**5. Inhibition of NB cell proliferation by mTOR inhibitor**

The PI3K/Akt/mTOR signaling cascade is one of the most important intracellular pathways, which is frequently activated in diverse cancers [38, 39]. The PI3K/Akt/ mTOR pathway can be activated by transmembrane tyrosine kinase growth factor receptors, such as IGF-1 receptor, fibroblast growth factor receptors, ErbB family receptors, and others [40, 41]. Both mTOR S2448 and mTOR S2481 were extensively phosphorylated in NB cell lines [13]. Cell proliferation of NB cell lines was inhibited by AZD8055, a potent dual mTORC1-mTORC2 inhibitor [13]. According to the IC50, the NB cell lines were divided into two groups, sensitive to AZD8055 group (IC50 < 0.5 μM) and insensitive to AZD8055 group (IC50 > 0.5 μM) [13]. We also found that insensitive group showed lower mTOR (p < 0.001) expression and lower activity of mTOR complex 1 (p = 0.013) and mTOR complex 2 (p = 0.023) [13]. Cell cycle distribution analysis of NB cell lines was performed by flow cytometry. Cell cycle was affected with an increase in G0/G1 phase in dose-dependent manner of AZD8055 [13]. Western blotting analysis revealed that Cyclin D1 and Cyclin D3 were downregulated in AZD8055-treated NB cells [13]. AZD8055 inhibited both mTOR S2448 and mTOR S2481 phosphorylation significantly in a concentration-dependent manner [13]. Phosphorylation of downstream targets of mTOR complex 1, P70S6K T389 and 4E-BP1 S65, was also inhibited by AZD8055 treatment [13]. AZD8055 inhibited mTOR complex 2 substrates, Akt S473 and Akt T308 [13]. Although phosphorylation of Akt S473 was persistently inhibited in response to AZD8055 treatment, phosphorylation of Akt at the T308 site was inhibited for only 3–6 hours. These results indicate that AZD8055 inhibits mTOR activity and its downstream proteins in vitro in NB cells. Interestingly, NB cell lines were induced autophagy by AZD8055 treatment via downregulation of Akt/mTOR signaling pathway [13]. Autophagy inhibitor, 3-methyladenine, treatment resulted in a significant decrease of the AZD8055-induced apoptosis [13]. These results suggest that AZD8055 inhibited cell growth and induced cell cycle arrest, autophagy, and apoptosis in NB cells. Moreover, NB tumor growth in athymic nude mice was significantly inhibited by AZD8055 without toxicity [13]. Taken together, our results highlight that mTOR is a promising target for NB treatment. AZD8055 might be investigated for treatment of patients with advanced and refractory NB.
