Phosphorylation-independent mTORC1 inhibition by the autophagy inducer Rottlerin

• Rottlerin inhibits proliferation and causes autophagic death in apoptosis-resistant MCF-7 breast cancer cells.
• Autophagy is induced by PKCδ-, PP2A, - ERK-, PI3K/AKT-, Bcl2/Beclin- and AMPK-independent mTORC1 inhibition.
• Rottlerin inhibits mTORC1 but not mTORC2 activity.
• Ex vivo pull-down assay demonstrated that Rottlerin directly binds mTOR.

We recently found that Rottlerin not only inhibits proliferation but also causes Bcl-2- and Beclin 1-independent autophagic death in apoptosis-resistant breast adenocarcinoma MCF-7 cells. Having excluded a role for canonical signaling pathways, the current study was aimed to investigate the contribution of the AMPK/mTOR axis in autophagy induction and to search for the upstream signaling molecules potentially targeted by Rottlerin. Using several enzyme inhibitors, Western blotting analysis, mTOR siRNA and pull down assay, we demonstrate that the Rottlerin-triggered autophagy is mediated by inhibition of mTORC1 activity through a novel AMPK and mTORC1 phosphorylation-independent mechanism, likely mediated by the direct interaction between Rottlerin and mTOR.