CCI-779 Inhibits Rhabdomyosarcoma Xenograft Growth by an Antiangiogenic Mechanism Linked to the Targeting of mTOR/Hif-1α/VEGF Signaling 1

Angiogenesis is one of the critical steps in tumor growth and metastasis. The goal of this study was to evaluate whether the antitumor activity of CCI-779 is related to antiangiogenic effects in vivo in tumors of mice bearing human rhabdomyosarcoma (RMS) xenografts. We now demonstrate that CCI-779 rapidly inhibits mTOR activity, as indicated by S6 reduction and eukaryotic initiation factor 4E-binding protein 1 (4E-BPi) phosphorylation in two xenograft models of RMS within 24 hours of treatment. Treatment with a single 20-mg/kg dose of CCI-779 suppressed S6 phosphorylation for more than 72 hours and 4E-BPi phosphorylation for more than 96 hours. Based on these data, an intermittent treatment schedule (every 3 days for 30 days) was chosen and displayed a significant suppression of both tumor growth and mTOR signaling. Western blot analysis and immunohistochemical studies demonstrated that the antitumor activity of CCI-779 was associated with antiangiogenesis, as indicated by impaired levels of hypoxiainducible factor-1α (Hif-1α) and vascular endothelial growth factor (VEGF) protein expression and by decreased microvessel density in Rh30 and RD xenografts. Together, these data suggest that CCI-779 inhibits human RMS xenograft growth by an antiangiogenic mechanism associated with the targeting of mTOR/Hif-1α/VEGF signaling.