Inhibition of mTOR and targeting insulin-like growth factor I synergistically enhance taxol-induced cytotoxicity in estrogen receptor positive breast cancer cells.

A54

Background: Estrogen receptors (ER) are expressed in approximately 65% of human breast cancer. Cumulative data from clinical trials suggest that some chemotherapeutic agents, such as Paclitaxel, may be less effective in patients with ER+ tumors than those with ER- tumors. Specifically, ERα mediates breast cancer resistance to paclitaxel through inhibition of apoptotic cell death in MCF-7 cell line (Sui M, Cancer Res, 2007). In addition, in hormone-dependent breast cancer cells, the insulin-like growth factor I receptor (IGF-IR) is overexpressed and engaged in a powerful functional cross-talk with the ER signaling pathway, leading to the activation of the phosphatidylinositol 3’-kinase (PI3K)/Akt and ERK, and the downstream mammalian target of rapamycin (mTOR). The IGF binding protein-3 (IGFBP-3) limits IGF bioavailability and its interaction with the receptor. IGF-IR signaling blockade prevents rapamycin (a specific inhibitor of mTOR)-inducedAkt activation and sensitizes tumor cells to inhibition of mTOR.
 This study was specifically designed to explore the ability of recombinant human IGFBP-3 (rhIGFBP-3) and rapamycin to enhance paclitaxel efficacy in ER + breast cancer cells.
 
 Material and methods: MCF-7 cells were treated with paclitaxel (1-100 nM) alone or in combination with rhIGFBP-3 (20µg/ml) and rapamycin (10 & 100 nM) for 72h. Cell survival was measured using the MTT assay. For Western blot analysis, MCF-7 cells were grown in serum-free medium for 24h and subsequently treated with IGF-I (50ng/ml), rhIGFBP-3 (20µg/ml), rapamycin (10nM) alone or in combination with paclitaxel (25nM) for 30min.
 
 Results: Treatment with taxol, rhIGFBP-3 and rapamycin, as single agents, inhibited the growth of MCF-7/Her2-18 cells in a dose-dependent manner. The IC50s were 7.5 nM, 20 µg/ml and 10 nM, respectively. The combination of various concentrations of paclitaxel with a constant dose of rhIGFBP-3 (20 µg/ml) or rapamycin (10nM) exhibited greater dose-dependent inhibition of cell proliferation than did each agent alone. The triple combination of paclitaxel, rhIGFBP-3 and rapamycin resulted in a greater enhancement of paclitaxel cytotoxicity. Synergistic interactions were obtained with the combinations of paclitaxel + rhIGFBP-3, paclitaxel + rapamycin and the triple combination as evaluated by the isobologram method (CI < 1). Western blot analysis revealed that the triple combination caused a further decrease of p-ERα, p-IGF-IR and the downstream p-AKT and p-MAPK.
 
 Conclusions: The different combinations showed strong synergistic interactions in the MCF-7 breast cancer cells, associated with a decrease in ER and IGF-IR signaling. Furthermore, we demonstrated that, rhIGFBP-3 abrogated rapamycin and paclitaxel-induced AKT and MAPK activation. The effects of these combinations on mTOR downstream components, 4E-BP1 and S6K1, apoptosis using FACS analysis, caspase activation, and cell cycle progression will be presented at the meeting.