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. 2013 Dec 6;288(49):35287-96.
doi: 10.1074/jbc.M113.495986. Epub 2013 Oct 16.

Pan-mammalian target of rapamycin (mTOR) inhibitor AZD8055 primes rhabdomyosarcoma cells for ABT-737-induced apoptosis by down-regulating Mcl-1 protein

Ellen Preuss  1 , Manuela Hugle, Romy Reimann, Marcel Schlecht, Simone Fulda
Affiliations

Pan-mammalian target of rapamycin (mTOR) inhibitor AZD8055 primes rhabdomyosarcoma cells for ABT-737-induced apoptosis by down-regulating Mcl-1 protein

Ellen Preuss et al. J Biol Chem. .

Abstract

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.

Keywords: Apoptosis; Cancer; Cell Death; Rhabdomyosarcoma; mTOR.

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Figures

FIGURE 1.
FIGURE 1.
AZD8055 and ABT-737 cooperate to induce cell death in RMS cells. RMS cell lines were treated for 48 h with 1 μm AZD8055 and/or indicated concentrations of ABT-737. Cell death was determined by PI staining and flow cytometry (A), and corresponding CI values are shown for 1 μm AZD8055 and 1 μm ABT-737 (B). In C, apoptosis was determined by analysis of DNA fragmentation of PI-stained nuclei. Mean + S.D. of three independent experiments performed in triplicate are shown; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 2.
FIGURE 2.
AZD8055 and ABT-737 cooperate to trigger caspase activation, mitochondrial perturbations and caspase-dependent apoptosis. TE671 and RMS13 cells were treated for indicated times (A and D) or for 48 h (B and C) with 1 μm AZD8055 and/or 1 μm ABT-737 in the presence or absence of 50 μm Z-VAD-fmk. In A, caspase activation and PARP cleavage were assessed by Western blotting; arrows indicate cleavage fragments, and an asterisk indicates unspecific bands. In B, cell death was determined by PI staining and flow cytometry. In C, apoptosis was determined by analysis of DNA fragmentation of PI-stained nuclei. In D, loss of mitochondrial membrane potential (MMP) was analyzed by flow cytometry using the dye tetramethylrhodamine methyl ester. Mean + S.D. of three independent experiments performed in triplicate are shown (B–D); **, p < 0.01; ***, p < 0.001.
FIGURE 3.
FIGURE 3.
Effect of AZD8055 on PI3K/mTOR pathway activation. TE671 and RMS13 cells were treated for indicated times with 1 μm AZD8055 (AZD) and/or 1 μm ABT737 (ABT). Protein expression of phospho-Akt (Ser-473), Akt, phospho-S6 ribosomal protein, S6 ribosomal protein, phospho-4E-BP1, and 4E-BP1 was analyzed by Western blotting.
FIGURE 4.
FIGURE 4.
Effect of PI3K/mTOR inhibitors on ABT-737-induced apoptosis. TE671 and RMS13 cells were treated for 48 h (A and B) or 6 h (C) with indicated concentrations of ABT-737 and/or AZD8055, RAD001, NVP-BEZ235, or NVP-BKM120. In A, apoptosis was determined by analysis of DNA fragmentation of PI-stained nuclei. Mean + S.E. of three independent experiments performed in triplicate are shown. In B, corresponding CI values are shown for combinations affecting > 20% of the fraction (1 μm ABT-737 plus 1 μm AZD8055 or 1 μm NVP-BEZ235 or 1 μm NVP-BKM120). In C, protein expression of phospho-Akt (Ser-473), Akt, phospho-S6 ribosomal protein, S6 ribosomal protein, phospho-4E-BP1, and 4E-BP1 was analyzed by Western blotting.
FIGURE 5.
FIGURE 5.
Down-regulation of Mcl-1 by mTORC1/2 or PI3K inhibition. A, TE671 and RMS13 cells were treated for indicated times with 1 μm AZD8055 and/or 1 μm ABT-737. Expression of pro- and antipoptotic proteins was analyzed by Western blotting. B, TE671 and RMS13 cells were treated for 6 h with 1 μm ABT-737 and/or 1 μm AZD8055 or 1 μm NVP-BEZ235 or 1 μm NVP-BKM120 or 1 μm RAD001. Expression of Mcl-1 protein was analyzed by Western blotting.
FIGURE 6.
FIGURE 6.
Down-regulation of Mcl-1 sensitizes for ABT-737-induced cell death. A–D, TE671 cells were stably transduced with two different shRNA sequences against Mcl-1 or control shRNA. In A, Mcl-1 expression was determined by Western blot analysis. In B and C, cells were treated for 48 h with 1 μm AZD8055 and/or 1 μm ABT-737, and cell death was determined by PI staining and flow cytometry (B) or apoptosis was determined by analysis of DNA fragmentation of PI-stained nuclei (C). Mean + S.D. of three independent experiments performed in triplicate are shown; **, p < 0.01; ***, p < 0.001. In D, cells were treated for 6 h with 1 μm AZD8055 and/or 1 μm ABT-737, and caspase activation was assessed by Western blotting; arrows indicate caspase cleavage fragments. E and F, TE671 cells were transiently transfected with three distinct siRNA against Mcl-1 (siMcl-1_1, siMcl-1_2, siMcl-1_3), or control siRNA (siControl). Mcl-1 expression was analyzed by Western blotting (E). Apoptosis was determined by analysis of DNA fragmentation of PI-stained nuclei and flow cytometry (F). Mean + S.D. of three independent experiments performed in triplicate are shown; *, p < 0.05; ***, p < 0.001.
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