Inhibition of the serine-threonine kinase mammalian target of rapamycin (mTOR) has been reported to enhance the efficacy of several cytotoxic agents. Recent findings suggest that mTOR may be involved in a signaling pathway activated by microtubule-damaging drugs. The PI3K and AKT kinase pathway is a direct upstream regulator of mTOR. Using cells containing constitutively active myristoylated AKT it has been shown that AKT markedly increases resistance to microtubule-directed agents and a significant component of this AKT-mediated protection is mTOR-dependent. Other downstream targets of mTOR include the insulin receptor substrate-1 (IRS-1), a key adapter transmitting signals from activated insulin/insulin-like growth factor-1 receptors (IGF-1R). When IRS-1 is phosphorylated on a serine by mTOR, it dissociates from IGF-1R resulting in its proteasomal degradation. Also, recent data has shown that a recombinant humanized anti-IGF-1R antibody enhances the antitumor activity of vinorelbine in a non small cell lung cancer cell line. The aim of this study was to determine the nature of the interactions between mTOR inhibitors and microtubule-directed agents. Experimental Design: MCF-7 breast cancer cells were exposed for 72 hours either to the mTOR inhibitor rapamycin, at concentrations ranging from 0.1 to 100 (nM), the microtubule-directed agent Vinorelbine, at concentrations ranging from 0.01 to 10 (μg/ml) or simultaneous combination of both agents at a fixed ratio . Growth inhibition was measured using the MTT assay and synergistic interactions between the drugs were measured using the median-effect plot analysis and calculations of the combination index (CI) by the method of Chou and Talalay. Expression of AKT, PTEN and IGF-1R was assessed using western blot analysis. Results: Treatment with rapamycin or vinorelbine, as single agents, induced dose dependent growth inhibition. Synergistic interaction was obtained with the combination of rapamycin and vinorelbine, as evaluated by median-effect plot analysis and a combination index isobologram method (CI < 1). Preliminary mechanistic studies showed that vinorelbine, as single agent, caused a dose-dependent decrease of the intracellular levels of AKT and IGF-IR. Its combination with rapamycin caused a further decrease of these levels. Rapamycin, as sinle agent, did not show any effect on the expression of the above proteins. Conclusions: Rapamycin synergistically potentiates the in vitro cytotoxicity of vinorelbine in breast cancer cells. Further studies are undertaken to elucidate the molecular mechanisms involved in these interactions. Our results suggest that the combination of vinorelbine with an mTOR inhibitor may have beneficial therapeutic effects in patients with breast cancer.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]