The mammalian target of rapamycin (mTOR) is a serine/threonine kinase and a key enzyme in the PI3K/AKT pathway which regulates cell growth, survival, metabolism and migration. mTOR interacts with several proteins to form two different complexes: mTORC1 (mTOR, Raptor and GBL) and mTORC2 (mTOR, Rictor, GBL and mSin1). mTORC1 regulates protein translation initiation via phosphorylation of ribosomal protein p70S6K and 4E-BP1. Both of these proteins in turn regulate translation of specific proteins involved in ribosome biogenesis, cell cycle, and apoptosis. In contrast to mTORC1, which is inhibited by rapamycin, mTORC2 is rapamycin-insensitive and was recently identified as the PDK2 responsible for phosphorylation / activation of AKT at Ser473. mTORC2 also regulates cytoskeleton organization. To further evaluate the different effects of inhibiting mTORC1 and mTORC2 on tumor cell growth and survival, we used small interfering RNAs (siRNAs) to down-regulate individual components of mTOR complexes. mRNA expression for mTOR and the associated proteins (Rictor, Raptor and GBL) was knocked down in the prostate cancer cell line PC3 (PTEN-null) and DU145 (PTEN wt) using specific siRNAs. Reduction in mRNA levels of all genes examined by ~80% was confirmed by real-time PCR and reduction for each corresponding protein by 80~90% was confirmed by Western blot analysis. The phosphorylation level of mTORC1 substrate, p70S6KT389, was reduced by ~90% after knocking down the components of mTORC1 (mTOR, Raptor) or treatment with rapamycin. The phosphorylation level of mTORC2 substrate, AKTS473, was reduced by 80~90% after knocking down the components of mTORC2 (mTOR, Rictor or GBL), but not Raptor or treatment with rapamycin. Furthermore, knockdown of mTOR or Rictor caused dramatic morphological changes in PC3 cells. Knockdown of mTOR, significantly reduced PC-3 cell growth rate by >80%, while knockdown of raptor or rictor individually had a less dramatic effect on proliferation. Our data suggest that inhibition of both rapamycin-sensitive pathway (mTORC1) and rapamycin-insensitive pathway (mTORC2) will have a more profound impact on tumor cell growth than inhibition of either pathway alone.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA