding designated concentrations of doxorubicin for 48 h. C, cells were grown in the presence of RAD001 (10 nmol/L) and/or erlotinib (3 µmol/L) for 48 h. NT, no treatment.

Figure 3

RAD001 plus erlotinib does not substantially alter cell death but does alter AKT signaling. A, MPNST cell lines grown in the presence of 10 nmol/L RAD001, 3 µmol/L erlotinib, or 0.05 to 5 µg/mL doxorubicin, alone or in combination for 3 d, were analyzed for cell death using a TUNEL assay. Cell death was determined by counting the TUNEL-positive nuclei compared with all 4′,6-diamidino-2-phenylindole – positive nuclei using a fluorescence microscope. Student’s t test showed significant increase in apoptosis for RAD001 versus carrier (P < 0.05), RAD001 versus carrier at doxorubicin concentrations of 0.05 µg/mL (P < 0.01) and 5 µg/mL (P < 0.5). RAD001 approached significance at 0.5 g/mL doxorubicin (P = 0.06). No significant difference was observed when erlotinib alone compared with carrier (P = 0.12) nor in RAD001/erlotinib compared with RAD001 alone (P = 0.09). B, Western blot of lysates from designated cell lines exposed to drugs shown after 48 h in vitro under standard growth conditions. Combo denotes 3 µmol/L erlotinib and 10 nmol/L RAD001. Blots were probed with anti-T389 phospho-S6K1 (pS6K), anti-total AKT (AKT), anti-S473 phospho-AKT (pAKT), and β-actin as a loading control.

Figure 4

RAD001 blocks growth of MPNST STS26T xenografts. Athymic nude mice were injected with the sporadic MPNST cell line STS26T. A, treatment designated began at day 3 and continued daily through day 21 postinjection. Tumor size was measured every 3rd day. B, gross photographic images of flanks of mice at day 15 treated with placebo, erlotinib, RAD001, and RAD001 in combination with erlotinib. C, heat maps of fluorescent stains of tumors marked by DsRed (tumor cells) or FITC-dextran (tumor perfusion). Significantly less dextran is taken up by RAD001-treated tumors compared with placebo-treated mice.

Figure 5

RAD001 and RAD001 with erlotinib improve outcome in established tumors. Athymic nude mice were injected with the sporadic MPNST cell line STS26T. Treatment was initiated 16 d after implantation, concurrently with a one-time injection of 8 mg/kg doxorubicin diluted in PBS, or PBS alone. Beginning at day 16, mice received RAD001, placebo, or erlotinib by daily gavage until day 30 when all placebo-, erlotinib-, or doxorubicin-treated mice were sacrificed. At day 31, the remaining mice were divided into two groups, one receiving continuous treatment (A) and one taken off treatment for the duration of the experiment (B). RAD, RAD001; erl, erlotinib.

Figure 6

Histopathology and signaling in RAD001-treated tumors. A, low magnification of H&E-stained photomicrograph of sections from a single tumor from a placebo-treated mouse (left) at day 30. Note rims of darkly stained growing tumor cells surrounding lighter necrotic core. RAD001-treated tumor at the same magnification showing dramatic size difference (right). B, photomicrographs taken at ×200 showing similarity in histology in growing cores of tumors in placebo- and RAD001-treated tumor. Sections were stained with H&E, MIB1 (proliferation), or CD31 (vessels) C. Western blot of lysates from tumors taken from mice treated as designated, probed with anti-phospho T389-S6K1 (pS6K), anti-total S6K (S6K), anti-total AKT (AKT), anti phospho S473-AKT (pAKT), and β-actin as a loading control. Combo, erlotinib + RAD001.