Abstract B15: Targeted inhibition of mTOR signaling attenuates the growth and progression of colorectal cancer

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the USA. The mammalian target of rapamycin (mTOR) acts downstream of PI3K to regulate cell growth, proliferation and survival. The mTOR kinase nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR‐RAPTOR complex (mTORC1) regulates translation initiation through the effectors S6K and 4E‐BP1, while the mTOR‐RICTOR‐mSin1 complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin inhibits the kinase activity of mTORC1; prolonged rapamycin treatment also inhibits mTORC2 assembly and Akt activation in certain cells. Furthermore, rapamycin mediated mTORC1 inhibition leads to loss of mTOR‐S6K‐IRS1 dependent feedback inhibition, thereby resulting in increased Akt activation. The purpose of the present study was to determine: (i) sensitivity of CRC cells to rapamycin treatment, and (ii) effect of targeting mTORC1 and mTORC2 upon CRC proliferation, apoptosis, cell cycle progression, migration, invasion and xenograft growth. Methods: HCT116, KM20, Caco‐2 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with siRNA/shRNA directed against mTOR, RAPTOR or RICTOR. Effects on cell proliferation (measured by Coulter counter cell counts), apoptosis (measured by detecting the level of histone‐associated DNA fragments), cell cycle progression (measured by fluorescence‐activated cell sorting), migration (measured by wound healing assay), invasion (measured by transwell matrigel assay) and subcutaneous growth were analyzed. Results: (i) Immunohistochemical analysis showed that the mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in primary CRCs and matched liver metastases compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the proliferation of HCT116 and KM20 cells (rapamycin sensitive); however, rapamycin did not alter SW480 or Caco‐2 proliferation (rapamycin resistant). (iii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116 and SW480 cells. (iv) Transient siRNA‐mediated knockdown of RAPTOR decreased proliferation of KM20 cells only, while knockdown of RICTOR decreased proliferation of all four cell lines. (v) Stable shRNA‐mediated knockdown of mTORC1 and mTORC2 components leads to decreased proliferation, increased apoptosis and inhibition of G1‐S phase cell cycle progression in HCT116 cells. (vi) Stable shRNA-mediated knockdown of mTORC1 and mTORC2 leads to decreased proliferation, but only mTORC2 knockdown leads to increased apoptosis in SW480 cells. (vii) Targeted inhibition of mTORC1 and mTORC2 attenuates growth of rapamycin‐sensitive and — resistant CRCs in xenografted nude mice. Conclusions: The mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in CRCs. Transient inhibition of mTORC2, but not mTORC1, decreased the proliferation of rapamycin‐sensitive and —resistant CRC cells. Stable inhibition of mTORC2 decreased proliferation and induced apoptosis in rapamycin‐sensitive and —resistant CRC cells. Finally, targeted inhibition of both mTORC1 and mTORC2 attenuates subcutaneous growth of CRCs in vivo . Thus, inhibition of mTORC2 activity represents a novel therapeutic strategy for treatment of rapamycin‐sensitive and, more importantly, rapamycin‐resistant CRCs. Citation Information: Cancer Res 2009;69(23 Suppl):B15.