The mTOR pathway inhibitor RAD001 (everolimus) is highly efficacious in tamoxifen-sensitive and -resistant breast cancer xenografts

Abstract

The rapamycin derivative RAD001 (everolimus) is presently in clinical trials. Preclinical studies have suggested preferential activity in antiestrogen resistant breast cancer cells. We investigated the response of breast cancer xenografts with different tamoxifen (TAM) sensitivity towards RAD001 and analyzed the regulatory machinery as well as the cross-talk between different signaling pathways. The ERα-positive and TAM-sensitive patient-derived breast carcinoma model 3366, its TAM-resistant counterpart 3366/TAM and 4049, a breast cancer with inherent TAM-resistance, were transplanted to immunodeficient nude mice and treated with RAD001 or TAM or the combination of both compounds. Shock frozen tumors were prepared for Western Blot and immunohistochemical analysis to semi-quantitatively evaluate the expression of the ERα and the ERα regulated IGF-IR as well as PTEN, pAkt, mTOR, (phospho)-p70S6K, (phospho)-4E-BP1 and cyclin D1. RAD001 significantly inhibited the growth of tamoxifen responding and non-responding xenografts. The highest efficacy was found for the combined treatment with TAM and RAD001. RAD001 modified the protein expression of mTOR and its downstream molecule 4E-BP1 as well as the level of PTEN and ERα, but independent of the tumors sensitivity towards TAM. The protein kinase Akt was found in the active phosphorylated form (pAkt) only in TAM-resistant xenografts, but not detectable in the TAM-responding 3366 line. All treatment modalities down-regulated pAkt expression in the TAM-resistant tumors. p70S6K and IGF-IR proteins were not significantly influenced by RAD001 treatment. Our findings document the linkage between different growth-controlling pathways. Due to its capability to be active in a TAM-resistant in vivo model, RAD001 could potentially serve as a promising second-line therapy in breast cancer.